text:
|Publication number
|Publication type
|Application number
|Publication date
|Oct 6, 1998
|Filing date
|Dec 27, 1994
|Priority date
|Mar 10, 1993
|Fee status
|Also published as
|Publication number
|Elias Meezan, Stephen M. Manzella
|Original Assignee
|The Uab Research Foundation
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (9), Non-Patent Citations (26), Referenced by (27), Classifications (21), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 08/028,970, filed Mar. 10, 1993, now abandoned.
1. Field of the Invention
The instant invention relates to a method of priming the incorporation of glucose into an oligosaccharide having α(1→4 ) glycosidic linkages. In particular, the invention relates to a method of treating diabetes mellitus.
2. Background Art
Diabetes mellitus is a common, widespread disease syndrome best characterized by hyperglycemia due to an absolute or relative lack of insulin (Diabetes Mellitus, Theory and Practice, 3d Ed., M. Ellenberg and H. Rifkin, eds., Medical Examination Publishing Co., New Hyde Park, 1983). Hyperglycemia is accompanied by altered metabolism of carbohydrates, lipids and proteins, and although the disease can be controlled by dietary treatment, insulin and oral hypoglycemic agents, there is a substantial incidence of vascular complications in long-term diabetes which continues to be a significant cause of morbidity and mortality in this disease. Diabetic patients can generally be clinically placed into one of two major subtypes of the disease, insulin-dependent diabetes mellitus (IDDM or Type-I diabetes) or non-insulin-dependent diabetes mellitus (NIDDM or Type-II diabetes). Although the incidence of these two types of diabetes varies widely throughout the world, in the United States about 90% of diabetic patients have Type II diabetes with most of the remainder being classified as Type-I. Both Type-I and Type-II diabetes have a complex pathogenesis which is contributed to by both genetic and environmental factors.
Type-I diabetes mellitus appears to be an autoimmune disease of the pancreatic B cell in which this cell type is largely destroyed leading to a chronic state of insulin deficiency and consequent hyperglycemia. In contrast, Type-II diabetics show no significant loss of pancreatic B cells and have normal or even elevated levels of plasma insulin. Nevertheless, these patients are relatively insulin-deficient, because their B cells do not respond adequately to hyperglycemia by releasing enough insulin to maintain euglycemia. Therefore, the central characteristic of both types of diabetes mellitus is hyperglycemia.
The consequences of chronic hyperglycemia to body metabolism are complex. It is becoming increasingly clear, however, that the major factor responsible for the development of most of the long-term complications of diabetes is the toxic effect of prolonged exposure of diabetic tissues to elevated levels of glucose. Tissues dispose of glucose primarily by oxidizing it to carbon dioxide and water or by converting it to lactate and/or glycogen by nonoxidative pathways. In humans, it has recently been shown (Shulman, G. I. et al., New Engl. J. Med., 322:223-228 (1990)) that the synthesis of muscle glycogen accounts for most of the total body glucose uptake and for all of the non-oxidative glucose metabolism in both normal and diabetic subjects. In the most prevalent form of diabetes mellitus, NIDDM, insulin resistance appears to be due to a defect in the pathway of glycogen synthesis in skeletal muscle (Bogardus, C. and Lillioja, S., New Engl. J. Med., 322:262-263 (1990)). Glycogen metabolism has also been shown to be impaired in the skeletal muscle of IDDM patients so that the synthesis of this glucose storage compound is inadequate to meet the demands of hyperglycemia, resulting in an accumulation of glucose and its metabolites in muscle cells (Vaag, A. et al., Diabetes, 41:174-182 (1992)). Therefore, impaired utilization of glucose via its incorporation into macromolecular glycogen appears to be a common metabolic defect found in both Type-I and Type-II diabetes mellitus.
The regulation of glycogen metabolism is complex and is influenced by a variety of factors (Hers, H. G., et al., The Metabolic Basis of Inherited Disease, C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Valle, eds. McGraw-Hill, N.Y., pp. 425-452 (1989)). Existing therapies for Type-I and Type-II diabetes mellitus are not specifically directed at influencing the glycogen biosynthetic pathway.
Glycogen storage disease is another group of disorders involving abnormalities in the metabolism of glycogen. The disease has various forms, each of which has a different enzymatic basis. The end result, however, is always accumulation of glycogen in the cells and its resulting pathologic consequences. The liver is a major organ of glycogen synthesis and storage. Because endogenous levels of glycogenin are significantly lower in liver than in muscle, liver glycogen is much larger than muscle glycogen. The pathologic consequences of glycogen storage disease are manifested most markedly in liver, even though muscle and other tissues are also involved in this disease. (Hers, H. G. et al., "Glycogen Storage Diseases" in The Metabolic Basis of Inherited Disease (C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Valle, eds.) McGraw-Hill, N.Y., pp. 425-452 (1989)). The large size of the liver glycogen molecule may contribute to the major involvement of this organ in glycogen storage disease. Although various therapeutic approaches have been used in the treatment of the different forms of glycogen storage disease, the major intervention in this disease remains dietary manipulation (Moses, S. W., J. Pediat. Gastroenterol. Nutr., 11:155-174 (1990)). Pharmacologic agents have also been used to a limited extent. There is a need for new types of agents to treat this severe disease syndrome.
Glycogenin is a self-glycosylating protein which serves an integral role in the biosynthesis of glycogen by creating a protein-linked malto-oligosaccharide primer upon which glycogen synthase and branching enzyme build the macromolecular glycogen molecule (Smythe, C. et al., Eur. J. Biochem., 200:625-631 (1991)). The glycogenin protein, by an unknown mechanism, becomes self-glycosylating and is capable of elongating the initial mono- or disaccharide moiety to a linear oligosaccharide chain using UDP glucose as the donor substrate. This fully glycosylated form of glycogenin then serves as the oligosaccharide primer to initiate chain elongation by glycogen synthase. Glycogenin also has the interesting ability to use UDP xylose as a donor substrate (Roden, E. et al., Abstracts of the 6th Congress of the Pan-American Association of Biochemical Societies, (1990)), and indeed xylose incorporation into glycogen has been reported in studies of proteoglycan synthesis in chick embryo chondrocyte microsomal preparations (Kimura, J. H. et al., Arch. Biochem. Biophys., 191:687-697 (1978)).
Although it is not clear whether the mechanism of self-glucosylation by glycogenin is intra or intermolecular, the enzyme is capable of transfer to exogenous substrates (Lomako, J. et al., FEBS Lett., 264:13-16(1990) using p-nitrophenyl glucoside and p-nitrophenyl linked maltooligosaccharides. Such p-nitrophenyl saccharides, however, have a lower affinity for glycogenin and can be hydrolyzed to potentially toxic products such as p-nitrophenol (Gressner, A. M. et al., Biochem. Pharm., 42:1987-1995 (1991)). Polymers having maltose and maltotriose side chains have also been shown to act as primers to elicit the de novo synthesis of glycogen (Zehavi, U. and Herchman, M., Carbohydr. Res. 151:371-378 (1986) and Zehavi, et al., Carbohydr. Res. 228: 255-263 (1992)). These polymers, however, are insoluble and cannot permeate cell membranes. Other stimulators of glycogen synthesis such as proglycosyn have been reported (Harris, R. A. et al., J. BioL Chem., 264:14674-14680 (1989), Guo, Z. K. et al., J. Biol. Chem., 266:22323-22327 (1991) and Yamanouchi, K. et al., Arch. Biochem. Biophys., 294:609-615 (1992)), but they do not act as primers.
Thus, there exists an important need for a treatment that increases glucose utilization via the glycogen biosynthetic pathway and thereby can be used to treat both Type-I and Type-II diabetes mellitus and glycogen storage disease. Such a treatment would be most beneficial if it was compatible and synergistic with existing therapies for these diseases and if it were nontoxic.
The present invention provides a method of priming the incorporation of cellular glucose into an oligosaccharide in a cell comprising administering to the cell a priming amount of a membrane-permeating nontoxic glycoside having a suitable hydrophobic organic group joined by a linkage to a suitable hydrophilic saccharide, wherein the hydrophilic saccharide has a nonreducing terminal sugar residue selected from the group consisting of glucose or xylose, thereby priming the incorporation of cellular glucose onto the glycoside to form an oligosaccharide.
The invention further provides a method of treating diabetes mellitus by priming the incorporation of cellular glucose into an oligosaccharide.
The present invention also provides a method of treating glycogen storage disease by priming the incorporation of cellular glucose into an oligosaccharide.
The invention further provides an alkyl glycoside having an 8 to 14 carbon alkyl chain joined by a linkage to a saccharide having from four to eight glucose residues, wherein the linkage is selected from the group consisting of a glycosidic linkage and a thioglycosidic linkage.
The invention also provides an alkyl glycoside having an 8 to 14 carbon alkyl chain joined by a linkage to a saccharide having from one to eight glucose residues and a nonreducing terminal sugar residue consisting of xylose, wherein the linkage is selected from the group consisting of a glycosidic linkage and a thioglycosidic linkage.
The present invention also provides a method for assaying for the presence of glycogen synthesis enzymatic activity in a sample comprising a) incubating the sample with a composition comprising an alkyl glycoside having a nonreducing terminal glucose joined in a anomeric linkage, and radiolabeled UDP-glucose, and b) determining the presence of radioactivity incorporated into the alkyl glycoside, the presence of radioactivity indicating the presence of glycogen synthesis enzymatic activity in the sample.
Accordingly, the present invention also provides a method of and compounds for priming the incorporation of cellular glucose into an oligosaccharide.
The instant invention also provides alkyl glycoside compounds having an 8 to 14 carbon alkyl chain joined by a linkage to a saccharide having from 4 to 8 glucose residues and alkyl glycoside compounds having an 8 to 14 carbon alkyl chain joined by a linkage to a saccharide having from 1 to 8 glucose residues and a non-reducing sugar residue consisting of xylose.
The present invention still further provides methods for treating diabetes mellitus and glycogen storage disease.
Finally, the instant invention provides a method for assaying the presence of glycogen synthesis activity in a sample.
The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples.
As used in the claims,"a" means one or more.
As used herein, a"suitable" group means one that, when a part of a particular glycoside molecule, will render the glycoside able to fulfill the limiting characteristics of the invention, i.e., that the glycoside is (1) able to prime the incorporation of glucose into an oligosaccharide within a cell; (2) nontoxic; and (3) membrane-permeating, i.e., having amphipathic properties which allow it to readily penetrate cells chemically intact so that it has access to glycogenin and glycogen synthase. Suitable compounds can be determined using the methods set forth in the examples."Nontoxic," as used herein, includes that the glycoside molecule has a sufficiently low toxicity to be suitable for human administration.
As used herein,"glycoside" refers to the condensation product of any sugar with any suitable hydrophobic organic group, involving the loss of at least the H of the hemiacetal OH of the sugar, and the formation of a linkage between the sugar and the organic group.
As used herein,"saccharide" is inclusive of monosaccharides, oligosaccharides or polysaccharides in straight chain or ring forms. Oligosaccharidcs are saccharides having two or more monosaccharide residues.
As used herein,"priming amount" means an amount or dose sufficient to prime the incorporation of cellular glucose into an oligosaccharide in a cell.
As used herein, methods performed"in a cell" indicate both in vitro and in vivo methods.
Maltose is a known disaccharide of glucose residues. Maltotriose has three glucose residues. Maltotetraose has four glucose residues. Other maltose derivatives are thusly named.
A"glycosidase inhibitor" refers to a compound which inhibits the hydrolysis of the glycosidic bond between two carbohydrates or a carbohydrate and the aglycone of a glycoside, an example of which is acarbose (Miles Laboratories, United States and Bayer, Germany).
A"hypoglycemic agent" refers to a compound which lowers plasma glucose levels, for example, tolbutamide and glyburide.
Also included herein is a method for assaying glycogen synthesis enzymatic activity in a sample. Such a"sample" can be a crude extract of a tissue or a cell culture or a more purified tissue or cell extract, as by affinity purification or by chromatofocusing. Glycosides useful for assaying include those useful for priming the incorporation of glucose into oligosaccharides, as it is the incorporation of radioactive forms of glucose into α(1→4) glycosidic linkages in oligosaccharides that indicates the presence of such enzymatic activity. Examples of novel uses of glycosides for assaying glycogen synthesis enzymatic activity include using non-p-nitrophenyl compounds. This use is the first use of nontoxic glycosides for assaying glycogen synthesis enzymatic activity.
