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1. A process for the preparation of L-amino acids, in particular L-threonine, which comprises carrying out the following steps: a) fermentation of microorganisms of the Enterobacteriaceae family which produce the desired L-amino acid and in which one or more of the genes chosen from the group consisting of sucC and sucD, or nucleotide sequences which code for these, is or are enhanced, in particular over-expressed, b) concentration of the desired L-amino acid in the medium or in the cells of the microorganisms, and c) isolation of the desired L-amino acid, constituents of the fermentation broth and/or the biomass in its entirety or portions (>0 to 100%) thereof optionally remaining in the product. 2. A process as claimed in claim 1, wherein microorganisms in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally enhanced are employed. 3. A process as claimed in claim 1, wherein microorganisms in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated are employed. 4. A process as claimed in claim 1, wherein the expression of the polynucleotide (s) which code(s) for one or more of the genes chosen from the group consisting of sucC and sucD is increased. 5. A process as claimed in claim 1, wherein the regulatory and/or catalytic properties of the polypeptides (proteins) for which the polynucleotides sucC and sucD code are improved or increased. 6. A process as claimed in claim 1, wherein, for the preparation of L-amino acids, microorganisms of the Enterobacteriaceae family in which in addition at the same time one or more of the genes chosen from the group consisting of: 6.1 the thrABC operon which codes for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase, 6.2 the pyc gene which codes for pyruvate carboxylase, 6.3 the pps gene which codes for phosphoenol pyruvate synthase, 6.4 the ppc gene which codes for phosphoenol pyruvate carboxylase, 6.5 the pntA and pntB genes which code for transhydrogenase, 6.6 the rhtB gene which imparts homoserine resistance, 6.7 the mgo gene which codes for malate:guinone oxidoreductase, 6.8 the rhtC gene which imparts threonine resistance, 6.9 the thrE gene which codes for the threonine export protein, 6.10 the gdhA gene which codes for glutamate dehydrogenase, 6.11 the hns gene which codes for the DNA-binding protein HLP-II, 6.12 the pgm gene which codes for phosphoglucomutase, 6.13 the fba gene which codes for fructose biphosphate aldolase, 6.14 the ptsH gene which codes for the phosphohistidine protein hexose phosphotransferase, 6.15 the ptsi gene which codes for enzyme I of the phosphotransferase system, 6.16 the crr gene which codes for the glucose-specific IIA component, 6.17 the ptsG gene which codes for the glucose-specific IIBC component, 6.18 the lrp gene which codes for the regulator of the leucine regulon, 6.19 the mopB gene which codes for 10 Kd chaperone, 6.20 the ahpC gene which codes for the small sub-unit of alkyl hydroperoxide reductase, 6.21 the ahpF gene which codes for the large sub-unit of alkyl hydroperoxide reductase, 6.22 the cysK gene which codes for cysteine synthase A, 6.23 the cysB gene which codes for the regulator of the cys regulon, 6.24 the cysJ gene which codes for the flavoprotein of NADPH sulfite reductase, 6.25 the cysi gene which codes for the haemoprotein of NADPH sulfite reductase, 6.26 the cysH gene which codes for adenylyl sulfate reductase, 6.27 the phoE gene which codes for protein E of outer cell membrane, 6.28 the malE gene which codes for the periplasmic binding protein of maltose transport, 6.29 the pykF gene which codes for fructose-stimulated pyruvate kinase I, 6.30 the pfkB gene which codes for 6-phosphofructokinase II, 6.31 the talB gene which codes for transaldolase B, 6.32 the rsea gene which codes for a membrane protein which acts as a negative regulator on sigmaE activity, 6.33 the rseC gene which codes for a global regulator of the sigmaE factor, 6.34 the soda gene which codes for superoxide dismutase, 6.35 the phoB gene which codes for the positive regulator PhoB of the pho regulon, 6.36 the phoR gene which codes for the sensor protein of the pho regulon, 6.37 the sucA gene which codes for the decarboxylase sub-unit of 2-ketoglutarate dehydrogenase, 6.38 the sucB gene which codes for the dihydrolipoyltranssuccinase E2 sub-unit of 2-ketoglutarate dehydrogenase is or are enhanced, in particular over-expressed, are fermented. 7. A process as claimed in claim 1, wherein, for the preparation of L-amino acids, microorganisms of the Enterobacteriaceae family in which in addition at the same time one or more of the genes chosen from the group consisting of: 7.1 the tdh gene which codes for threonine dehydrogenase, 7.2 the mdh gene which codes for malate dehydrogenase, 7.3 the gene product of the open reading frame (orf) yjfA, 7.4 the gene product of the open reading frame (orf) ytfP, 7.5 the pckA gene which codes for phosphoenol pyruvate carboxykinase, 7.6 the poxB gene which codes for pyruvate oxidase, 7.7 the aceA gene which codes for isocitrate lyase, 7.8 the dgsA gene which codes for the DgsA regulator of the phosphotransferase system, 7.9 the fruR gene which codes for the fructose repressor, 7.10 the rpos gene which codes for the sigma38 factor is or are attenuated, in particular eliminated or reduced in expression, are fermented. |
<SOH> FIELD OF THE INVENTION <EOH>This invention relates to a process for the preparation of L-amino acids, in particular L-threonine, using strains of the Enterobacteriaceae family in which at least one or more of the genes chosen from the group consisting of sucC and sucD is (are) enhanced. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a process for the fermentative preparation of L-amino acids, in particular L-threonine, using microorganisms of the Enterobacteriaceae family which in particular already produce L-amino acids and in which at least one or more of the nucleotide sequence(s) which code(s) for the sucC and sucD genes is (are) enhanced. detailed-description description="Detailed Description" end="lead"? |
Bipolar plate of fuel cell |
In a bipolar plate of a fuel cell including a plate having a certain area and thickness; inflow and outflow buffer grooves respectively formed at both sides of the plate so as to have a certain area and depth; plural channels for connecting the inflow buffer groove and the outflow buffer groove; plural buffer protrusions formed in the inflow and outflow buffer grooves so as to have a certain height; an inflow path formed on the plate so as to be connected to the inflow buffer groove; and an outflow path formed on the plate so as to be connected to the outflow buffer groove, it is possible to uniformize flux distribution and reduce flow resistance of fuel and air respectively flowing into a fuel electrode and an air electrode of a fuel cell. |
1. A bipolar plate of a fuel cell, comprising: a plate having a certain area and thickness; inflow and outflow buffer grooves respectively formed at both sides of the plate so as to have a certain area and depth; plural channels for connecting the inflow buffer groove and the outflow buffer groove; an inflow path formed on the plate so as to be connected to the inflow buffer groove; and an outflow path formed on the plate so as to be connected to the outflow buffer groove. 2. The bipolar plate of claim 1, wherein the channels are linearly formed. 3. The bipolar plate of claim 2, wherein channel width is increased gradually from a channel arranged on the middle to a channel arranged on the edge. 4. The bipolar plate of claim 2, wherein width of the channels is uniform, and a projected buffer portion is formed at an inlet side of each channel so as to reduce a width of the inlet. 5. The bipolar plate of claim 1, wherein the inflow path and the outflow path is respectively constructed as at least one through hole. 6. The bipolar plate of claim 1, wherein the inflow path and the outflow path are formed at a side of the plate. 7. The bipolar plate of claim 1, wherein a distribution means is formed in the inflow path in order to give flow resistance to a fluid flowing into the inflow path. 8. The bipolar plate of claim 7, wherein the distribution means is formed as a shape having an area corresponded to the section of the inflow path and a certain thickness, and it is made of a porous material. 9. A bipolar plate of a fuel cell, comprising: a plate having a certain area and thickness; inflow and outflow buffer grooves respectively formed at both sides of the plate so as to have a certain area and depth; plural channels for connecting the inflow buffer groove and the outflow buffer groove; plural buffer protrusions formed in the inflow and outflow buffer grooves so as to have a certain height; an inflow path formed on the plate so as to be connected to the inflow buffer groove; and an outflow path formed on the plate so as to be connected to the outflow buffer groove. 10. The bipolar plate of claim 9, wherein the buffer protrusions are linearly arranged between the channels. 11. The bipolar plate of claim 9, wherein the buffer protrusions are linearly arranged on the channels. 12. The bipolar plate of claim 9, wherein the buffer protrusions are irregularly arranged. 13. The bipolar plate of claim 9, wherein the buffer protrusions have the same height, and the height of the buffer protrusion is the same with the depth of the inflow buffer groove or the outflow buffer groove. 14. The bipolar plate of claim 9, wherein the buffer protrusion has a rectangular section. 15. The bipolar plate of claim 9, wherein the channels are linearly formed. 16. The bipolar plate of claim 15, wherein channel width is increased gradually from a channel arranged on the middle to a channel arranged on the edge. 17. The bipolar plate of claim 15, wherein width of the channels is uniform, and a projected buffer portion is formed at an inlet side of each channel so as to reduce a width of the inlet. 18. The bipolar plate of claim 9, wherein the length of the inflow buffer groove and the outflow buffer groove is not less than ⅕ of the length of the channel. 19. The bipolar plate of claim 9, wherein a distribution means is formed in the inflow path in order to give flow resistance to a fluid flowing into the inflow path. 20. The bipolar plate of claim 19, wherein the distribution means is formed as a shape having an area corresponded to the section of the inflow path and a certain thickness, and it is made of a porous material. |
<SOH> BACKGROUND ART <EOH>A fuel cell is generally environment-friendly energy, and it has been developed in order to substitute for the conventional fossil energy. As depicted in FIG. 1 , the fuel cell includes a stack to be combined with at least one unit cell 11 in which electron-chemical reaction occurs; a fuel supply pipe 20 connected to the stack 10 so as to supply fuel; an air supply pipe 30 connected to the stack 10 so as to supply air; and discharge pipes 40 , 50 for discharging by-products of fuel and air passing the reaction respectively. The unit cell 11 includes a fuel electrode (anode) (not shown) in which fuel flows; and an air electrode (cathode) (not shown) in which air flows. The operation of the fuel cell will be described. First, fuel and air are supplied to the fuel electrode and the air electrode of the stack 10 through the fuel supply pipe 20 and the air supply pipe 30 respectively. Fuel supplied to the fuel electrode is ionized into positive ions and electrons (e-) through electrochemical oxidation reaction in the fuel electrode, the ionized positive ions are moved to the air electrode through an electrolyte layer, and the electrons are moved to the fuel electrode. The positive ions moved to the air electrode perform electrochemical reduction reaction with air supplied to the air electrode and generate by-products such as reaction heat and water, etc. In the process, by the movement of the electrons, electric energy is generated. The fuel through the reaction in the fuel electrode, and water and additional by-products generated in the air electrode are respectively discharged through the discharge lines 40 , 50 . The fuel cell can be classified into various types according to kinds of electrolyte and fuel, etc. used therein. In the meantime, as depicted in FIG. 2 , the unit cell 11 constructing the stack 10 includes two bipolar plates 100 having an open channel 101 in which air or fuel flows; and a M.E.A (membrane electrode assembly) 110 arranged between the two bipolar plates 100 so as to have a certain thickness and area. The two bipolar plates 100 and the M.E.A 110 arranged therebetween are combined with each other by additional combining means 120 , 121 . A channel formed by a channel 101 of the bipolar plate 100 and a side of the M.E.A 110 constructs a fuel electrode, and oxidation reaction occurs while fuel flows through the channel of the fuel electrode. And, a channel formed by a channel 101 of the other bipolar plate 100 and the other side of the M.E.A 110 constructs an air electrode, and reduction reaction occurs while air flows through the channel of the air electrode. A shape of the bipolar plate 100 , in particular, a shape of the channel 101 affects contact resistance generated in flowing of fuel and air and flux distribution, etc., and contact resistance and flux distribution affect power efficiency. And, the bipolar plates 100 have a certain shape appropriate to processing facilitation and mass production. As depicted in FIG. 3 , in the conventional first bipolar plate, through holes 131 , 132 , 133 , 134 are respectively formed at each edge of the plate 130 having a certain thickness and a rectangular shape. Among the four through holes, the diagonally arranged two through holes 131 , 133 are fuel paths, and the diagonally arranged two through holes 132 , 134 are air paths. Hexagonal channel 135 in which a fluid flows is respectively formed at both sides of the plate 130 , and plural straight channels 136 are horizontally formed along the whole internal area of the hexagonal channel 135 . And, the hexagonal channel 135 formed at the side of the plate 130 and the plural straight connection channels 136 are connected to the diagonally arranged two through holes 131 , 133 through plural straight channels 137 . And, the hexagonal channel 135 formed at the other side of the plate 130 and the plural straight channels 136 are connected to the diagonally arranged two through holes 132 , 134 through plural straight connection channels 137 . In more detail, in the plate 130 , fuel flows on the side, and air flows on the other side. FIG. 3 is a plane view illustrating a side of the conventional bipolar plate. The operation of the conventional bipolar plate will be described. Fuel or air flows into the through holes 131 , 132 , the fuel or air flows into the hexagonal channel 135 and the plural straight channels 136 through the connection channels 137 , and it flows into the connection channels at the other side. The fuel or air flowing into the connection channels 137 are discharged through the through holes 133 , 134 at the other side. In the meantime, in another structure of the conventional second bipolar plate, as depicted in FIG. 4 , through holes 141 , 142 , 143 , 144 are respectively formed at edges of the plate 140 having a certain thickness and a rectangular shape. And, curved plural channels 145 are formed on a side of the plate 140 so as to connect the diagonally arranged two through holes 141 , 143 . And, curved plural channels 145 are formed on the other side of the plate 140 so as to connect the diagonally arranged two through holes 142 , 144 . The operation of the second bipolar plate will be described. Fuel and air respectively flow into the through holes 141 , 142 , fuel or air respectively flowing into the through holes 141 , 142 passes the plural channels 145 and is discharged through the other through holes 143 , 144 . However, in the conventional first bipolar plate, because the number of the connection channels 137 for connecting the through holes 131 , 132 , 133 , 134 , the hexagonal channel 135 and the straight channels 136 is very little in comparison with the number of the straight channels 136 formed in the hexagonal channel, flux distribution of a fluid flowing into the through holes 131 , 132 is not good, and it is inappropriate to using the conventional first bipolar plate in flowing of great amount of fluid. In the meantime, in the conventional second bipolar plate, because the channels 145 of fuel and air are formed as a curved shape, flow resistance is increased in flowing of fuel and air, and accordingly pressure loss for flowing the fluid is increased. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention. In the drawings: FIG. 1 shows the conventional fuel cell system; FIG. 2 is an exploded-perspective view illustrating a stack of the conventional fuel cell; FIG. 3 is a plane view illustrating an example of a bipolar plate of the conventional fuel cell; FIG. 4 is a plane view illustrating another example of a bipolar plate of the conventional fuel cell; FIG. 5 is a plane view illustrating a first embodiment of a bipolar plate of a fuel cell in accordance with the present invention; FIG. 6 is a sectional view taken along a line A-B in FIG. 5 ; FIGS. 7 and 8 are plane views respectively illustrating channels of the bipolar plate of the fuel cell in accordance with the first embodiment of the present invention; FIG. 9 is a plane view illustrating distribution means of the bipolar plate of the fuel cell in accordance with the first embodiment of the present invention; FIG. 10 is a plane view illustrating a second embodiment of a bipolar plate of a fuel cell in accordance with the present invention; FIG. 11 is a sectional view taken along a line C-D in FIG. 10 ; FIGS. 12 and 13 are plane views respectively illustrating modifications of buffer protrusions of the bipolar plate of the fuel cell in accordance with the second embodiment of the present invention; FIGS. 14 and 15 are plane views respectively illustrating other examples of channels of the bipolar plate of the fuel cell in accordance with the second embodiment of the present invention; FIG. 16 is a plane view illustrating distribution means of the bipolar plate of the fuel cell in accordance with the second embodiment of the present invention; FIG. 17 is an exploded-perspective view illustrating a stack of the bipolar plate of the fuel cell in accordance with the second embodiment of the present invention; FIG. 18 is a plane view illustrating an operational state of the bipolar plate of the fuel cell in accordance with the first embodiment of the present invention; and FIG. 19 is a plane view illustrating an operational state of the bipolar plate of the fuel cell in accordance with the second embodiment of the present invention. detailed-description description="Detailed Description" end="lead"? |
Laminate-controlling light autonomously and window using the same |
There are provided laminated bodies or laminated body-containing windows, which comprise isotropic aqueous solutions obtained by dissolving a water-soluble polysaccharide derivative having nonionic amphipathic functional groups in an aqueous medium composed of water and an amphipathic substance, laminated between plates that are at least partially transparent and allow direct vision of the isotropic aqueous solutions, wherein there are added to the isotropic aqueous solutions in appropriate amounts ultraviolet absorbers comprising nonionic or ionic benzophenone derivatives or benzotriazole derivatives which are highly weather resistant and uniformly dissolve in the isotropic aqueous solutions. The isotropic aqueous solutions are transparent and become opaque upon irradiation with light, and exhibit stable reversible change, in order to provide vastly improved weather resistance to the laminated bodies against exposure to sunlight rays over prolonged periods of time. |
1. A laminated body comprising an isotropic aqueous solution obtained by dissolving 100 parts by weight of a water-soluble polysaccharide derivative having a weight-average molecular weight of about 10,000 to about 200,000 and having a nonionic amphipathic functional group, in about 100 to about 2000 parts by weight of an aqueous medium composed of water in an amount of about 25 to about 450 with respect to 100 parts by weight of said polysaccharide derivative and an amphipathic substance with a molecular weight of about 60 to about 5000, laminated between plates that are partially transparent and allow direct vision of said aqueous solution, wherein there is added in an amount of 0.01-10 parts by weight with respect to 100 parts by weight of said isotropic aqueous solution at least one compound selected from the group consisting of nonionic benzophenone derivatives and benzotriazole derivatives having solubility of 1 g or greater in the amphipathic substance at 20° C. and ionic benzophenone derivatives and benzotriazole derivatives with an ionic functional group bonded to the benzene ring via a chain portion and having solubility of 1 g or greater in water at 2° C. 2. A laminated body according to claim 1, wherein said nonionic benzophenone derivative or benzotriazole derivative has 2° C. solubility of 1 g or greater in the amphipathic substance polyoxypropylene trimethylolpropane having a molecular weight of about 400. 3. A laminated body according to claim 1, wherein said nonionic benzophenone derivative or benzotriazole derivative is a compound represented by the following general formula 1 or 3. (wherein R1 and R2 each represent hydrogen or hydroxyl, with at least one of R1 and R2 being hydroxyl, and R3−Rio each represent hydrogen, C1-4 alkyl, C1-4 alkoxy, hydroxyl, polyglycerin, polyethylene oxide or O—(R11)n-A (where A represents an unprotected sugar residue (a residue lacking one hydroxyl group from, for example, a monosaccharide such as glucose or galactose, a disaccharide such as trehalose or maltose or a trisaccharide such as maltotriose), and R11 represents a direct bond (n=0), C1—4 alkylene or C1—4 alkyleneoxide (where n is an integer of 1 to 6)), with at least one from among R3 to Rio being hydroxyl, polyglycerin, polyethylene oxide or 0-(R11)n-A); (wherein R1 represents hydroxyl, and R3—R6 each represent hydrogen, C1-4 alkyl, C1-4 alkoxy, hydroxyl, polyglycerin, polyethylene oxide or 0-(R11),,-A (where A represents an unprotected sugar residue (a residue lacking one hydroxyl group from, for example, a monosaccharide such as glucose or galactose, a disaccharide such as trehalose or maltose or a trisaccharide such as maltotriose), and R11 represents a direct bond (n=0), C1-4 alkylene or C1-4 alkyleneoxide (where n is an integer of 1 to 6)), with at least one from among R3 to R6 being hydroxyl, polyglycerin, polyethylene oxide or O—(R11)n-A). 4. A laminated body according to claim 1, wherein one from among R3 to R6 and one from among R, to Rio are hydroxyl groups. 5. A laminated body according to claim 4, wherein the remaining groups of R3 to R6 and R, to Rio are hydrogen atoms, methoxy groups or ethoxy groups. 6. A laminated body according to claim 1, wherein the ionic functional group is a sulfonic acid group, carboxylic acid group, phosphoric acid group or ammonium group. 7. A laminated body according to claim 1, wherein a temperature shifting agent is further added to said isotropic aqueous solution. 8. A laminated body according to claim 1, wherein two or more different isotropic aqueous solution layers are provided. 9. A laminated body according to claim 1, wherein an additional substrate is situated on at least one side to provide a gas layer. 10. A window containing a laminated body which comprises an isotropic aqueous solution obtained by dissolving 100 parts by weight of a water-soluble polysaccharide derivative having a weight-average molecular weight of about 10,000 to about 200,000 and having a nonionic amphipathic functional group, in about 100 to about 2000 parts by weight of an aqueous medium composed of water in an amount of about 25 to about 450 with respect to 100 parts by weight of said polysaccharide derivative and an amphipathic substance with a molecular weight of about 60 to about 5000, laminated between plates that are partially transparent and allow direct vision of said aqueous solution, wherein there is added in an amount of 0.01-10 parts by weight with respect to 100 parts by weight of said isotropic aqueous solution at least one compound selected from the group consisting of nonionic benzophenone derivatives and benzotriazole derivatives having solubility of 1 g or greater in the amphipathic substance at 20° C. and ionic benzophenone derivatives and benzotriazole derivatives with an ionic functional group bonded to the benzene ring via a chain portion and having solubility of 1 g or greater in water at 20° C. 11. A window according to claim 10, wherein said nonionic benzophenone derivative or benzotriazole derivative has 2° C. solubility of 1 g or greater in the amphipathic substance polyoxypropylene trimethylolpropane having a molecular weight of about 400. 12. A window according to claim 10, wherein said nonionic benzophenone derivative or benzotriazole derivative is a compound represented by the following general formula 1 or 3. (wherein R1 and R2 each represent hydrogen or hydroxyl, with at least one of R1 and R2 being hydroxyl, and R3-R10 each represent hydrogen, C1—, alkyl, C1—, alkoxy, hydroxyl, polyglycerin, polyethylene oxide or O—(R1) n-A (where A represents an unprotected sugar residue (a residue lacking one hydroxyl group from, for example, a monosaccharide such as glucose or galactose, a disaccharide such as trehalose or maltose or a trisaccharide such as maltotriose), and R II represents a direct bond (n=0), C1—, alkylene or C1—, alkyleneoxide (where n is an integer of 1 to 6)), with at least one from among R3 to Rio being hydroxyl, polyglycerin, polyethylene oxide or O—(R11)n-A); (wherein R1 represents hydroxyl, and R3-R6 each represent hydrogen, C1—, alkyl, C1—, alkoxy, hydroxyl, polyglycerin, polyethylene oxide or O-(R11)n-A (where A represents an unprotected sugar residue (a residue lacking one hydroxyl group from, for example, a monosaccharide such as glucose or galactose, a disaccharide such as trehalose or maltose or a trisaccharide such as maltotriose), and R11 represents a direct bond (n=0), C1—, alkylene or C1—, alkyleneoxide (where n is an integer of 1 to 6)), with at least one from among R3 to R6 being hydroxyl, polyglycerin, polyethylene oxide or 0-(R11),-A). 13. A window according to claim 10, wherein one from among R3 to R6 and one from among R, to R10 are hydroxyl groups. 14. A window according to claim 13, wherein the remaining groups of R3 to R6 and R, to R10 are hydrogen atoms, methoxy groups or ethoxy groups. 15. A window according to claim 10, wherein the ionic functional group is a sulfonic acid group, carboxylic acid group, phosphoric acid group or ammonium group. 16. A window according to claim 10, wherein a temperature shifting agent is further added to said isotropic aqueous solution. 17. A window according to claim 10, wherein two or more different isotropic aqueous solution layers are provided. 18. A window according to claim 10, wherein an additional substrate is situated on at least one side to provide a gas layer. |
