The present invention provides novel polynucleotides encoding HGPRBMY23 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel HGPRBMY23 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly renal diseases and/or disorders, colon cancer, breast cancer, and diseases and disorders related to aberrant NFKB modulation. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

Regulation of cell proliferation, differentiation, and migration is important for the formation and function of tissues. Regulatory proteins such as growth factors control these cellular processes and act as mediators in cell-cell signaling pathways. Growth factors are secreted proteins that bind to specific cellsurface receptors on target cells. The bound receptors trigger intracellular signal transduction pathways which activate various downstream effectors that regulate gene expression, cell division, cell differentiation, cell motility, and other cellular processes. Some of the receptors involved in signal transduction by growth factors belong to the large superfamily of G-protein coupled receptors (GPCRs) which represent one of the largest receptor superfamilies known.
GPCRs are biologically important as their malfunction has been implicated in contributing to the onset of many diseases, which include, but are not limited to, Alzheimer's, Parkinson, diabetes, dwarfism, color blindness, retinal pigmentosa and asthma. Also, GPCRs have also been implicated in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure and in several cardiovascular, metabolic, neuro, oncology and immune disorders (F Horn, G Vriend, J. Mol. Med. 76: 464-468, 1998.). They have also been shown to play a role in HIV infection (Y Feng, C C Broder, P E Kennedy, E A Berger, Science 272:872-877, 1996).
GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antiparallel alpha (a) helices. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. These proteins range in size from under 400 to over 1000 amino acids (Strosberg, A. D. (1991) Eur. J. Biochem. 196: 110; Coughlin, S. R. (1994) Curr. Opin. Cell Biol. 6: 191-197). The amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops of GPCRs alternate with intracellular loops and link the transmembrane domains. Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account for structural and functional features of the receptor. In most G-protein coupled receptors, the bundle of a helices forms a ligand-binding pocket formed by several G-protein coupled receptor transmembrane domains.
The TM3 transmembrane domain has been implicated in signal transduction in a number of G-protein coupled receptors. Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the b adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
The extracellular N-terminal segment, or one or more of the three hydrophilic extracellular loops, have been postulated to face inward and form polar ligand binding sites which may participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. In turn, the large, third intracellular loop of the activated receptor interacts with an intracellular heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or ion channel proteins. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines have also been implicated in ligand binding (See, e.g., Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego Calif., pp. 2-6; Bolander, F. F. (1994) Molecular Endocrinology, Academic Press, San Diego Calif., pp. 162-176; Baldwin, J. M. (1994) Curr. Opin. Cell Biol. 6: 180-190; F Horn, R Bywater, G Krause, W Kuipers, L Oliveira, A C M Paiva, C Sander, G Vriend, Receptors and Channels, 5:305-314, 1998).
Recently, the function of many GPCRs has been shown to be enhanced upon dimerization and/or oligomerization of the activated receptor. In addition, sequestration of the activated GPCR appears to be altered upon the formation of multimeric complexes (AbdAlla, S., et al., Nature, 407:94-98 (2000)).
Structural biology has provided significant insight into the function of the various conserved residues found amongst numerous GPCRs. For example, the tripeptide Asp(Glu)-Arg-Tyr motif is important in maintaining the inactive confirmation of G-protein coupled receptors. The residues within this motif participate in the formation of several hydrogen bonds with surrounding amino acid residues that are important for maintaining the inactive state (Kim, J. M., et al., Proc. Natl. Acad. Sci. U.S.A., 94:14273-14278 (1997)). Another example relates to the conservation of two Leu (Leu76 and Leu79) residues found within helix II and two Leu residues (Leu 128 and Leu131) found within helix III of GPCRs. Mutation of the Leu128 results in a constitutively active receptor—emphasizing the importance of this residue in maintaining the ground state (Tao, Y. X., et al., Mol. Endocrinol., 14:1272-1282 (2000); and Lu. Z. L., and Hulme, E. C., J. Biol. Chem., 274:7309-7315 (1999). Additional information relative to the functional relevance of several conserved residues within GPCRs may be found by reference to Okada et al in Trends Biochem. Sci., 25:318-324 (2001).
GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, bombesin, bradykinin, endothelin, y-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, C5a anaphylatoxin, folliclestimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropinreleasing hormone (TRH), and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors.
GPCRs are implicated in inflammation and the immune response, and include the EGF modulecontaining, mucin-like hormone receptor (Emrl) and CD97p receptor proteins. These receptors contain between three and seven potential calcium-binding EGF-like motifs (Baud, V. et al. (1995) Genomics 26: 334-344; Gray, J. X. et al. (1996) J. Immunol. 157: 5438-5447). These GPCRs are members of the recently characterized EGF-TM7 receptors family. In addition, post-translational modification of aspartic acid or asparagine to form erythro-p-hydroxyaspartic acid or erythro-p-hydroxyasparagine has been identified in a number of proteins with domains homologous to EGF. The consensus pattern is located in the N-terminus of the EGF-like domain. Examples of such proteins are blood coagulation factors VII, IX, and X; proteins C, S, and Z; the LDL receptor; and thrombomodulin.
One large subfamily of GPCRs are the olfactory receptors. These receptors share the seven hydrophobic transmembrane domains of other GPCRs and function by registering G protein-mediated transduction of odorant signals. Numerous distinct olfactory receptors are required to distinguish different odors. Each olfactory sensory neuron expresses only one type of olfactory receptor, and distinct spatial zones of neurons expressing distinct receptors are found in nasal pasages. One olfactory receptor, the RAlc receptor which was isolated from a rat brain library, has been shown to be limited in expression to very distinct regions of the brain and a defined zone of the olfactory epithelium (Raming, K. et al., (1998) Receptors Channels 6: 141-151). In another example, three rat genes encoding olfactory-like receptors having typical GPCR characteristics showed expression patterns exclusively in taste, olfactory, and male reproductive tissue (Thomas, M. B. et al. (1996) Gene 178: 1-5).
Another group of GPCRs are the mas oncogene-related proteins. Like the mas oncogenes themselves, some of these mas-like receptors are implicated in intracellular angiotensin II actions.
Angiotensin II, an octapeptide hormone, mediates vasoconstriction and aldosterone secretion through angiotensin II receptor molecules found on smooth vascular muscle and the adrenal glands, respectively.
A cloned human mas-related gene (mrg) mRNA, when injected into Xenopus oocytes, produces an increase in the response to angiotensin peptides. Mrg has been shown to directly affect signaling pathways associated with the angiotensin II receptor, and, accordingly, affects the processes of vasoconstriction and aldosterone secretion (Monnot, C. et al. (1991) Mol. Endocrinol. 5: 1477-1487).
GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Rhodopsin is the retinal photoreceptor which is located within the discs of the eye rod cell. Parma, J. et al. (1993, Nature 365: 649-651) reported that somatic activating mutations in the thyrotropin receptor cause hyperfunctioning thyroid adenomas and suggested that certain GPCRs susceptible to constitutive activation may behave as protooncogenes.
Purines, and especially adenosine and adenine nucleotides, have a broad range of pharmacological effects mediated through cell-surface receptors. For a general review, see Adenosine and Adenine Nucleotides in The G-Protein Linked Receptor Facts Book, Watson et al. (Eds.) Academic Press 1994, pp. 19-31.
Some effects of ATP include the regulation of smooth muscle activity, stimulation of the relaxation of intestinal smooth muscle and bladder contraction, stimulation of platelet activation by ADP when released from vascular endothelium, and excitatory effects in the central nervous system. Some effects of adenosine include vasodilation, bronchoconstriction, immunosuppression, inhibition of platelet aggregation, cardiac depression, stimulation of nociceptive afferants, inhibition of neurotransmitter release, pre-and postsynaptic depressant action, reducing motor activity, depressing respiration, inducing sleep, relieving anxiety, and inhibition of release of factors, such as hormones.
Distinct receptors exist for adenosine and adenine nucleotides. Clinical actions of such analogs as methylxanthines, for example, theophylline and caffeine, are thought to achieve their effects by antagonizing adenosine receptors. Adenosine has a low affinity for adenine nucleotide receptors, while adenine nucleotides have a low affinity for adenosine receptors.