The assay method comprises incubating the sample with a composition comprising an alkyl glycoside having a nonreducing terminal glucose residue joined in α anomeric linkage, and radiolabeled UDP-glucose, followed by determining the presence of radioactivity incorporated into the alkyl glycoside, the presence of radioactivity indicating the presence of glycogen synthesis enzymatic activity in the sample. To limit the enzymatic activity measured to glycogenin, the concentration of UDP-glucose can be relatively low, for example, 10 μM or less, and the glucose-6-phosphate needed for the activation of glycogen synthase is omitted. To limit the enzymatic activity measured to glycogen synthase activity, the concentration can be relatively high, for example, 1 mM or greater, and glucose-6-phosphate (around 5-10 mM) is included.
The invention also provides a method of priming the incorporation of ccllular glucose into an oligosaccharide in a cell comprising administering to the cell a priming amount of a membrane-permeating nontoxic glycoside having a suitable hydrophobic organic group joined by a linkage to a suitable hydrophilic saccharide, wherein the hydrophilic saccharide has a nonreducing terminal sugar residue selected from the group consisting of glucose or xylose, thereby priming the incorporation of cellular glucose onto the glycoside to form an oligosaccharide. Such priming of the incorporation of glucose into an oligosaccharide can be used to treat diabetes mellitus and glycogen storage disease, as described herein.
Glycosides useful for priming the incorporation of glucose into oligosaccharides have amphipathic properties due to their hydrophobic organic groups and their hydrophilic saccharide. These amphipathic properties enable them to readily penetrate cells chemically intact, so they have access to glycogenin and glycogen synthase. They can then serve as acceptors for the addition of multiple residues of glucose by these enzymes to create oligosaccharides having α(1→4) glycosidic linkages. Glycosides preferentially have up to around eight monosaccharide residues in order to readily permeate the cell.
Any compound chosen should be of minimal or non-toxicity to the cell, such as not to cause damage to the cell. Toxicity for any given compound may vary with the concentration of compound used. It is also beneficial if the compound chosen is metabolized or eliminated by the body and if this metabolism or elimination is done in a manner that will not be harmfully toxic.
The hydrophobic organic group can be, for example, an alkyl chain, an aralkyl group, an aryl group, or a steroid group. An alkyl chain can be chosen of any desired size, depending on the hydrophobicity desired and the hydrophilicity of the saccharide moiety. A preferred range of alkyl chains is from 8 to 14 carbon atoms. An aryl group can consist of, e.g., a phenyl group, a naphthyl group, an anthracene group, a phenanthrene group, or a flavonoid group. An aralkyl group can consist of, e.g., a benzyl group, a tolyl group, a 2-methylazulene group or a methylumbelliferyl group. A steroid group can be chosen from, for example, sapogenin, estradiol, cholesterol or cortisol.
The saccharide can be chosen, for example, from any currently commercially available saceharide species or can be synthesized. The saccharide can be a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide, or a combination thereof to form a saccharide chain. Some examples of the many possible saccharides to use include glucose, maltose, maltotriose, maltotetraose, sucrose and trehalose. One particularly useful saccharide would be one in which the end residue is xylose, as a compound comprising this structure would be more stable once administered and less susceptible to rapid hydrolytic cleavage by glycohydrolases such as α-glucosidase and amylase, and thus increase its therapeutic effect. Additionally, various oxygen atoms within the compounds can be substituted for by sulfur in order to decrease susceptibility to glycohydrolases. For example, the heteroatom of the sugar ring can be either oxygen or sulfur, or the linkage between monosaccharides in an oligosaccharide can be oxygen or sulfur. (Horton, D. and Wander, J. D., "Thio Sugars and Derivatives," The Carbohydrates: Chemistry and Biochemistry, 2d. Ed. Vol. IB, (W. Reyman and D. Horton eds.), pp. 799-842, (Academic Press, N.Y.), (1972)). Oligosaccharides can have either α (alpha) or β (beta) anomeric configuration but the linkage joining the last monosaccharide residue should preferably be in α anomeric configuration.
Many alkyl glycosides can be synthesized by known procedures, i.e., chemically, as described, e.g., in Rosevear et al., Biochemistry 19:4108-4115 (1980) or Koeltzow and Urfer, J. Am. Oil Chem. Soc., 61: 1651-1655 (1984), U.S. Pat. No. 3,219,656 and U.S. Pat. No. 3,839,318 or enzymatically, as described, e.g., in Li et al., J. Biol. Chem., 266:10723-10726 (1991) or Gopalan et al., J. Biol. Chem. 267:9629-9638 (1992). Many useful saccharides can be synthesized from a commercially available saccharide and UDP-glucose or UDP-xylose catalyzed by glycogenin, as described herein.
The linkage between the hydrophobic organic group and the hydrophilic saccharide can include, among other possibilities, a glycosidic, thioglycosidic (Horton), amide (Carbohydrates as Organic Raw Materials, F. W. Lichtenthaler ed., VCH Publishers, N.Y., 1991) carbon to carbon, or ester linkage (Sugar Esters: Preparation and Application, J. C. Colbert ed., (Noyes Data Corp., N.J.), (1974)).
Examples from which useful compounds can be chosen for the therapeutic composition include:
(a) a saccharide joined with an alkyl group: alkyl glycosides, such as octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-, and octadecyl α- or β-D-maltoside, α-glucoside or -sucroside (linked to the fructose moiety of the sucrose) (synthesized according to Koeltzow and Urfer; Anatrace Inc., Maumee, Ohio; Calbiochem, San Diego, Calif.; Fluka Chemie, Switzerland); alkyl thiomaltosides, such as heptyl, octyl, dodecyl-, tridecyl-, and tetradecyl-β-D-thiomaltoside (synthesized according to Defaye, J. and Pederson, C., "Hydrogen Fluoride, Solvent and Reagent for Carbohydrate Conversion Technology" in Carbohydrates as Organic Raw Materials, 247-265 (F. W. Lichtenthaler, ed.) VCH Publishers, N.Y. (1991)); alkyl thioglucosides, such as heptyl- or octyl 1-thio α- or β-D-glucopyranoside (Anatrace, Inc., Maumee, Ohio; see Saito, S. and Tsuchiya, T. Chem. Pharn. Bull. 33:503-508 (1985)); alkyl maltotriosides (synthesized according to Koeltzow and Urfer); derivatives of palatinose and isomaltamine linked by amide linkage to an alkyl chain (synthesized according to Kunz, M., "Sucrose-based Hydrophilic Building Blocks as Intermediates for the Synthesis of Surfactants and Polymers" in Carbohydrates as Organic Raw Materials, 127-153); derivatives of isomaltamine linked by urea to an alkyl chain (synthesized according to Kunz); long chain aliphatic carbonic acid ureides of sucrose β-amino-alkyl ethers (synthesized according to Gruber and Greber,"Reactive Sucrose Derivatives" in Carbohydrates as Raw Materials, pp. 95-116); long chain aliphatic carbonic acid amides of sucrose β-amino-alkyl ethers (synthesized according to Austrian Patent 382,381 (1987), Chem. Abstr., 108:114719 (1988) and Gruber and Greber, pp. 95-116); alkyl glycosides provided by this invention, such as octyl-α-D-glucopyranosyl-α-D-xylopyranoside (synthesized from octyl-α-D-glucopyranoside and UDP-xylose catalyzed by glycogenin, as described herein), dodecyl-β-D-glucopyranosyl-α-D-glucopyranosyl-α-D-glucopyranosyl-α-D-glucopyranoside (dodecyl maltotetraoside) (synthesized from dodecyl-β-D-maltotrioside and UDP-glucose catalyzed by glycogenin, as described herein), dodecyl-β-D-glucopyranosyl-α-Dglucopyranosyl-α-D-xylopyranoside (synthesized from dodecyl-β-D-maltoside and UDP-xylose catalyzed by glycogenin); alkyl thioglycosides provided by this invention, such as octyl-1-thio-α-maltotrioside (synthesized from octyl-1-thio-α-maltoside and UDP-glucose, as described herein);
(b) a saccharide joined with an aryl group: phenyl α-D-glucopyranoside (synthesized according to Carbohydrates, P. M. Collins ed., p. 414 (Chapman and Hall, London, 1987)); 1-naphthyl α, β-maltoside (synthesized according to Matsubara, S. J., Biochem (Tokyo), 49:226-231 (1961)); phenyl sucrose (phenyl group linked to fructose moiety of sucrose) (synthesized according to Hough, L., Application of the Chemistry of Sucrose in Carbohydrates as Organic Raw Materials, 33-55); carminic acid (Natural Red 4) (Sigma Chemical Co., St. Louis, Mo.);
(c) a saccharide joined to an aralkyl group: 4-nonylumbelliferyl-α-glucoside (synthesized according to Bieberich, E. and Legler, G., Biol. Chem. Hoppe-Seyler, 370:809-817 (1989)); 2-hydroxymethyl-azulene glucopyranoside (synthesized according to Daub et al., "From Carbohydrates to Pigments: An Exercise in Molecular Material Science and Material Transformation," Carbohydrate as Organic Raw Materials, pp. 340-350).
(d) a saccharide joined to a fatty acid group: trehalose-6-mycolate (isolated according to Haferburg, et al., Extracellular Microbial Lipids as Biosurfactants in Advances in Biochemical Engineering Biotechnology, A. Fiechter, ed., Vol. 33, 53-93 (Springer-Verlag, Berlin, 1986)); stearyl sucrose (stearyl group linked to fructose moiety of sucrose) (manufactured by Mitsubishi Food Corp., Japan; derivatives synthesized according to Benson, F. R., Polyol Surfactants in Nonionic Surfactants, M. J. Schick, ed., pp. 247-299 (Marcel Dekker, N.Y., 1967) and references therein);
Some preferred glycosides include maltose, maltotriose, and maltotetraose linked by glycosidic linkage to an alkyl chain of 8, 10, 12 or 14 carbon atoms, i.e., octyl-, decyl-, dodecyl- and tetradecyl maltoside, maltotrioside, and maltotetraoside. These compositions are nontoxic, since they are degraded to an alcohol and an oligosaccharide, and amphipathic so they readily permeate cells. Also preferred are mixtures of glycosides wherein at least one glycoside in the mixture is optimal for priming glucose incorporation by glycogenin and at least one glycoside in the mixture is optimal for glycogen synthase.
The above examples are illustrative of the types of glycosides to be used in the methods claimed herein; the list is not exhaustive. Derivatives of the above compounds which fit the criteria of the claims should also be considered when choosing a glycoside. All of the compounds can be screened for efficacy following the methods taught in the examples.
The method can comprise administering, in addition to the glycoside, compounds and/or compositions that will also aid in relief of the symptoms of diabetes mellitus, such as proglycosyn, hypoglycemic agents (for example, tolbutamide and glyburide) and glucosidase inhibitors (for example, acarbose) as well as dietary or pharmacologic agents used in the treatment of glycogen storage disease. Dosages for the above-mentioned additional compounds are established and known to those skilled in the art (Kahn, C. R. et al., "Insulin, Oral Hypoglycemic Agents and the Pharmacology of the Exocrine Pancreas" in The Pharmacological Basis of Therapeutics, Goodman and Gilman 8th ed., pp. 1463-1495, Pergamon Press, N.Y. (1990)). Additionally, the composition can comprise the glycoside in liposome form, wherein the liposome contains an additional compound(s), as listed above.
Compounds for priming the incorporation of glucose into oligosaccharides are provided which comprise an alkyl glycoside having an 8 to 14 carbon alkyl chain joined by a linkage to a saccharide having from four to eight glucose residues. Such compounds are formed by the addition of glucose residues from UDP-glucose to a known alkyl glycoside by the glycogen synthesis enzymes, as described herein. Such compounds can also comprise an 8 to 14 carbon alkyl chain joined by a linkage to a saccharide having from one to eight glucose residues and a non-reducing terminal sugar residue consisting of xylose. Such a compound can be synthesized by the addition of glucose residues from UDP-glucose, by glycogenin or glycogen synthase, and the addition of a final xylose residue, by glycogenin, to a known alkyl glycoside. The linkages between each added monosaccharide residue will thus be α(1→4); the linkage between the alkyl chain and the first saccharide residue is determined by the chosen known starter alkyl glycoside. Preferable linkages include glycosidic and thioglycosidic linkages.
Compositions which can be used for treating diabetes are provided which comprise a compound useful for priming the incorporation of cellular glucose into an oligosaccharide, as described herein, and an additional antidiabetes agent, specifically, proglycosyn; a hyperglycemic agent, such as tolbutamide or glyburide; or a glucosidase inhibitor, such as acarbose.
The compounds or compositions may be administered orally, by inhalation, topically, parenterally (e.g., intravenously), by intramuscular injection, transdermally, or the like, although oral administration is typically preferred and intravenous administration is not recommended for compositions including acarbose. The amount of active compound administered will, of course, be dependent on the subject being treated, the subject's weight, the severity of symptoms, the manner of administration and the judgment of the prescribing physician. Generally, however, dosage will approximate that which is typical for the administration of compounds such as tolbutamide. Dosage optimally would be that required to reduce intracellular glucose levels, for example, to a normal range of about 5-6.7 mM.