<SOH> BACKGROUND ART <EOH>In recent years, light regulating glass capable of controlling penetration of sunlight rays has become a topic of interest for energy conservation, comfort, etc. The present specification will refer mainly to window glass to be used for windows in buildings, automobiles and the like, but the laminated bodies of the invention are widely applicable, with no limitation to windows. The present inventors focused on the fact that windows are directly exposed to sunlight rays. By effectively utilizing the temperature difference between the presence and absence of solar radiation and between seasons, it became possible to develop revolutionary self-responding light regulating laminated bodies which, when exposed to sunlight rays in the high temperature summer season, naturally become opaque and block the sunlight rays. More specifically, for example, U.S. Pat. No. 5,615,040 (corresponding to Japanese Unexamined Patent Publication HEI No. 6-255016) is cited in Journal of Japan Solar Energy Society, Taiyo Energy, Vol. 27, No. 5 (2001), pp. 14-20. The basic structure of the invention described therein is a laminated body in which an isotropic aqueous solution is sealed between a pair of plates. The isotropic aqueous solution comprises at least a water-soluble polysaccharide derivative, an amphipathic substance and water. The principle depends on a stably reversible temperature-dependent sol-gel phase transition. At low temperature, the molecules are uniformly dissolved to produce an isotropic aqueous solution (sol state), while at high temperature a phase transition occurs as the dissolved molecules aggregate into a flocculated state (gel state). In the gel state, the difference in density between the solvent and the fine aggregates creates opacity due to light scattering, thereby blocking about 80% of light. When the laminated body is used to construct a window, the transparent state is maintained to permit penetration of sunshine when the temperature of the laminated body remains lower in the winter season, while heating by direct sunlight rays during the summer season produces opacity which cuts approximately 80% of the sun's rays, thereby providing an energy-conserving, light-regulating window glass. The laminated body satisfies the following fundamental conditions also listed in the aforementioned document. 1) Phase changes between the transparent and opaque state must be reversible. 2) Reversible changes must be able to be repeated without phase separation. 3) The material must be weather resistant. This laminated body has already been tested as window glass by the present inventors, but it was found that the weather resistance needed to be further improved for it to be suitable for common use as window glass which is exposed to constant sunlight. The results of actual rooftop exposure testing in a Tokyo district using a laminated body assembled with a satisfactory sealed structure indicated an increase in the initial opacity temperature already within about 3 years, even with 5 mm-thick float glass. The present inventors diligently examined methods of adding ultraviolet absorbers to the isotropic aqueous solution and as a result succeeded in developing a laminated body exhibiting revolutionary high weather resistance having features 1) and 2) above, and adequately satisfying condition 3) above. Window glass must exhibit high weather resistance for use over long periods of 10 years or more and even 20 or 30 years. It should also be as light and thin as possible for reduced load on building frames and compatibility with window frames, as well as for more advantageous manufacturing, transport, construction and the like. The present inventors had also previously examined methods of imparting glass panels with ultraviolet-blocking functions, but because of problems of such as coloring and weight increase and the need for special working, such methods were not generally suitable. The present inventors therefore conducted more detailed examination focusing on various ultraviolet absorbers in order to vastly improve the weather resistance of the isotropic aqueous solution itself. Previously, there have existed only written references to the general concept of adding ultraviolet absorbers that dissolve in isotropic aqueous solutions for improved weather resistance (benzophenone derivatives, benzotriazole derivatives, salicylic acid ester derivatives, etc.), as also referred to by the present inventors in the aforementioned document, and the patent document mentions only Sumisorb•110S (2-hydroxy-4-methoxybenzophenone-5-sulfonic acid) by Sumitomo Chemical Co., Ltd. as a water-soluble ultraviolet absorber. We therefore tested two types of laminated bodies, comprising an isotropic aqueous solution containing no ultraviolet absorber or an isotropic aqueous solution containing Sumisorb•110S by Sumitomo Chemical Co., Ltd., by an ultraviolet exposure test as described in the examples, and found that air bubbles were generated in both cases from about 50 hours to 100 hours, producing unrecoverable irregularities. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a cross-sectional view of an embodiment of a laminated body according to the invention. FIG. 2 is a cross-sectional view of an embodiment of a laminated body of the invention having a gas layer additionally situated therein. FIG. 3 is a cross-sectional view of an embodiment of a laminated body of the invention having isotropic aqueous solution layers with different compositions. FIG. 4 is a graph showing the change in transmittance of a laminated body of the invention between the transparent state and the opaque state. detailed-description description="Detailed Description" end="lead"? |
Method and support for biological analysis using oligonucleotides comprising a marker capable of enzymatic activation |
The invention concerns a method for analyzing biological targets of DNA or RNA type, which comprises the following steps: a) contacting the targets to be analyzed (ICAM-20) with oligonucleotide probes (ISIS) labeled with a cofactor A of an enzyme E; b) adding, to the reaction medium, enzyme E corresponding to cofactor A and a substrate S of enzyme E, substrate S being converted by enzyme E into a compound C; c) measuring a signal representative of the activity of enzyme E on substrate S, for example the fluorescence intensity (I) of substrate S; and d) comparing this signal with the signal obtained when the oligonucleotide probes labeled with cofactor A (ISIS) are contacted with enzyme E and substrate S, under the same conditions, but in the absence of the targets, the difference between the two signals indicating the presence of complementary targets (ICAM-20) of the oligonucleotide probes. |
1. A method for analyzing biological targets of DNA or RNA type, which comprises the following steps: a) contacting the targets to be analyzed with oligonucleotide probes labeled with a cofactor A of an enzyme E, the cofactor being such that it is recognized by enzyme E when it is fixed onto a free oligonucleotide and is less recognized by enzyme E when the oligonucleotide on which it is fixed is hybridized with a complementary oligonucleotide; b) adding, to the reaction medium, enzyme E corresponding to cofactor A and a substrate S of enzyme E, substrate S being converted by enzyme E into a compound C; c) measuring a signal representative of the activity of enzyme E on substrate S; and d) comparing the signal with the signal obtained when the oligonucleotide probes, labeled with cofactor A, are contacted with enzyme E and substrate S, under the same conditions, but in the absence of the targets, the difference between the two signals indicating the presence of complementary targets of the oligonucleotide probes. 2. The method according to claim 1, wherein cofactor A is no longer or almost no longer recognized by enzyme E when the oligonucleotide probes labeled with cofactor A are hybridized with complementary biological targets. 3. The method according to claim 2, wherein the signal measured in step c) represents no more than 50% of the signal obtained in the absence of biological targets. 4. The method according to claim 1, wherein the signal representative of the enzymatic activity of enzyme E on substrate S is an optic signal. 5. The method according to claim 1, wherein enzyme E and cofactor A are such that cofactor A does not fix itself onto enzyme E by covalent bond. 6. The method according to claim 1, wherein enzyme E is such that its substrate S or conversion compound C of the substrate S is used to determine the enzymatic activity of enzyme E by a variation in the light absorbing or fluorescence properties due to consumption of substrate S or production of compound C. 7. The method according to claim 5, wherein cofactor A is flavin or one of its derivatives. 8. The method according to claim 7, wherein the enzyme is a flavoprotein. 9. The method according to claim 8, wherein enzyme E is a flavin reductase or a NAD(P)H oxydase and substrate S is NADPH or NADH. 10. The method according to claim 9, wherein the signal measured is the intensity of NADH fluorescence at 460 nm or the light absorption of NADH at 340 nm. 11. The method according to claim 1, wherein cofactor A being is flavin, enzyme E is a flavin reductase and substrate S is NADH or NADPH, the signal representative of the activity of enzyme E is obtained by using a second enzyme and an aldehyde, the second enzyme being able to catalyze the reaction of the flavin reduced with the aldehyde and oxygen, the reaction being accompanied by luminescence representative of the activity of enzyme E. 12. The method according to claim 11, wherein the second enzyme is luciferase. 13. The method according to claim 1, wherein the comparison of the two signals enables determination of the extent of complementarity between the target and the oligonucleotide probe labeled with cofactor A. 14. The method according to claim 1, wherein the quantities of substrate used are such that the signal representative of the activity of enzyme E is amplified. 15. The method according to claim 1, wherein the oligonucleotide probes labeled with cofactor A are fixed onto a solid support. 16. An analysis support for biological targets of DNA or RNA type comprising oligonucleotide probes fixed onto a solid support, wherein the oligonucleotide probes are labeled with a cofactor A of an enzyme E such that the cofactor A fixed on the probe is recognized by enzyme E, but is less recognized by enzyme E when the oligonucleotide probe is hybridized with a complementary oligonucleotide, enzyme E having a substrate S or conversion compound C of the substrate which enables determination of the enzymatic activity of enzyme E by an optic, signal representative of the consumption of substrate S or of the production of compound C. 17. The analysis support according to claim 16, wherein cofactor A is flavin or one of its derivatives. 18. The analysis support according to claim 16, wherein the oligonucleotide probe is labeled with a group of formula —(CH2)n—R1 in which n is a whole number ranging from 2 to 8 and R1 represents the group of formula II: |
Packaging with venting holes for containing a particulate product |
A packaging for containing a particulate product is disclose in preferred forms of a canister (10) and a bag (100). The packaging includes a body (11, 110) and at least one microhole (40, 400) formed in the body (11, 110). Other than the microhole(s) (40, 400), the body (11, 110) is hermetically sealed. In this regard, the body (11, 110) defines an internal storage region (20, 200) configured to contain a particulate product (22). The microhole(s) (40, 400) are sized to allow passage of air from the internal storage region (20, 200), as well as to limit passage of the particulate product (22). During use, a decrease in atmospheric pressure applied to the packaging, such as during shipping, results in air being vented from the internal storage region (20, 200) via the microhole(s) (40, 400). Due to this air flow, an internal pressure of the body (11, 110) maintains substantial equilibrium with atmospheric pressure such that the body (11, 110) will not unexpectedly expand or in the case of bag (100) burst. |
1. Packaging for containing a particulate product, the packaging comprising: a body defining an internal storage region; and at least one microhole formed in the body sized for allowing air communication with the internal storage region due to changes in atmospheric pressure as a result of an altitude change and for limiting passage of the particulate product from the internal storage region; wherein except for the at least one microhole, the body is constructed for sealing of the internal storage region about the particulate product while the air communication occurs. 2. The packaging of claim 1, wherein the body has an internal pressure, and further wherein the at least one microhole is configured such that upon a decrease in atmospheric pressure, a volume of air vents through the at least one microhole. 3. The packaging of claim 1, wherein the at least one microhole is configured to vent air from the internal storage region at a rate to maintain pressure equilibrium as the canister is raised from a minimum altitude to a maximum altitude. 4. The packaging of claim 1, wherein the at least one microhole is sized to prevent passage of contaminants into the internal storage region. 5. The packaging of claim 1, wherein the at least one microhole includes a plurality of microholes which are uniformly sized. 6. The packaging of claim 1, wherein the at least one microhole has a diameter in the range of approximately 10-100 micrometers. 7. The packaging of claim 1, wherein the at least one microhole has a diameter of approximately 70 micrometers. 8. The packaging of claim 1, wherein a total cross-sectional area of the at least one microhole is related to a volume of the internal storage region. 9. The packaging of claim 8, wherein the total cross-sectional area of the at least one microhole is further related to a compressed volume of particulate product contained within the internal storage region. 10. The packaging of claim 1, wherein the internal storage region has a volume in the range of approximately 2,000-4,000 cm3, the particulate product has a volume in the range of approximately 200-800 cm3, and the at least one microhole has a total cross-sectional area in the range of approximately 0.001-0.004 cm2. 11. The packaging of claim 1, wherein the internal storage region has a volume of approximately 3,145 cm3, the at least one microhole has a total cross-sectional area of approximately 0.0024 cm2 and the air flow rate from the internal storage region is about 0.31 cm3/sec. 12. The packaging of claim 1, wherein the internal storage region has a volume in the range of approximately 2,000-4,000 cm3, the particulate product has a compressed volume in the range of approximately 200-800 cm3, and the at least one microhole includes approximately 40-100 microholes. 13. The packaging of claim 1, wherein the body includes: opposing face panels; opposing side panels connected to the opposing face panels to define an upper opening and a lower opening; a bottom panel connected to the opposing face panels and the opposing side panels so as to encompass the lower opening; and a top panel connected to the opposing face panels and the opposing side panels so as to encompass the lower opening. 14. The packaging of claim 13, wherein each of the panels includes a plastic material configured to maintain integrity of particulate product disposed within the internal storage region. 15. The packaging of claim 1, wherein the body is configured to contain a dry food product. 16. The packaging of claim 15, wherein the food product is a ready-to-eat cereal. 17. The packaging of claim 1, wherein the body includes: opposing face panels having their sides integrally connected and including lower and upper portions; and lower and upper seals closing the opposing face panels at the upper and lower portions, with the face panels formed of flexible material to create a bag. 18. The packaging of claim 17, wherein the flexible material creating the bag has a tendency to return to an inflated shape after a volume of air is vented from the internal storage region due to a reduction of atmospheric pressure and then an increase in atmospheric pressure. 19. The packaging of claim 18, wherein the body is configured to contain a dry food product in the form of a snack. 20. A packaged good article comprising: a packaging including: a body defining an internal storage region, at least one microhole formed in the body sized to allow air communication with the internal storage region due to changes in atmospheric pressures as a result of an altitude change; and a particulate product disposed within the internal storage region; wherein the at least one microhole is sized to prevent release of the particulate product with the body being constructed to seal the internal storage region about the particulate product except for the at least one microhole. 21. The packaged good article of claim 20, wherein the body has an internal pressure, and further wherein the at least one microhole is configured such that upon a decrease in atmospheric pressure, a volume of air vents through the at least one microhole. 22. The packaged good article of claim 20, wherein the at least one microhole is configured to vent air from the internal storage region at a rate to maintain pressure equilibrium as the canister is raised from a minimum altitude to a maximum altitude. 23. The packaged good article of claim 20, wherein the at least one microhole is sized to prevent passage of contaminants into the internal storage region. 24. The packaged good article of claim 20, wherein the at least one microhole includes a plurality of microholes which are uniformly sized. 25. The packaged good article of claim 20, wherein the at least one microhole has a diameter of approximately 10-100 micrometers. 26. The packaged good article of claim 20, wherein the at least one microhole has a diameter of approximately 70 micrometers. 27. The packaged good article of any one of claims claim 20, wherein a total cross-sectional area of the at least one microhole is related to a volume of the internal storage region. 28. The packaged good article of claim 27, wherein the total cross-sectional area of the at least one microhole is further related to a volume of air contained within the internal storage region. 29. The packaged good article of claim 20, wherein the internal storage region has a volume in the range of approximately 2,000-4,000 cm3 of which air occupies approximately 80-95 percent, and the at least one microhole has a total cross-sectional area in the range of approximately 0.001-0.004 cm2. 30. The packaged good article of claim 20, wherein the internal storage region has a volume of approximately 3,145 cm3 and the at least one microhole has a total cross-sectional area of approximately 0.0024 cm2. 31. The packaged good article of claim 20, wherein the internal storage region has a volume in the range of approximately 2,000-4,000 cm3 of which air occupies approximately 80-95 percent, and the at least one microhole includes approximately 40-100 microholes. 32. The packaged good article of claim 20, wherein the body includes: opposing face panels; opposing side panels connected to the opposing face panels to define an upper opening and a lower opening; a bottom panel connected to the opposing face panels and the opposing side panels so as to encompass the lower opening; and a top panel connected to the opposing face panels and the opposing side panels so as to encompass the lower opening. 33. The packaged good article of claim 32, wherein each of the panels include a plastic material configured to maintain integrity of the particulate product. 34. The packaged good article of claim 20, wherein the particulate product is a dry food product. 35. The packaged good article of claim 34, wherein the food product is a ready-to-eat cereal. 36. The packaged good article of claim 20, wherein the body includes: opposing face panels having their sides integrally connected and including lower and upper portions; and lower and upper seals closing the opposing face panels at the upper and lower portions, with the face panels formed of flexible material to create a bag. 37. The packaged good article of claim 36, wherein the flexible material creating the bag has a tendency to return to an inflated shape after a volume of air is vented from the internal storage region due to a reduction of atmospheric pressure and then an increase in atmospheric pressure. 38. The packaged good article of claim 37, wherein the body is configured to contain a dry food product in the form of a snack. 39. A method of manufacturing a packaged good article, the method comprising: forming a sealable packaging having an internal storage region; imparting at least one microhole into the packaging, the at least one microhole extending from an exterior of the packaging to the internal storage region; and partially filling the internal storage region with a particulate product, a majority of a remaining volume of the internal storage region being air; with the air within the internal storage region generating an internal pressure, and further with a change in atmospheric pressure as a result of an altitude change, the at least one microhole providing air communication with the internal storage region. 40. The method of claim 39, wherein imparting the at least one microhole includes: determining a volume of air required to be vented from the internal storage region to maintain pressure equilibrium when the packaged good article is raised from a minimum altitude to a maximum altitude; and determining a required number of microholes based upon the volume of air required to be vented. 41. The method of claim 40, wherein determining a required number of microholes further includes: determining a flow rate of air from the internal storage region required to maintain pressure equilibrium. 42. The method of claim 39, wherein imparting the at least one microhole includes: determining a total cross-sectional area of microholes required to maintain pressure equilibrium when the packaged good article is raised from a minimum altitude to a maximum altitude; and determining a required number of microholes based upon the total cross-sectional area. 43. The method of claim 39, wherein imparting the at least one microhole includes: forming the at least one microhole each having a diameter of approximately 70 micrometers. 44. The method of claim 39, wherein forming the sealable packaging includes: connecting opposing face panels and opposing side panels to form a tubular body having an upper opening and a lower opening; connecting a top panel to the opposing face panels and the opposing side panels so as to encompass the upper opening; and connecting a bottom panel to the opposing face panels and the opposing side panels so as to encompass the lower opening. 45. The method of claim 44, wherein the internal storage region is partially filled with the particulate product prior to connecting the bottom panel. 