There are four accepted subtypes of adenosine receptors, designated A1, A2A, A2B, and A3. In addition, an A4 receptor has been proposed based on labeling by 2 phenylaminoadenosine (Cornfield et al. (1992) Mol. Pharmacol. 42: 552-561).
P2x receptors are ATP-gated cation channels (See Neuropharmacology 36 (1977)). The proposed topology for PZX receptors is two transmembrane regions, a large extracellular loop, and intracellular N and C-termini.
Numerous cloned receptors designated P2y have been proposed to be members of the G-protein coupled family. UDP, UTP, ADP, and ATP have been identified as agonists. To date, P2Y1-7 have been characterized although it has been proposed that P2Y7 may be a leukotriene B4 receptor (Yokomizo et al. (1997) Nature 387: 620-624).
It is widely accepted, however, that P2Y 1, 2,4, and 6 are members of the G-protein coupled family of P2y receptors.
At least three P2 purinoceptors from the hematopoietic cell line HEL have been identified by intracellular calcium mobilization and by photoaffinity labeling (Akbar et al. (1996) J. Biochem. 271: 18363-18567).
The Ai adenosine receptor was designated in view of its ability to inhibit adenylcyclase. The receptors are distributed in many peripheral tissues such as heart, adipose, kidney, stomach and pancreas. They are also found in peripheral nerves, for example intestine and vas deferens. They are present in high levels in the central nervous system, including cerebral cortex, hippocampus, cerebellum, thalamus, and striatum, as well as in several cell lines. Agonists and antagonists can be found on page 22 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. These receptors are reported to inhibit adenylcyclase and voltage-dependent calcium chanels and to activate potassium chanels through a pertussis-toxin-sensitive G-protein suggested to be of the G/Go class. Ai receptors have also been reported to induce activation of phospholipase C and to potentiate the ability of other receptors to activate this pathway.
The A2A adenosine receptor has been found in brain, such as striatum, olfactory tubercle and nucleus accumbens. In the periphery, A2 receptors mediate vasodilation, immunosuppression, inhibition of platelet aggregation, and gluconeogenesis. Agonists and antagonists are found in The G-Protein Linked Receptor Facts Book cited above on page 25, herein incorporated by reference. This receptor mediates activation of adenylcyclase through Gs.
The A2B receptor has been shown to be present in human brain and in rat intestine and urinary bladder. Agonists and antagonists are discussed on page 27 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. This receptor mediates the stimulation of cAMP through Gg.
The A3 adenosine receptor is expressed in testes, lung, kidney, heart, central nervous system, including cerebral cortex, striatum, and olfactory bulb. A discussion of agonists and antagonists can be found on page 28 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. The receptor mediates the inhibition of adenylcyclase through a pertussis-toxin-sensitive G-protein, suggested to be of the Gi/Go class.
The P2Y purinoceptor shows a similar affinity for ATP and ADP with a lower affinity for AMP. The receptor has been found in smooth muscle, for example, and in vascular tissue where it induces vasodilation through endothelium-dependent release of nitric oxide. It has also been shown in avian erythrocytes.
Agonists and antagonists are discussed on page 30 of The G-Protein Linked Receptor Facts Book cited above, herein incorporated by reference. The receptor function through activation of phosphoinositide metabolism through a pertussis-toxin insensitive G-protein, suggested to be of the Gi/Go class.
Characterization of the HGPRBMY23 polypeptide of the present invention led to the determination that it is involved in NFkB pathway through modulation of the IkB protein, either directly or indirectly."
The fate of a cell in multicellular organisms often requires choosing between life and death. This process of cell silicide, known as programmed cell death or apoptosis, occurs during a number of events in an organisms life cycle, such as for example, in development of an embryo, during the course of an immunological response, or in the demise of cancerous cells after drug treatment, among others. The final outcome of cell survival versus apoptosis is dependent on the balance of two counteracting events, the onset and speed of caspase cascade activation (essentially a protease chain reaction), and the delivery of antiapoptotic factors which block the caspase activity (Aggarwal B. B. Biochem. Pharmacol. 60, 1033-1039, (2000); Thornberry, N. A. and Lazebnik, Y. Science 281, 1312-1316, (1998)).
The production of antiapoptotic proteins is controlled by the transcriptional factor complex NF-kB. For example, exposure of cells to the protein tumor necrosis factor (TNF) can signal both cell death and survival, an event playing a major role in the regulation of immunological and inflammatory responses (Ghosh, S., May, M. J., Kopp, E. B. Annu. Rev. Immunol. 16, 225-260, (1998); Silverman, N. and Maniatis, T., Genes & Dev. 15, 2321-2342, (2001); Baud, V. and Karin, M., Trends Cell Biol. 11, 372-377, (2001)). The anti-apoptotic activity of NF-kB is also crucial to oncogenesis and to chemo- and radio-resistance in cancer (Baldwin, A. S., J. Clin. Inves. 107, 241-246, (2001)).
Nuclear Factor-kB (NF-kB), is composed of dimeric complexes of p50 (NF-kB1) or p52 (NF-kB2) usually associated with members of the Rel family (p65, c-Rel, Rel B) which have potent transactivation domains. Different combinations of NF-kB/Rel proteins bind distinct kB sites to regulate the transcription of different genes. Early work involving NF-kB suggested its expression was limited to specific cell types, particularly in stimulating the transcription of genes encoding kappa immunoglobulins in B lymphocytes. However, it has been discovered that NF-kB is, in fact, present and inducible in many, if not all, cell types and that it acts as an intracellular messenger capable of playing a broad role in gene regulation as a mediator of inducible signal transduction. Specifically, it has been demonstrated that NF-kB plays a central role in regulation of intercellular signals in many cell types. For example, NF-kB has been shown to positively regulate the human beta-interferon (beta-IFN) gene in many, if not all, cell types. Moreover, NF-kB has also been shown to serve the important function of acting as an intracellular transducer of external influences.
The transcription factor NF-kB is sequestered in an inactive form in the cytoplasm as a complex with its inhibitor, IkB, the most prominent member of this class being IkBa. A number of factors are known to serve the role of stimulators of NF-kB activity, such as, for example, TNF. After TNF exposure, the inhibitor is phosphorylated and proteolytically removed, releasing NF-kB into the nucleus and allowing its transcriptional activity. Numerous genes are upregulated by this transcription factor, among them IkBa. The newly synthezised IkBa protein inhibits NF-kB, effectively shutting down further transcriptional activation of its downstream effectors. However, as mentioned above, the IkBa protein may only inhibit NF-kB in the absence of IkBa stimuli, such as TNF stimulation, for example. Other agents that are known to stimulate NF-kB release, and thus NF-kB activity, are bacterial lipopolysaccharide, extracellular polypeptides, chemical agents, such as phorbol esters, which stimulate intracellular phosphokinases, inflammatory cytokines, IL-1, oxidative and fluid mechanical stresses, and Ionizing Radiation (Basu, S., Rosenzweig, K, R., Youmell, M., Price, B, D, Biochem, Biophys, Res, Commun., 247(1):79-83, (1998)). Therefore, as a general rule, the stronger the insulting stimulus, the stronger the resulting NF-kB activation, and the higher the level of IkBa transcription. As a consequence, measuring the level of IkBa RNA can be used as a marker for antiapoptotic events, and indirectly, for the onset and strength of pro-apoptotic events.
The upregulation of IkBa due to the downregulation of HGPRBMY23 places this GPCR protein into a signalling pathway potentially involved in apoptotic events. This gives the opportunity to regulate downstream events via the activity of the protein HGPRBMY23 with antisense polynucleotides, polypeptides or low molecular chemicals with the potential of achieving a therapeutic effect in cancer, autoimmune diseases. In addition to cancer and immunological disorders, NF-kB has significant roles in other diseases (Baldwin, A. S., J. Clin Invest. 107, :3-6 (2001)). NF-kB is a key factor in the pathophysiology of ischemia-reperfusion injury and heart failure (Valen, G., Yan. Z Q, Hansson, G K, J. Am. Coll. Cardiol. 38, 307-14 (2001)). Furthermore, NF-kB has been found to be activated in experimental renal disease (Guijarro C, Egido J., Kidney Int. 59, 415-425 (2001)). As HGPRBMY23 is highly expressed in kidney there is the potential of an involvement in renal diseases.
Using the above examples, it is clear the availability of a novel cloned G-protein coupled receptor provides an opportunity for adjunct or replacement therapy, and are useful for the identification of G-protein coupled receptor agonists, or stimulators (which might stimulate and/or bias GPCR action), as well as, in the identification of G-protein coupled receptor inhibitors. All of which might be therapeutically useful under different circumstances.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of HGPRBMY23 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the HGPRBMY23 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