Depending on the intended mode of administration, the pharmaceutical compounds or compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, aerosols, liposomes, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compounds or compositions, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, E. W. Martin, (ed.), Mack Publishing Co., Easton, Pa.
Liposomes can be prepared according to Szoka and Papahadjopoulos, Ann. Rev. Biophys. Bioeng., 9:467 (1980) and Kiwada et al., Chem. Pharm. Bull. 33:753 (1985). The surfactant liposome can be administered without an additional permeability enhancing compound or as a carrier for an additional permeability enhancing compound. Liposomes can be administered, for example, orally or by aerosol inhalation.
For oral administration, fine powders or granules may contain diluting or dispersing agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension, wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup (see, e.g., Weber, N. and Benning, H. J Nutr. 114:247-254 (1984)). Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules may be coated.
For inhalation administration, the composition can be dissolved or dispersed in liquid form, such as in water or saline, preferably at a concentration at which the composition is fully solubilized and at which a suitable dose can be administered within an inhalable volume. Delivery can be repeated several times a day, depending upon the specific dosage chosen and the rate at which the chosen composition is cleared from the airways, with the goal being to reduce intracellular glucose levels.
Parenteral administration, if used, could also be by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference herein.
The exact amount of such compounds required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact activity promoting amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine testing given the teachings herein.
Any material added to the priming agent should be pharmaceutically acceptable. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.
Glycogenin Assay
Glycogenin is a self-glycosylating glycosyltransferase which can be obtained from muscle and which can also be obtained from rat and beef kidney. The enzyme is capable of transferring glucose and xylose from the respective UDP-sugar substrates to itself and also of transferring these sugars to exogenous acceptors, such as dodecyl-β-D-maltoside, tetradecyl-β-D-maltoside, octyl-β-D-maltoside, octyl-α-D-glucoside and other glycosides, as described herein.
Fresh beef kidney was purchased from a local slaughter house. Purification of glycogenin from kidney involves the following steps: chromatography of a 105,000�g supernatant from a homogenate of beef kidney on DE-53 cellulose, chromatofocusing and chromatography on a column of UDP-glucuronic acid-agarose, which was eluted with cytidine 5'-diphosphate (CDP). This procedure resulted in an almost 38,000 fold purification of the enzyme after UDP-glucuronic acid-agarose chromatography. The protein concentration of the affinity purified enzyme was determined to be 10 μg/ml with the Quantigold protein reagent (Diversified Biotech, Newton Centre, Mass.).
The following materials were obtained from the sources indicated: EN3 HANCE, liquid and surface spray, UDP- 1-3 H! glucose (specific activity, 7.8 Ci/mmole or 11.7 Ci/mmole (kinetic studies only) and UDP- 1-3 H! xylose (specific activity, 8.9 Ci/mmole) DuPont (Boston, Mass.); SepPak C18 cartridges (Millipore Corporation, Milford, Mass.); tetradecyl maltoside and dodecyl maltoside, Anatrace (Maumee, Ohio); octyl maltoside and dodecyl sucrose, Calbiochem (San Diego, Calif.); p-nitrophenyl maltoside, Boehringer Mannheim (Indianapolis, Ind.) and all other chemicals, Sigma (St. Louis, Mo.). Radioactivity was measured in a Wallac model 1410 liquid scintillation spectrometer.
An appropriate sample of UDP- 3 H! glucose or UDP- 3 H! xylose (supplied in 70% ethanol by the manufacturer) was evaporated to dryness with a gentle stream of filtered air and dissolved in 50 mM Tris Cl, pH 7.4/75 mM BAL/1.5 mM ATP. Reaction mixtures, in 1.5 ml polypropylene tubes, had a total of 75 μl and contained the following components: UDP- 3 H! glucose or UDP- 3 H! xylose (5 μl, 0.15 μCi), enzyme protein (19 μg of chromatofocusing purified or 10 ng of affinity purified enzyme), DBM (.01 mg/ml, 0.1 mg/ml, 1 mg/ml, 10 mg/ml, and 20 mg/ml) or other glycoside at such concentrations, and 50 mM Tris-Cl/6 mM MnCl2 /12 mM MgCl2 /5 mM BAL/0.1 mM ATP, pH 7.4, with either 2 mM CHAPS (TCA precipitation) or 1 mM CHAPS (SepPak C18 isolation). After incubation for 60 min at 37� C. product formation was measured by either of the two procedures described below.
Product isolation by precipitation with trichloracetic acid. After completed incubation, 1% bovine serum albumin (50 μl) and 10% trichloracetic acid/4% phosphotungstic acid (150 μl) were added, and the mixture was vortexed and kept on ice for at least 15 min. The precipitate was sedimented by centrifugation in a microfuge and washed three times with 5% trichloroacetic acid. The bottom of the tube, containing the pellet, was clipped off with a dog nail clipper and dropped into a 6 ml scintillation vial, the protein was dissolved in 250 μl of 2M NaOH, and, after neutralization with 250 μl of 2M HCl, 4.5 ml of Ecolume was added, and the radioactivity was measured.
Product isolation by adsorption to SepPak C18 cartridges. The reaction mixture was brought to a, volume of 0.5 ml by addition of water and was heated to 100� C. for 1 min. After cooling to room temperature, the mixture was centrifuged, and a 10 μl aliquot was removed for measurement of total radioactivity. The remaining 490 μl was applied with the use of a 10 ml syringe to a SepPak C18 cartridge, which had been preconditioned by washing with 8 ml of 95% ethanol, 8 ml of 70% ethanol, and 20 ml of water. After sample application, the cartridge was washed with 2�9 ml of water, followed by an additional 2 ml portion of water, and adsorbed radioactive material was then eluted with 3�4 ml of 70% ethanol. Typically, no significant radioactivity was found in the last water wash, and most of the bound radioactive material was found in the first ethanol eluate.
Gel electrophoresis and autoradiography. SDS-PAGE was carried out with slight modification of Laemmli (Laemmli, U. K., Nature, 227:680-685 (1970)). A Hoefer vertical slab gel electrophoresis unit (Hoefer Scientific Instruments, San Francisco, Calif.) was used for casting and electrophoresing with a 10% (w/v) acrylamide separating gel and a 4.5% (w/v) acrylamide stacking gel. Reaction mixtures, with total volumes of 75 μl, containing 0.15 μCi (0.26 μM) UDP- 3 H! glucose, 10 ng of affinity purified enzyme (intact or heat denatured), in the absence and presence of increasing concentrations of DBM were incubated at 37� C. for 60 min. The reaction was terminated by addition of 5 μl of water and 20 μl of sample reducing buffer (5� concentrated) to give final concentrations of 40 mM Tris-Cl, pH 6.8, 1.5% (w/v) SDS, 6% (v/v) glycerol, 100 mM DTT and 0.002% (w/v) bromophenol blue and heated to 100� C. for 3 min. The supernatant was applied to the gel and electrophoresed at 7.5 mA for 16 hours. The gel was soaked in EN3 HANCE, dried and autoradiography was performed using Kodak AR film (Eastman Kodak, Rochester, N.Y.) at -70� C.
Inhibition of glucose transfer to DBM by cytidine 5'-diphosphate. It has been shown previously that cytidine 5'-diphosphate is a potent inhibitor of the self-glucosylation of glycogenin (Manzella et al., The Phannacologist, 33:206 (1991)). It has now been determined that this is also true for the transfer to exogenous acceptor. Cytidine 5'-diphosphate (0.1-0.5 mM) inhibited the transfer of 3 H!glucose to DBM by glycogenin in a dose dependent manner.
Results with DBM. Glycogenin transfers glucose from UDP-glucose to dodecyl-β-D-maltoside (DBM) in a dose-dependent fashion as the concentration of the acceptor substrate is increased from 0.0001-10 mg/ml DBM. The enzymatic transfer of glucose from UDP-glucose to DBM by glycogenin is dependent on the amount of enzyme protein present (0.1-0.4 μg/ml), the time of incubation (0-150 minutes) and the concentration of the donor substrate UDP-glucose (0-15 μM). Transfer of glucose to DBM by glycogenin can be demonstrated by three different independent assay procedures: TCA precipitation, adsorption to SepPak C18 and biphasic liquid scintillation counting. That the transfer is dependent on both the presence of enzyme and DBM is indicated by the markedly reduced amount or absence of product in incubation mixtures lacking enzyme or DBM (0.1 mM or 0.2 mM). That glycogenin is the enzyme catalyzing the transfer of glucose from UDP-glucose to DBM is indicated by the dose-dependent inhibition of this reaction with CDP, a nucleotide which we have shown to be a potent inhibitor of glycogenin. Therefore DBM is capable of acting as an artificial acceptor substrate for glucose transfer from UDP-glucose by glycogenin. This results in a marked increase in the amount of glucose that can be transferred in the presence of enzyme alone by self-glycosylation and therefore makes DBM valuable in conditions where increased disposal of excess glucose is beneficial, e.g., diabetes mellitus. Furthermore, by providing many more primer sites for de novo synthesis of glucose chain molecules, DBM can be used to treat glycogen storage disease by creating more molecules of a much smaller and more manageable size, rather than the accumulation of large glycogen molecules that characterize this disease.
Characterization of the Product of Transfer. Size exclusion chromatography of a reaction mixture containing DBM showed much more 3 H-glucose radiolabeled product eluting in the void volume (Vo) of a Sephadex G-50 column than identical chromatography of a reaction mixture lacking DBM.
That this increased product formation represented transfer of 3 H!-glucose to DBM rather than self-glycosylation by glycogenin was indicated by electrophoresis of standard reaction mixtures containing increasing amounts of DBM on SDS-polyacrylamide gels. Autoradiography of the resulting gel showed a decrease of 3 H!-glucose incorporation into the 32-38 kDa products representing glycogenin as the DBM in the reaction mixture was increased, and an increase in incorporation into a radiolabeled product which moved at the bottom of the gel and represented DBM to which 3 H-glucose had been transferred. This product was identified as DBM-glucose by thin layer chromatography and autoradiography of the reaction mixture which had first been passed through a SepPak C18 cartridge and eluted with 70% ethanol to separate the labeled product from unreacted and degraded UDP- 3 H!glucose substrate. The formation of DBM-glucose (dodecyl-β-D-maltotrioside) was further demonstrated by separation of the new product from the DBM starting material by high pressure liquid chromatography. When UDP- 3 H!xylose was used as the starting donor substrate, then DBM-xylose was formed (dodecyl-β-D-glucosyl-α-(1→4)glucosyl-α-(1→4)xyloside. Under appropriate reaction conditions (low DBM concentration, longer incubation times), more than one glucose residue could be transferred to DBM to form DBM-glucose-glucose (dodecyl-β-D-maltotetraoside). Synthesis of this compound was also demonstrated by its separation from DBM and dodecyl-β-maltotrioside on high pressure liquid chromatography. By isolating the newly formed products by HPLC and reincubating them with glycogenin and UDP-glucose, it is possible to form products containing larger oligosaccharides attached to DBM, i.e., dodecyl-β-D-maltopentaoside, dodecyl-β-D-maltohexaoside, etc. Each of these products represents a new compound which may potentially serve as an acceptor substrate for glycogenin and/or glycogen synthase. Therefore, under appropriate conditions, DBM is capable of serving as an acceptor for several glucose and/or xylose residues producing dodecyl-oligosaccharide products, which can serve as further substrates for glycogenin and/or glycogen synthase demonstrating the ability of this compound to facilitate disposal of excess glucose into glycogen-like products.
Results with other alkyl glycosides. Other alkylglycosides were also capable of serving as acceptor substrates for the transfer of glucose and xylose from their respective UDP-sugar donor substrates by glycogenin. These included octyl-β-D-maltoside, decyl-β-D-maltoside and tetradecyl-β-D-maltoside which all could be converted to their respective maltotriosides by addition of one glucose residue. Of these substrates, DBM was the best acceptor for glucose, being twice as active as tetradecyl-β-D-maltoside and four times better than octyl-β-D-maltoside. In contrast dodecyl-β-D-sucrose showed a much lower activity as a glucose acceptor indicating the importance of the saccharide moiety in the activity of the alkyl glycosides. Most importantly, DBM was 29 times more effective as a glucose acceptor than p-nitrophenyl-α-maltoside, the best exogenous acceptor for glycogenin reported prior to this invention. In addition, DBM and the other alkylglycosides are metabolized to nontoxic products, whereas, metabolism of p-nitrophenyl-α-maltoside, would give rise to the toxic p-nitrophenol. Octyl-α-D-glucoside also served as an effective glucose acceptor for glycogenin and was much better in this regard than octyl-β-D-glucoside and octyl-β-D-thioglucoside, showing the importance of the α-linkage of the carbohydrate to the alkyl moiety when alkyl monosaccharides are used as acceptors. It should be noted that this linkage is of much less importance when examining the activity of alkyl disaccharides, such as dodecylmaltoside; the β-linked compound (DBM) is the most active compound thus far tested, and the α-linked compound would be expected to have equal or greater activity. Note also that even though the activities are low, octyl-β-D-thioglucoside is more active as a glucose acceptor than octyl-β-D-glucoside, indicating that substitution of a thioether for the ether linkages in the alkylglycosides would likely make them better acceptors as well as more resistant to enzymatic degradation.