46. The method of claim 39, wherein the particulate product is a ready-to-eat cereal. 47. The method of claim 39, wherein forming the sealable packaging includes: connecting opposing face panels to form a tubular body having upper and lower portions; sealing the upper portions; and sealing the lower portions. 48. The method of claim 47, wherein the internal storage region is partially filled with the particulate product after sealing one of the upper and lower portions and prior to sealing the other of the upper and lower portions. 49. The method of claim 47, with connecting opposing face panels comprising connecting opposing face panels formed of flexible material such that the shape of the packaging being formed into a final shape relying upon the air in the internal storage region. 50. A method of shipping a particulate product, the method comprising: sealing the particulate product within an internal storage region of a packaging; imparting at least one microhole adjacent an upper portion of the packaging, with the at least one microhole being sized to provide air communication with the internal storage region due to changes in atmospheric pressure as a result of an altitude change; packing a plurality of the sealed packaging in a pack pattern in a shipper with the packaging each being a vertical orientation; stacking a plurality of shippers with the packaging in each of the shippers being in the vertical orientation; and shipping the stack of shippers from a first altitude to a second altitude different from the first altitude causing air to flow through the at least one microhole. 51. The method of claim 50, with sealing the particulate product comprising sealing the particulate product with a volume of air within the internal storage region of the packaging formed of flexible material and relying upon the volume of air to form a final shape; and with shipping the stack of shippers comprising shipping the stack of shippers from a high altitude to a low altitude. 52. The method of claim 51, with shipping the stack of shippers comprising shipping the stack of shippers from the high altitude to a higher altitude and then to the low altitude. 53. The method of claim 47, with shipping the stack of shippers comprising shipping the stack of shippers from a low altitude to a high altitude. 54. The method of claim 51, with shipping the stack of shippers comprising shipping the stack of shippers from the high altitude to an altitude less than the high altitude. 55. The method of claim 53, with sealing the particulate product comprising sealing the particulate product with a volume of air within the internal storage region of the packaging formed of flexible material and relying upon the volume of air to form a final shape. 56. The method of claims 55, with sealing the particulate product comprising sealing the particulate product in a bag formed by opposing face panels having sides integrally interconnected and sealing closed at upper and lower portions; and with imparting at least one microhole comprising imparting at least one microhole in the interconnection between the sides of the face panels. 57. The method of any one of claims 50, with imparting at least one microhole comprising imparting at least one microhole having a diameter in the range of approximately 10-1000 micrometers. 58. The method of any one of claims 50, with imparting at least one microhole comprising imparting at least one microhole having a total cross sectional area providing an air vent rate to prevent a pressure differential between the internal storage region and the atmosphere surrounding the packaging to exceed a bursting strength of the packaging. 59. The method of any one of claims 50, with shipping the stack of shippers comprising trucking the stack of shippers. 60-62. (cancelled). |
<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention relates to packaging for containing a particulate product. More particularly, it relates to packaging having venting holes for containing a particulate product, the venting holes facilitating pressure equilibrium at high altitudes. Extremely popular forms of packaging for dry, particulate products sold to consumers are a paper carton or a printed plastic bag. The paper carton normally is rectangular in shape, constructed of one or more layers of paper, and may or may not include an additional plastic liner. A wide variety of products are packaged in this form, ranging from consumable items such as cereals and baking goods, to non-consumable items such as laundry detergents and de-icing salt pellets. Paper cartons present a number of advantages for manufacturers, retailers and consumers. For example, paper cartons are relatively inexpensive to manufacture and provide a number of flat surfaces onto which product or promotional information can be displayed. Due to the rectangular shape, cartons are readily stackable. Thus, a retailer can maximize shelf space while fully displaying the product. Consumers likewise find the stackability characteristic desirable for home storage. Finally, paper cartons are typically sized in accordance with consumer preferences such that a desired amount or volume of product is provided with each individual carton. Certain types of products are amenable to storage within a paper carton alone. Generally speaking, however, a paper carton cannot, in and of itself, adequately maintain product integrity. For example, a paper carton likely will not prevent aroma, moisture, contaminants, small insects, etc. from passing through to the contained product. Thus, paper cartons for virtually all particulate-type products require an additional container or liner disposed within the paper carton. This is especially true for consumable/food products. A widely accepted technique for maintaining product integrity is to place the product into an inner container or bag, that in turn is stored in the carton (commonly referred to as a “bag in a box”). The bag is typically made of a plastic or glassine material and is, in theory, sealed about the product. In this sealed form, the bag maintains product freshness and provides protection against insect infestation, whereas the outer paper carton provides packaging strength and display. Alternatively, a double packaging machine (DPM) technique may be employed to form a plastic or glassine liner within a paper carton. Regardless of the exact manufacturing process, the resulting packaging configuration includes a box with an inner liner that serves as a barrier material. For virtually all applications, a large volume of air will be “contained” within the inner liner in addition to the particulate-type product. That is to say, the particulate-type product will not encompass the entire internal volume of the inner liner, and may include spacing between individual product particles. As described above, a concerted attempt is made to hermetically seal the inner liner about the particulate-type product. On a mass production basis, however, current packaging technology cannot consistently meet this goal. For example, small openings may remain at an apex of two inner liner film sheets joined to one another. In short, manufacturers accept the fact that some leakage will occur into and out of the inner liner through one or more small openings. Although unexpected, these openings normally are not large enough for passage of contaminants or discharge of product. In fact, the openings may provide a benefit during shipping. Packaged product is typically shipped via truck from the manufacturer to retailers at various locations. The location (e.g., city or town) of a particular retailer often is at a greater altitude than that of the manufacturer, or the route traveled by the truck may include a relatively drastic change in altitude. With increasing altitude, the atmospheric pressure exerted on the carton decreases. Because the carton/inner liner is not hermetically sealed, the pressure differential causes air to vent from the inner liner, thereby bringing an internal pressure of the packaging into equilibrium with the now lower atmospheric pressure. Were the inner liner hermetically sealed, this release of air could not occur, resulting in expansion of the inner liner. This expansion may damage the inner liner/carton. Additionally, where a quantity of cartons are closely packed within a corrugated shipping container, expansion of the inner liners may cause the cartons to tightly lodge against one another, rendering removal of the packages from the shipping container extremely difficult. From a manufacturer's standpoint, a paper carton with an inner liner packaging satisfies a number of important criteria including low cost, stackability, and large, flat surfaces for displaying product and promotional information. Unfortunately, however, consumers may encounter several potential drawbacks. These possible disadvantages are perhaps best illustrated by reference to a ready-to-eat cereal product, although it should be understood that a wide variety of other products are similarly packaged. Most ready-to-eat cereal products are sold to consumers in a paper carton with an inner liner packaging format. To consume the cereal, the user must first open the paper carton. In this regard, a top portion of the carton typically forms at least two flaps folded on top of one another. The flaps are initially at least partially adhered to one another with an adhesive. By pulling or otherwise tearing one flap away from the other, a consumer can then access the inner bag. An all too common problem is that the selected adhesive creates too strong of a bond between the flaps, making flap separation exceedingly difficult. Once the carton has been opened, the consumer must then open the inner bag. Once again, this may be a cumbersome procedure. More particularly, an elongated seal is typically formed and extends along a top portion of the bag. This seal is broken (or “opened”) by pulling apart opposite sides of the bag. In some instances, the so-formed seal is too rigid for simple opening. Even further, a person with reduced dexterity and strength, such as a child or elderly individual, may have difficulty in breaking an even relatively light seal. As a result, attempts at opening the inner bag or liner often result in an undesirable tear along a side of the bag, causing unacceptable product displacement from the bag, or an uneven opening. The consumer may resort to using a knife or scissors, possibly resulting in bodily harm. Once the carton and bag or liner has been opened, the consumer is then ready to pour the contents from the package. Due to the flexible nature of the inner bag, the actual opening through which the product flows is unpredictable. That is to say, the opening formed in the bag is not uniform or fixed. As a result, a larger than expected volume of product may unexpectedly pour from the container. Alternatively, where the inner bag has not been properly opened, product flow may be unacceptably slow. Further, an inherent bias or bend typically causes the flaps to extend upwardly relative to a top of the carton. Thus, the flaps will impede a user from visually confirming acceptable product volume and flow. Additionally, the inner bag typically is not secured to the carton. During a subsequent pouring operation, then, the entire bag may undesirably release from the carton. A further consumer concern relating to box with an inner liner packaging stems from attempts to reclose the package for subsequent storage of remaining product. Again with reference to widely employed ready-to-eat cereal packaging, following dispensing of a portion of the cereal from the package, the user is then required to roll or fold the top portion of the bag or liner over onto itself so as to “close” the bag. It is not uncommon for a user to simply forget to perform this operation. Alternatively, even where an attempt is made, the bag cannot be resealed and thus remains at least partially open. Similarly, the bag may subsequently unroll. Individual cereal pieces may be undesirably released from the bag and/or contaminants can enter into the bag. Regardless, a reclosure feature normally associated with the carton normally does not provide an effective barrier to unexpected product displacement and/or contamination due to removal, poor design, misuse, lack of use, etc. These concerns are exacerbated when attempting to store a previously-opened package on its side or when the package is accidentally dropped. In either case, because neither the carton nor the bag provides a complete closure, unanticipated release of cereal from the container may occur. Viewed as a whole, concerns relating to standard box with an inner liner packaging present numerous opportunities for consumer dissatisfaction. Essentially, consumer preferences for improvements to particulate-type product packaging can be separated into four categories. Consumers prefer that the packaging be easy to open, easily and satisfactorily reclosed, facilitate consistent and easy pouring and is acceptable for “clean” use by a child or others with limited dexterity. Obviously, consumers further prefer that product costs be as low as possible, and that certain other beneficial attributes associated with the existing box with inner liner packaging continue to be implemented. These existing properties include package strength, product damage protection, use of high volume commercially available materials, visual display of product and promotional material, recycleability, stackability, and moisture, aroma, contaminant and insect protection. Certain other packaging schemes are available that address, at least in part, several of the above-listed consumer preferences. Unfortunately, however, these packaging techniques entail other drawbacks, thereby limiting their usefulness. For example, rigid plastic containers having removable, sealable lids are available. The greatly increased costs associated with this packaging configuration prohibit its implementation on a mass production basis. Similarly, it may be possible to provide the inner bag with a “zip-lock” sealing feature. While this technique may alleviate several of the reclosure issues previously described, the zip-lock design is expensive and often times does not provide a complete seal importantly, with these and other envisioned packaging schemes, consistent formation of a hermetic seal will result in the above-described expansion concerns when the package is shipped to a high altitude location. Once again, because the packaging technique does not account for necessary venting, an increase in altitude may cause problematic package expansion. Consumers continue to express a high demand for particulate-type products sold in a paper cartons. However, various problems associated with use of standard packaging, and in particular box with an inner liner packages, may diminish purchasing enthusiasm. Alternative packaging designs may satisfy some consumer concerns, but in fact create new problems, such as deleterious package expansion during shipment to higher altitude locations. The printed plastic bag or pouch without the use of a carton or canister is also very popular for a wide variety of products ranging from consumables such as potato chips and candy bars to non-consumables such as fertilizer and water softener salt. The bag or pouch is made up of one or more polymers and is typically made up of multiple layers of one or more polymers. It may also contain aluminum foil, metallized films, or other barrier materials that have been condensed or coated onto one or more layers of the plastic film. Plastic pouches have several characteristics that make them desirable for manufactures, retailers and consumers. For example, pouches are inexpensive, use fewer unit operations, and have high gloss appearance. Pouches can be displayed on the retail shelf or hung on pegs. Pouches are easily sized to fit consumer purchase desires and create less waste for the consumer to dispose of. Certain products are pouched and sent through distribution channels without the structural support of a paperboard carton, corrugated container or other materials used to minimize damage to the product. However, for consumable food products that do not use a paperboard carton around the pouch, it is most common to utilize a corrugated container to protect the contents of the pouch from the physical rigors of product distribution. Typically, the food product is put into a preformed or “form, fill, seal” pouch that has been printed with the necessary graphics prior to being formed into a pouch. The pouch provides barriers to elements that may either egress or ingress the pouch. Examples are moisture, flavors, aromas, oxygen, insects, and dirt. With most consumable food products, the majority of the volume of the pouch is air, including the air that surrounds and is between discrete pieces and the air that is contained within the lattice or cellular structure of the food itself. In some instances, pouches have enough air or other gas to provide a cushion that can prevent breakage of larger particulate materials in handling or distribution. In others, the air is either mechanically or chemically removed from the pouch resulting in a very tight compressed appearing package. Pouches sold without the added protection of a paperboard carton must be robust. These pouches must withstand the rigors of distribution and give the consumer confidence that the product is high quality and wholesome. Typically, these pouches have very high seal integrity with virtually no air leaks. This is especially true of salty snack packaging. However, the consumer also expects to be able to easily open snack packaging with minimal effort, typically utilizing what is described in the industry as a peelable seal. Very high seal integrity with a peelable seal creates a special problem for those particulate products that are shipped from a lower elevation to a higher elevation. As the elevation of the pouch increases, the differential pressure between elevations causes the volume of the air in the pouch to increase. The increase in volume of air forces the pouch to expand primarily in the front to back dimension of the pouch. Once the constraints of the corrugated container are met and the pouch continues to increase in elevation, the pouch will develop internal air pressure. If it develops enough pressure, it will burst through the weakest portion of the pouch, typically the seal area and especially the peelable seal. There are several potential remedies for this situation. Most of which have serious drawbacks ranging from cost to consumer usage. As an example, the seal strength could be increased enough to eliminate bursting, but the package is not convenient for consumers and the material costs would be greater. Vacuum packaging the product would remove the breakage cushion and would result in a less desirable appearance which would adversely affect market appeal as well as increase packaging costs. Therefore, a need exists for a particulate product packaging configured to address consumer use preferences while providing adequate venting upon shipment over high elevations. |
<SOH> SUMMARY OF THE INVENTION <EOH>One aspect of the present invention provides a packaging for containing a particulate-type product. The packaging includes a body and microholes formed in the body. The body defines an internal storage region. The microholes formed in the body are sized for allowing air flow from the internal storage region, while limiting passage of particulate-type product from the internal storage region. With this configuration, other than the microholes, the body is substantially hermetically sealed. As the packaging is physically moved from a low altitude to a high altitude, atmospheric pressure acting upon the body decreases. The microholes compensate for this decrease in atmospheric pressure by allowing a sufficient volume of air to vent from the internal storage region. Thus, an internal pressure of the body remains in substantial equilibrium with atmospheric pressure such that the body does not overly expand. In one preferred embodiment, the packaging is configured as a canister to maintain a food product such as a ready-to-eat cereal. Another aspect of the present invention relates to a packaged good article comprising packaging and a particulate-type product. The packaging includes a body and microholes formed in the body. The body defines an internal storage region. The microholes are configured to allow air flow from the internal storage region. Other than the microholes, the body is substantially hermetically sealed. The particulate-type product is disposed within the internal storage region. With this in mind, the microholes are sized to limit, preferably prevent, release of the particulate-type product from the internal storage region. In one preferred embodiment, the particulate-type product is a dry, ready-to-eat cereal. Yet another aspect of the present invention relates to a method of manufacturing a packaged good article. The method includes forming a hermetically sealable packaging having an internal storage region. Microholes are imparted into the packaging, extending from an exterior of the packaging to the internal storage region. The internal storage region is then partially filled with a particulate-type product. A majority of the remaining volume of the internal storage region not otherwise occupied by the particulate-type product is filled with air. This air within the internal storage region imparts an internal pressure onto the packaging. Upon a decrease in atmospheric pressure acting upon the packaging, the microholes allow venting of a sufficient of air from the internal storage region to equilibrate the internal pressure with atmospheric pressure. In one preferred embodiment, the internal storage region is partially filled with a ready-to-eat cereal. |
Novel human proton-gated channels |
The present invention provides a novel human proton-gated ion channel (hASIC1B) and polynucleotides which identify and encode hASIC1B. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding hASIC1B and a method for producing hASIC1B. The invention also provides for use of hASIC1B, and agonists, antibodies or antagonists specifically binding hASIC1B, in the prevention and treatment of diseases associated with expression of hASIC1B. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding of hASIC1B for the treatment of diseases associated with the expression of hASIC1B. The invention also provides diagnostic assays, which utilize the polynucleotides, or fragments or the complements thereof, and antibodies specifically binding hASIC1B. |