Methods of diagnosing colon adenocarcinoma using mRNA encoding the human g-protein coupled receptor, HGPRBMY23


A track system for providing a movable component in a vehicle interior may comprise a track assembly comprising a track for the movable component and a base comprising a chassis for the movable component to be movable along the track. The track assembly may comprise a track cover. The chassis may move the track cover to uncover the track under the chassis. The track cover may comprise a front section, a rear section and a middle section. The front section and the rear section may cover the track. The chassis may comprise a ramp configured to raise the front section as the chassis moves in a forward direction. The middle section may extend below the front section, the rear section and the chassis. The track cover may slide along a pulley between the front section and the middle section.

It is known to provide a track system for providing a movable component in an interior of a vehicle.
It would be advantageous to provide an improved track system for a movable component of an interior of a vehicle.
It would be advantageous to provide an improved track system for a movable component of an interior of a vehicle comprising a track with a track cover configured to fit within a track opening.
It would be advantageous to provide an improved track system for a movable component of an interior of a vehicle comprising a track with a track cover configured to fit within a track opening as the movable component is moved along the track.

SHANGHAI YANFENG JINQIAO AUTOMOTIVE TRIM SYSTEMS CO. LTD.


A leaching conduit comprising: a high aspect ratio channel; a first pipe in fluid communication with the high aspect ratio channel; and where the aspect ratio of the high aspect channel is configured to allow for aerobic conditions in and around the leaching conduit. A wastewater system comprising: a processing tank; a leaching conduit in fluid communication with a dosing device; wherein the leaching conduit comprises: a high aspect ratio channel; a dosing pipe in fluid communication with the high aspect ratio channel; and where the aspect ratio of the high aspect channel is configured to allow for aerobic conditions in and around the leaching conduit. A high aspect ratio form comprising: an open bottomed and open topped generally rectangular container comprising: a first end wall; a front wall adjoining the first end wall with the top of the front wall and the top first end wall being generally coplanar; a rear wall adjoining the first end wall; a second end wall adjoining the front wall and the rear wall having a first height with the top of the second end wall, the top of the front wall, and the top of the rear wall being generally coplanar; a plurality of interior walls having a second height, and the plurality of interior walls adjoin the front wall and the rear wall with the tops of the interior walls, front wall and rear wall being generally coplanar; at least one lifting member attached at least one of the following: the first end wall, the front wall, the rear wall, the second end wall; and where the interior walls are configured to form at least one volume of a first width, and at least one volume of a second width, and wherein the first width corresponds to a width of a geonet volume and the second width corresponds to a width of a granular volume. A cover for a high aspect ratio form comprising: a cover configured to fit onto a high aspect ratio form; a plurality of openings in the cover configured to not cover a plurality of granular volumes in the high aspect ratio form.

Known leaching conduits, such as arch shape cross section molded plastic chambers, or stone filled trenches with perforated pipe, used for domestic and commercial wastewater systems provide interior void space, based on the thinking that a buffer space or flow equalization is thus provided for variations of inflow of wastewater. The sidewalls of conduits, where they interface with the surrounding soil, are also commonly conceived as providing surface area for percolation of wastewater, in addition to the bottom surface of the conduit. A familiar crushed stone filled trench, having a modest (4 inch) diameter perforated pipe running along its length may have about 50% void space. Currently, arch shape cross-section molded plastic leaching chambers have entirely open interiors, open bottoms and sloped and perforated sidewalls. A common cross section shape for each typical conduit has a width of about 30 to 36 inches and a height of about 12 to 18 inches. Thus this conduit may have from about 12 inches to about 18 inches of water depth at any one time. It has been seen that in these prior art conduits, a biomat will often form on the bottom and sides of the conduit, thereby lessening the effectiveness of the leaching conduits to properly infiltrate the wastewater into the soil. Drip irrigation lines are usually approximately one half inch in diameter and are typically buried 12 to 6 inches below grade.
Leaching conduits are typically covered with 6 to 12 inches or more of soil, for several reasons. One is to protect the conduits from damage. Another is to prevent contact of humans and animals with potentially deleterious microorganisms associated with the wastewater being treated. Still another is to prevent odors. The dimensions of the conduits discussed in the preceding paragraph would lead to the fact that the bottom surface of the conduits are typically at about 24 inches or more below the soil surface.
Generally, it is an aim to have aerobic treatment of the wastewater in the soil. Current thinking with prior art systems is that there is an air-soil gas interchange, so that oxygen is continuously supplied to the soil, to enable good microbiological treatment. However, the soil depths at which prior art conduits operate are disadvantaged in this respect. Since the bottom surface of the conduits are typically about 18 to 24 inches below the soil surface, the bottom surfaces of the conduits are often in an anaerobic condition since the oxygen demand exceeds the oxygen supply. One improvement with such systems is to force air serially through the conduit and soil influence zone which surrounds the conduit, as described in U.S. Pat. No. 6,485,647 to David Potts, issued on Nov. 26, 2002, and which is incorporated herein by reference in its entirety.
Therefore, a wastewater system is needed that provides for greater aerobic conditions in leaching conduits, thereby allowing for greater processing of the wastewater prior and during absorption into the soil.


An optical head apparatus wherein a lens holder which holds an objective lens for condensing a light beam on an optical recording medium is turned in a plane perpendicular to an optical axis of the objective lens and one portion of construction members of turning unit for performing a tracking control for the optical recording medium is fixed to a fixed body. One or some of construction members of the turning unit for tracking control is not mounted to a moving body for supporting the lens holder but is mounted to the fixed body, as a result, weight of a movable unit including the moving body and its loading members is to be reduced to realize high-speed access with respect to the optical recording medium.