Glycogen Synthase Assay
The assay of glycogen synthase in vitro depends on the presence of polymeric glycogen as an acceptor for glucose transfer. Oligosaccharides such as maltose, maltotriose and maltotetraose serve as substrates for glycogenin and as primers for glycogen synthase but have only a low affinity for these enzymes. Glycosides, on the other hand, in addition to serving as an acceptor substrate for glucose and xylose transfer by glycogenin, can also serve as a glucose acceptor for transfer catalyzed directly by glycogen synthase and are active in stimulating the synthesis of glycogen-related oligosaccharides linked to the starter glycoside.
Rabbit muscle glycogen synthase obtained from Sigma Chemical Co., (7.6 μg) was incubated with 0.45 μCi UDP- 3 H!glucose (specific activity of 10.4 Ci/mmol), 1 mM UDP-glucose (glucose donor), 10 mM glucose-6-phosphate (the natural activator of glycogen synthase) and 1 mg glycogen (glucose acceptor) in 25 mM EDTA/5 mM DTT/100 mM glycine. NaOH buffer, pH 8.6 in a total volume of 100 μl for 30-240 minutes at 37� C. At different time points, glycogen was precipitated from the reaction mixtures with ethanol and the precipitate washed to remove any unincorporated radiolabel. The glycogen precipitate was then solubilized and counted in a liquid scintillation counter to quantitate the amount of 3 H!glucose incorporated into glycogen. By this method, glucose transfer to glycogen can be demonstrated.
Results. When DBM (0.5 mM) replaces glycogen as the glucose acceptor in the incubation mixture, a linear transfer of glucose to DBM with time is seen up to 4 hours, with the glucose transfer amounting to about 20% of the maximal value seen with glycogen (using the SepPak C18 assay). It should be noted however, that the glucose transfer to DBM showed no signs of plateauing at 4 hours incubation time and that longer incubation would have resulted in even greater glucose transfer to DBM. When glycogen synthasecatalyzed glucose transfer to DBM was plotted as a function of the acceptor substrate, i.e., from 0.1 to 10 mM DBM for a 60 min incubation, near-maximal activity was seen at less than 0.5 mM DBM, indicating that this substrate has a high affinity for glycogen synthase. In these experiments glucose transfer to DBM was totally dependent on the presence of glucose-6-phosphate (G-6-P), indicating that under these conditions it was mediated by glycogen synthase and not by glycogenin, since G-6-P activates only the former enzyme. Interestingly, when octyl-, dodecyl-, and tetradecyl- maltosides were compared under these conditions, transfer of glucose to all three acceptor substrates was dependent on G-6-P, and octylmaltoside proved to be the best acceptor, followed by DBM and tetradecylmaltoside. Therefore, the glucose acceptor specificities of glycogenin and glycogen synthase differ somewhat so that mixtures of alkylglycosides, e.g., octylmaltoside and dodecylmaltoside may prove optimum in serving as initiators of glycogen synthesis enzymatic activity and glucose acceptor substrates for both enzymes.
Of the greatest importance is the demonstration that DBM can serve as an acceptor for glucose addition by glycogen synthase to produce DBM-linked oligosaccharides which are greater than seven glucose residues in length, showing that this compound can act as a reservoir for glucose disposal. When glycogen synthase was incubated for 24 hours with G-6-P, UDP- 3 H!glucose and 0.5 mM DBM, several products of glucose transfer to DBM could be visualized following separation by thin layer chromatography and autoradiography. These products corresponded to the dodecyl-maltotrioside, -tetraoside, -pentaoside, -hexaoside, -heptaoside and higher oligosaccharide derivatives of DBM. Note that considerably more product is seen at the origin of the TLC plate in the incubation mixture containing DBM than in that lacking it, indicating that glucose incorporation into DBM-linked products with greater than seven glucose residues is appreciable. Therefore, DBM and other suitable glycosides can serve as glucose acceptors for both glycogen synthase and glycogenin to promote glucose disposal in conditions such as hyperglycemia and diabetes mellitus. Providing an abundance of acceptor substrates in tissues, such as liver, which has a smaller number of extremely large glycogen molecules can alleviate the problem of excessive glycogen deposition in the glycogen storage diseases by promoting glucose disposal into more readily handled smaller glucose polymers built on DBM or other glycosides.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.
|Cited Patent
|Filing date
|Publication date
|Sep 3, 1968
|Dec 15, 1970
|Rohm & Haas
|Alkyl oligosaccharides and their mixtures with alkyl glucosides and alkanols
|Mar 19, 1969
|Jun 22, 1971
|Eisai Co Ltd
|Xylose derivatives and preparation thereof
|Mar 7, 1983
|Jun 12, 1984
|Seikagaku Kogyo Co. Ltd.
|O-xylopyranoside series compounds and methods of use
|Apr 25, 1985
|Sep 1, 1987
|Research Corporation
|Emulsion-based gel and process for preparing same
|Jan 13, 1986
|Sep 19, 1989
|Symbicom Aktiebolag
|Glycosidic derivatives
|Apr 25, 1988
|Oct 31, 1989
|Fournier Innovation Et Synergie
|Novel β-D-phenylthioxylosides, their method of preparation and their use as therapeutics
|Feb 27, 1991
|Nov 24, 1992
|Huels Aktiengesellschaft
|Process for the preparation of carbohydrate surfactants
|Jun 24, 1985
|Dec 2, 1986
|A. E. Staley Manufacturing Company
|Branched chain glycosides
|Title not available
|Bogardus, C. et al., "Where all the Glucose Doesn't go in Non-insulin-dependent Diabetes Mellitus," N. Eng. J. Med., 322(4):262-263 (Jan. 25, 1990).
|Bogardus, C. et al., Where all the Glucose Doesn t go in Non insulin dependent Diabetes Mellitus, N. Eng. J. Med. , 322(4):262 263 (Jan. 25, 1990).
|Durette et al, "Structure-Activity Relationships of Aminalkyl and -aryl Glycosides Having Insulin-like activity," Jour. Med. Chem. 21(9): pp. 854-859 (1978).
|Durette et al, Structure Activity Relationships of Aminalkyl and aryl Glycosides Having Insulin like activity, Jour. Med. Chem. 21(9): pp. 854 859 (1978).
|Gressner, A. M., "Questioning The Reliability of ρ-Nitrophenyl-β-D-xyloside as Probe to Study the Metabolic Effects of Abrogated Proteoglycan Synthesis in Cultured Cells," Biochem. Pharm., 42:1987-1995 (1991).
|Gressner, A. M., Questioning The Reliability of Nitrophenyl D xyloside as Probe to Study the Metabolic Effects of Abrogated Proteoglycan Synthesis in Cultured Cells, Biochem. Pharm. , 42:1987 1995 (1991).
|Guo, Z. K. et al., "Stimulation of Glycogen Synthesis by Proglycosyn (LY177507) by Isolated Hepatocytes of Normal and Streptozotocin Diabetic Rats," J. Biol. Chem., 266:22323-22327 (1991).
|Guo, Z. K. et al., Stimulation of Glycogen Synthesis by Proglycosyn (LY177507) by Isolated Hepatocytes of Normal and Streptozotocin Diabetic Rats, J. Biol. Chem. , 266:22323 22327 (1991).
|Harris, R. A. et al., "Stabilization of Glycogen Stores and Stimulation of Glycogen Synthesis in Hepatocytes by Phenacyl Compounds," J. Biol. Chem., 264:14674-14680 (1989).
|Harris, R. A. et al., Stabilization of Glycogen Stores and Stimulation of Glycogen Synthesis in Hepatocytes by Phenacyl Compounds, J. Biol. Chem. , 264:14674 14680 (1989).
|Lomako, J. et al., "Substrate Specificity of the Autocatalytic Protein that Primes Glycogen Synthesis," FEBS Lett., 264:13-16 (1990).
|Lomako, J. et al., Substrate Specificity of the Autocatalytic Protein that Primes Glycogen Synthesis, FEBS Lett. , 264:13 16 (1990).
|Shulman, G. I. et al., "Quantitation of Muscle Glycogen Synthesis in Normal Subjects and Subjects with Non-insulin-dependent Diabetes by 13 C Nuclear Manetic Resonance Spectroscopy," N. Eng. J. Med., 322(4):223-228 (Jan. 25, 1990).
|Shulman, G. I. et al., Quantitation of Muscle Glycogen Synthesis in Normal Subjects and Subjects with Non insulin dependent Diabetes by 13 C Nuclear Manetic Resonance Spectroscopy, N. Eng. J. Med. , 322(4):223 228 (Jan. 25, 1990).
|Smythe, C. et al., "The Discovery of Glycogenin and the Priming Mechanism for Glycogen Biogenesis," Eur. J. Biochem., 200:625-631 (1991).
|Smythe, C. et al., The Discovery of Glycogenin and the Priming Mechanism for Glycogen Biogenesis, Eur. J. Biochem., 200:625 631 (1991).
|Vaag, A. et al., "Effect of Acute Hyperglycemia on Glucose Metabolism in Skeletal Muscles in IDDM Patients," Diabetes, 41:174-182 (Feb. 1992).
|Vaag, A. et al., Effect of Acute Hyperglycemia on Glucose Metabolism in Skeletal Muscles in IDDM Patients, Diabetes , 41:174 182 (Feb. 1992).
|Weber, N. et al., "Metabolism of Orally Adminstered Alkyl βGlycosides in the Mouse," J. Nutr., 114:247-254 (1984).
|Weber, N. et al., Metabolism of Orally Adminstered Alkyl Glycosides in the Mouse, J. Nutr. , 114:247 254 (1984).
|Yamanouchi, K. et al., "Metabolic Effects of Proglycosyn," Arch. Biochem. Biophys., 294(2):609-615 (May 1, 1992).
|Yamanouchi, K. et al., Metabolic Effects of Proglycosyn, Arch. Biochem. Biophys., 294(2):609 615 (May 1, 1992).
|Zhehavi U. et al., "Polymers having (1→4)-and (1→6)-linked α-D-glucopyranosyl Groups as Acceptors in the Glycogen Synthase Reaction," Carbohydr. Res., 228:225-263 (1992).
|Zhehavi U. et al., Polymers having (1 4) and (1 6) linked D glucopyranosyl Groups as Acceptors in the Glycogen Synthase Reaction, Carbohydr. Res. , 228:225 263 (1992).
|Zhehavi, U. et al., "Probing Acceptor Specificity in the Glycogen Synthase Reaction with Polymer-Bound Oligosacchrides," Carbohydr. Res., 151:371-378 (1986).
|Zhehavi, U. et al., Probing Acceptor Specificity in the Glycogen Synthase Reaction with Polymer Bound Oligosacchrides, Carbohydr. Res. , 151:371 378 (1986).
|Citing Patent
|Filing date
|Publication date
|Jan 15, 2002
|Oct 28, 2003
|John-Claude Savoir
|Stable shaped particles of crystalline organic compounds
|May 29, 2002
|Dec 16, 2003
|John-Claude Savoir
|Stable shaped particles of crystalline organic compounds
|Feb 26, 2003
|May 18, 2004
|John-Claude Savoir
|Stable shaped particles of crystalline organic compounds
|Dec 22, 2003
|Oct 31, 2006
|Regents Of The University Of Minnesota
|Method of detecting equine glycogen storage disease IV
|Aug 18, 2003
|Aug 5, 2008
|Amylin Pharmaceuticals, Inc.
|Methods for regulating postprandial blood glucose
|Feb 26, 2003
|Sep 23, 2008
|Skendi Finance Ltd.
|Stable shaped particles of crystalline organic compounds
|Sep 12, 2003
|Nov 17, 2009
|Institut National De La Recherche Agronomique
|Use of prebiotics, preferably glucooligosaccharide, for the prevention of the onset of type II diabetes
|Jun 14, 2004
|Aug 30, 2011
|The Trustees Of The University Of Pennsylvania
|Vaccines, immunotherapeutics and methods for using the same
|Dec 22, 2008
|Sep 18, 2012
|Aegis Therapeutics, Llc.
|Alkylglycoside compositions for drug administration
|Dec 22, 2009
|May 14, 2013
|Aegis Therapeutics, Llc
|Compositions for drug administration
|Aug 11, 2008
|Oct 8, 2013
|Aegis Therapeutics Llc
|Absorption enhancers for intranasal interferon administration
|Jun 13, 2012
|Nov 25, 2014
|Hale Biopharma Ventures, Llc
|Administration of benzodiazepine compositions
|Feb 10, 2012
|Jan 6, 2015
|Aegis Therapeutics, Llc.
|Absorption enhancers for intranasal administration
|Feb 17, 2009
|Jan 20, 2015
|Premier Nutrition Corporation
|Carbohydrate gel
|May 13, 2013
|Mar 15, 2016
|Aegis Therapeutics, Llc
|Compositions for drug administration
|May 1, 2003
|Feb 19, 2004
|Schwartz Arthur G.
|7-Hydroxy-16alpha-fluoro-5-androsten-17-ones and 7-hydroxy-16alpha-fluoro-5-androstan-17-ones and derivatives thereof
|Aug 18, 2003
|May 20, 2004
|Kolterman Orville G.
|Methods for regulating gastrointestinal motility
|Feb 26, 2003
|Aug 26, 2004
|John-Claude Savoir
|Stable shaped particles of crystalline organic compounds
|May 11, 2005
|Mar 2, 2006
|Aegis Therapeutics Llc
|Absorption enhancers for drug administration
|Sep 12, 2003
|May 11, 2006
|Institut National De La Recherche Agronomique
|Use of prebiotics, preferably glucooligosaccharide, for the prevention of the onset of type II diabetes
|Jun 29, 2006
|Nov 2, 2006
|Brown Truman R
|Methods for alleviating deleterious effects of 3-deoxyglucosone
|Aug 6, 2007
|Nov 22, 2007
|Brown Truman R
|Methods for alleviating deleterious effects of 3-deoxyglucosone
|Aug 20, 2008
|Feb 19, 2009
|Aegis Therapeutics, Inc.
|Compositions for Drug Administration
|Dec 22, 2008
|Jun 25, 2009
|Maggio Edward T
|Compositions for Drug Administration
|Dec 22, 2009
|Jun 24, 2010
|Aegis Therapeutics Llc
|Compositions for drug administration
|Feb 17, 2009
|Jan 13, 2011
|Asker Jeukendrup
|Carbohydrate gel
|Sep 12, 2003
|May 13, 2004
|Agronomique Inst Nat Rech
|Use of prebiotics, preferably glucooligosaccharide, for the prevention of the onset of type ii diabetes
|U.S. Classification
|514/24, 536/18.6, 514/866, 514/61, 536/123, 536/123.1, 536/4.1, 536/17.2, 514/54, 514/25, 536/123.13
|International Classification
|Cooperative Classification
|European Classification
|Apr 23, 2002
|Maintenance fee reminder mailed
|Oct 2, 2002
|Fee payment
Year of fee payment: 4
|Oct 2, 2002
|Surcharge for late payment
|Mar 28, 2006
|Fee payment
Year of fee payment: 8
|May 10, 2010
|Maintenance fee reminder mailed
|Oct 6, 2010
|Lapse for failure to pay maintenance fees
|Nov 23, 2010
|Expired due to failure to pay maintenance fee
Effective date: 20101006