1. A purified and isolated human proton-gated ion channel protein (hASIC1B) selected from the following: a hASIC1B having at least 80% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 85% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 90% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 95% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 98% identity with the amino acid sequence defined in SEQ ID NO:2 and a hASIC1B having at least 99% identity with the amino acid sequence defined in SEQ ID NO:2. 2. The protein of claim 1, which is characterized by the activation of protons (acids, low pH solutions). 3. The protein of claim 2 which has the amino acid sequence defined in SEQ ID NO:2. 4. A nucleic acid which encodes a protein as defined in claim 1. 5. The nucleic acid of claim 4, which is capable of hybridizing to SEQ ID NO: 1. 6. The nucleic acid of claim 4, which has the sequence defined in SEQ ID NO: 1. 7. A recombinant vector, comprising the nucleic acid of claim 4. 8. The recombinant vector of claim 7, which is an expression vector. 9. A host comprising the recombinant vector of claim 7. 10. A host cell comprising the recombinant vector of claim 8. 11. A process for producing a human proton-gated ion channel protein (hASIC1B) comprising culturing the host cell of claim 10 under conditions sufficient for the production of said protein and recovering said protein from the culture. 12. A process for producing a cell which produces a protein as defined in claim 1. 13. A process as defined in claim 12, further comprising transforming or transfecting a host cell with a recombinant expression vector comprising a nucleic acid which encodes a protein, wherein the protein (hASIC1B) is selected from the following: a hASIC1B having at least 80% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 85% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 90% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 95% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 98% identity with the amino acid sequence defined in SEQ ID NO:2 and a hASIC1B having at least 99% identity with the amino acid sequence defined in SEQ ID NO:2. 14. An antibody immunospecific for a hASIC1B protein as defined in claim 1. 15. A hybridoma producing an antibody as defined in claim 14. 16. A method for the treatment of a subject in need of enhanced activity or expression of a hASIC1B protein, wherein the protein is selected from the following: a hASIC1B having at least 80% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 85% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 90% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 95% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 98% identity with the amino acid sequence defined in SEQ ID NO:2 and a hASIC1B having at least 99% identity with the amino acid sequence defined in SEQ ID NO:2, comprising administering to the subject a therapeutically effective amount of an agonist to said hASIC1B protein; or providing to the subject a nucleic acid of claim 4 in a form so as to effect production of said hASIC1B protein activity in vivo. 17. A method for the treatment of a subject having a need to inhibit activity or expression of a hASIC1B protein of claim 1 comprising: (a) administering to the subject a therapeutically effective amount of an antagonist to said hASIC1B protein; or (b) administering to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said hASIC1B protein; or (c) administering to the subject a therapeutically effective amount of a protein that competes with said hASIC1B protein for its ligand. 18. A process for diagnosing a disease or a susceptibility to a disease in a subject related to the expression or activity of a hASIC1B protein as defined in any of claim 1 in a subject comprising: (a) determining the presence or absence of a mutation in the nucleotide sequence encoding said hASIC1B protein in the genome of said subject; or (b) analyzing for the presence or amount of hASIC1B protein expression in a sample derived from said subject. 19. A method for identifying agonists to a hASIC1B protein, wherein the hASIC1B protein is selected from the following: a hASIC1B having at least 80% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 85% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 90% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 95% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 98% identity with the amino acid sequence defined in SEQ ID NO:2 and a hASIC1B having at least 99% identity with the amino acid sequence defined in SEQ ID NO:2, comprising: (a) putting cells produced by the process of claim 11 in contact with candidate compound(s); and (b) determining whether the candidate compound induces or modulates a biological activity or signal transduced by the hASIC1B receptor; or (c) determining whether the candidate compound induces inward currents or modulates proton-induced inward currents transduced by the hASIC1B receptor. 20. An agonist identified by the method of claim 19. 21. An agonist of claim 20, which is, or is an adjuvant to, an antidepressant, a desensitizing agent, an antipruritic, an analgesic, a chemotherapeutic or antineoplastic agent, an antipsychotic, a psychotherapeutic agent, a respiratory and cerebral stimulant, a cognitive stimulant, memory stimulant, a promoter of neuronal regeneration, a stimulant of cell growth or proliferation, an insecticide, a pesticide or an anthelmintic, or any combination thereof. 22. The method for identifying antagonists to a hASIC1B protein, wherein the hASIC1B protein is selected from the following: a hASIC1B having at least 80% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 85% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 90% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 95% identity with the amino acid sequence defined in SEQ ID NO:2, a hASIC1B having at least 98% identity with the amino acid sequence defined in SEQ ID NO:2 and a hASIC1B having at least 99% identity with the amino acid sequence defined in SEQ ID NO:2, comprising: (a) putting cells produced by the process of claim 11 in contact with a low pH solution (pH<7.4) or any other agonist; and (b) determining whether the signal generated by protons or said agonist is modulated, diminished or abolished in the presence of candidate compound(s). 23. An antagonist identified by the method of claim 22. 24. An antagonist as defined in claim 23, which is, or is an adjuvant to, an analgesic, an antipyretic, an antipruritic, an anxiolytic, sedative or hypnotic, a psychotherapeutic agent, an anticonvulsant, a neuroprotectant, a general anesthetic, a local anesthetic, a hypotensive agent, a muscle relaxant, an antidiarrhea agent, an antacid, or any combination thereof. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Tissue acidosis is associated with a number of painful physiological (e.g. cramps) and pathological conditions (e.g. inflammation, intermittent claudication, myocardial infarction). Experimentally, similar painful events can be reproduced by infusing low pH solutions into skin or muscle. Furthermore, the prolonged intradermal infusion of low pH solutions can mimic the characteristic hyperalgesia of chronic pain. To further charaterize the effects of protons and their relation to pain, low pH solutions were applied to patch-clamped central and peripheral sensory neurons. Inward currents were induced when pH was dropped to acidic values, providing evidence for the existence of proton-activated ion channels. Several types of native currents were observed in sensory neurons from rat and human trigeminal and dorsal root ganglia: rapidly inactivating currents; non-inactivating currents; and biphasic currents displaying a rapidly inactivating current followed by non-inactivating current. Other differences regarding ion selectivities were also reported. These results suggested the existence of several proton-gated ion channels. The prolonged pain induced by tissue acidification is most likely associated with a non-inactivating proton-gated ion channel. Cloned Proton-Gated Ion Channels The mammalian proton-gated cation channels have recently been cloned and named <<ASIC>> for Acid Sensing Ion Channels. Sequence analysis identifies them as members of the DEG/ENaC superfamily of ion channels. The putative membrane topology of ASIC receptors predicts two transmembrane spanning domains with both N— and C— termini in the intracellular compartment, as shown for the epithelial sodium channels. Four sub-classes of ASIC receptors have been identified: 1. ASIC1 ion channels display rapidly inactivating inward currents (Waldmann et al., Nature 1997; 386: 173) 2. ASIC2 ion channels display slowly inactivating inward currents (Brassilana et al., J Biol Chem 1997; 272: 28819). 3. ASIC3 ion channels display biphasic inward currents with an initial rapidly inactivating component, followed by a sustained non-inactivating current (Waldmann et al., J Biol Chem 1997; 272: 20975; Babinski et al., J Neurochem 1999; 72: 51) 4. ASIC4 ion channels contain characteristic ASIC-like sequence motifs but do not appear to be activated by protons in homomultimeric association. Families of ASIC Receptors Created by Alternative Splicing of mRNAs A common feature of these ion channels is the existence of alternative splice variants, which display important functional differences. Indeed, the replacement of the first 185 amino acids of ASIC1 (hereinafter named ASIC1A) by a distinct new sequence of 172 amino acids generates a new channel, ASIC1B, which has similar current kinetics as ASIC1A but needs lower pH values for activation (pH 50 of 6.2 and 4.5, respectively for ASIC1A and ASIC1B). Also, it appears that ASIC1B is specifically expressed in rat dorsal root ganglia. A similar situation is also observed with rat ASIC2 (hereinafter named ASIC2A), where the replacement of the first 185 amino acids by a distinct new sequence of 236 amino acids generates another ASIC ion channel subunit, ASIC2B. When expressed alone as a homomultimer in mammalian cells or Xenopus oocytes, ASIC2B does not appear to be activated by low pH solutions. However, coexpression of ASIC2B with other ASIC subunits (e.g. ASIC2A, ASIC3) gives rise to heteropolymeric ion channels with distinct properties such as novel ion selectivities or pH 50 values (Lingueglia et al., J Biol Chem 1997: 272: 30 29778). ASIC3, which has been identified in human, also appears to exist in various forms. Indeed, DRASIC is an ASIC3-like channel identified in rat, which displays 85% identity with the human ASIC3 sequence and has similar biphasic current kinetics. However, important differences regarding tissue distribution, ion selectivities and pH 50 suggest that DRASIC might not be the human orthologue of ASIC3 but rather a different subtype. Furthermore, the existence of two 3′ splice variants of human ASIC3 (ASIC3B and 3C, recently submitted to GenBank) have been reported but differences in function have yet to be documented. Alternative splicing, therefore, appears like an important mechanism for increasing the diversity of ASIC receptors, which most probably assume critical roles in the nervous system, such as neurotransmission, nociception or mechanosensation (see below). Because of the great, differences between the existing splice variants, the actual functional characteristics of the new splice variants is unpredictable and might prove to be completely different from any known ASIC receptor. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention reports the discovery of the human ASIC1B receptor (hereinafter referred to as hASIC1B), which shows distinct features from the previously published rat ASIC1B. Also contemplated within the scope of this invention is the potential involvement of this new subunit in neurotransmission and/or nociception and/or mechanosensation and/or any other neurological and/or metabolic processes in normal and pathophysiological conditions. This invention seeks also to cover any uses of this new subunit as a therapeutic target, including but not limiting to drug screening technologies (i.e. screening for channel antagonists, agonists and/or modulators), diagnostic marker, gene therapies. Also within the scope of the present invention is the heteropolymerization of the hASIC1B subunits with each other and/or with one or more subunits of the ASIC family from any species, including but not limiting to ASIC1, ASIC1A, BNaC2, ASIC1B, ASIC2A, ASIC2B, MDEG, MDEG1, MDEG2, BNC1, BNaC1, DRASIC, ASIC3, ASIC4, SPASIC or any variants thereof, as well as heteropolymerization of hASIC1B with any other members of the Degenerin and EnaC family from any species. The object of this invention is to provide the preferred primary sequence of the polynucleotide molecule (SEQ ID No.1) encoding the full length hASIC1B polypeptide molecule (SEQ ID No.2). Still another object of this invention is to provide a partial genomic polynucleotide sequence of hASIC1B deduced from the non-characterized sequences deposited in GenBank under Accession NO: AC025154, AC074032, and AC025361. In particular, the sequence of the characteristic and distinctive first exon of hASIC1B contained within one uninterrupted contig in clone AC025154. The invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode hASIC1B. The present invention also features antibodies which bind specifically to hASIC1B, and pharmaceutical compositions comprising substantially purified hASIC1B. The invention also features use of agonists and antagonists of hASIC1B. |
Optical recording/reproducing medium-use substrate, production method for optical recording/reproducing medium reproducing stamper and optical recording/reproducing medium producing stamper |
An optical recording/reproducing medium having grooves (2) formed along recording tracks and used for recording/reproducing when irradiated with light L with a specified wavelength λ, wherein the track pitch p of a groove (2) is at least 200 nm and up to 350 nm, and a ratio wg/p between the width wg of a groove (2) and a track pitch p is at least 0.24 and up to 0.67. |
1. In an optical recording and reproducing medium substrate having a groove formed along a recording track, said groove being a wobbling groove, said optical recording and reproducing medium substrate characterized in that said groove has a track pitch selected in a range of from 200 nm to 350 nm, said groove having a width wg and said track pitch p, a ratio wg/p being selected in a range of from 0.24 to 0.67, said groove having a surface roughness selected in a range of from 0.4 nm to 0.85 mm. 2. An optical recording and reproducing medium substrate according to claim 1, wherein said groove has a width selected in a range of from 47 nm to 235 nm. 3. An optical recording and reproducing medium substrate according to claim 1, wherein said groove has a groove side surface of which inclination angle is selected in a range of from 15.4° to 40°. 4. An optical recording and reproducing medium substrate according to claim 1, wherein said groove has a depth or height selected in a range of from 15 nm to 30 nm. 5. In a method of manufacturing an optical recording and reproducing medium manufacturing stamper for molding an optical recording and reproducing medium substrate having a groove formed along a recording track, said groove being a wobbling groove, said method of manufacturing an optical recording and reproducing medium manufacturing stamper comprising the steps of manufacturing an optical recording and reproducing medium manufacturing master by developing a photoresist on a master substrate after said photoresist has been exposed with a pattern corresponding to a predetermined uneven pattern and etching a stamper transferred from said optical recording and reproducing medium manufacturing master to microminiaturize a width of a groove pattern corresponding to said groove. 6. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 5, wherein said stamper is etched by plasma etching or reactive ion etching. 7. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 6, wherein said plasma etching or reactive ion etching uses Ar gas or gas which results from mixing oxygen gas into said Ar gas. 8. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 5, wherein said groove pattern corresponding to said groove of said optical recording and reproducing medium has a track pitch selected in a range of from 200 nm to 350 nm, said groove pattern has a width wg′ and said groove pattern has a track pitch p whose ratio wg′/p′ is selected in a range of from 0.24 to 0.67 and said groove pattern has a surface roughness selected in a range of from 0.4 nm to 0.85 nm. 9. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 5, wherein said stamper transferred from said optical recording and reproducing medium manufacturing master by said etching has a groove pattern of which width is selected in a range of from 47 nm to 235 nm. 10. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 5, wherein said groove pattern has a groove pattern side surface of which inclination angle is selected in a range of from 15.4° to 40°. 11. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 5, wherein said groove pattern has a depth or height selected in a range of from 15 nm to 30 nm. 12. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 5, wherein when said optical recording and reproducing medium manufacturing master is manufactured, said photosensitive layer on said master substrate uses high gamma photoresist of which γ characteristic value is greater than 4.5. 13. A method of manufacturing an optical recording and reproducing medium manufacturing stamper according to claim 5, wherein said photosensitive layer on said master substrate is exposed by exposure light having a wavelength ranging from 266 nm to 413 nm. 14. In an optical recording and reproducing medium manufacturing stamper for molding an optical recording and reproducing medium substrate having a groove formed along a recording track, said groove being a wobbling groove, an optical recording and reproducing medium manufacturing stamper characterized in that a groove pattern corresponding to the groove of said optical recording and reproducing medium has a track pitch selected in a range of from 200 nm to 350 nm, a ratio wg′/p′ between a width wg′ of said groove pattern and a track pitch p′ of said groove pattern being selected in a range of from 0.24 to 0.67, said groove pattern having a surface roughness being selected in a range of from 0.4 nm to 0.85=n. 15. An optical recording and reproducing medium manufacturing stamper according to claim 14, wherein said groove pattern width wg′ is selected in a range of from 47 nm to 235 nm. 16. An optical recording and reproducing medium manufacturing stamper according to claim 14, wherein said groove pattern has a groove side surface and an inclination angle selected in a range of from 15.4° to 40°. 17. An optical recording and reproducing medium manufacturing stamper according to claim 14, wherein said groove pattern has a depth or height selected in a range of from 15 nm to 30 nm. 18. An optical recording and reproducing medium manufacturing stamper according to claim 14, wherein said optical recording and reproducing medium manufacturing stamper is formed such that an optical recording and reproducing medium manufacturing master is manufactured after a photoresist on a master substrate has been exposed with a pattern corresponding to a predetermined uneven pattern and developed, a stamper transferred from said optical recording and reproducing medium manufacturing master being formed by etching, said groove pattern width of said stamper being selected to be smaller than a width of a groove pattern of said optical recording and reproducing medium manufacturing master. 19. An optical recording and reproducing medium manufacturing stamper according to claim 18, wherein said stamper is etched by plasma etching or reactive ion etching. 20. An optical recording and reproducing medium manufacturing stamper according to claim 18, wherein said plasma etching or reactive ion etching uses Ar gas or gas which results from mixing oxygen gas into said Ar gas as its gas. |
<SOH> BACKGROUND ART <EOH>Various kinds of optical disks shaped like discs for optically recording and/or reproducing information are put into practical use as optical recording and reproducing mediums. A read-only optical disk having embossed pits formed on a disk substrate beforehand, a magneto-optical disk for recording data by magneto-optical effect and a phase-change optical disk for recording data by phase-change of a recording film are available as such optical disks. Of these optical disks, in the optical disk in which data can be written, such as a magneto-optical disk and a phase-change optical disk, it is customary to form grooves extending along recording tracks on a disk substrate. The grooves are so-called guide grooves formed along recording tracks in order to make mainly tracking servo, and an opening end between the grooves is referred to as a land. In the optical disk with the grooves being formed thereon, it is customary to make tracking servo by using a tracking error signal based upon a push-pull signal obtained from light reflected and deflected on the grooves. The push-pull signal is calculated as a difference between outputs of two photo-detectors located symmetrically across the center of the track, for example, after the two photo-detectors have detected light reflected and diffracted on the groove. In these optical disks, high recording density has been achieved so far by improving reproduction resolution of an optical pickup mounted on a reproducing apparatus. Then, improvement of the reproduction resolution of the optical pickup has been optically realized so far by reducing a wavelength λ of laser light for use in mainly reproducing data or by increasing a numerical aperture NA of an objective lens for converging laser light on the optical disk. In the respective conventional formats of a so-called CD-R available as a write-once type CD (Compact Disc), an MD (Mini Disc) available as a rewritable magneto-optical disk, a DVD-R available as a write-once DVD (Digital Versatile Disc) or a DVD+RW or DVD-RW available as a rewritable DVD (these trade names are all registered trademarks of optical disks), the groove widths most suitable for the recording and reproducing characteristics are different depending upon factors such as properties of recording films and characteristics of servo signals. In the ordinary optical disk manufacturing process, when a stamper for use in molding its substrate is manufactured, a photoresist is coated on a master substrate and the above-mentioned pits and grooves are formed by so-called photolithography using pattern exposure and development. Hence, the groove width is determined by a diameter of a beam spot of exposure beam. When a master of an optical recording and reproducing medium such as the above-mentioned CD-R and CD-RW is recorded by one exposure beam, pattern exposure is made by an optical recording apparatus of which schematic arrangement is shown in FIG. 13 . In FIG. 13 , reference numeral 20 denotes a light source formed of a He-Cd laser of gas laser using gas, for example, as an amplification medium. Laser light L emitted from this light source 20 is deflected 90° in its traveling direction by a mirror M 1 and introduced into a modulation optical system 25 . In the optical modulation system 25 , the laser light L is reduced in diameter of beam by a condenser lens L 1 and introduced into an AOM (Acousto Optical Modulator; acousto-optic modulator) 28 , in which it is modulated in light intensity in response to ultrasonic waves that were modulated based upon a recording signal supplied to the AOM 28 . Reference numeral 27 denotes a driving driver for inputting a signal such as an EFM signal. The laser light L modulated by this AOM 28 is enlarged or reduced in beam diameter by a beam enlargement lens or a beam reduction lens L 2 , is traveled as the parallel beam and reflected by a mirror M 2 , thereby being introduced into a moving optical table 40 in the horizontal direction. The moving optical table 40 includes a lens L 3 , for example, as a focusing and diffraction light correction optical system, a mirror M 3 for directing the direction of the optical axis to the irradiated surface and an objective lens L 4 . The lens L 3 is located on a light incident side convergence surface P 2 , formed at the position conjugating to the focusing condenser surface P 1 of the objective lens L 4 , at its position in which the laser light L is to be focused. Thereafter, the laser light L is focused on the surface of a photoresist 12 on the master substrate 11 through this focusing and diffracted light correction lens L 3 and the objective lens L 4 and thereby the photoresist is exposed with a predetermined pattern. The master substrate 11 is rotated as shown by an arrow b by a rotary drive means, though not shown. A dot-and-dash line c denotes a center axis of the substrate 11 . In such optical recording apparatus, the above-mentioned beam relay optical system is located between the light source 20 and the objective lens L 4 to change the focal distance of the lens L 2 or the lens L 3 such that the objective lens L 4 may focus light on the photoresist 12 and that the effective numerical aperture NA relative to the objective lens L 4 may change to change the diameter of the exposure beam. In the above-mentioned CD-R and CD-RW, concave and convex patterns of the groove are recorded by a He-Cd laser (wavelength is 442 nm), and an optimum groove width falls within a range of from 550 nm to 600 nm. Since the DVD+RW, DVD-R and DVD-RW of the high density optical disks have a recording capacity of 4.7 GB, which is high recording density as high as about 7.2 times the recording density of the CD-R and CD-RW, the optimum groove width thereof is smaller than that of the above-mentioned CD-R and the like and falls within a range of from 300 to 330 nm. Therefore, by using a Kr laser (wavelength is 413 nm) with short wavelength, the spot diameter d of exposure beam can be reduced, and hence the optimum groove width of the DVD+RW, DVD-R and DVD-RW can be realized. The spot diameter d of the exposure beam is expressed by the following equation (1): in-line-formulae description="In-line Formulae" end="lead"? d= 1.22 ×λ/NA (1) in-line-formulae description="In-line Formulae" end="tail"? (λ: recording wavelength, NA: numerical aperture) In a cited patent reference 1 (official gazette of Japanese laid-open patent application No. 10-241214), a groove width that falls within a range of from about 600 nm to 800 nm can be realized by using an Ar laser (wavelength is 458 nm). Recording wavelengths λ, track pitches, groove widths and ratios between the groove width and recording wavelength of the above-mentioned respective optical disks are shown on the following table 1. TABLE 1 Groove Recording width/ wavelength Groove Rec. λ Track pitch width wavelength Cited patent 458 nm 600-800 nm 1.31-1.75 reference 1 CD − R 442 nm 1600 nm 600 nm 1.36 CD − RW 442 nm 1600 nm 550 nm 1.24 DVD + RW 413 nm 740 nm 300 nm 0.73 DVD − R 413 nm 740 nm 330 nm 0.80 DVD − RW 413 nm 740 nm 310 nm 0.75 A study of this table 1 reveals that the ordinary density optical disks of CD-R and CD-RW have the groove widths larger than the recording wavelength (442 nm), i.e., the ratios between the groove width and the recording wavelength larger than 1.0, which is enough to expose, i.e. sensitize the photoresist in most part of the spot of exposure beam so that these groove widths can be formed relatively easily. However, the high density optical disks of the DVD+RW, DVD-R, DVD-RW and the like have the groove widths smaller than