1. Field of the Invention
The present invention relates to an optical head apparatus which records or reproduces data in or from an optical recording medium, such as an optical disk.
2. Description of Related Art
There is a great demand that an optical disk apparatus, as an outer storage device of a computer, for example, should be put into practical use or heightened its performance, because it has a large capacity and is capable of non-contact recording, reproducing, and the like. As compared with a magnetic disk apparatus that is widely known and used, the optical disk apparatus is inferior in its high-speed accessibility. However, if with a new type of the optical disk apparatus introduced, the inferior high-speed accessibility will be improved to be superior to the magnetic disk apparatus, in addition to its exchangeability with a recording medium, it will easily be understood that the optical disk apparatus is to replace the magnetic disk apparatus.
One type of improving the high-speed accessibility in the optical head apparatus is that a light source, a light-receiving unit, and the like are fixedly provided, and an actuator for objective lens, separately provided therein alone is made access with respect to a disk so that weight of a movable unit can be reduced. Naturally, in this actuator separation type, it is important to lighten the weight of the movable unit. During a high-speed access, the other type, that will be described later, of performing focusing control and tracking control for the disk by sliding or turning the movable unit around a pole hardly generates unnecessary vibration owing to unbalance and the like as compared with other types. So, this type is advantageous for the high-speed access.
FIG. 1 is a plan view to illustrate the optical head apparatus of prior art, FIG. 2 is a sectional view taken generally along the line II--II of FIG. 1, and FIG. 3 is a connection diagram of a tracking control coil of the optical head apparatus shown in FIG. 1. FIG. 2 also illustrates a disk that will be referred to later. In these figures, reference numeral 1 designates a moving body which is comprised of a base 2, a pole 3 mounted at right angles to the center of the base 2, a damper fixing pin 4 provided at both ends of the base 2, and a bearing 5 provided at four corners of the base 2.
Reference numeral 6 designates first moving means, which is comprised of a guide 7 made of iron being fit into the bearing 5, a linear motor coil 8 fixedly provided to both ends of the base 2 in FIG. 1 in a manner of surrounding the guide 7, a linear motor magnet 9 provided in parallel to the guide 7 and in proximity to the coil 8, and a linear motor yoke 10 provided adjacent to the magnet 9. Meanwhile, fine lines shown in the coil 8 designate the direction to which the coil 8 is rolled (each coil that will be referred to later is the same as this). And characters N and S shown in the magnet 9 designate polarities (each magnet that will be referred to later is the same as this). The guide 7, magnet 9, and yoke 10 are fixed to a fixed body (not shown) which is comprised of a frame body, a box body or the like, and the moving body 1 is guided by the guide 7 and is movable in the direction A. The guide 7 is connected to the yoke 10 in the upper and the lower portions of FIG. 1 (not shown).
Reference numeral 11 designates second moving means, which is comprised of a cylindrical focusing control yoke 12 with a circular ditch 13 being provided in the central portion of the base 2, a cylindrical focusing control magnet 14 provided at a side surface of the ditch 13, and a cylindrical focusing control coil 17 provided at the lower portion in FIG. 2 of a lens holder 15 that will be referred to later and in a position confronting the magnet 14 inside the ditch 13. There is provided the lens holder 15 around the pole 3 via a cylindrical movable bearing 16. The lens holder 15 is rotatable around the pole 3 in the direction B and is also slidable along the pole 3 in the direction C. Reference numeral 18 designates turning means, which is comprised of a tracking control magnet 19 provided to the base 2 above and below the lens holder 15 in FIG. 1, a tracking control yoke 20 provided to the base 2 being adjacent to the magnet 19, and a tracking control coil 21 provided in the lens holder 15.
The lens holder 15 is provided with a damper 22, which is locked by the damper fixing pin 4, generating a directional force of putting the lens holder 15 back to a neutral position when the lens holder 15 is shifted from the position. At one end of the lens holder 15, there is held an objective lens 23 whose optical axis is in parallel to the pole 3. And at the other end of the lens holder 15, there is provided a counter weight 24 which sustains balance between the lens holder 15 and the pole 3. On the base 2, below the objective lens 23 in FIG. 1 there is provided a reflecting mirror 25 which allows a light beam being incident in the direction D to reflect upon the objective lens 23. Above the pole 3 is positioned a disk 26 which is an optical recording medium. The center of the disk 26 is supported by the fixed body, and the disk 26 is adapted to rotate in a plane perpendicular to the optical axis of the objective lens 23.
Now will be described below the operation of the optical head apparatus of prior art. When data is recorded or reproduced, the disk 26 is rotated, and light beam emitted from a fixed optical system (not shown) including a light source becomes incident on the optical head apparatus in the direction of the arrow D and is reflected by the reflecting mirror 25, and, then, is turned 90 degrees, following which being focused on the disk 26 via the objective lens 23. While the access with respect to a desired track (not shown) on the disk 26 is performed, various kinds of control are done to allow the objective lens 23 to follow the disk 26 to cope with the surface deflection and decentering of the disk 26. Tracks on the disk 26 are provided above the objective lens 23 in a depth direction in FIG. 2. When it is desired to perform the access of the objective lens 23 with respect to the desired track, the linear motor coil 8 is energized. Because the coil 8 is located in a magnetic field of the linear motor magnet 9, a force is generated by energizing and the moving body 1 on which the coil 8 is mounted is moved in the direction A. There is no problem if the polarities N and S of the magnet 9 are opposite to those shown in the figure (the same as polarities of other magnets).
After that, fine adjustment is done for accurate tracking control against the decentering and the like of the disk 26. When a DC voltage V is applied to energize the tracking control coil 21 in a magnetic field of the tracking control magnet 19 as shown in the connection diagram of FIG. 3, couple of force in the direction of the arrow E, for example, is generated and then the lens holder 15 is rotated around the pole 3 against the force of the damper 22, moving the objective lens 23 in the direction B. In addition to this, adjustment of the position of the objective lens 23 is done in the direction C so that the light beam can correctly be focused in spite of the surface deflection and the like of the disk 26. To perform this focusing control, the focusing control coil 17 in a magnetic field of the focusing control magnet 14 is energized, and then a force acting in the direction C is generated to move the lens holder 15 on which the objective lens 23 is mounted.
Such an optical head apparatus of prior art has thus been constructed. As it is provided with the whole turning means 18 for tracking control on the moving body 1, weight of the movable unit including the moving body 1 and its loading members becomes larger. Accordingly, this larger weight becomes an obstacle to the high-speed access with respect to the disk 26.

Optical head apparatus with light weight movable lens holder


A splicer for light waveguides is disclosed, including a receptacle including a first body of a predetermined length, having first recesses cut lengthwise through the center of the contact surface, and a second body of length the same as that of the first body, having second recesses cut lengthwise through the center of the surface corresponding to the first recesses cut through the first body; expanding means for expanding space separating the first and second bodies in order to easily insert light waveguides into the recesses; and bending means surrounding the receptacle's outer surface that securely hold first and second bodies together and provide the necessary clamping force.

1. Field of the Invention
The present invention relates to a splicer for connecting light waveguides. More particularly, it relates to a splicer for connecting light waveguides that provides double clamping.
2. Discussion of Related Art
A communications network, constructed with light waveguides, needs splicing of fiber optic cable under certain circumstances. The methods of extending light waveguides includes fusion splicing and mechanical splicing technics. According to the mechanical splicing technique, two optical fibers are mechanically joined to each other by making cores of two fibers meet in a V-shaped centering groove. In contrast with fusion splicing techniques, this mechanical splicing technique does not employ electric power, and can be easily carried out in the field. Since a conventional splicer for light waveguides does not clamp coated or jacketed light waveguide regions effectively, stress concentrates on cladded regions, thus easily breaking the fiber optic filament. To prevent breakage of the optical fiber, it is necessary to clamp and hold outer layers of a jacket so that the stress applied to the fiber optic cable is not directly applied to the cladded regions.
A conventional splicer for fiber optic cable is disclosed in U.S. Pat. No. 4,850,671. Japanese Patent Application No. 89-174977 (filed on Jul. 6, 1989) has proposed a splicer for fiber optic cable having the clamping function. In the conventional splicer, a cover plate is laid on a base plate while light waveguides are tuned therein, and the cladded and jacketed filaments of the light waveguides are clamped simultaneously. When the cover plate is laid on the base plate, the connecting condition of the light waveguides may vary. Whenever the connecting condition of the light waveguides inserted into the splicer is inferior, there are problems tuning the light waveguides again after disassembling the splicer. Thus, according to more effective tuning mechanism, the cladded or jacketed fiber optic elements are first clamped, before the remaining parts of the fiber optic cable are clamped.