text:
Is your resume in need of a little disruption?
Startups are all about
making things work better.
Our resume writing service is all about doing that for your CV.
StartUpHire is the place to find Director,-Solution-Design jobs in and near . Review the list below of Director,-Solution-Design jobs. These Director,-Solution-Design jobs represent some of the most rewarding careers available. You can refine your job search for Director,-Solution-Design jobs in by adding a job title, company name, or keyword to the search box above … or click the "Advanced Search" link for more options. Don't see the employment opportunity you're looking for? Check back soon for more Director,-Solution-Design jobs in , or sign up for an email alert (link in upper left) to receive periodic updates on new Director,-Solution-Design jobs in , .
Department: Sales Location: 710 North Drive - Melbourne, FL 32934 Avidyne Corporation is leading the general aviation (GA) industry in the design and manufacture of innovative and easy-to-use avionics systems. Avidyne is uniquely focused developing avionics systems that make1/30/2017 Melbourne, FL Save This Job
Sales Boston, Massachusetts Apply Associate Director of Business Development Who You Are You are a connector who has experience developing ?Trusted Advisor? relationships with clients to explore new ideas, develop solutions to critical problems, and recognize organizational1/17/2017 Boston, MA Save This Job
NORTH AMERICA SALES DIRECTOR North America We are currently seeking a dynamic, driven sales leader for Xtera products and services within the assigned region. This individual must have sales experience with a strong business acumen. They will be responsible for sales activities1/14/2017 Allen, TX Save This Job
Portland, OR, OR Full Time Sales Experienced About Opal At Opal, we believe enterprise software should be beautiful, simple and designed for the job at hand. Opal is a collaboration platform, built for brand marketing teams to produce, organize and govern creative1/11/2017 Portland, OR Save This Job
The Director of Marketing works closely with the VP of Marketing to develop all marketing, advertising, and communications initiatives, both internal and external, across multiple platforms and formats to drive customer acquisition, subscription sales, engagement, retention and1/11/2017 Westerville, OH Save This Job
Playa Vista ApplyApply with LinkedIn Job Description: Job Summary (General Responsibilities): The Global Director of Procurement role is a vital strategic position for Belkin operating within a fast moving and dynamic global market, requiring strategic and innovative procurement1/10/2017 Playa Vista, CA Save This Job
New York, United States Sales DESCRIPTION JOB DESCRIPTION Director of Marketing is responsible for all activities related to conceptualizing, implementing and executing STAQ's market strategy and branding efforts. Specific responsibilities include both strategic development of1/4/2017 New York, NY Save This Job
Location Cleveland, OH 44115 Other Location Description OnShift is the premier provider of unique, tailored human capital management software for the long-term post-acute care and senior living industries. Our solutions are deployed in over 2500 care settings ranging from1/3/2017 Cleveland, OH Save This Job
Department: Clinical Research Job Location: South San Francisco Cytokinetics is a late-stage biopharmaceutical company focused on discovering, developing and commercializing first-in-class muscle activators as potential treatments for debilitating diseases in which muscle1/3/2017 South San Francisco, CA Save This Job
Job Description: Works closely with the Clinical Development team for the oversight and execution of assigned clinical studies ?May be responsible for one or more concurrent clinical studies ?Contribute in the development of the clinical study design and writing of the study1/3/2017 Lexington, MA Save This Job
Are you passionate about putting patients at the center of medical research? Do you have the ability to capture the patient?s voice through custom research instruments in such a way that you can literally change the fortunes of a business as well as shape the success of bringing1/1/2017 San Francisco, CA Save This Job
We are recruiting an engineering director to play a key leadership role in building our next-gen Big Data predictive analytics platform & products with a focus on B2B marketing/sales. The person will join a group filled with talented people, including many with background from12/27/2016 San Mateo, CA Save This Job
Job reference:js-3298-2398k Location:California - San Francisco Salary:Negotiable Job type:Permanent Sector:Big Data & Cloud Computing Date posted:22 Nov 2016 Contact:Mary KypreosinShare Apply for this job Add to shortlist Print job Send this job by email Email me jobs like12/26/2016 San Francisco, CA Save This Job
at PatientsLikeMe (View all jobs) Cambridge As the Director of Scientific Operations you will work in partnership with the Head of Biology and Bioinformatics as well as with PatientsLikeMe senior management and working teams from Research and Policy, Project Management, User12/24/2016 Cambridge, MA Save This Job
Department:420 - Analytics Location:Alpharetta, GA Click Here to Complete Your Application Seeking a Healthcare Business Intelligence Analyst Guru! Improving healthcare business performance, turning quality, risk and compliance efforts into opportunities, and creating, enhancing11/15/2016 Alpharetta, GA Save This Job
Creative Services Austin, Texas Apply Description Essential Functions: Establish art direction and design for a variety of digital and interactive marketing deliverables, including emails, landing pages, websites, banner ads, social media, webinars, case studies, infographics1/23/2017 Austin, TX Save This Job
Aptara is seeking a talented and innovative user interface and experience designer/developer to join our new Sales Consulting team, reporting to the SVP, Sales Consulting. You will work with our clients to create web and mobile solutions that fit their content, goals and budgets.1/4/2017 Falls Church, VA Save This Job
Talent Mountain View, CA Coursera is scaling a global platform to provide universal access to the world?s best education, and we?re driven by the passion and mission to let people learn without limits. To date, we?ve brought over 1800 courses to 24 million learners worldwide,2/22/2017 Mountain View, CA Save This Job
TARIS Biomedical is a unique therapeutically-focused urology company developing drug-device combination products for the treatment of genitourinary diseases. The individual will be responsible for supporting development activities such as analytical method transfer, test method2/13/2017 Lexington, MA Save This Job
Sunnyvale, CA December 26, 2016 Enlighted Inc., founded in 2009, is the leading provider of Internet of Things (IoT) solutions in the commercial real estate space. Enlighted develops innovative wireless sensor technology, big data cloud solutions, and enables a variety of1/24/2017 Sunnyvale, CA Save This Job