the recording wavelength (413 nm), i.e., the ratios between the groove width and the recording wavelength smaller than 1.0 so that these groove widths cannot be formed relatively easily. Further, with respect to the high recording density optical disks, there is proposed a format by which a groove width can be much more microminiaturized up to approximately less than 200 nm. In a DVR (Digital Video Recordable) that is under development as an ultra-high density optical disk, as FIG. 14 shows a schematic plan arrangement in a partly enlarged-scale, its format is discussed such that a groove is formed as a wobble groove, a track pitch being selected to be 325 nm and a groove width being selected to be about 150 nm. However, there has not yet been proposed an ultra-high density optical disk manufacturing method which is not only excellent in productivity in actual practice but also satisfactory in yield. In FIG. 14 , reference numeral 2 denotes a groove and reference numeral 8 denotes a land. A cited patent reference 2 (Japanese patent No. 3104699) has reported a molded substrate having a groove width less than 100 nm manufactured by a manufacturing method in which a land portion and a groove portion are inverted by using a so-called mother stamper whose concavities and convexities are inverted to those of a stamper by a duplicate of a stamper. However, the example described in the above-described cited patent reference 2 has an extremely large land width as compared with a groove width. The following table 2 shows groove widths, land widths, track pitches and ratios between groove width and track pitch of inventive examples 1 to 3 of this cited patent reference 2 and the CD-R, CD-RW, DVD+RW, DVD-R, DVD-RW and MD, respectively. TABLE 2 Groove Groove width/ width Land width Track pitch Track pitch Cited patent 40 nm 360 nm 400 nm 0.10 reference 2 Inventive examples 1, 2 Cited patent 60 nm 290 nm 350 nm 0.17 reference 2 Inventive example 3 CD − R 600 nm 600 nm 1600 nm 0.38 CD − RW 550 nm 600 nm 1600 nm 0.34 DVD + RW 300 nm 440 nm 740 nm 0.41 DVD − R 330 nm 410 nm 740 nm 0.46 DVD − RW 310 nm 430 nm 740 nm 0.42 MD 1200 nm 400 nm 1600 nm 0.75 As is clear from the above-described table 2, in the above-described cited patent reference 2, since the groove width is extremely small as compared with the land width, accordingly, the ratio between the groove width and the track pitch is as very small as 0.10 to 0.17 and amplitude amounts of a push-pull signal serving as a tracking servo signal and a cross-track signal (Cross Track Signal: CTS) decrease, stable tracking servo cannot be realized. Therefore, this conventional manufacturing method cannot be directly applied to an optical disk that intends to increase recording density by reducing the track pitch to less than about 350 nm. The amplitude amount of the push-pull signal is maximized when the ratio between the groove width and the track pitch is ½, and the amplitude amount of the CTS signal is maximized when the ratio between the groove width and the track pitch is approximately {fraction (1/3)} or approximately {fraction (2/3)}. As shown on the above-described table 2, in the commercially-available optical disks such as the CD-R, CD-RW, DVD+RW, DVD-R, DVD-RW, the ratio between the groove width and the track pitch falls within a range of from approximately 0.34% to 0.75%. Moreover, the above-described cited patent reference 2 can realize the narrow groove width by using the inverted pattern in which an area which might serve as a future land portion is formed as the groove by the mother stamper as described above. In this case, when recording light is wobbled and the pattern is exposed to form a wobble groove, since a portion that might be formed as a future land portion is formed as a groove, different wobble signals are recorded on the left and right of the groove. There is then a risk that a leakage of a signal will occur when a wobble signal is reproduced. Thus, a problem arises, in which it will be difficult to form wobbling grooves at a level suitable for actual practice. In view of the aforesaid aspects, it is an object of the present invention to provide an optical recording and reproducing medium, an optical recording and reproducing medium manufacturing stamper and its manufacturing method suitable for the application to high recording density optical disks such as the aforementioned DVR and which is excellent in tracking servo characteristics, in reproducing characteristics of wobble signals and which can achieve high recording density at a level suitable for actual practice. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 is a schematic cross-sectional view showing an arrangement of a main portion of an example of an optical recording and reproducing medium substrate according to the present invention; FIG. 2 is a schematic cross-sectional view showing an arrangement of a main portion of an example of an optical recording and reproducing medium; FIGS. 3A, 3B , 3 C and 3 D are manufacturing process diagrams showing an example of a manufacturing method of an optical recording and reproducing medium manufacturing mother stamper according to the present invention, respectively; FIG. 4 is a diagram used to explain a γ characteristic value; FIG. 5 is a schematic block diagram showing an arrangement of an example of an optical recording apparatus; FIG. 6 is a schematic diagram showing an arrangement of an example of an etching system; FIG. 7 is a diagram used to explain a surface roughness of a groove surface and an inclination angle of a groove side surface; FIG. 8 is an explanatory diagram showing a relationship between a wavelength of recording laser light and a groove width; FIG. 9 is a diagram showing changes of the surface roughness of the groove surface relative to an etching time; FIG. 10 is a diagram showing changes of the inclination angle of the groove side surface relative to an etching time; FIG. 11 is a schematic cross-sectional view showing arrangement of a main portion of other example of an optical recording and reproducing medium substrate according to the present invention; FIG. 12 is a schematic cross-sectional view showing an arrangement of a main portion of other example of an optical recording and reproducing medium; FIG. 13 is a schematic diagram showing an arrangement of an example of an optical recording apparatus; and FIG. 14 is a plan view showing an arrangement of a main portion of an example of a conventional optical recording and reproducing medium. detailed-description description="Detailed Description" end="lead"? |
Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced suca or sucb gene |
The invention relates to a process for the preparation of L-amino acids, in particular L-threonine, in which the following steps are carried out: a) fermentation of microorganisms of the Enterobacteriaceae family which produce the desired L-amino acid and in which at least one or more of the genes chosen from the group consisting of sucA and sucB, or nucleotide sequences which code for these, is or are enhanced, in particular over-expressed, b) concentration of the desired L-amino acid in the medium or in the cells of the bacteria, and c) isolation of the desired L-amino acid. |
1. A process for the preparation of L-amino acids, in particular L-threonine, which comprises carrying out the following steps: a) fermentation of microorganisms of the Enterobacteriaceae family which produce the desired L-amino acid and in which one or more of the genes chosen from. the group consisting of sucA and sucB, or nucleotide sequences which code for these, is or are enhanced, in particular over-expressed, b) concentration of the desired L-amino acid in the medium or in the cells of the microorganisms, and c) isolation of the desired L-amino acid, constituents of the fermentation broth and/or the biomass in its entirety or portions (>0 to 100%) thereof optionally remaining in the product. 2. A process as claimed in claim 1, wherein microorganisms in which further genes of the biosynthesis pathway of the desired L-amino acid are additionally enhanced are employed. 3. A process as claimed in claim 1, wherein microorganisms in which the metabolic pathways which reduce the formation of the desired L-amino acid are at least partly eliminated are employed. 4. A process as claimed in claim 1, wherein the expression of the polynucleotide (s) which code(s) for one or more of the genes chosen from the group consisting of sucA and sucB is increased. 5. A process as claimed in claim 1, wherein the regulatory and/or catalytic properties of the polypeptides (proteins) for which the polynucleotides sucA and sucB code are improved or increased. 6. A process as claimed in claim 1, wherein, for the preparation of L-amino acids, microorganisms of the Enterobacteriaceae family in which in addition at the same time one or more of the genes chosen from the group consisting of: 6.1 the thrABC operon which codes for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase, 6.2 the pyc gene which codes for pyruvate carboxylase, 6.3 the pps gene which codes for phosphoenol pyruvate synthase, 6.4 the ppc gene which codes for phosphoenol pyruvate carboxylase, 6.5 the pntA and pntB genes which code for transhydrogenase, 6.6 the rhtB gene which imparts homoserine resistance, 6.7 the mqo gene which codes for malate:quinone oxidoreductase, 6.8 the rhtC gene which imparts threonine resistance, 6.9 the thrE gene which codes for the threonine export protein, 6.10 the gdhA gene which codes for glutamate dehydrogenase, 6.11 the hns gene which codes for the DNA-binding protein HLP-II, 6.12 the pgm gene which codes for phosphoglucomutase 6.13 the fba gene which codes for fructose biphosphate aldolase, 6.14 the ptsH gene which codes for the phosphohistidine protein hexose phosphotransferase, 6.15 the ptsI gene which codes for enzyme I of the phosphotransferase system, 6.16 the crr gene which codes for the glucose- specific IIA component, 6.17 the ptsG gene which codes for the glucose- specific IIBC component, 6.18 the lrp gene which codes for the regulator of the leucine regulon, 6.19 the mopB gene which codes for 10 Kd chaperone, 6.20 the ahpC gene which codes for the small sub- unit of alkyl hydroperoxide reductase, 6.21 the ahpF gene which codes for the large sub- unit of alkyl hydroperoxide reductase, 6.22 the cysK gene which codes for cysteine synthase A, 6.23 the cysB gene which codes for the regulator of the cys regulon., 6.24 the cysJ gene which codes for the flavoprotein of NADPH sulfite reductase, 6.25 the cysI gene which codes for the haemoprotein of NADPH sulfite reductase, 6.26 the cysH gene which codes for adenylyl sulfate reductase, 6.27 the phoE gene which codes for protein E of outer cell membrane, 6.28 the malE gene which codes .for the periplasmic binding protein of maltose transport, 6.29 the pykF gene which codes for fructose- stimulated pyruvate kinase I, 6.30 the pfkB gene which codes for 6-phosphofructokinase II, 6.31 the talB gene which codes for transaldolase B, 6.32 the rseA gene which codes for a membrane protein which acts as a negative regulator on sigmaE activity, 6.33 the rseC gene which codes for a global regulator of the sigmaE factor, 6.34 the soda gene which codes for superoxide dismutase, 6.35 the phoB gene which codes for the positive regulator PhoB of the pho regulon, 6.36 the phOR gene which codes for the sensor protein of the pho regulon, 6.37 the sucC gene which codes for the β-sub-unit of succinyl-CoA synthetase, 6.38 the sucD gene which codes for the α-sub-unit of succinyl-CoA synthetase, is or are enhanced, in particular over-expressed, are fermented. 7. A process as claimed in claim 1, wherein, for the preparation of L-amino acids, microorganisms of the Enterobacteriaceae family in which in addition at the same time one or more of the genes chosen from the group consisting of: 7.1 the tdh gene which codes for threonine dehydrogenase, 7.2 the mdh gene which codes for malate dehydrogenase, 7.3 the gene product of the open reading frame (orf) yjfA, 7.4 the gene product of the open reading frame (orf) ytfP, 7.5 the pckA gene which codes for phosphoenol pyruvate carboxykinase, 7.6 the poxB gene which codes for pyruvate oxidase, 7.7 the aceA gene which codes for isocitrate lyase, 7.8 the dgsA gene which codes for the DgsA. regulator of the phosphotransferase system, 7.9 the fruR gene which codes for the fructose repressor, 7.10 the rpoS gene which codes for the sigma38 factor is or are attenuated, in particular eliminated or reduced in expression, are fermented. |
<SOH> FIELD OF THE INVENTION <EOH>This invention relates to a process for the preparation of L-amino acids, in particular L-threonine, using strains of the Enterobacteriaceae family in which at least one or more of the genes chosen from the group consisting of sucA and sucB is (are) enhanced. |
<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a process for the fermentative preparation of L-amino acids, in particular L-threonine, using microorganisms of the Enterobacteriaceae family which in particular already produce L-amino acids and in which at least one or more of the nucleotide sequence(s) which code(s) for the sucA and sucB genes is (are) enhanced. |
Fatty acid desaturase gene obtained from pomegranate and method for the production of unsaturated fatty acids |
The present invention relates to a process for the production of unsaturated or saturated fatty acids and to a process for the production of oils and/or triglycerides with an increased content of unsaturated or saturated fatty acids. The invention furthermore relates to nucleic acid sequences, nucleic acid constructs, vectors and organisms comprising the nucleic acid sequences, nucleic acid constructs and/or vectors. Moreover, the invention relates to fatty acid mixtures and to triglycerides with an increased content of unsaturated fatty acids, and to their use. |
1. An isolated nucleic acid that encodes a polypeptide with a desaturase activity, wherein said nucleic acid contains a sequence selected from the group consisting of: the sequence of SEQ ID NO: 2; the sequence of SEQ ID NO: 7; a sequence derived from the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 8 by back translation of the respective amino acid sequences, owing to the degeneracy of the genetic code; and a derivative of the sequence of SEQ ID NO: 2 or SEQ ID NO: 7 wherein said derivative encodes a peptide that has at least 75 % identity at the amino acid level to said polypeptide, wherein the desaturase activity of the peptide is not substantially reduced as compared with the desaturase activity of said polypeptide. 2. An amino acid sequence encoded by the nucleic acid of claim 1. 3. The amino acid sequence of claim 2, which contains the sequence of SEQ ID NO: 3 or SEQ ID NO: 8. 4. A nucleic acid construct comprising the nucleic acid of claim 1 linked to one or more regulatory signal sequences. 5. A vector comprising the nucleic acid construct of claim 4. 6. An organism comprising the vector of claim 5. 7. The organism of claim 6, which is a plant, a microorganism or an animal. 8. A transgenic plant comprising the nucleic acid of claim 1. 9. A process for production of oils or triglycerides with an increased content of unsaturated fatty acids, which comprises: introducing the nucleic acid of claim 1 to an oil-producing organism; culturing said organism; and isolating the oils or triglycerides. 10. The process of claim 9, further comprising liberating said oils or triglycerides from said organism. 11. The process of claim 9, further comprising introducing at least one further nucleic acid to the organism, wherein said at least one further nucleic acid encodes a polypeptide with a desaturase activity, which is functional or non-functional, and said at least one further nucleic acid contains a sequence selected from the group consisting of: a sequence that encodes a Δ-5-desaturase, a Δ-6-desaturase, a Δ-8-desaturase or Δ-12-desaturase; the sequence of SEQ ID NO: 5; a sequence derived from the amino acid sequence of SEQ ID NO: 6 by back translation of said amino acid sequence, owing to the degeneracy of the genetic code; and a derivative of the sequence of SEQ ID NO: 5 wherein said derivative encodes a peptide that has at least 90% identity with the amino acid sequences of SEQ ID NO: 6 and the desaturase activity of said peptide is not substantially reduced as compared to the desaturase activity of said polypeptide. 12. The process of claim 9, wherein the unsaturated fatty acids have an increased content of punicic acid. 13. The process of claim 9, wherein the unsaturated fatty acids have an increased content of octadecaconjudienoic fatty acids. 14. The process of claim 9, wherein the unsaturated fatty acids have an increased content of octadecaconjutetraenoic fatty acids. 15. The nucleic acid of claim 1, wherein the polypeptide has a functional desaturase activity. 16. The process of claim 11, wherein the polypeptide has a functional desaturase activity. 17. The process of claim 9, wherein the organism is a plant or a microorganism. 18. A composition comprising oils or triglycerides with an increased content of unsaturated fatty acids prepared by the process of claim 9. 19. A composition comprising oils or triglycerides with an increased content of unsaturated fatty acids prepared by the process of claim 11. 20. A process for generating transgenic plants comprising introducing the construct of claim 4 to a plant. 21. A process for isolating a genomic sequence via homology screening comprising screening a genome with the nucleic acid of claim 1 or a fragment thereof. 22. A process for production of foods, animal feed, cosmetics or pharmaceuticals comprising introducing the nucleic acid of claim 1 to an organism. 23. An isolated nucleic acid sequence that encodes a protein that converts a fatty acid of structure I and has two double bonds separated from each other by a methylene group, to a triunsaturated fatty acid of structure II in which the three double bonds of the fatty acid are conjugated and the variables in structure I and structure II have the following meanings: R1=hydrogen, substituted or unsubstituted, unsaturated or saturated, branched or unbranched C1-C10-alkyl-, R2=substituted or unsubstituted, unsaturated or saturated C1-C9-alkyl-, R3 and R4 independently of one another, hydrogen, substituted or unsubstituted, saturated or unsaturated, branched or unbranched C1-C22-alkylcarbonyl- or phospho-, and n=1 to 14. 24. An isolated nucleic acid that encodes a polypeptide with a desaturase activity, wherein said nucleic acid contains a sequence selected from the group consisting of: the sequence of SEQ ID NO: 5; a sequence derived from the amino acid sequence of SEQ ID NO: 6 by back translation of said amino acid sequence, owing to the degeneracy of the genetic code; a derivative of the sequence of SEQ ID NO: 5 that encodes a peptide which has at least 90% identity at the amino acid level with the amino acid sequence of SEQ ID NO: 6, wherein the desaturase activity of said peptide is not substantially reduced as compared with the desaturase activity of said polypeptide. 25. An amino acid sequence encoded by the nucleic acid of claim 24. 26. A nucleic acid construct comprising the nucleic acid of claim 24, linked to one or more regulatory signal sequences. 27. A vector comprising the nucleic acid construct of claim 26. 28. An organism comprising the vector of claim 27. 29. A transgenic plant comprising the nucleic acid of claim 24. 30. A process for isolating a genomic sequence via homology screening comprising screening a genome with the nucleic acid of claim 24 or a fragment thereof. 31. The process of claim 11, wherein the nucleic acid encodes a non-functional polypeptide. 32. The process of claim 31, wherein the nucleic acid is an antisense nucleic acid. 33. An isolated nucleic acid that encodes a polypeptide with a non-functional desaturase activity, wherein said nucleic acid contains a sequence selected from the group consisting of: the sequence of SEQ ID NO: 2; the sequence of SEQ ID NO: 7; a sequence derived from the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 8 by back translation of the respective amino acid sequences, owing to the degeneracy of the genetic code; and a derivative of the sequence of SEQ ID NO: 2 or SEQ ID NO: 7 wherein said derivative encodes a peptide that has at least 75 % identity at the amino acid level to said polypeptide. 34. The nucleic acid of claim 33, which is an antisense nucleic acid. 35. A nucleic acid construct comprising the nucleic acid of claim 33 linked to one or more regulatory signal sequences. 36. A vector comprising the nucleic acid construct of claim 35. 37. An organism comprising the vector of claim 36. 38. The organism of claim 37, which is a plant, a microorganism or an animal. 39. A transgenic plant comprising the nucleic acid of claim 33. 40. A process for production of oils or triglycerides with an increased content of unsaturated fatty acids, which comprises: introducing the nucleic acid of claim 33 to an oil-producing organism; culturing said organism; and isolating the oils or triglycerides. 41. The process of claim 40, further comprising liberating said oils or triglycerides from said organism. 42. The process of claim 40, further comprising introducing at least one further nucleic acid to the organism, wherein said at least one further nucleic acid encodes a polypeptide with functional or non-functional desaturase activity and said at least one further nucleic acid contains a sequence selected from the group consisting of: a sequence that encodes a Δ-5desaturase, a Δ-6-desaturase, a Δ-8-desaturase or Δ-12-desaturase; the sequence of SEQ ID NO: 5; a sequence derived from the amino acid sequence of SEQ ID NO: 6 by back translation of said amino acid sequence, owing to the degeneracy of the genetic code; and a derivative of the sequence of SEQ ID NO: 5 wherein said derivative encodes a peptide that has at least 90% identity with the amino acid sequences of SEQ ID NO: 6. 43. The process of claim 40, wherein the oils or triglycerides have an increased content of punicic acid. 44. The process of claim 40, wherein the oils or triglycerides have an increased content of octadecaconjudienoic fatty acids. 45. The process of claim 40, wherein the oils or triglycerides have an increased content of octadecaconjutetraenoic fatty acids. 46. A process for production of oils or triglycerides with an increased content of saturated fatty acids, which comprises: introducing a nucleic acid to an oil-producing organism wherein said nucleic acid encodes a polypeptide with a non-functional desaturase activity, wherein said nucleic acid contains a sequence selected from the group consisting of: the sequence of SEQ ID NO: 2; the sequence of SEQ ID NO: 7; a sequence derived from the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 8 by back translation of the respective amino acid sequences, owing to the degeneracy of the genetic code; and a derivative of the sequence of SEQ ID NO: 2 or SEQ ID NO: 7 wherein said derivative encodes a peptide that has at least 75 % identity at the amino acid level to said polypeptide; culturing said organism; and isolating said oils or triglycerides. 47. The process of claim 46, wherein the nucleic acid is an antisense nucleic acid. 48. The process of claim 46, further comprising liberating said oils or triglycerides. 49. The process of claim 46, wherein the oil-producing organism is a plant or a microorganism. 50. A composition comprising oils or triglycerides with an increased content of unsaturated fatty acids prepared by the process of claim 46. 51. A process for isolating a genomic sequence via homology screening comprising screening a genome with the nucleic acid of claim 33 or a fragment thereof. 52. A process for production of foods, animal feed, cosmetics or pharmaceuticals comprising introducing the nucleic acid of claim 33 to an organism. 53. An isolated nucleic acid that encodes a polypeptide with a non-functional desaturase activity, wherein said nucleic acid contains a sequence selected from the group consisting of: the sequence of SEQ ID NO: 5; a sequence derived from the amino acid sequence of SEQ ID NO: 6 by back translation of said amino acid sequence, owing to the degeneracy of the genetic code; and a derivative of the sequence of SEQ ID NO: 5 that encodes a peptide which has at least 90% identity at the amino acid level with the amino acid sequence of SEQ ID NO: 6. 54. The nucleic acid of claim 53, which is an antisense nucleic acid. 55. A nucleic acid construct comprising the nucleic acid sequence of claim 53, which is linked to one or more regulatory signal sequences. 56. A vector comprising the nucleic acid construct of claim 55. 57. An organism comprising the vector of claim 56. 58. A transgenic plant comprising the nucleic acid of claim 53. |
Leather degreasing agent |