Systems and methods are provided for a content addressable memory. A system includes a common memory module configured to store a plurality of entries, ones of the entries being defined by a string of bits. A first parallel compare logic unit is configured to compare a first lookup key against a plurality of entries stored in the memory module in a first memory operation cycle and to output a match indication indicating a match between the first lookup key and the string of bits of an entry from among the plurality of entries. A second parallel compare logic unit is configured to compare, in the first memory operation cycle, a second lookup key against the plurality of entries stored in the memory module and to output a match indication indicating a match between the second lookup key and the string of bits of an entry from among the plurality of entries.

A typical memory, such as a random access memory (RAM), accepts an address as an input and, in response, outputs one or more data values corresponding to that address as an output. A content addressable memory (CAM) operates in a different manner, where the content addressable memory receives a data value as an input and outputs an indication of whether that data value is present at any address in the content addressable memory.
The description above is presented as a general overview of related art in this field and should not be construed as an admission that any of the information it contains constitutes prior art against the present patent application.

Method and apparatus for content addressable memory parallel lookup


An enclosing cup includes a base, an enclosing container for holding liquid, a handle and a mouth. The enclosing container is supported by the base. The mouth is supported by the enclosing container. The enclosing container incorporates a cavity for containing liquid, and an aperture. The mouth communicates with the cavity via the aperture. Liquid flows into and out of the cavity via the aperture and the mouth. The handle is disposed within the enclosing container. The liquid is disposed around the handle and a user's hand that holds the handle. The handle makes contact with the inner side of the enclosing container at one or more points. The handle allows a user to hold and operate the enclosing cup, while the mouth of the enclosing cup allows the user to drink the liquid out of the enclosing cup.

Drinkware, such as bear glasses, coffee mugs and water cups, is a common type of devices in people's daily life. These apparatuses are made in different shapes and sizes. However, a conventional drinkware device includes a container part that is adapted to contain and hold beverage or liquid food. A user of the device retrieves the contained liquid (such as water, orange juice, apple juice, bear, etc.) from the container for consumption. When a handle is attached to the container, the user operates the handle to use the drinkware. The handle is on a side of the container. With or without a handle, the container can also be held between the user's fingers and palm. Regardless of the amount of the liquid deposited inside the container, it presents only a single surface that makes contact with the air. For example, whether a glass is ten percent (10%), eighty percent (80%) or one hundred percent (100%) full with water, there is only a single water surface that makes contact with the air.
Accordingly, there is a need fora new drinkware with a liquid container wrapping around the hand of a user when the user holds a handle of the drinkware. The handle is disposed within the enclosing container. When the new drinkware is not fully filled, the liquid inside the container can provide two liquid surfaces making contact with the air. Furthermore, the liquid inside the new drinkware is present around the user's hand holding the handle of the drinkware. In other words, the drinkware's container encloses the user holding hand.


The object of the invention is to provide a means to enhance the drying property of a coating film formed as a vibration damper and repressing the occurrence of crack and expansion in the surface of the coating film. The task mentioned above is accomplished by using a thickener for vibration damper, which contains a polymer comprising an alkali-soluble monomer unit and an associating monomer unit. The alkali-soluble monomer unit may possess a carboxyl group, a sulfonic acid group, or a phosphoric acid group, or a salt thereof. The associating monomer unit may possess in a side chain thereof a group represented by the formula, —(R1—O)n—X—R2, (wherein R1 denotes methylene group, ethylene group, propylene group or butylene group, n denotes a number in the range of 10-300, X denotes a direct bond, —C(═O)—, or —C(═O)NH—, and R2 denotes a hydrocarbon group of 6-30 carbon atoms).

1. Field of the Invention
This invention relates to a thickener to be contained in a coating material for forming a vibration damper and more particularly to a thickener which is capable of enhancing the drying property of a coating film and repressing the occurrence of a crack and an expansion of the surface of the coating film.
2. Description of the Related Art
The coating material is expected to possess various qualities to suit purposes such as protection, insulation, and beautification. The vibration damper is one of the uses found for the coating material. The term "vibration damper" refers to a material which is disposed on the surface of a material for the purpose of blocking transmission of sound and preventing vibration. The vibration damper is preferred to be disposed on the surface of the chassis of an automobile, for example, with the object of keeping the interior of the automobile quiet.
The sound insulating sheet may be cited as the material which can be utilized as-the vibration damper. The disposal of the sound insulating sheet, however, consumes labor hour. Particularly when this disposal is effected on such a member as the chassis of an automobile which possesses a complicated surface contour, marked decline of productivity cannot be avoided. It is not easily automated and this fact obstructs efforts to cut the cost of production.
The coating film which possesses the quality of a vibration damper, therefore, has been attracting attention. The technique concerning the coating film which functions as a vibration damper has been embodied, for example, by copolymer latex for vibration damper obtained by emulsion polymerizing a monomer mixture comprising a conjugate diene type monomer (a), an ethylenically unsaturated monomer possessing an epoxy group (b), an ethylenically unsaturated carboxylic acid amide monomer (c), and an ethylenically unsaturated monomer (d) other than (b) and (c) at a specific weight ratio (see, the official gazette of JP-2000-178499). The vibration damper is formed by using the coating material incorporating the latex therein. The formation of the vibration damper which is attained by using such a coating material as this excels in productivity and permits automation.
To function effectively as the vibration damper, the coating film is required to:possess a uniform wall thickness. Incidentally, when a thick coating film is to be dried, since it tends to dry gradually from the first surface onward, the part of the coating film in the proximity of the first surface is completely hardened while the inner part of the coating film still retains water. Thus, the vaporization of the water inside the coating film poses the problem of suffering the part of the coating film already hardened in the proximity of the first surface to expand outward and the problem of causing the coating film to sustain a crack. When the coating film expands or cracks, the quality thereof as the vibration damper will be greatly degraded. This mishap deprives the elaborate formation of the coating material with the object of being utilized as the vibration damper of its significance. These problems gain particularly in prominence when the coating film is formed by using an emulsion coating material. The reason for this prominence is that the emulsion coating material tends to succumb to fusion quickly and form a film when the water content encompassing particles is decreased.

Thickener for water-based vibration damper


In a common rail operating method and system, an arrangement for controlling the rail pressure is provided with a rail pressure controller including a current control circuit for controlling a suction throttle valve operating current (i) which valve is arranged in the fuel supply line to a high pressure pump supplying high pressure fuel to the common rail. The suction valve operating current control circuit includes a preliminary control value generator which serves also as an emergency control signal generator for the control of the suction valve if an error occurs in the system at least to permit an orderly engine shutdown procedure.