subset: commoncrawl


text:
Bounce Guesthouse is centrally located easily within 7-8mins by walk to Hongik University subway station, which is... More Info »
Uhbu's Guest House offers advantages for tourist that are looking for unique experiences in Korea. 1.... More Info »
ISAK Guest House is the best hostel in hostel. ISAK is located in the centre of various transportation links,... More Info »
저희는 외국인 전용 게스트하우스로 지정을 받은 곳이라서 내국인은 숙박할 수가 없습니다. "Five star hostel" "Five star people" "Five star insiders"... More Info »
Great memories come from the 'Pencil' ★★ TOURISM MECCA Hongdae (means Hongik university - area) in Seoul ★★... More Info »
I, Bong Do Lee, would like to welcome you to my guesthouse, Bong House. I have rooms to suit every type of visitor to... More Info »
JK House in Seoul(home stay style) is located in heart of Shin-chon, university town. Within 5 minutes by walk... More Info »
* Comfortable and safe guesthouse * Awesome location, just 2 mins walking distance from Hongik univ station exit... More Info »
Clean and modern guestrooms with free Wi-Fi and a washing machine are offered at Twin Rabbit Hostel, just 10 minutes'... More Info »
We are opening on 15th of December. Please don't worry for the reason that there isn't any review for now. We are... More Info »
=!!! We offer about 57%~36% Discount Now !!! = The Superior 5 Bed Family Room Ensuite, Superior 4 Bed Private... More Info »
The Excellent base for your Seoul trip. It\\\'s only maxim one hour from Incheon Airport to Birdsnest. (It\\\'s... More Info »
Welcome to Chingu Guesthouse, your sweet home away from home!!! Feel at home with our warm atmosphere and caring... More Info »
Cheap & Fun Hostel for Travelers & Backpackers in Seoul, Korea Kimchi Seoul G.H. provides a truly unique travelers... More Info »
Guaranteed the best location by majority of guests!! Instantaneous selection influences your happy trip!! I am MIN!... More Info »
COME INN is named after 'Come in!' We would like you to visit freely and stay comfortably at our accommodation.... More Info »
Home is where the heart is, Another home sweet home, House740. Modern classical space will give you real rest. and... More Info »
Our rooms are cosy with special bunk beds, which you can fold. This means that when there are no other guests, your... More Info »
K-Guesthouse Myeongdong 1 also offers many facilities to enrich your stay in Seoul. For the comfort and convenience... More Info »
In explaining about PHILSTAY,PHIL means 'friend, beloved and favorite', STAY means 'remaining'.In other words,... More Info »
Video guides, travel podcasts, city maps & free city guides. What to eat, where to stay and things to do and see - our travel guides have it all.Find a guide »
We want to make sure that you get to explore as much of Paris as possible and that includes the nightlife scene. We've compiled some of the best places in Paris to check out if ...Read more »
We offer the most comprehensive selection of hostels on the internet with over 33,000 great places to stay in 170 countries. The ultimate online resource for hostels everywhere.Find great value hostels »


text:
Is RHB Bank Jalan Tan Sri Teh Ewe Lim, Jelutong incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
Are these details correct?
Is RHB Bank Jalan Tan Sri Teh Ewe Lim, Jelutong in No 112, Jalan Tan Sri Teh Ewe Lim, 11600 George Town, Penang your business? Claim your listing and attract more leads by adding more content, photos and other business details.
We have more Financial services available on Hotfrog MALAYSIA. You can update your search for Financial by location, keyword or service options.
Small Business Hub
Sharing tips, ideas and advice to help grow your small business.
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub


text:
Is this attraction free to the public?
Is gratuity suggested at this attraction?
Would this be a good rainy day activity?
Is there food available at this attraction?
Is a car required to access this attraction?
Is this attraction accessible using public transportation?
Is this attraction closed during certain parts of the year?
Is this attraction wheelchair accessible?
Does this activity require advanced planning, ticketing or reservations?
Is this activity accessible without advanced planning or reservations?
Thanks for helping!
Never expected to find these bowling lanes in Bielefeld. Crowded and good service.
I,ve bowled here for 15years regulary & can say i enjoy going there. Prices are right, food is good & the personal friendly. Not only for adults but they allocate for children & birthday party's.
The lanes are in good condition & high scores are often . There are 20 lanes', of which 6 are new & can be used...
Own or manage this property? Claim your listing for free to respond to reviews, update your profile and much more.