The invention relates to a degreaser for treating hides, skins, pelts and other intermediates in leather and fur manufacture and also wool or related proteinaceous materials that is based on nonionic surfactants of the type of alcohol alkoxylates, comprising alcohol alkoxylates obtained by reaction of at least one alcohol ROH with n mol of at least one alkylene oxide per mole of alcohol ROH, where R is an alkyl radical of 5 to 30 carbon atoms which has a main chain, of 4 to 29 carbon atoms that has at least one C1— to C10-alkyl branch attached in the chain middle; the alkylene oxide has 2 to 6 carbon atoms, and n is an integer from 1 to 100. The invention further relates to a process for degreasing hide, skins, pelts and other intermediates of leather and fur manufacture and wool or related proteinaceous materials, which comprises using a degreaser according to the present invention, and to the use of the degreasers. |
1-13. (canceled) 14. A degreaser for treating hides, skins, pelts and other intermediates in leather and fur manufacture and also wool or related proteinaceous materials that is based on nonionic surfactants of the type of alcohol alkoxylates, comprising alcohol alkoxylates obtained by reaction of at least one alcohol ROH with n mol of at least one alkylene oxide per mole of alcohol ROH, where R is an alkyl radical of 5 to 30 carbon atoms which has a main chain, which is the longest alkyl chain of the radical R, of 4 to 29 carbon atoms that has at least one C1- to C10-alkyl branch attached in the chain middle, beginning at the carbon atom C#2, the numbering starting with the carbon atom (C#1) that is attached directly to the oxygen atom adjacent to the radical R, and ending with the carbon atom ω-2, where w is the terminal carbon atom of the main chain, including C#2 and the carbon atom ω-2, the alkylene oxide has 2 to 6 carbon atoms, and n is an integer from 1 to 100. 15. A degreaser as claimed in claim 14, comprising a mixture of alcohol alkoxylates based on 1 to 3 different alcohols ROH. 16. A degreaser as claimed in claim 14, wherein the main chain has at least one C2- to C4-alkyl radical branch attached in the chain middle. 17. A degreaser as claimed in claim 14, wherein the radical R has 10 to 20 carbon atoms, of which 9 to 19 carbon atoms form the main chain. 18. A degreaser as claimed in claim 14, wherein the alkylene oxide is ethylene oxide. 19. A degreaser as claimed in claim 14, wherein n is an integer from 3 to 15. 20. A degreaser as claimed in claim 14, being a mixture of alcohol alkoxylates based on at least one alcohol alkoxylate obtained by reaction of an alcohol ROH with n>6 mol of at least one alkylene oxide per mole of alcohol ROH and on at least one further alcohol alkoxylate obtained by reaction of an alcohol ROH with n=1 to 6 mol of at least one alkylene oxide per mole of alcohol ROH, the alcohol ROH and the alkylene oxide in the at least two alcohol alkoxylates being identical or different. 21. A degreaser as claimed in claim 14, comprising as well as the alcohol alkoxylates >1 to 25% by weight, based on the amount of alcohol alkoxylates used, of unconverted alcohol ROH. 22. A process for degreasing hide, skins, pelts and other intermediates of leather and fur manufacture and wool or related proteinaceous materials, which comprises using a degreaser as claimed in claim 14. 23. A process as claimed in claim 22, wherein the alcohol alkoxylates are used in an amount of from 0.5 to 5% by weight, based on the weight of the hides, skins, pelts or other intermediates of leather and fur manufacture or wool or related proteinaceous materials. 24. A process as claimed in claim 22, further comprising setting a temperature of from 15 to 45° C. 25. A degreaser as claimed in claim 14, wherein the alcohol alkoxylate is obtained by reaction of an alcohol with ethylene oxide and propylene oxide. |
Human type antihuman ige receptor antibody and fragment |
An antibody or an antibody fragment is provided that is efficacious for treating allergic diseases in which IgE is involved. A human antibody and a fragment thereof to a receptor (FcεRI) having high affinity to Fc portion of IgE was obtained using phage antibody display technique. The human anti-IgE receptor antibody and a fragment thereof of the invention has an activity to inhibit the binding between IgE and IgE receptor and hence is expected to be useful as a medicament for treating allergic diseases caused by the binding between IgE and IgE receptor. |
1. A gene fragment encoding VH chain of human anti-IgE receptor antibody that binds to a human IgE receptor and has an activity to inhibit the binding between the IgE receptor and IgE. 2. The gene fragment of claim 1 wherein said human IgE receptor is a receptor for Fc portion of IgE (herein after referred to as “FcεR”), preferably FcεRI. 3. The gene fragment of claim 1 wherein complimentary determining regions (CDR1 to CDR3) in said VH chain have the following amino acid sequences: CDR1: Ser Asn Tyr Met Ser (SEQ ID NO: 5); CDR2: Val Ile Tyr Arg Gly Gly Ser Gly Asp Asn Thr Tyr Tyr Ala Gly Ser Val Lys Gly (SEQ ID NO: 6); CDR3: Ser Ser Asp Val Gly Tyr Gly Ile Leu Arg Gly Tyr Met Asp Val (SEQ ID NO: 7). 4. The gene fragment of claim 1 wherein said VH chain gene consists of a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 2. 5. A gene fragment encoding VL chain of human anti-IgE receptor antibody that binds to a human IgE receptor and has an activity to inhibit the binding between the IgE receptor and IgE. 6. The gene fragment of claim 5 wherein said human IgE receptor is a receptor for Fc portion of IgE (herein after referred to as “FcεR”), preferably FcεRI. 7. The gene fragment of claim 5 or 6 wherein complimentary determining regions (CDR1 to CDR3) in said VL chain have the following amino acid sequences: CDR1: Ser Gly Gln Asp Leu Thr Asn Lys Tyr Val Ser (SEQ ID NO: 8); CDR2: Glu Asp Ser Lys Arg Pro Ser (SEQ ID NO: 9); CDR3: Gln Ala Tyr Asp Thr Asn Gly Trp Val (SEQ ID NO: 10). 8. The gene fragment claims 5 wherein said VL chain gene consists of a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 4. 9. A single-chain Fv gene comprising the VH chain gene of any one of claims 1 to 4 bound to a gene fragment encoding VL chain of human anti-IgE receptor antibody that binds to a human IgE receptor and has an activity to inhibit the binding between the IgE receptor and IgE. 10. A gene fragment encoding a human anti-IgE receptor antibody comprising the VH chain gene of any one of claims 1 to 4 and a chain gene fragment encoding VL chain of human anti-IgE receptor antibody that binds to a human IgE receptor and has an activity to inhibit the binding between the IgE receptor and IgE, said VH chain gene and said VL chain gene being bound to a human antibody CH chain gene and a human antibody CL chain gene, respectively. 11. A gene fragment encoding a human anti-IgE receptor antibody fragment comprising the VH chain gene of any one of claims 1 to 4 and/or a VL chain gene fragment encoding VL chain of human anti-IgE receptor antibody that binds to a human IgE receptor and has an activity to inhibit the binding between the IgE receptor and IgE, said VH chain gene and said VL chain gene being bound to a human antibody CH chain gene or a portion thereof and a human antibody CL chain gene or a portion thereof, respectively. 12. The gene fragment of claim 11 wherein said antibody fragment is selected from Fab, Fab′, or F(ab′)2. 13. A gene fragment encoding a human anti-IgE receptor antibody fragment comprising the single-chain Fv gene of claim 9 bound to a human antibody CH chain gene or a portion thereof or a human antibody CL chain gene or a portion thereof. 14. A human anti-IgE receptor antibody or a human anti-IgE receptor antibody fragment that is prepared by the genetic recombination technique with an expression vector in which the gene fragment of claim 1 is incorporated. 15. An inhibitor to the IgE-IgE receptor binding comprising the human anti-IgE receptor antibody or the human anti-IgE receptor antibody fragment of claim 14. 16. A medicament for treating allergic diseases comprising the human anti-IgE receptor antibody or the human anti-IgE receptor antibody fragment of claim 14. |
<SOH> BACKGROUND ART <EOH>Fcε receptor I (hereinafter also referred to as “FcεRI”), one of receptors (FcεR) for Fc portion (Fcε) of immunoglobulin E (IgE), has high affinity to IgE. FcεRI is a glycoprotein molecule expressed principally on the cellular membrane of mast cells and basophiles and plays an important role in type I allergic reaction for activation of these cells. Upon crosslinkage of antigen-specific IgE with corresponding multivalent antigens, i.e. allergens, FcεRI aggregates and signal transduction mechanism begins to act to thereby activate mast cells. As a result, a cellular degranulation occurs to thereby release chemical mediators such as histamine and serotonin, inducing novel synthesis and release of leukotrienes, prostaglandins and the like to provoke type I allergic reaction. Human FcεRI consists of three distinct subunits, i.e. an IgE binding factor a chain, a signal amplifying factor β chain, and a signal transmitting factor Υ chain, forming either a tetramer consisting of each one α and β chains and two Υ chains, or a trimer consisting of one α chain and two Υ chains. On the surface of the cellular membrane of mast cells and basophiles, tetrameric FcεRI is principally expressed and plays an important role in type I allergic reaction for activation of these cells as described above. On the cellular membrane of skin Langerhans cells, monocytes, eosinophiles, dendritic cells, and platelets, expression of trimeric FcεRI is principally observed though at a lower level than that of tetrameric FcεRI and is suggested to contribute to antigen display and production of chemical mediators. It is believed that the a chain alone in FcεRI directly interacts with IgE and its binding region to IgE spans overall the extracellular region of the α chain (Nature, vol. 406 (2000), p. 259). As for function of FcεRI within the living body, analysis with the α chain-knockout mouse suggested that FcεRI may contribute to protection mechanism from infection with certain parasite. However, a phenotype is not found under normal conditions in the knockout mouse and hence an FcεRI gene is not a gene indispensable to survival in mice. As described above, interaction between IgE and FcεRI is important for onset of disease in case of allergic diseases. It is also known that FcεRI-expressing cells increase in patient blood. Besides, it is known that expression of FcεRI is enhanced in eosinophiles, monocytes and basophiles in peripheral blood of patients suffering from atopic asthma, allergic rhinitis and atopic dermatitis, suggesting its involvement in onset of diseases. It is reported that autoantibody against FcεRI α chain occurs in serum from some patients with chronic hives. Thus, activation of FcεRI-expressing cells due to crosslinkage of FcεRI with anti-FcεRI autoantibody has been postulated as a mechanism for onset of disease. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 shows reactivity of the obtained scFv clone (αFcR51) with various antigens (human FcεRI α chain, human serum albumin, human TNFRI, or human serum) as measured in ELISA. FIG. 2 shows an inhibitory activity of scFv clone (αFcR51) to the IgE-FcεRI binding as measured in ELISA. FIG. 3 shows an inhibitory effect of scFv clone (αFcR51) to histamine release by inhibiting the IgE-FcεRI binding. detailed-description description="Detailed Description" end="lead"? |
Novel glucans and novel glucansucrases derived from lactic acid bacteria |
The invention pertains to glucans capable of being produced by glucosyltransferase activity of a lactic acid bacterium on a sucrose substrate, the glucan having an average molecular weight between 10 kDa and 1 GDa, consisting essentially of alpha (1,3)- and alpha (1,6)-linked anhydroglucose units (AGU) and to glucansucrases capable of producing these glucans from sucrose. The glucans have thickening and anti-corrosive properties. The glucans can be chemically modified. |
1-20. (cancelled) 21. A process of producing a glucan having at least 10 anhydroglucose units, having a backbone consisting essentially of α(1,3)- and/or α(1,6)-linked anhydroglucose units (AGU), comprising subjecting sucrose to the activity of a glucosyltransferase produced by a Lactobacillus strain capable of producing α(1,3)- and/or α(1,6)-linked glucans, or to the Lactobacillus strain capable of expressing the glucosylfransferase. 22. A Lactobacillus strain capable of producing, in the presence of sucrose, a glucan having at least 10 anhydroglucose units (AGU) having a backbone consisting essentially of α(1,3)- and/or α(1,6)-linked AGU. 23. A glucan capable of being produced by glucosyltransferase activity of a lactic acid bacterium on a sucrose substrate, the glucan having an average molecular weight between 10 kDa and 1 GDa, and having a backbone consisting essentially of α(1,3)- and α(1,6)-linked anhydroglucose units (AGU). 24. The glucan according to claim 23, which has an average molecular weight between 10 kDa and 50 Mda. 25. The glucan according to claim 23, which is capable of being produced by glucosyltransferase activity of a Lactobacillus species. 26. The glucan according to claim 25, comprising 15-80% of α(1,3)-linked AGU, 2-80% of α(1,6)-linked AGU, and 2-25% of α-(1,3,6)-linked AGU. 27. The glucan according to claim 26, having an average molecular weight of 50 kDa-1 MDa and comprising 30-45% of α(1,3)-linked AGU, 30-45% of α(1,6)-linked AGU, and 3-13% of α(1,3,6)-linked AGU. 28. The glucan according to claim 26, having an average molecular weight of 10-50 MDa and comprising 15-26% α(1,3)-linked AGU, 30-50% of α(1,6)-linked AGU, 5-20% of α(1,3,6)-linked AGU. 29. The glucan according to claim 26, having an average molecular weight of 1-50 MDa and comprising 45-60% of α(1,3)-linked AGU, 4-10% of α(1,6)-linked AGU, and 10-20% of α(1,3,6)-linked AGU. 30. A glucan capable of being produced by glucosyltransferase activity of a lactic acid bacterium on a sucrose substrate, having an average molecular weight of 10-50 MDa and comprising 80-99% of α(1,6)-linked AGU and 0-15% of α(1,3)-linked AGU. 31. A protein having glucosyltransferase activity, capable of producing, in the presence of sucrose, a glucan according to claim 23. 32. The protein according to claim 31, comprising an amino acid sequence of at least 100 amino acids, the sequence exhibiting at least 80% amino acid identity with any one of the amino acid sequences of SEQ ID No. 2, 4, 8, 10, 12, 14, 16, 18, 20 and 22, and/or having a stretch of 100 amino acids having at least 90%, amino acid identity with any one of the said amino acid sequences. 33. The protein according to claim 31, comprising an amino acid sequence having at least 99% amino acid identity with the amino acid sequence of SEQ ID No. 6, and/or having a stretch of 100 amino acids having 100% amino acid identity with the amino acid sequence of SEQ ID No. 6. 34. A nucleic acid sequence encoding the protein according to claim 31. 35. A recombinant host cell containing one or more copies of a nucleic acid construct comprising a nucleic acid sequence according to claim 34 and capable of expressing a protein having glucosyl-transferase activity. 36. A Lactobacillus strain, capable of producing the glucan according to claim 23. 37. The Lactobacillus strain according to claim 36, corresponding to strain 33, 180 or ML1 as described herein. 38. A Leuconostoc strain, capable of producing the glucan according to claim 30. 39. The Leuconostoc strain according to claim 38 corresponding to strain 86, deposited under accession number LMG P-20350. 40. A chemically modified glucan, which is obtained by 2,3-oxidation, 6-oxidation, phosphorylation, acylation, alkylation, hydroxyalkylation, carboxymethylation, aminoalkylation of one or more AGU of a glucan according to claim 23. 41. A method of thickening a water-based composition, coprising incorporating in said composition the glucan according to claim 23. 42. A method of controlling corrosion of a substrate, comprising applying onto the substrate the glucan according to claim 23. 43. A method of improving the condition of the gastrointestinal tract, modulating immune properties, controlling ulcers or lowering cholesterol levels, comprising administering an effective amount of the glucan according to claim 23. 44. A method of improving the condition of the gastrointestinal tract, comprising administering an effective amount of a Lactobacillus bacterium capable of producing the glucan according to claim 23 as a probiotic agent, or together with an indigestible glucan, as a synbiotic agent. 45. A nutritional or pharmaceutical composition comprising the glucan of claim 23 as a prebiotic or as a bioactive agent. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Several bacteria are known to produce exopolysaccharides, i.e. polysaccharides secreted into the culture medium. Well-known examples of bacterial exopolysaccharides include xanthan from Xanthomonas campestris , gellan from Sphingomonas paucimobilis and pullulan from Aureobasidium pullulans . Lactic acid bacteria known to produce exopolysaccharides include Leuconostoc mesenteroides strains producing dextrans, α(1→6)-linked poly-anhydroglucose, and alternans i.e. poly-anhydroglucoses having alternating α(1→6) and α(1→3)-linkages, oral Streptococcus strains producing glucans responsible for dental plaque formation, and a particular Lactobacillus reuteri strain producing α(1,6)- and α(1,4)-linked anhydroglucose (Van Geel-Schutten, et al., Appl. Environ. Microbiol . (1999) 65, 3008-3014). The properties of exopolysaccharides depend on the type of monosaccharide units, the type of linkages, the degree and type of branching, the length of the polysaccharide chain, the molecular weight and the conformation of the polymers. Argüello-Morales et al. ( FEMS Microbiol. Lett. 182 (2000) 81-85) describe an alternansucrase from Leuconostoc mesenteroides NRRL B-1355. Monchois et al, ( Gene 182 (1996) 23-32; FEMS Microbiol. Lett. 159 (1998) 307-315) for instance describe two different dextransucrases from Lc. mesenteroides NRRL B-1299. A method for selecting Leuconostoc mesenteroides strains that produce a high proportion of alternan to dextran is described in U.S. Pat. No. 5,789,209. The prior art does not disclose or suggest other lactic acid bacteria than Leuconostoc or Streptococcus that are capable of producing glucans having both α(1→6) and α(1→3)-linkages. |
<SOH> SUMMARY OF THE INVENTION <EOH>Several lactic acid bacteria strains were found, according to the invention, to be capable of producing a particular class of glucans. These glucans have in common that their anhydroglucose units (AGU) are linked α(1,3)- and/or α(1,6)-glucosidic bonds, i.e. they are α-glucans largely or completely devoid of α(1,4)-bonds. These glucans may be of the alternan (alternating α(1,3) and α(1,6) linkages), mutan (mixed α(1,3) and α(1,6) linkages, usually α(1,3) predominant) or dextran (mainly α(1,6) linkages, some α(1,3)) type, or other type. The glucans can be produced from sucrose, using sucrase enzymes which are active in the lactic acid bacteria. They can be produced on a large scale and isolated in a commercially feasible way, as the glucans are produced outside the bacterial cell, or even in the absence of the bacteria, using isolated sucrase enzymes. The glucans are produced by food-grade strains and have interesting properties, such as prebiotic utility or thickening of water-based compositions. The invention is concerned with these novel glucans, with the lactic acid bacterial, especially Lactobacillus strains and their enzymic proteins that produce these glucans from sucrose, as well as with methods for producing the glucans using the strains and/or their enzymes, with nucleotide sequences encoding these enzymic proteins which convert sucrose, with the use of the glucans as thickeners, prebiotics, anticorrosives, etc., and as starting materials for modified glucans. |
Casting preforms for optical fibres |
This invention relates to a method of preparing a preform for an optical fibre, and more particularly to a method of preparing a preform for a polymer holey optical fibre. The invention provides a method of preparing a preform for manufacture of a polymer holey optical fibre comprising casting a preform body in a mould from a suitable material, said mould including at least one protrusion adapted to form a corresponding hole within the preform, and subsequently separating the preform body and mould. The invention also provides a method of preparing a preform for manufacture of a polymeric holey optical fibre comprising separately casting one or more elements of a preform in respective mould(s) from a suitable material, and separating said elements from said respective mould(s) and combining said elements to construct a preform having a plurality of holes therein, each hole being formed in an element or formed by the combination of two or more elements. |
1. A method of preparing a preform for manufacture of a polymeric holey optical fibre comprising casting a preform body in a mould from a suitable material, said mould including a plurality of protrusions adapted to form a corresponding plurality of holes within the preform, and subsequently separating the preform body and mould. 2. A method of preparing a preform for manufacture of a polymeric holey optical fibre comprising separately casting a plurality of elements of a preform in respective mould(s) from a suitable material, and separating said elements from said respective mould(s) and combining said elements to construct a preform having a plurality of holes therein, each hole being formed in an element or formed by the combination of two or more elements. 3. The method as claimed in claim 1 or 2, wherein the material from which the preform is cast comprises a suitable monomeric or mixed polymeric/monomeric material. 4. The method as claimed in any one of claims 1 to 3, wherein the said plurality of holes in the preform pass through the preform. 5. The method as claimed in any one of claims 1 to 4, wherein the said plurality of holes have parallel axes and are parallel to the principal axis of the preform. 6. The method as claimed in any one of claims 1 to 5, wherein the thermal expansion coefficients of the mould and the polymer are sufficiently different so that heating or cooling causes dimensional changes in the mould relative to the polymer to facilitate removal of the preform from the mould. 7. The method as claimed in any one of claims 1 to 6, wherein said mould is a sacrificial mould. 8. The method as claimed in any one of claims 1 to 7, wherein one or more surfaces of the mould are provided with a sacrificial surface coating in order to facilitate the separation of the mould from the body of the cast preform upon completion of the casting process. 9. The method as claimed in any one of claims 1 to 6, wherein one or more surfaces of said mould are coated with an adhesion reducing material in order to facilitate the separation of the mould and the preform after casting has occurred. 10. The method as claimed in claim 9, wherein said adhesion reducing material is PTFE. 11. The method as claimed in any one of claims 1 to 10, wherein the mould is heated to facilitate removal of the body from the mould. 12. The method as claimed in claim 7 wherein after casting the mould is liquefied and removed in a liquid state. 13. The method as claimed in claim 7 wherein a solvent is used to dissolve the mould after casting is complete. 14. The method as claimed in claim 7 wherein the mould comprises a particulate material and a binder that is dissolved or melted upon the completion of the casting process so as to facilitate destruction of the mould and the removal of the casted preform. 15. The method as claimed in any one of claims 1 to 5 wherein the mould is inflated by means of a liquid or gas whilst casting occurs and subsequently deflated upon completion of the casting process so as to facilitate the removal of the casting from the mould. 16. The method as claimed in any one of claims 1 to 5 wherein lubricant is used to reduce the adhesion between the mould and the body of the cast preform so as to facilitate separation upon completion of the casting process. 17. The method as claimed in claim 1 or 2 wherein the mould is formed from a shape memory metal so as to facilitate separation of the mould from the body of the cast preform upon the completion of the casting process. 18. The method as claimed in claim 16 wherein the shape memory alloy is an alloy of nickel and titanium which is used to form a rod around which the body of a preform is cast, such that after casting the rod of shape memory alloy is cooled, resulting in a contraction in its shape and facilitating its removal from the surrounding cast body. 19. A preform for manufacture of a polymeric holey optical fibre comprising a preform body cast from a suitable material, said preform body including a plurality of holes. 20. A preform for manufacture of a polymeric holey optical fibre comprising a plurality of elements cast from a suitable material, said elements being combined to construct a preform having a plurality of holes therein, each hole being formed in an element or formed by the combination of two or more elements. 21. A preform as claimed in claim 19 or 20 wherein the material from which the preform is cast comprises a monomeric or mixed polymeric/monomeric material. 22. A preform as claimed in any one of claims 19 to 21 wherein the said plurality of holes in the preform pass through the preform. 23. A preform as claimed in any one of claims 19 to 22 wherein the said plurality of holes have parallel axes and are parallel to the principal axis of the preform. |