The invention relates to a method and an apparatus for controlling the fuel pressure in a common rail fuel injection system, wherein a rail pressure deviation is determined by a comparison of the desired and the actual rail pressure and wherein a rail pressure control value for controlling a throttle valve by way of a rail pressure controller is calculated from the rail pressure control deviation and the fuel supply to a high pressure pump and, consequently, the rail pressure is controlled.
In a common rail fuel injection system, the fuel is pumped by a low-pressure pump from the fuel tank to a high-pressure pump. The high-pressure pump supplies the fuel with an increased pressure to a rail (high pressure storage). In the flow path between the low pressure pump and the high pressure pump, there is a controllable suction throttle valve by way of which the fuel admission to the high pressure pump is controlled.
DE 103 30 466 B3 discloses such a common rail system wherein the rail pressure is controlled by an electronic control unit disposed in a rail pressure control circuit providing a control value corresponding to the rail pressure. By a filter arranged in a feedback branch, noise signals are suppressed such as signals which have the same frequency as the injection frequency or the pumping frequency of the high pressure pump. The filtered rail pressure signal is compared as rail pressure actual value with a desired rail pressure value resulting in a rail pressure control deviation. From the rail pressure control deviation, the rail pressure controller determines a control value, that is a desired volume flow. This control value is then converted to a pulse-width modulated signal (PWM). This signal is applied to the suction throttle valve for controlling the rail pressure.
The ohmic resistance of the suction throttle valve winding however changes with the temperature. This means that the rail pressure controller calculates different control values for the same stationary operating point, for example, different integration components. During stationary engine operation, the integration component of the rail pressure controller is additionally deposited in a leakage performance graph. Upon failure of the rail pressure sensor then, instead of the control value computed by the rail pressure controller, a value from the leakage performance graph is used. However, this may be problematic as the quality the rail pressure control may then suffer upon failure of the pressure sensor.
A measure for decreasing the temperature dependency of a rail pressure control circuit is known from DE 198 02 583 A1. Here, the rail pressure controller is provided with a current control circuit. The guide value of the current control circuit corresponds to a desired electric current, which is provided by the rail pressure controller as a control value. By way of a current sensor, the electric current which flows through the winding of a pressure control valve is determined from the actual current value. From the control deviation between the desired current value and the actual current value the current controller determines a control value. The current control of the pressure valve is absolutely necessary since the pressure control valve is arranged at the high pressure side and controls the fuel release from the rail back to the fuel tank. Since a pressure of about up to 180 bar is present in the rail, during a throttling control to a pressure of 0 bar, a large amount of heat is released. From this as well as by the application of the electric current the temperature of the winding is increased. With the control circuit shown, a pulse width modulated signal is applied to current controller as input signal. Since a current controller must be highly dynamic the application of an unfiltered PWM signal may result in an instability of the current control circuit. There is no backup for the current control circuit in case of a current measurement error or failure.
It is therefore the object of the present invention to provide a stable and temperature-independent rail pressure control circuit with a suction throttle valve which additionally includes an error protection.

Method and apparatus for controlling the pressure in a common rail system


A moving image reproducing apparatus is provided with CPU 8 and a displaying unit 7. A moving image including plural frames of image is displayed on the display unit 7. CPU 8 sets a display position in one of the frames of image and displays a guide-indicator at the display position, wherein the guide-indicator is used for analyzing a specific motion of a person. CPU 8 displays the guide-indicator on each of the frames of image following the frame of image, in which the display position is set, in an overlapping manner. CPU 8 obtains displacement information of a field angle of each of the reproduced frames of image following the frame of image, in which the display position is set. CPU 8 adjusts the display position based on the displacement information, such that the change of the field angle of each frame of image is cancelled.

1. Field of the Invention
The present invention relates to a moving image reproducing apparatus and a moving image reproducing method, which can reproduce a previously recorded moving image for the purpose of analyzing a specific motion, and to recording medium storing a program used for the same purpose.
2. Description of the Related Art
Japanese Patent No. Hei10-201898 (1998-201891) discloses an image capturing/reproducing apparatus, which displays reference lines on a moving image in an overlapping manner, when reproducing the moving image, that is obtained by capturing a golfer image, which swings a golf club, wherein the reference lines are used as a guide-indicator for analyzing a golf-club swinging form. In the image capturing/reproducing apparatus, a user finds and displays a frame of image representing a learner-golfer taking an address position by a frame-by-frame playback operation and manually sets display positions of the reference lines appropriate for analyzing the golf-club swinging form, and then playbacks the moving image to analyze the golf-club swinging form.
But the image capturing/reproducing apparatus disclosed in the above patent document can invite the following drawback in the case that the moving image captured with a hand-held digital camera using no tripod stand is used for analyzing the swinging form.
In the moving image captured with a hand-held digital camera using no tripod stand, the field angle (capturing range) can often change from frame of image to frame of image due to camera shake.
Therefore, in the case of analyzing the golf-club swinging form using the moving image imaged or obtained with the hand-held digital camera using no tripod stand, a positional relationship between the learner-golfer and the reference lines, the display positions of which have been set by the user before reproducing the moving image, can change before starting the golf-club swing motion, resulting in disrupting a precise analyze of the swinging form.

Moving image reproducing apparatus capable of adjusting display position of indicator for motion analysis based on displacement information of frames, and moving image reproducing method and recording medium for same


A system that buffers an application image reduces bandwidth requirements for accessing memory. The application image may be logically separated into tiles. A viewport may identify a visible portion of the application image, where the visible portion is smaller than the application image. The tiles overlapped by the viewport may be buffered in a front buffer and a back buffer. The tiles not overlapped by the viewport may be buffered in the back buffer but not in the front buffer. A composition manager, with knowledge of the viewport and at least two noncontiguous tile buffers in the front buffer, may extract the visible portion of the application image directly from the noncontiguous tile buffers.

1. Technical Field
This disclosure relates to graphics and, in particular, to graphics buffers.
2. Related Art
Graphical user interfaces (GUI), Picture in Picture (PiP), and other types of graphical images may be composited from multiple independently rendered images. For example, a GUI may contain a background image and one or more rendered graphic layers on top of the background image. The graphic layers may comprise applications, task bars, or other type of graphic elements. Each of the graphic layers may be rendered in an application buffer independently of the other graphic layers by individual software applications. The graphic layers may be composited together into a single composite image in a display buffer, which may be presented on a display device, transmitted over a network, or otherwise processed. The application buffer may represent an image that is larger than the corresponding graphic layer composited into the single composite image.


An apparatus and a method for isolating a biologic product, such as plasmid DNA, from cells. The method involves lysing cells in a controlled manner separate insoluble components from a fluid lysate containing cellular components of interest, followed by membrane chromatographic techniques to purify the cellular components of interest. The process utilizes a unique lysis apparatus, ion exchange and, optionally, hydrophobic interaction chromatography membranes in cartridge form, and ultrafiltration. The process can be applied to any biologic product extracted from a cellular source. The process uses a lysis apparatus, including a high shear, low residence-time mixer for advantageously mixing a cell suspension with a lysis solution, a hold time that denatures impurities, and an air-sparging bubble mixer that gently yet thoroughly mixes lysed cells with a neutralization/precipitation buffer and floats compacted precipitated cellular material.