text:
Posted on 25 July 2005
- 12:00
Reto started his pre-season preparations late after playing for Switzerland in the World Under-20 Championships in June.
That meant he missed out on our trip to the Peace Cup in South Korea - a tournament we won with a 3-1 win against Lyon in the final on Sunday.
Instead, Reto has been getting into shape with the reserve group at Spurs Lodge for the last couple of weeks and returned to action for an hour as skipper of our Spurs XI side in the 1-1 draw at Boreham Wood last Thursday.
He followed that up with 90 minutes and a goal in Saturday's 3-1 friendly win against Conference South outfit Cambridge City.
Coach Clive reported: "Reto was a week behind in terms of his pre-season programme so he had an hour on Thursday and then played the full 90 minutes at Cambridge City.
"I thought he looked very strong and very comfortable on Saturday and that's a good sign.
"He's trained hard and the pleasing thing is that he's making the progress you'd want to see - he's not far off the main group in terms of his physical capabilities."
24 November 2016
Our Official Supporters' Clubs in the USA are once again acting as a force for good in their communities for Thanksgiving.
15 November 2016
This is your chance to take a tour of White Hart Lane with an all-time Tottenham Hotspur great.
26 August 2016
To assist our fans at the beginning of the new season we have launched an updated interactive map to illustrate best access routes into the stadium following the removal of sections of the North and East stands, the closure of Paxton Road to general access and the changes to the West Stand Car Park during the close season.
View mobile site
© Tottenham Hotspur Ltd 2017. All rights reserved.
All photography copyright Getty Images (unless otherwise stated)


text:
Exploring how to adapt the visual styles of the full/desktop site to the mobile-optimised version with consideration for interaction and small-screen legibility.
Pro-tip: test mobile designs on real devices - I've already noted several elements that need larger touch targets.
NB: I've removed the client logo.
keyboard shortcuts:
L or F like
post comment
Show and tell for designers
What are you working on? Dribbble is a community of designers sharing screenshots of their work, process, and projects.
Copyright © 2009–2017 Dribbble LLC. All screenshots © their respective owners. Shipped from Salem, Mass. USA.


text:
Cute shower curtains
Explore related topics
LC Lauren Conrad Ella Ruffle Fabric Shower Curtain
http://www.modularhomepartsandaccessories.com/contemporarybathroomaccessories.php has some info on the perfect accessories for a bathroom.
This website has a TON of cute shower curtains. For the future place.
66"(Width) x 72"(Height) Turquoise Block Chevron Zigzag Infinity Anchor Quotes I refuse to Sink Theme Design 100% Polyester Bathroom Shower Curtain Shower Rings Included -Best Visual Enjoyment For You Chevron Shower-curtain http://www.amazon.com/dp/B00OQ0L6JE/ref=cm_sw_r_pi_dp_J549ub1EQ3W14
Quick and Cheap Bathroom Mini Makeover | HGTV Design Blog – Design Happens
neapolitan ruffles- very cute any ideas what this might be? shower curtain?
4040 Locust Toures Shower Curtain - Urban Outfitters
Magical Thinking Pompom Shower Curtain - Urban Outfitters
Amazon.com: Shower Curtain Hooks Rings - Oval Blue Cute Decorative Crystal Gems Bling Rhinestones Bathroom Bath Set Women Girl Gift (Blue): Home & Kitchen
shower curtain from anthro with woven Portuguese bathmat... http://www.anthropologie.com/anthro/product/home-bath/093677.jsp


text:
Science & Medicine, Natural Sciences, Society & CultureFollow
|Science & Medicine
|Natural Sciences
|Top 200
Deep in the back of your mind, you've always had the feeling that there's something strange about reality. There is. Join Robert, Joe and Christian as they examine neurological quandaries, cosmic mysteries, evolutionary marvels and our transhuman future on Stuff To Blow Your Mind, a podcast from HowStuffWorks.com.
June 16th, 2011
Episode 151 of 798 episodes
What makes a dog a lousy father and a wolf a stellar patriarch? Join Robert and Julie as they anthropomorphize the animal kingdom in an effort to find out why some species are more like Cliff Huxtable while some are more like Darth Vader.
You'll need to sign in to write a comment.
Welcome to the daily audio workout for your head.


text:
Is Nbi Satellite Office incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
Are these details correct?
Hotfrog Philippines provides information regarding Nbi Satellite Office in Manila Metro Manila. Nbi Satellite Office is located at Tutuban Center Mall, Claro M. Recto Avenue, Manila Metro Manila and provides Utility Companies services. Contact them on (02) 200 0000.
Is Nbi Satellite Office in Tutuban Center Mall, Claro M. Recto Avenue, Manila Metro Manila your business? Claim your listing and attract more leads by adding more content, photos and other business details.
We have more Utility Companies services in Manila Metro Manila available on Hotfrog Philippines. You can update your search for Manila Utility Companies by location, keyword or service options.
Small Business Hub
Sharing tips, ideas and advice to help grow your small business.
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub


text:
22 January 2008
And the winner is ...
Congratulations Diana. Please email your postal address and I'll get the book in the post as soon as I can.
|Reactions:funny, int
7 comments:
- AnonymousJanuary 22, 2008 7:31 am
Bummer, I was out of town yesterday and didn't read your blog till today so I couldn't comment. But I'm going to see if I can get the book from the library. Congrats Diana!!!ReplyDelete
WOW! I'm completely bowled over, thank you so much. I'm blushing with excitement!!!I will pass on when read in the same way.... Diana xReplyDelete
Congratulations Diana!! I'm sure you'll enjoy it. ;o)ReplyDelete
Congratulations, Diana!ReplyDelete
congrats Pebbledash!!ReplyDelete
ohh, bolderdash, but congrads to pebbledash!ReplyDelete
Congratulations Pebbledash!ReplyDelete
Thank you for your comment today. I love reading your opinions and thoughts. We have built up a wonderfully diverse community here that I'm very proud to be a part of.
A link to your blog will be automatically added to your comment. Please don't add another link to your blog in your comment. Those comments will not be published.


text:
"We are integral to a global supply chain that supports electronics," Qualcomm CEO Steve Mollenkopf told CNBC. » Read More
By: Chris Morris
Sony officials announced Sunday that sales of the company's PlayStation VR have actually outpaced projections. » Read More
By: Alexandra Gibbs, Karen Tso
"We're definitely seeing demand from the most-connected customers that they want some simplicity in a device," EE's CEO told CNBC. » Read More
By: Dan Frommer
A decade after the iPhone launched, the BlackBerry platform is all but dead, Recode reports. » Read More
Get the best of CNBC in your inbox
Matt Hunter is the senior technology editor at CNBC.com.
CNBC.com news associate
Michelle Castillo is a reporter for CNBC Digital, covering advertising and media.
Senior Tech Reporter
Harriet Taylor is a CNBC.com technology reporter based in San Francisco.
Working from Los Angeles, Boorstin is CNBC's media and entertainment reporter and editor of CNBC.com's Media Money section.
Jon Fortt is an on-air editor. He covers the companies, start-ups, and trends that are driving innovation in the industry.
Josh Lipton is CNBC's technology correspondent, working from CNBC's Silicon Valley bureau.


text:
The engineers club online services - www.engineers.com
Engineering jobs
for alumni with professional degrees from the top engineering schools.
Web Links
|Engineering Job
Engineering Central features job ads for chemical, civil, electrical, industrial, manufacturing and mechanical engineers. There is also a special job bank for entry level engineers and you can add your resume to their database for free.
|Six Sigma
Six Sigma Training and Lean Manufacturing Training Resources for all Organizations
| Bai Engineering
The best source for engineering!
|Seal and o-ring design
Problem Solving Products offers engineering help with o-rings and seals.
|Strahanrecruiting.com - Website to Strahan Associates - Strahan Associates is an employment recruiting firm that focuses on engineering and manufacturing jobs with high end companies.
Quality Engineer
|Autotech Engineering
Build anything with aluminum t-slots!
|Big Business Web.com - Your one-stop for business!
Systems International
|Andy's Best CAD
Provides AutoCAD design and drafting services for HVAC, plumbing, architectural, structural steel detailing, mechanical fabrication, electrical, fire protection, landscape architectural, 3D solid modeling, CAD standards creation and CAD conversion projects
|BPA Industrial Air Solutions; Portable Dust Collectors
Description: Features the most popular brands of all types of industrial dust collectors (portable, jet, cartridge & cyclone).
logo design services by Halcyon Logo Design
Halcyon Logo Design creates original eye-catching company logos and business stationery. Our high quality design service is fast, efficient and affordable.
|Webmaster Forums -
Webmaster Resources
Global online community for webmasters. Webmaster forums, resources and much more.
Friendly Products
Molecular-Tech Coatings Inc. - Canadian industrial paints and coatings manufacturer. Developers of an environmentally friendly paint stripper that works!
|Engineers International - www.engineers-international.com
|Arcspace Architectural Online - www.arcspace.com
|Ritter Engineering, a source for Palm OS solutions
|Amsoil synthetic oil dealer Amsoil synthetic oil dealer also offering transmission fluid, filters, fuel additive, cooler motorcycle, hydraulic, compressor, 2 cycle and gear oil. - www.lubeoilsales.com
|Robots tracking and analyzing
|Business Industrial Network - Provides online solution database, engineering books, e-books and more.
|NY Large Format Drawing Scanning Cad Conversion - Affordable document imaging, pdf ocr services, large format scanning, bluepint drawing scanning, auto cad drawing conversion, cad drafting, raster to vector service.
|Internet Marketing
Seoprosoftware.com offers great Internet Maketing Services.
|Laser marking systems Laser
Alase Technologies manufactures industrial laser
|Electronic and mechanical industrial power transmission products - Designer, manufacturer and marketer of electronic and mechanical industrial power transmission products including gear motors, jaw coupling, variable speed drives, bushings and sheaves.
|Job Listings
JobPier.com's Search Engine visits Corporate websites, indexes and lists Jobs found in them. JobPier.com also features direct Employer Job Listings
Your mortgage guide - www.mortgageguide.co.uk
|Personalized Baby Blankets
|Gift Baskets - Gift Basket Directory - Gund Teddy Bears
|Holiday Gift Baskets - Gift Baskets Canada
|Ruhim Websearch
|URL.biz - Business
Other Useful Information:
Absolutely Everything in Shipping and Packaging - Tape, Corrugated Boxes, chipboard boxes, pallet wrap film, packaging equipment.
Portable Garage - We offer portable garages and storage structures for equipment and miscellaneous storage, helping protect everything from the elements; cars, trucks, RVs, plants, equipment, etc.
Schutzhund Training - South Central Region schutzhund clubs and German shepherd dog training.
Offshore Software Development Company - Cost/quality effective offshore custom software and web development solutions, offshore software outsourcing.
California Solar Electric Company - Serving Northern California, Specializes in solar power systems design, sales, and installation for your home or business.
Amsoil - Amsoil synthetic oil dealer offering wholesale prices on Amsoil products. Synthetic motor oils, transmission fluid, filters, fuel additive, cooler motorcycle, hydraulic, compressor, 2 cycle and gear oil. Free Amsoil Catalog.
117-101 - Great Certification Success for 117-101 Exam Using Our 117-101 Materials.
Web Hosting - Provide reviews and ratings of the cheap web hosting services with superior quality and support.
Online Mortgage Advice- Online Mortgage Advice: Your online guide to getting the best mortgage and buying/selling your home
Packaging Services, Solutions and Supplier in the world - Find out the outstanding resources at packaging-global.com, for packaging products like packaging equipment, machineries, materials and laser, digital printing machines etc.
Jokes - A repository of jokes on various popular categories. Features Bar jokes, College jokes, Sports jokes and lots more.
Affordable software hardware
Web Design Canada
Free articles, tools and games. Biometric devices with time and attendance software. Search engine optimization and Internet Marketing services.
Corporate Seals and Supplies purchase corporate supplies online, Minute Book Kits, corporate seals and more.
The perfect place for a Texas Wedding - Grace Church Wedding Chapel
Bigall Search Engine - Search engine, web directory, online calculators and more.
Job marketing UK - Charity jobs UK including not for profit fundraising, finance, admin, secretarial, HR and medical recruitment from TPP.
German Shepherd Dogs GSD - Zwinger Vom Godinghofer Weg
Last Modified October 11, 2006
Daily Quote
Not everything that can be counted counts, and not everything that counts can be counted. Albert Einstein
926 S. Cedar Ridge Rd.
Duncanville, TX 75137
Phone: 800-633-3191 Fax: 800-648-7244