<SOH> BACKGROUND TO TH INVENTION <EOH>Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. In the late 1990's, Philip Russell from the University of Bath, United Kingdom and his co-workers developed optical fibres which comprised micro structured silica with a series of several hundred air holes running along its length. These fibres were sometimes referred to as holey fibres and more lately as crystal fibres due to the complex lattice microstructure of the air holes. Technically, such holey or crystal fibres do not include a “core” or “cladding” as the terms are used when referring to conventional graded index optical fibres. In the art, however, the term “cladding” is sometimes used to refer to the microstructure or lattice of air holes, of the “core” being a reference to the defect or irregularity in this microstructure lattice, ie. absence of an air hole through which the fibre transmits light. The first generation of fibres used a simple repeating triangular arrangement of air holes, with a single missing air hole forming the defect through which light was transmitted. More complex structures have now been developed. Originally, Russell and his team developed the fibres to exploit photonic band gap effect. However, it was soon realised that the fibres also operated by simple index guidance due to the high refractive index of the core region or defect compared to the effective index of the surrounding air holes or cladding microstructure. While the performance of crystal fibres via index guiding is well known, their use for transmission via the photonic band gap effect is not as well known. In particular, the size, shape and layout of the air holes must be strictly controlled to first realise and enhance transmission by photonic band gap. Accordingly, it would be useful to have an improved production method for producing optical fibre which not only provides consistent results but which allows more varied arrangement of the fibre. It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. |
<SOH> SUMMARY OF THE INVENTION <EOH>To this end one aspect of the present invention provides a method of preparing a preform for manufacture of a polymeric holey optical fibre comprising casting a preform body in a mould from a suitable material, said mould including a plurality of protrusions adapted to form a corresponding plurality of holes within the preform, and subsequently separating the preform body and mould. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. A further aspect of the present invention provides a method of preparing a preform for manufacture of a polymeric holey optical fibre comprising separately casting a plurality of elements of a preform in respective mould(s) from a suitable material, and separating said elements from said respective mould(s) and combining said elements to construct a preform having a plurality of holes therein, each bole being formed in an element or formed by the combination of two or more elements. A further aspect of the present invention provides a preform for manufacture of a polymeric holey optical fibre comprising a preform body cast from a suitable material, said preform body including a plurality of holes. A further aspect of the present invention provides a preform for manufacture of a polymeric holey optical fibre comprising a plurality of elements cast from a suitable material, said elements being combined to construct a preform having a plurality of holes therein, each hole being formed in an element or formed by the combination of two or more elements. Typically, the material from which the preform is cast comprises a suitable monomeric or mixed polymeric/monomeric material. Preferably, the holes in the preform pass through the preform. Preferably, the holes have parallel axes and are parallel to the principal axis of the preform. Advantageously, the present invention allows the casting of preforms, capillaries and canes for photonic crystal fibres. The casting method of the present invention can be used to produce the preform as a unitary body, or as a series of separate interconnectable elements. The preform can be separated from the mould as a unitary body for later drawing into a fibre. Alternatively, in some cases it may be preferable to draw the optical fibre directly from the preform while it remains in the mould. The above described technique and its preferred embodiments provides a number of significant advantages over the prior art. They include the opportunity to produce holey fibre preforms with discrete elements, eg. air holes, of various shapes and sizes, complex fibre shapes which are currently difficult or expensive to produce using conventional techniques, eg. multiple core structures, ability to produce holey fibres from a wide range of optically suitable materials than is currently used, a more efficient mechanism for producing holey optical fibres and preforms, and the opportunity to provide continuous production of such products. |
Fabric softener composition |
The invention relates to a fabric softener composition comprising an aqueous phase comprising a cationic softener and a hydrophobic phase comprising a fragrance. The composition should be shaken prior to use for adapting a dispersion which separates into an aqueous phase and a hydrophobic phase after use. The composition may be used as fabric softener to give a pleasant feet, and to prolong the release of fragrance on fabrics up to 7 days. |
1. A fabric softener composition having an upper hydrophobic phase comprising a fragrance and a lower aqueous phase comprising a cationic softener wherein the pH value of the aqueous phase is below 4. 2. A fabric softener composition according to claim 1 wherein the cationic softener is selected from the group of imidazolinium salts; amido amines; and ester-linked quaternary ammonium salts. 3. A fabric softener composition according to claim 1 comprising up to 18% (w/w) of the cationic softener. 4. A fabric softener composition according to claim 1 wherein the upper hydrophobic phase further comprises an oil is selected from the group consisting of hydrocarbon oil, silicone oil and ester oil. 5. A fabric softener composition according to claim 4 wherein the oil has a hydrophilic-lipophilic balance less than 5. 6. A fabric softener composition according to claim 1 wherein the upper hydrophobic phase further comprises an oil and wherein the fabric softener composition comprising from 1% to 50% (w/w) of the oil. 7. A fabric softener composition according to claim 1 comprising from 2% to 18% (w/w) of cationic softener; from 1% to 50% (w/w) of oil; from 0.1% to 3% (w/w) of fragrance; and from 29% to 96.9% (w/w) of water. 8. A fabric softener composition according to claim 1 wherein the fragrance is characterised by a clogP greater than 2. 9. A fabric softener composition according to claim 1 adapted to disperse upon agitation, wherein the dispersion separates into a hydrophobic phase and an aqueous phase from about 10 to about 40 minutes after agitation. 10. A method of prolonging the release of fragrance from a fabric up to 7 days comprising the step of treating a fabric with the fabric softener composition of claim 1. 11. A method of prolonging the release of fragrance from a fabric up to 7 days comprising the step of treating a fabric with a fabric softener composition of claim 1 comprising: from 2% to 18% (w/w) of cationic softener; from 1% to 50% (w/w) of oil; from 0.1% to 3% (w/w) of fragrance; and from 29% to 96.9% (w/w) of water. 12. A packaged fabric softener composition comprising of: (1) a package; (2) the fabric softener composition of claim 1. 13. A packaged fabric softener composition of claim 12 comprising from 2% to 18% (w/w) of cationic softener; from 1% to 50% (w/w) of oil; from 0.1% to 3% (w/w) of fragrance; and from 29% to 96.9% (w/w) of water. 14. A fabric softener composition according to claim 7 wherein the cationic softener is selected from the group of imidazolinium salts; amido amines; and ester-linked quaternary ammonium salts. 15. A fabric softener composition according to claim 14 wherein the fragrance is characterised by a clogP greater than 2. 16. A fabric softener composition according to claim 15 wherein the fragrance is characterised by a clogP greater than 3. 17. A fabric softener composition according to claim 8 wherein the fragrance is characterised by a clogP greater than 3. |
Rotary hearth furnace for use in the iron and steel industry |
A rotary hearth furnace for use in the iron and steel industry comprises a furnace (12, 112) with plan in the shape of an annulus, closed at the bottom by a rotary hearth (14, 114), lined at the top with refractory material (15, 115), and a base (28, 128; 30, 130) of the furnace (12, 112). Said hearth (14,114) comprises a plurality of sectors of an annulus (17, 117; 17′, 117′), all the same as one another and connected to form an annulus, complementary to that of the internal plan of the furnace (12, 112), which rotate around the central axis of the annulus, by means of two concentric sets of wheels (26, 126) arranged according to two circumferences, set at equal instances, with supports (25,125), fixed to the base (28) or below the hearth (114), complementary to two circular rails (20, 120), fixed respectively below the hearth (14) or the base (128). According to the invention, both said sets of wheels (26, 126) and said two rails (20, 120) are positioned in such a way as to have an equal load distribution. |
1) Rotary hearth furnace for use in the iron and steel industry, comprising a furnace (12, 112) with plan in the shape of an annulus, closed at the bottom by a rotary hearth (14, 114), lined at the top with refractory material (15, 115), and a base (28, 128; 30, 130) of the furnace (12, 112), wherein said hearth (14, 114) comprises a plurality of sectors of an annulus (17, 117; 17′, 117′), all the same as one another and connected to form an annulus, complementary to that of the internal plan of the furnace (12, 112), which rotate around the central axis of the annulus, by means of two concentric sets of wheels (26, 126) arranged according to two circumferences, set at equal distances, with supports (25, 125), fixed to the base (28) or below the hearth (114), complementary to two circular rails (20, 120), fixed respectively below the hearth (14) or the base (128), characterised in that both said sets of wheels (26, 126) and said two rails (20, 120) are positioned in such a way as to have an equal load distribution. 2) Rotary hearth furnace according to claim 1, characterised in that both said sets of wheels (26, 126) and said two rails (20, 120) are positioned symmetrically with respect to a circumference which divides the annulus of the hearth (14, 114) into two concentric annuli loaded in the same way, said circumference being larger than the median circumference of said annulus of the hearth (14, 114). 3) Rotary hearth furnace according to claim 1, characterised in that both said wheels (26, 126) of the two sets are the same as one another, and that said rails (20, 120) have the same section. 4) Rotary hearth furnace according to claim 1, characterised in that both said wheels (26, 126) are aligned, two by two, on the same radii of the two concentric circumferences of the two sets of wheels (26, 126), and are of the same number as the sectors (17, 117). 5) Rotary hearth furnace according to claim 1 or 4, characterised in that said sectors (17, 117) are divided into two semi-sectors (17′, 117′) along arcs (18) of an intermediate circumference between the two end circumferences, internal and external, of the annulus of the hearth (14, 114), said arches (18) dividing the sectors (17, 117) into two equally loaded semi-sectors (17′, 117′). 6) Rotary hearth furnace according to claim 5, characterised in that said semi-sectors (17′, 117′) are connected to the rails (20), or, respectively, to the supports (125) of the wheels (126) by means of vertical uprights (23, 123), positioned corresponding or close to their centre of gravity. 7) Rotary hearth furnace according to claim 1, characterised in that said base (28, 128) is held up by a support structure (16, 116), comprising concentric circumferential sets of columns (30, 130), aligned in groups on the same radii of said circumferences, where said radii are spaced at equal distances from one another, and having the same number of sectors (17, 17′). 8) Rotary hearth furnace according to claim 7, characterised in that said groups of columns (30, 130) are of the same number as the sectors (17, 117) of the hearth (14, 114), said columns (30, 130) having the same deflection under the load of the hearth (14, 114). 9) Rotary hearth furnace according to claim 8, characterised in that said sectors (17, 117) are divided into two semi-sectors (17′, 117′) along arcs (18) of an intermediate circumference between the two end circumferences, internal and external, of the annulus of the hearth (14, 114), where said arcs (18) divide the sectors (17, 117) into two equally loaded semi-sectors (17′, 117′), said semi-sectors (17′, 117′) being connected to the rails (20), or, respectively, to the supports (125) of the wheels (126) by means of vertical uprights (23, 123), positioned corresponding or close to their centre of gravity. 10) Rotary hearth furnace according to claim 1, characterised in that the furnace (12, 112) has a plan in the shape of an annulus with large dimensions. |
Educational game |
An educational game is provided, comprising a plurality of qualification cards least one subject, said qualification cards having a qualification question and a corresponding qualification answer; and a plurality of preparatory cards for at least one subject, said preparatory cards having a preparatory question and a corresponding preparatory answer wherein a player of the game, when presented with said qualification question and on providing said qualification answer, is presented with said preparatory question and on providing said preparatory answer, receives a score. A method for playing an educational game is provided, wherein a player's turn comprises receiving a qualification question; providing a qualification response to said qualification question; if said qualification response is the corresponding answer to said qualification question, receiving a preparatory question; providing a preparatory response to said preparatory question; and if said preparatory response is the corresponding answer to said preparatory question, receiving a score. |
1. An educational game, comprising: (a) a plurality of qualification cards for at least one subject, said qualification cards having a qualification question and a corresponding qualification answer; and (b) a plurality of preparatory cards for at least one subject, said preparatory cards having a preparatory question and a corresponding preparatory answer; wherein a player of the game, when presented with said qualification question and on providing said qualification answer, is presented with said preparatory question and on providing said preparatory answer, receives a score. 2. The game of claim 1 wherein each of said qualification cards include a plurality of qualification questions and corresponding qualification answers for a variety of skill levels. 3. The game of claim 2 wherein each of said qualification cards further include a plurality of qualification questions and corresponding qualification answer for a variety of age groups. 4. The game of claim 3 wherein each of said preparatory cards include a plurality of preparatory questions and corresponding preparatory answer for a variety of skill levels. 5. The game of claim 4 wherein each of said preparatory cards further include a plurality of preparatory questions and corresponding preparatory answer for a variety of age groups. 6. The game of claim 5 wherein said qualification questions and preparatory questions are based on standardized examination questions. 7. The game of claim 6 wherein said score received for providing said preparatory answer is at least partially random. 8. The game of claim 1 further comprising a rotatable drum for display of and access to said qualification cards and said preparatory cards and a stationary base for holding said rotatable drum. 9. The game of claim 8 further comprising a magnetic mascot for said player positionable on said stationary base. 10. The game of claim 9 wherein a metallic strip is positioned on said rotatable drum such that when said rotatable drum is rotated, said metallic strip will be biased to said magnet. 11. The game of claim 10 wherein said preparatory cards, when not in use are storable within said stationary base below said rotatable drum. 12. The game of claim 11 wherein said preparatory cards are organizable by subject matter in a plurality of slots on said rotatable drum. 13. The game of claim 12 wherein said qualification cards are positioned within a qualification card holder slot on said rotatable drum. 14. The game of claim 13 further comprising score counters organizable in a score counter holder slot on said rotatable drum. 15. The game of claim 14 wherein a plurality of elongated slots are positioned on the perimeter of said stationary base. 16. A method for playing an educational game wherein a player's turn comprises: (a) receiving a qualification question; (b) providing a qualification response to said qualification question; (c) if said qualification response is the corresponding answer to said qualification question, receiving a preparatory question; (d) providing a preparatory response to said preparatory question; and (e) if said preparatory response is the corresponding answer to said preparatory question, receiving a score. 17. The method of playing an educational game of claim 16 wherein said score is multiplied by a number randomly generated by a roll of a die to determine a value of points received by said player. 18. The method of playing an educational game of claim 16 wherein said player, when receiving said value of points, receives score counters equal to said value of points organizable in a plurality of elongated slots positioned on the perimeter on a stationary base. 19. The method of playing an educational game of claim of claim 17, wherein said player wins when said player receives points at least equal to a predetermined winning number. |
<SOH> BACKGROUND <EOH>In a society that values education, tools to assist in learning are in demand for all age groups in all subject areas. For example, elementary school children use pictorial workbooks to learn basic mathematics. Tutoring programs are offered to high school students who may need extra help in chemistry. Preparatory courses and practice exam workbooks are available for university students preparing for qualification examinations to enrol in postgraduate programs. Regardless of age, skill level, and subject area, an effective method to learn is to make the learning process enjoyable. and fun. A number of educational games disclosed in the art target players of a specific age group. Such games are based on the limited scope of knowledge of players within the specific age group and therefore limit the extent to which skills may be developed in any given subject area. Furthermore, because most educational games in the art are so limited, simultaneous participation by players of different age and skill levels is not possible. For example, younger individuals in a group of players may feel left out when other older individuals are playing because the game is too difficult for them to play. Other board games in the art utilize a game board and related parts for use in playing the game. The use of such board and related parts may make it difficult for a group of individuals to play because the game board often has to be moved to the face the players, resulting in many pieces of the game being disturbed. Alternatively, the game may be played with the information on the game board positioned upside down for some of the players. Thus, it is an object of this invention to provide a means for individuals to learn the subject matter of a wide range of subject areas in an enjoyable and relaxed manner. More particularly, the present invention provides a means for individuals to learn about various subject areas in accordance with their academic needs. It is a further object of this invention to enable individuals of differing age groups and skill levels to play simultaneously such that each player has a similar chance of winning the game. It is a further object of this invention to permit the game to be played with ease by mounting the game board on a rotating drum, thereby allowing players to rotate the game board without disturbing the game pieces. |
<SOH> SUMMARY OF THE INVENTION <EOH>An educational game is provided, comprising a plurality of qualification cards for at least one subject, said qualification cards having a qualification question and a corresponding qualification answer; and a plurality of preparatory cards for at least one subject, said preparatory cards having a preparatory question and a corresponding preparatory answer wherein a player of the game, when presented with said qualification question and on providing said qualification answer, is presented with said preparatory question and on providing said preparatory answer, receives a score. A method for playing an educational game is provided, wherein a player's turn comprises receiving a qualification question; providing a qualification response to said qualification question; if said qualification response is the corresponding answer to said qualification question, receiving a preparatory question; providing a preparatory response to said preparatory question; and if said preparatory response is the corresponding answer to said preparatory question, receiving a score. |
Methods of polyvalent bacteriophage preparation for the treatment of bacterial infections |
The multivalent strains of bacteriophages, methods of obtaining these and their use in the treatment of bacterial infections, particularly those of drug-resistant bacterial strains, especially arising in mucoviscidosis patients, are provided. |