The present invention relates to an apparatus and scalable methods of lysing cells. The invention also relates to methods of isolating and purifying cellular components from lysed cells. The invention is particularly suited for scalable lysis of plasmid-containing bacterial cells, and subsequent preparation of large quantities of substantially purified plasmid. The resulting plasmid is suitable for a variety of uses, including but not limited to gene therapy, plasmid-mediated hormonal supplementation or other therapy, DNA vaccines, or any other application requiring substantial quantities of purified plasmid. Over the last five years, there has been an increased interest in the field of plasmid processing. The emergence of the non-viral field has caused researchers to focus on a variety of different methods of producing plasmids.
Because plasmids are large and complex macromolecules, it is not practical to produce them in large quantities through synthetic means. Instead, they must be initially produced in biological systems, and subsequently isolated and purified from those systems. In virtually all cases, biological production of plasmids takes the form of fermenting Escherichia coli (E. coli) cells containing the plasmid of interest. A number of techniques for fermenting plasmid-containing E. coli cells have been known by those skilled in the art for many years. Many fermentation processes have been published, are well known and are available in the public domain.
Cell lysis and the subsequent treatment steps used to prepare a process stream for purification are the most difficult, complex and important steps in any plasmid process. It is in this process step where yield and quality of the plasmid of interest are primarily determined for each run. The search for an optimal method, one that is continuous and truly scalable, has been an obstacle in getting acceptable processes with commercial applicability.
There are a variety of ways to lyse bacterial cells. Well-known methods used at laboratory scale for plasmid purification include enzymatic digestion (e.g. with lysozyme), heat treatment, pressure treatment, mechanical grinding, sonication, treatment with chaotropes (e.g. guanidinium isothiocyante), and treatment with organic solvents (e.g. phenol). Although these methods can be readily practiced at small scale, few have been successfully adapted for large-scale use in preparing plasmids.
Methods such as pressure treatment, mechanical grinding, or sonication can be difficult to implement at large scale. Moreover, Carlson et al. (1995, Biotechnol. Bioeng. 48, 303–315) have shown that such mechanical methods can lead to unacceptable plasmid degradation. Methods involving chaotropes and/or organic solvents are problematic to scale up because these chemicals are typically toxic, flammable, and/or explosive. Handling and disposing of such chemicals is manageable at small scale, but generally creates substantial problems at large scale. U.S. Pat. No. 6,197,553 describes a large-scale lysis technique involving treatment with lysozyme and heat. However, this technique requires carefully controlled heating and cooling of the enzymatically-treated bacterial cells to achieve lysis. The technique also has disadvantages in that it requires the use of an animal-derived enzyme (lysozyme), which can be expensive and is a potential source of biological contamination. Using animal-derived materials is quickly becoming unacceptable when preparing plasmids or other cellular components of interest for human or veterinary applications.
Currently, the preferred method for lysing bacteria for plasmid purification is through the use of alkali and detergent. This technique was originally described by Birnboim and Doly (1979, Nucleic Acids Res. 7, 1513–1523). A commonly used variation of this procedure, as described on pp. 1.38–1.39 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), is to suspend bacterial cells in 10 mL of a resuspension solution, consisting of 50 mM glucose, 25 mM Tris, pH 8.0, 10 mM EDTA. The suspension is mixed with 20 mL of a lysis solution, consisting of 0.2 N NaOH, 1% sodium dodecylsulfate (SDS) and incubated for 5–10 minutes. During this period, the cells lyse and the solution becomes highly viscous. The high pH denatures both the host genomic DNA and the plasmid DNA. The SDS forms complexes with cellular proteins, lipids, and membrane components, some of which are tightly associated with the host genomic DNA. The lysate-mixture is next treated with 15 mL of an ice-cold neutralization/precipitation solution, consisting of 3 M potassium acetate that has been adjusted to pH 5.5 with acetic acid. This acidified mixture is incubated on ice for 5–10 minutes, in part to allow plasmid DNA to renature. During this time, a white flocculent precipitate is formed. The precipitate comprises potassium SDS, which is poorly soluble under these conditions. In addition, the precipitate contains host genomic DNA, proteins, lipids, and membrane components, which remain bound to the SDS. The precipitate is subsequently removed by filtration or centrifugation, yielding a clarified lysate containing the desired plasmid, which can be subjected to various purification procedures.
This lysis method has very distinct advantages over those described above. In addition to providing efficient release of plasmid molecules from the cells, this procedure provides substantial purification of the plasmid by removing much of the host protein, lipids, and genomic DNA. Removal of genomic DNA is particularly valuable, since it can be difficult to separate it from plasmid DNA by other means. These advantages have made this a preferred method for lysing bacterial cells during plasmid purification at laboratory scale.
Unfortunately, this method presents significant challenges for scaling up. First, thorough mixing of suspended cells with lysis solution is easily managed at small scale by simply vortexing or repeatedly inverting the vessel containing the cells. However, this is impractical at large scale, where volumes may be in the range of tens or hundreds of liters. Common techniques for mixing large volumes of liquid, such as batch impeller mixing, are problematic because as some cells begin to lyse after initial mixing, they release genomic DNA that dramatically increases solution viscosity. This increase in viscosity significantly interferes with further mixing.
A second challenge is that excessive incubation at high pH after addition of alkaline lysis solution can lead to permanent denaturation of the plasmid, making it unsuitable for most subsequent uses. It is therefore necessary to ensure that the lysed cells are thoroughly mixed with neutralization/precipitation solution within a relatively narrow time frame, typically within 5–10 minutes. It is also well known that mixing at this step must be gentle (i.e. low shear). Vigorous (i.e. high shear) mixing at this step releases substantial amounts of material from the flocculent precipitate into the plasmid-containing solution. This includes large amounts of host genomic DNA and endotoxins. These substances are difficult to separate from the plasmid during subsequent purification. Thus, while complete mixing is required to precipitate all of the SDS-associated impurities and renature all of the plasmid, mixing should also be as gentle as possible. This is easily accomplished at small scale by timed addition of neutralization/precipitation solution using hand mixing techniques such as gentle swirling or inversion of the containers. In contrast, rapid yet gentle mixing is difficult to achieve at large scale. Low shear stirring or impeller mixing in batch mode requires relatively long times to achieve complete mixing, which could result in unacceptably high levels of permanently denatured plasmid. More rapid techniques such as high speed impeller mixing are likely to result in unacceptably high levels of genomic DNA and endotoxin in the plasmid-containing solution.
It has previously been believed that mixing a cell suspension and a lysis solution must be performed at very low shear. This has been particularly claimed in regard to mixing suspensions of plasmid-containing bacteria with lysis solutions comprising alkali and detergent. For example, Wan et al., in U.S. Pat. No. 5,837,529, in discussing methods of lysing plasmid-containing cells with alkali or enzymes, contend that it is crucial to handle such lysates very gently to avoid shearing genomic DNA. Similarly, Nienow et al., in U.S. Pat. No. 6,395,516, in discussing the challenges of alkaline lysis, claim that too vigorous mixing at any stage of the procedure may lead to fragmentation of genomic DNA, which may substantially contaminate the final purified product. Yet again, Bridenbaugh et al., in U.S. Patent Application No. 2002/0198372, emphasize the need for gentle mixing of cells with lysis solution. These concerns have led such investigators to develop ostensibly scalable means to gently mix suspended cells with lysis solutions. For instance, U.S. Pat. No. 5,837,529 and U.S. Patent Application No. 2002/0198372 each contemplate using static mixers to achieve continuous low shear mixing, while U.S. patent application Ser. No. 6,395,516 contemplates using a designed vessel for controlled mixing in batch mode. Such methods have clear drawbacks. In one regard, while striving to minimize excessive shear, mixing of the cell suspension with the lysis solution may be incomplete. In another regard, using static mixers limits process flexibility. As described in U.S. Patent No. 2002/0198372, it is necessary to optimize the number of static mixing elements, as well as the flow rates of the fluids passing through the elements. Such optimization restricts the amount of material that may be processed in a given time with the optimized static mixing apparatus. This limits the ability to increase process scale, unless a new, higher-capacity static mixing apparatus is constructed and optimized. Use of batch mixing vessels, as described in U.S. Pat. No. 6,395,516, has comparable drawbacks. Achieving complete mixing in all regions of a batch mixing vessel is well known by those of skill in the art to be challenging. Furthermore, batch mixing vessels are poorly suited for applications that require a controlled exposure time wherein the cell suspension is contacted with the lysis solution. In particular, it is well known that prolonged exposure of plasmid-containing cells to alkali may lead to the formation of excessive amounts of permanently denatured plasmid, which is generally inactive, undesirable, and difficult to subsequently separate from biologically active plasmid. Typically, it is desirable to limit such exposure times to about 10 minutes or less. Achieving such limited exposure times is difficult or impossible using large scale batch mixing.