text:
- Compatible XF Versions:
- 1.0
- 1.1
- 1.2
- 1.3
- 1.4
- License:
- An active subscription is required to access premium features
- Visible Branding:
- Yes
Administrators (or other trusted members) who have been granted the 'Login as different user' permission can click on a link on a user's member card to log in as that user. The admin can then see exactly what the user can see, for example to see what difficulty they are having in performing a task such as uploading a file and, if necessary, complete that task on behalf of the user.
A link in the admin bar will allow them to quickly return to their own account at any time.
To log in as a different user, that user must be a member of one of the permitted user groups.
We recommend that this permission should only be granted to known, trusted and responsible admins or other such members.
Alternatively, by purchasing a subscription at waindigo.org and installing the Install and Upgrade by Waindigo add-on, you can also completely remove all branding and activate any premium features on all your add-ons. You can also remove all branding and get the premium features for this add-on if you make a significant contribution to this add-on in any way.
This add-on would not have been possible without the incredibly generous contributions of Morgain, Rumano, Tefka, Velli, our premium members, and the commercial site owners who have kindly donated. Big thanks also goes to those who have helped others in the discussion thread, liked and reviewed this add-on.
- You must like to download this resource
[Free Add-on ] Login As User by Waindigo 1.2.1
Gives trusted admins permission to login as any member, without having to know their password.
Recent Updates
- Version 1.2.1 released Jul 4, 2015


text:
I will be a PROFESSIONAL virtual assistant for 3 hours
About This Gig
I will for $5:
-Web Research
-Data Entry
-Translation english or spanish.
-Any other time consuming task that you want to delegate.
High experience in Excel, PowerPoint and Word.
If you have a big project:
You can hire my services, I
will send you a price quote for the entire project.
Contact my INBOX before ordering.


text:
11 Teacher-Approved Crafts for School
Bonus: Get our newsletter & special offers for free. We will not share or sell your email address. View our Privacy Policy
Latest Comments
"I like this vintage style Santa graphic, but I read that the designer used a cutting machine to cut a red vinyl decal to afix to the cream painted wood board. I don't know what kind of cutting machine she is talking about and I wonder how pricey it is? I'd like an alternative to getting that Santa on there."
"This wall decor is very chic with any message you write on it! And it couldn't be easier. However, another option to using foam core board would be to actually buy an old thrift store bulletin board and spray paint it black. I think it would give the wall art a little bit more of a lived-in rustic look as the cork board adds texture and interest."
"This DIY holiday project is something that I would like to do at home myself. This craft is not for everyone's taste, but if you like a certain minimalist decorating approach and you've got a creative eye then this is for you! At first I did not even notice the picture frame in the background. It's used to attach the branch to it. Cute idea!"
Denim Shorts Chore Chart
By: Pauline Molinari
We are adding the craft to your Craft Projects.
The project was added to your Craft Projects.
Click here to view your Craft Projects.
Vist Pauline at Club Chica Circle.
If you need help motivating kids to pitch in around the house, consider creating your own Denim Shorts Chore Chart. This denim crafts DIY project will leave kids wanting to perform chores so they can move the decorated sticks from one pocket to another. With materials such as paint, jewels and sparkle tape, you can completely customize the jeans to fit your style and match your decor.
You can find this project, as well as many other fabulous blogger projects, in our new eBook, The Best Blogger Craft Ideas 2014: Decoupage, Sewing, Jewelry Making, and More free eBook. Download your own free copy today!
- Lion Brand Yarn Sparkly variety pack
- Aleene's Fabric Fusion
- Sparkle Tape
- Tulip Fashion Glitter Bond and Glitter
- Folk Art Chalkboard Paint
- Martha Stewart Self-Adhesive Stencils
- Martha Stewart Multi-Surface Paint - metallic silver
- Martha Stewart pouncers
- Tulip Slick neon blue fabric paint
- David Tutera self-adhesive jewels
- Fiskars scissors
- Denim shorts
- Colored craft sticks
- Glue gun
- Painter's tape
- Sponge brush
- Mask off top 1/3 portion of colored large craft sticks with painters tape. Using a sponge brush, apply chalkboard paint on the rest of the stick and let dry.
- Create a decorating hanging yarn braid with approx. 18" of multicolored cut strands of yarn. With a pair of denim shorts (slightly used and grown out of from my daughter) glue the front and back of the shorts together with a glue gun. Attach decorative glitter sparkle tape along the top replicating a belt. Using fabric fusion glue, attach your braided yarn along the side seams from starting from the bottom of one end the other and let dry.
- Using self-adhesive letter stencils, apply the words "DO" and "DONE" just below the left and right front pockets with contrasting metallic paint with paint pouncer and let dry.
- Using a book for stability and a flat surface, use the decorative chevron self-adhesive stencil and apply Fashion glitter bond glue. While still wet, sprinkle on colored glitter, then carefully pull off stencil to reveal glittery design and let dry. Add polka dot details along the seams of the pockets and loops with Slick dimensional fabric paint.
- When craft sticks are dry, remove painters tape and apply small single strand of self-adhesive decorative jewels.
- Use chalk to write to do list or chores on each craft stick and put in the "DO" pocket. When the task is done, move the stick to the "DONE" pocket, and so on. Use the yarn to hang on a wall with a removable hook.
Tags / Related Topics
Your Recently Viewed Projects
Images from other crafters
Love these projects? Get MORE delivered to your Inbox – FREE Click Here!
Mar 04, 2014
This is a fun rainy day project. My only issue is using chalk. It does rub off and smudge a bit if you're not careful. I saved the chalk for "occasional" chores and used a white paint pen to write regular chores.
Report Inappropriate Comment
Are you sure you would like to report this comment? It will be flagged for our moderators to take action.
Thank you for taking the time to improve the content on our site.
What's Hot
- Sharpie Marker Pillow Case
- Buddy Up With Your Finger Friends
- Pine Cone Christmas Trees for Kids
- 17 Kids Sunday School Crafts
- Eco Friendly Plastic Cup Flowers
- Houses Coloring Page
- Mini Q-Tip Flowers
- Homemade I Spy Tube
- "Family Fun Crafts: 16 Craft Ideas for Kids" eBook
- "22 Summer Crafts for Kids" eBook
Something worth saving?
- SAVE all your favorite crafts
- ADD personal notes
- QUICKLY reference your crafts
Free eBooks
- 11 Crochet Shawl Patterns: Crochet Poncho Patterns, Free Easy Crochet Patterns and More
- 12 Knitted Scarf Patterns: Fabulous Free Knitting Patterns for Beginners
- 14 Free Crochet Patterns for Babies and Toddlers
- 16 Free Crochet Hat Patterns, Scarves, and Gloves
- 22 Free Crochet Patterns: Afghan Patterns, Crochet Hats, and More
- 24 Quick and Easy Knitting Patterns
- 7 Adult Coloring Pages
- 8 Free Apron Sewing Patterns
- Easy Lace Knitting Patterns
- Quick & Easy Decoupage: 12 Fabulous Mod Podge Projects for Your Home
Our Newest Projects & Articles
- 11 Easter Crafts for Kids
- 100+ Easter Crafts
- Duo of Bears Unisex Baby T-Shirt
- Cross My Heart Treat Bag
- How to Sew a Zipper into a Lined Skirt
- Tiny Valentine Gift Boxes
- Pretty Hearts Hot Pad
- DIY Mini Pallet Photo Magnets
- Padded Denim Hearts for Decoration or Keyring
- Faux Topiary Heart Place Card Holders


text:
Woodbury County Public Records
County Public Records - County Directory Edit
Directory of departments, services and officials within Woodbury County.
Physical Address:
Woodbury County Recorder 620 Douglas St. Sioux City, Iowa 51101
Contact: P: 712.279.6528
County Public Records - Woodbury County Official Website Edit
Get information on Woodbury County government and services.
Physical Address:
Woodbury County Board of Supervisors 620 Douglas St. Sioux City, Iowa 51101
Contact: P: 712.279.6525 F: 712.279.6577
County Trends in Iowa
Additional Woodbury County Records by Category
Trending Categories in Iowa
Recommend New Listing
Suggest a public record to add to this page.
Recommend New Listing
Search Public Records
About Woodbury County Public Records
Moving to a new city? There are a few things one must look for before making the final decision. If you are moving to Woodbury County, it would be wise to check the property records relating to ownership on real estate before making any purchases. Usually Woodbury County government maintains these records. Also, check for the Woodbury County criminal records to know about any sex offenders in the area.
Search for any county on PublicRecords360 and we search through tons of public records to display relevant listings. As per your needs, you can find a variety of county public records - Court records, Tax records, Education & School records, County Background Checks and other County Vital records.
If you do get suspicious about someone in your neighborhood in Woodbury County, you can also retrieve a consolidated report on all public records for that person with just a Single Search. Start by simply typing a name in search box above and find information like phone numbers, address, background history, criminal history & more. With our unlimited subscription product, you can perform quick, easy and unlimited public record searches.
Connect With Us
Link to this page
Copy and paste text in the box below to your blog or website.


text:
On Wed, 6 Nov 1996, Dana Brown wrote:
> I can't imagine iris blooming in July. Must have been some year. Did
> you get to see the blooms at all? What do they look like?
I couldn't resist saying something about `iris blooming in July' -
here in Zone 3 in northern New Hampshire, USA, I had irises blooming
in June, July, August and September.
The peak Siberian bloom here was around June 27th. The first Siberian
bloomed June 13th and the last Japanese took its leave on Sept.10th
this year.
We did have a space of 'down time' after the Siberians and waiting
for the Japanese. We sure saw some blooms and it was a very good
We consider iris to be a summer bloomer here rather than a spring
flower...in '97, I'll have some some MDBs blooming and maybe some
reticulatas in '98 and maybe we can try for five months of iris
bloom (or more). One more thing about colder climates and bloom.
The flowers last a long time due to the chilly nights, etc.
It is our compensation for not having reblooming TBs. :-)
Ellen Gallagher email@example.com Lancaster, NH,USA
Zone 3a