1. A method of obtaining multivalent bacteriophage strain charactarised in that: a) a sufficient number (n) of pathogenic bacterial strains of a specific species appearing randomly in a given region is accumulated, by which the isolated bacterial stains are possibly drug-resistant strains, b) a sufficient number of bacteriophage strains specific to at least one of the bacterial stains of the given species is accumulated, c) the lytic activity of the accumulated bacteriophage strains on each accumulated bacterial strain is determined, the value p, which is the proportion of the number of strains lysed by a given phage to the number of all the accumulated bacterial strains, is estimated, d) from the resultant value of p, it is tested whether n fulfills the condition: n ≥ pq d 2 z 1 - α 2 where: q=1−p d is a constant no larger than 0.1, optimally no larger that 10% p z1−α is a random variable of the normal distribution dependent on the confidence factor 1−α, which is not less that 0.95 e) the bacteriophage strains is selected which fulfils the criterion in d) and has a value of p not less than 2/n, optimally not less than 0.5. 2. The method of claim 1, wherein in step b) the bacteriophage strain is isolated from a sample originating from the environment, by passing through a membrane filter of pore size 0.2-0.4 μm, culture medium is added to the obtained filtrate and mixed with a broth culture of bacteria of a defined genus, incubated at a temperature of about 37° C. for approximately 1 hour, a portion of the suspension is removed and applied to plate with solid culture medium, incubated at approx. 37° C. for 2 to 24 hours, a sample of the medium surrounding an individual bald spot is isolated, transferred to the broth culture of bacteria of the defined genus and incubated until the culture brightens up, and the bacteriophage product is obtained by passing the lysate through a membrane filter of pore size 0.2-0.4 μm, whereby preferably the inoculation of the solid culture medium and re-isolation of the bald spots is repeated 5 times. 3. The method of claim 1 or 2, wherein in step c) the bacterial strain under study is inoculated onto the respective solid medium, onto which a portion of the bacteriophage preparation obtained in step b) is transferred, incubated at a temperature of approximately 37° C. for approximately 4 hours, after which it is left at a temperature of approximately 4° C. for about 2 to 24 hours, after which the lytic activity of the bacteriophage strain is evident by the appearance of at least individual bald spots. 4. The method of claim 1, wherein the pathogenic bacterial strains are of the genera Staphylococcus or Pseudomonas. 5. The method of claim 1 or 4, wherein selected in step c) multivalent bacteriophage strain specific to drug resistant bacterial strains of the genus Staphylococcus in Poland is bacteriophage strain from among S1 (PCM F/00001), S2 (PCM F/00002), S3 (PCM F/0003), S4 (PCM F/00004), S5 (PCM F/00006), S6 (PCM F/00006) and S7 (PCM F/00007). 6. The method of claim 5, wherein selected in step e) multivalent bacteriophage strain specific to drug resistant bacterial strains of the genus Staphylococcus appearing in mucoviscidosis patient is bacteriophage strain from among S2 (PCM F/00002), S4 (PCM F/00004) and S7 (PCM F/00007). 7. The method of claim 1 or 4, wherein selected in step e) multivalent bacteriophage strain specific to drug resistant bacterial strains of the genus Pseudomonas appearing in Poland is bacteriophage strain from among P1 (PCM P/00008), P2 (PCM F/00009), P3 (PCM F/00010), P4 (PCM F/00011), P5 (PCM F/00012), P6 (PCM F/00013), P7 (PCM F/00014), P8 (PCM F/00015), P9 (PCM F/00016), P10 (PCM F/00017), P11 (PCM F/00018), P12 (PCM F/00019), P13 (PCM F/00020), P14 (PCM F/00021), P15 (PCM F/00022), P16 (PCM F/00023), P17 (PCM F/00024), P18 (PCM F/00025), P19 (PCM F/00026), and P20 (PCM F/00027). 8. The method of claim 7, wherein selected in step e) multivalent bacteriophage strain specific to drug resistant bacterial strains of the genus Pseudomonas appearing in mucoviscidosis patients is bacteriophage stain among P3 (PCM F/00010), P6 (PCM F/00013) and P11 (PCM F/00018). 9. The method of claim 1 or 2, comprising further purification of the lysate, especially from endotoxin, whereby the mixture containing bacteriophages is contacted with a substrate containing cellulose or at least partially esterified derivative thereof, which is than washed with a solution which removes impurities, especially endotoxin, after which purified bacteriophages are eluted. 10. The method of claim 9, wherein the elution of endotoxin is carried out by means of water, a solution of a non-dissociating substance, or saline solution of a concentration not greater than 0.1 M, especially buffered. 11. The method of claim 9, wherein the elution of bacteriophages is carried out by means of a solution of a non-dissociating substance, or any buffer, or a saline solution of a concentration great an 0.05 M, especially buffered. 12. The method of claim 9, wherein the elution of endotoxin and bacteriophages is carried out at temperatures from −25° C. to 100° C. 13. The method of claim 9, wherein the elution of endotoxin and bacteriophages is carried out using an aqueous solution of salt containing organic solvent. 14. The method of claim 13, wherein the organic solvent is chosen from the group comprising dimethyl sulphoxide, dimethylformamide, isopropanol and acetone. 15. The method of claim 9, wherein cellulose partially esterified with organic or inorganic acid is used as a substrate. 16. The method of claim 9, wherein cellulose partially esterified with acetic, nitric, sulphurous or phosphoric acid is used. 17. The method of claim 9 is characterized by: cellulose in which from 0.01% to 5% of glucose molecules, especially from 0.25% to 1% of glucose molecules, are esterified is used. 18. A medicament for the treatment or prevention of infection arising from bacterial pathogen comprising the multivalent bacteriophage strain obtainable by the method according to claim 1-17 specific to bacteria belonging to genus of the bacterial pathogen. 19. The medicament of claim 18, wherein said multivalent bacteriophage strain specific to bacteria of the genus Staphylococcus, possibly drug resistant bacterial strains of the genus Staphylococcus appearing in Poland, is bacteriophage strain selected from among S1 (PCM F/00001), S2 (PCM F/00002), S3 (PCM F/00003), S4 (PCM F/00004), S5 (PCM F/00006), S6 (PCMF/00006) and S7 (PCMF00007), especially the phages S1, S2, and S4 or S5. 20. The medicament of claim 18 is characterized by: it serves in the treatment or prevention of infections arising in mucoviscidosis patients and, as a multivalent bacteriophage strain specific to bacteria of the genus Staphylococcus, contains at least one bacteriophage strain from among F/00002, F/00004 and F/00007. 21. The medicament of claim 18 wherein said multivalent bacteriophage strain specific to bacteria of the genus Pseudomonas, possibly drug resistant bacterial strains of the genus Staphylococus appearing in Poland, is bacteriophage strain selected from among P1 (PCM F/00008), P2 (PCM F/00009), P3 (PCM F/00010), P4 (PCM F00011), P5 (PCM F/00012), P6 (PCM F/00013), P7 (PCM F/00014), P8 (PCM F/00015), P9 (PCM F/00016), P10 (PCM F/00017), P11 (PCM F/00018), P12 (PCM F/00019), P13 (PCM F/00020), P14 (PCM F/00021), P15 (PCM F/00022), P16 (PCM F/00023), P17 (PCM F/00024), P18 (PCM F/00025), P19 (PCM F/00026), and P20 (PCM F/00027), especially the phages P7 , P20 and P6. 22. The medicament according to claim 18 is characterized by: it serves in the treatment or prevention of infections arising in mucoviscidosis patients and, as a multivalent bacteriophage strain specific to bacteria of the genus Pseudomonas, it contains at least one bacteriophage strain selected from among F/00010, F/00013 and F/00018. 23. Use of multivalent bacteriophage strain obtained by the method according to claim 1-17 in the production of medication for the treatment or prevention of infections arising from pathogenic bacteria. 24. The use of claim 23, wherein for the production of medication for the treatment or prevention of infections caused by bacteria of the genus Staphylococcus, possibly caused by drug resistant bacterial strains of the Staphylococcus appearing in Poland, at least on bacteriophage strain selected from among S1 (PCM F/00001), S2 (PCM F/00002), S3 (PCM F/00003), S4 (PCM F/00004), S5 (PCM F/00006), S6 (PCM F/00006) and S7 (PCM F/00007), especially the phages S1, S2, and S4 or S5, is used. 25. The use of claim 23, wherein for the production of medication for the treatment or prevention of infections arising in mucoviscidosis patient caused by bacteria of the genus Staphylococcus, at least one bacteriophage strain from among F/00002, F/00004 and F/00007 is used. 26. The use of claim 23, wherein for the production of medication for the treatment or prevention of infections arising from bacteria of the genus Pseudomonas, possibly caused by drug resistant bacterial strains of the genus Pseudomonas appearing in Poland, at least on bacteriophage strain from among P1 (PCM F/00008), P2 (PCM F/00009), P3 (PCM F/00010), P4 (PCM F/00011), P5 (PCM F/00012), P6 (PCM F/00013), P7 (PCM F/00014), P8 (PCM F/00015), P9 (PCM F/00016), P10 (PCM F/00017), P11 (PCM F/00018), P12 (PCM F/00019), P13 (PCM F/00020) P14 (PCM F/00021), P15 (PCM F/00022), P16 (PCM F/00023), P17 (PCM F/00024), P18 (PCM F/00025), P19 (PCM F/00026), and P20 (PCM F/00027), especially the phages P7, P20 and P6, is used. 27. The use of claim 23, wherein for the production of medication for the treatment or prevention of infections arising in mucoviscidosis patients caused by bacteria of the genus Pseudomonas, at least on bacteriophage strain selected from among F/00010, F/00013 and F/00018 is used. 28. The multivalent bacteriophage strain selected from among S1 (PCM F/00001), S2 (PCM F/00002), S3 (PCM F/00003), S4 (PCM F/00004), S5 (PCM F/00006), S6 (PCM F/00006) and S7 (PCM F/00007), wherein the strain characterised as specific to bacteria of the genus Staphylococcus possibly specific to drug resistant bacterial strains of the genus Staphylococcus, appearing in Poland. 29. The multivalent bacteriophage strain selected from among P1 (PCM F/00008), P2 (PCM F/00009), P3 (PCM F/00010), P4 (PCM F/00011), P5 (PCM F/00012), P6 (PCM F/00013), P7 (PCM F/00014), P8 (PCM F00015), P9 (PCM F/00016), P10 (PCM F/00017), P11 (PCM F/00018), P12 (PCM F/00019), P13 (PCM F/00020), P14 (PCM F/00021), P15 (PCM F/00022), P16 (PCM F/00023), P17 (PCM F/00024), P18 (PCM F/00025), P19 (PCM F/00026), and P20 (PCM F/00027), wherein the strain is characterised as specific to bacteria of the genus Pseudomonas, possibly specific to drug resistant bacterial strains of the genus Pseudomonas appearing in Poland. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The bacteriophages (phages) are a diverse group of viruses whose life cycle is connected exclusively with bacteria cells. Bacteriophages are characterized by a lysogenic or lytic life cycle. As anti-bacterial agents, lytic bacteriophages are especially useful which, after infection by bacteria cells to which they are sensitive, they replicate within them, leading to their total destruction (by lysis) and the release of new phages which attack and destroy subsequent bacteria cells. This process may occur both in vitro and in vivo. One of the essential characteristics of bacteriophages is the well-known high specificity of their lytic activity. This feature is exploited in, for example, species determination (phage typing) of various bacteria (see, for example, patent descriptions GB 2285684, U.S. Pat. No. 5,824,468 and SU 543260, as well as the international patent notifications WO 0100786 and WO 0109370). Other known applications of bacteriophages include their usefulness as tools in molecular biology, for example in the expression and selection of specific proteins (e.g. patent description U.S. Pat. No. 6,027,930), and in sterilization and cleansing media (e.g. patent descriptions EP 0414304, EP 0290295 and GB 2253859, as well as the international patent notifications WO 9808944 and WO 9003122). Modified phages are used in the production of vaccines (e.g. WO 9505454). Certain proteins of bacteriophage origin are also used (e.g. EP 0510907, U.S. Pat. No. 5,470,573, WO 9607329). The methods of isolating bacteriophages and obtaining phage preparations are well known and are constantly being perfected (e.g. GB 829266, CS 192212, RU 2109055). Phage therapy has been employed on a wide scale since the Second World War at the Institute of Microbiology and Virology of Tbilisi, Georgia. A bank of various phage preparations is used there in the treatment of bacterial infections and in prophylaxis. Available data indicate a great effectiveness of phage therapy. Similar research has been conducted in Poland for over 25 years. At the Bacteriophage Laboratory of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wroclaw the phage therapy is used treatment of infections caused by drug-resistant forms of bacteria and those not susceptible to antibiotics (see: Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1981, 31, 293; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1983, 31, 267; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1984, 32, 317; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1985, 33, 219; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1985, 33, 241; Stefan Ślopek et al., Archivum Immunologiae et Therapiae Experimentalis, 1987, 35, 569; Beata Weber-Dabrowska et al., Archivum Immunologiae et Therapiae Experimentalis, 2000, 48, 31-37; Beata Weber-Dabrowska et al., Archivum Immunologiae et Therapiae Experimentalis, 2000, 48, 547-551). The phage therapy carried out there over the last 14 years, which has included 1473 patients with purulent infections of different tissues and organs, indicates the high efficacy of phage therapy. Complete abatement of disease symptoms and return to health was noted in 1289 cases. It must be emphasized that in at least a dozen or so cases phage therapy presented the only possibility of eliminating the life-threatening infection. The therapy conducted concerned individual patients. It consisted of: a) the growth and identification of bacterial strains isolated from material obtained from the patient, b) determination of the susceptibility of the strain to specific phages and selection of the phage showing the highest lytic activity towards the strain, c) preparation of a phage lysate with a large number of phage particles, d) production of a sterile phage preparation for treatment. This procedure is rather costly, laborious and time consuming. At times, 7-10 days pass from the moment of obtaining the research material to the availability of the finished, sterilized phage preparation for treatment. Such a delay is too long in certain disease states. Phage therapy on a broad scale cannot be carried out with the procedure employed thus far. Infections accompanying mucoviscidosis present a particular problem. This is a hereditary, systemic disease consisting of dysfunction of mucous-secreting glands, predisposing the patient to chronic bronchial and pulmonary diseases. Secreted mucous is thick and sticky, making its elimination by natural routes (coughing) difficult. The accumulation and lingering of mucous in the bronchi and the impairment in clearing it create favorable conditions for bacterial infections, leading to chronic bronchitis and pneumonia. The most threatening pathogens causing infections accompanying mucoviscidosis are Staphylococcus aureus and bacilli of the genus Pseudonomas. Permanently recurring infections by these micro-organisms lead to serious pathological changes in the respiratory system and premature death. Few patients survive more than 25 years. Treatment of such infections with the available antibiotics has created a serious therapeutic problem worldwide, especially in the past few years. Antibiotic therapy of these infections has become less and less effective because the vast majority of the bacterial strains show resistance to all antibiotics, including the antibiotic of last resort—vancomycin. There is, therefore, an urgent need to introduce an alternative therapeutic method into medical practice for the treatment of refractory and highly dangerous bacterial infections. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention provides a method of obtaining multivalent bacteriophage strain, by which: a) a sufficient number (n) of different strains of bacterial pathogens of a defined genus accumulates, appearing randomly in a given region, whereby the isolated bacterial strains are preferably drug-resistant, b) a sufficient number of different bacteriophage strains, specific to at least one of the bacterial strains of the given genus, accumulates, c) the lytic activity of an accumulated bacteriophage strain on each accumulated bacterial strain is determined, then the value p, which is the proportion of the number of strains lysed by a given phage to the number of all the accumulated bacterial strains, is calculated. d) for the resultant value of p, it is tested whether n fulfils the condition: n ≥ pq d 2 z 1 - α 2 where: q=1−p d is a constant no larger than 0.1, optimally no larger that 10% p z 1-α is a random variable of the normal distribution dependent on the confidence factor 1−α, which is not less that 0.95, e) the bacteriophage strain is selected which fulfils the criterion defined in d) and has a value of p not less than 2/n, preferably not less than 0.5. The range of the lytic activity of each isolated bacteriophage is determined with regard to the population of pathogenic bacterial strains present in the given region. To this end, a sufficiently large collection of randomly chosen drug-resistant bacterial strains must be accumulated. With reference to the established large sample of bacteria of a population size of n strains (in the example described, n was 845 and 880, respectively, for the drug-resistant strains of Pseudomonas and Staphylococcus isolated in Poland), the frequency of success is calculated, or the proportion of the number of strains lysed by a given phage to the total number of strains studied. This frequency is regarded as the probability of success p. The probability of failure is then q=1−p. It is essential to choose the size n that a sample must have so that it can be regarded as representative of the general population of the strains of the given genus (e.g. Pseudomonas or Staphylococcus ). To this end, the following criterion for the sample to be representative is established: in-line-formulae description="In-line Formulae" end="lead"? P ( |ν−p|<d )= P (− d<ν−p<d )=1−α in-line-formulae description="In-line Formulae" end="tail"? The probability that the absolute value of the difference between the sample frequency ν and the probability of success p is less than the pre-assigned value of d is 1−α, which corresponds with a confidence level of 1−α. The value of 1−α is usually set at 0.95 or 0.98. The number of samples n should fulfil the condition: n ≥ pq d 2 z 1 - α 2 where z 1-α is a random variable of the normal distribution. For 1−α=0.95, z 1-α =1.96, and for 1−α=0.98, z 1-α =2.33. Based on this criterion and on the value of the frequency p obtained for the characterized phage, by assigning reasonable values to d and the confidence range 1−α it is possible to verify the magnitude n used to determine the range of lytic activity of a given phage and ascertain whether the lytic activity obtained for this n, with the assigned values of d and 1−α, may be regarded as correct with respect to the general population of the bacterial strains of the given genus. The strain of bacteriophages is preferably isolated in step b) from a sample originating from the environment by passing it through a membrane filter of pore size 0.2-0.4 μm, adding culture medium to the filtrate and mixing it with a broth culture of bacteria of the defined genus, incubating this at a temperature of about 37° C. for approximately one hour, removing a portion of the suspension and smearing it onto plates with solid culture medium, incubating at approx. 37° C. for 2 to 24 hours, isolating a sample of the medium surrounding the individual bald spot, transferring it to the broth culture of bacteria of defined genus, and incubating until the culture clears, and obtaining the bacteriophage preparation by passing the lysate through a membrane filter of pore size 0.2-0.4 μm, whereby it is preferred to repeat the inoculation of the solid culture medium and re-isolation of the bald spots 5 times. The bacterial strains in step c) are preferably inoculated onto solid culture medium, onto which a portion of the bacteriophage preparation obtained in step b) is deposited, incubated at about 37° C. for around 4 hours, after which the temperature is set at about 4° C. for around 2 to 24 hours, by which the lytic activity of the bacteriophage strain is evidenced by the appearance of, at least, individual bald spots. It is possibly to carry out then further purification of the lysate, particularly with regard to endotoxin, whereby the mixture containing bacteriophages is in contact with a substrate containing cellulose or a partially esterified derivative of it, then rinsed with a solution that removes impurities, especially endotoxin, after which the purified bacteriophages are washed out. The endotoxin can be effectively eluted with water, a solution of a non-dissociating substance, or a saline solution of concentration no greater than 0.1 M, possibly buffered. The bacteriophage fraction can also be effectively eluted with a solution of a non-dissociating substance, or any buffer, or a saline solution of concentration greater than 0.05 M, possibly buffered. Also, elution of endotoxin and bacteriophages are carried out at temperatures between −25° C. and +100° C. Preferably, the endotoxin and bacteriophage elution can be done using an aqueous saline solution containing organic solvent. The organic solvent is best selected from a group comprising dimethyl sulphoxide, dimethylformamide, isopropanol and acetone, and as a substrate cellulose partially esterified with organic or inorganic acid can be used. It may be used a substrate of cellulose partially esterified with acetic, nitric, sulphurous or phosphoric acid, in particular cellulose may be used as a substrate of which 0.01 to 5% of the glucose molecules have been esterified, preferably 0.25 to 1%, more preferably from 0.5 to 1% of the glucose molecules. Preferably the pathogenic bacterial strains are of the genera Staphylococcus or Pseudomonas. A further aspect of the invention is a medication for the treatment or prevention of infections caused by bacterial pathogens which contains an active agent and a possible pharmaceutically admissible carrier, such that the active agent is comprised of a multivalent bacteriophage strain specific to the bacteria of the genus and obtained using the method of this invention. In accordance with the invention, the medication may have the characteristic that, as a multivalent bacteriophage strain specific to bacteria of the genus Staphylococcus , it contains at least one strain of bacteriophages selected from among S1 (PCM F/00001), S2 (PCM F/00002), S3 (PCM F/00003), S4 (PCM F/00004), S5 (PCM F/00006), S6 (PCM F/00006) and S7 (PCM F/00007), preferably the phages S1, S2, and S4 or S5. This medication produced in accordance with the one aspect of the invention is to serve in the treatment or prevention of infections arising in persons afflicted with mucoviscidosis and, as a multivalent bacteriophage strain specific to bacteria of the genus Staphylococcus, it contains at least one bacteriophage strain selected from among F/00002, F/00004 and F/00007. In accordance with the invention, the medication should have the characteristic that, as a multivalent bacteriophage strain specific to bacteria of the genus Pseudomonas, it contains at least one strain of bacteriophages selected from among P1 (PCM F/00008), P2 (PCM F/00009), P3 (PCM F/00010), P4 (PCM F/00011), P5 (PCM F/00012), P6 (CM F/00013), P7 (PCM F/00014), P8 (CM F/00015), P9 (PCM F/00016), P10 (PCM F/00017), P11 (PCM F/00018), P12 (PCM F/00019), P13 (PCM F/00020), P14 (PCM F/00021), P15 (PCM F/00022), P16 (PCM F/00023), P17 (PCM F/00024), P18 (PCM F/00025), P19 (PCM F/00026), and P20 (PCM F/00027), optimally phages P7, P20 and P6. The medication produced in accordance with certain aspect of the invention is to serve in the treatment or prevention of infections arising in persons afflicted with mucoviscidosis and, as a multivalent bacteriophage strain specific to bacteria of the genus Pseudomonas , it contains at least one bacteriophage strain selected from among F/00010, F/00013 and F/00018. A further object of this invention is the application of a multivalent bacteriophage strain obtained according to the method of the invention to the development of a medication for the treatment or prevention of bacterial infections caused by bacterial pathogens. Advantageous for the production of a medication for the treatment or prevention of infections caused by bacteria of the genus Staphylococcus is the employment of at least one bacteriophage strain selected from among S1 (PCM F/00001), S2 (PCM F/00002), S3 (PCM F/00003), S4 (PCM F/00004), S5 (PCM F/00006), S6 (PCM F/00006) and S7 (PCM F/00007), optimally phages S1, S2, and S4 or S5. Advantageous for the creation of a medication for the treatment or prevention of infections caused by bacteria of the genus Staphylococcus in persons suffering from mucoviscidosis is the use of at least one bacteriophage strain chosen from among F/00002, F/00004 and F/00007. Also advantageous for the creation of a medication for the treatment or prevention of infections caused by bacteria of the genus Pseudomonas is the use of at least one bacteriophage strain selected from among P1 (PCM P/00008), P2 (PCM F/00009), P3 (PCM F/00010), P4 (PCM F/00011), P5 (PCM F/00012), P6 (PCM F/00013), P7 (PCM F/00014), P8 (PCM F/00015), P9 (PCM F/00016), P10 (PCM F/00017), P11 (PCM F/00018), P12 (PCM F/00019), P13 (PCM F/00020), P14 (PCM F/00021), P15 (PCM F/00022), P16 (PCM F/00023), P17 (PCM F/00024), P18 (PCM F/00025), P19 (PCM F/00026), and P20 (PCM F/00027), optimally the phages P7, P20 and P6. Advantageous for the creation of a medication for the treatment or prevention of infections caused by bacteria of the genus Pseudomonas in persons suffering from mucoviscidosis is the use of at least one bacteriophage strain chosen from among F/00010, F/00013 and F/00018. A further object of the invention is a multivalent bacteriophage strain specific to bacteria of the genus Staphylococcus selected from among S1 (PCM F/00001), S2 (PCM F/00002), S3 (PCM F/00003), S4 (PCM F/00004), S5 (PCM F/00006), S6 (PCM F/00006) and S7 (PCM F/00007), optimally from among S1, S2, S4 and S5. An object of the invention is also a multivalent bacteriophage strain specific to bacteria of the genus Pseudomonas, selected from among P1 (PCM F/00008), P2 (PCM F/00009), P3 (PCM F/00010), P4 (PCM F/00011), P5 (PCM F/00012), P6 (PCM F/00013), P7 (PCM F/00014), P8 (PCM F/00015), P9 (PCM F/00016), PlO (PCM F/00017), P11 (PCM F/00018), P12 (PCM F/00019), P13 (PCM F/00020), P14 (PCM F/00021), P15 (PCM P/00022), P16 (PCM F/00023), P17 (PCM F/00024), P18 (PCM F/00025), P19 (PCM F/00026), and P20 (PCM F/00027), optimally from among P7, P20 and P6. Our own collection of phages active against species of bacteria which are the most frequent etiologic factors in bacterial infection in humans was used in the research. The practical examples described concern multivalent bacteriophages acting lytically on strains of staphylococci ( Staphylococcus aureus ), including the methicillin-resistant strains thereof, and the blue pus bacillus ( Pseudomonas aeruginosa ), which appear in those afflicted with mucoviscidosis in particular. These species are currently the cause of serious infections causing high mortality. |
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