Removal of the flocculent precipitate is yet another challenge in scaling up alkaline lysis. Complete removal is desirable to eliminate the genomic DNA and other impurities trapped in the precipitate. At the same time, the precipitate must not be subjected to excessive shear. Otherwise, large amounts of genomic DNA, endotoxins, and other impurities are released from the precipitate and contaminate the plasmid-containing solution. At laboratory scale, the precipitate is readily removed by simple filtration, batch centrifugation, or both. However, batch centrifugation is highly impractical at large scale. Continuous centrifugation at large scale is also unsuitable because it subjects the precipitate to high shear stress, releasing unacceptable levels of impurities. Filtration at large scale is problematic due to the somewhat gelatinous, cheese-like consistency of the precipitate, which readily clogs even depth or bag filters.
Notwithstanding the above challenges, a variety of investigators have developed claimed improvements of the alkaline lysis method, or otherwise attempted to adapt it into a scalable production process. Kresheck and Altschuler, in U.S. Pat. No. 5,625,053, describe the use of non-ionic alkyldimethylphosphine oxide detergents in place of SDS. Use of these detergents is claimed to offer certain advantages relevant to large-scale preparation of pharmaceutical grade plasmid. However, the claimed improvements do not address the scalability issues described above.
Thatcher et al., in U.S. Pat. No. 5,981,735, describe a modification where the amount of NaOH added to the suspended cells is carefully controlled to ensure that the pH remains approximately 0.1 pH units below the point that results in substantial permanent denaturation of plasmid. This approach may address the issue of time-dependent generation of permanently denatured plasmid, but requires very precise pH control, which can be difficult at large scale. Furthermore, the preferred pH level must be determined in advance for each plasmid and host cell combination. Most importantly, this approach does not address the challenges of handling and mixing large liquid volumes.
Wan et al., in U.S. Pat. No. 5,837,529, describe a process of lysing cells, comprising the use of static mixers to mix suspended cells with a lysis solution (e.g. 0.2 N NaOH, 1% SDS), as well as to mix lysed cells with a precipitating solution (e.g. 3 M potassium acetate, pH 5.5). Static mixers are claimed to be particularly advantageous by providing a high degree of mixing at a relatively low shear, and are also amenable to a continuous flow-through process. A similar process using static mixers is described by Bridenbaugh et al. in WO 00/05358. Such procedures offer certain advantages, but drawbacks remain. As shown in WO 00/05358, both the number of static mixing elements and the solution linear flow rates must be carefully controlled at each stage. Using too few mixing elements or a low linear flow rate leads to inadequate mixing and poor plasmid yields. Using too many elements or a high linear flow rate leads to excessive shearing and release of genomic DNA into solution. These parameters must be experimentally optimized, and any efforts to increase process scale require re-optimization of element number and flow rate, limiting process flexibility and the robustness of this method for routine use.
Marquet et al. (1995, Biopharm 8, 26–37) describe the use of batch mixers originally designed for use in the food industry. They claim that these mixers can provide thorough mixing at low shear rates, making them suitable for use during large-scale alkaline lysis of plasmid-containing cells. However, batch mixing of large fluid volumes in tanks is often very difficult to scale up, particularly when there are dramatic differences in fluid viscosity, or when mixing itself leads to dramatic increases in viscosity. Batch mixing is also problematic when coupled with short, time-sensitive incubation steps. All of these concerns pertain to alkaline lysis, making batch mixing particularly unsuitable.
Thus, despite the efforts of previous investigators, there is still a clear need for new and improved procedures to perform alkaline lysis at large scale. A preferred process would address a series of key challenges, including: (1) thorough, rapid, and robust mixing of cells and lysis solution, to efficiently lyse cells and release plasmid; (2) time-controlled incubation of lysed cells in alkali, to prevent permanent plasmid denaturation; (3) thorough, rapid, and gentle mixing of alkaline lysate with neutralization/precipitation solution, to efficiently precipitate contaminating cellular components without releasing excess genomic DNA and endotoxin into the plasmid-containing solution; and (4) efficient yet gentle removal of the flocculent precipitate, again without releasing excess genomic DNA and endotoxin into the plasmid-containing solution. Furthermore, such a preferred process would be readily scalable, robust, suitable for use in all applications, would contain no animal derived products, and would be cost effective.
There is also a need for improved procedures for purifying plasmids from large-scale microbial cell lysates. In particular, the emerging fields of non-viral gene therapy, plasmid-mediated therapy and DNA vaccines require gram or even kilogram amounts of purified plasmid suitable for pharmaceutical use. It is thus necessary to purify plasmids away from the primary impurities remaining in the lysate, including residual genomic DNA, RNA, protein, and endotoxin. An ideal process should provide substantially pure material in high yield, be easy to scale up, involve a minimal number of steps, and be simple and inexpensive to perform. Any use of enzymes or animal-derived products should be avoided, as such reagents tend to be expensive and more importantly, are potential sources of contamination. Similarly, use of alcohols and organic solvents is to be avoided, as they are generally toxic, flammable, explosive, and difficult to dispose of in large quantities. Known or suspected toxic, mutagenic, carcinogenic, teratogenic, or otherwise harmful compounds should not be used. Finally, the process should avoid the need for expensive equipment such as large scale or continuous centrifuges, or gradient producing chromatography skids.
Various attempts have been made to develop a plasmid purification process that meets these ideals. For example, Horn et al., in U.S. Pat. No. 5,707,812, describe an integrated process involving alkaline lysis, filtration with diatomaceous earth, concentration and desalting by ultrafiltration/diafiltration (UF/DF), overnight precipitation of plasmid with 8% polyethylene glycol (PEG) 8000, centrifugation, resuspension, precipitation of impurities with 2.5 M ammonium acetate, centrifugation, precipitation of plasmid with isopropanol, centrifugation, resuspension, anion exchange column chromatography in the presence of 1% PEG 8000 on Q Sepharose™ (Amersham Biosciences Corp., Piscataway, N.J.) with step elution, plasmid precipitation with isopropanol, centrifugation, resuspension, and gel filtration column chromatography on Sephacryl™ S-1000 (Amersham Biosciences Corp., Piscataway, N.J.). Plasmid yields, quality, and purity were not described. Similar processes are disclosed by Marquet et al. in U.S. Pat. No. 5,561,064. These processes are not easily scaled, due to the multiple plasmid precipitations and centrifugations. In addition, achieving adequate resolution with gel filtration column chromatography typically requires relatively large columns. Use of isopropanol in multiple steps is another disadvantage of this process.
U.S. Pat. No. 5,990,301, issued to Colpan et al., describes an integrated process involving alkaline lysis, clarification by centrifugation and filtration, incubation with salt (NaCl) and nonionic detergent, anion exchange by DEAE column chromatography, isopropanol precipitation, centrifugation, and resuspension. The resulting plasmid was reported to contain "no detectable" RNA, genomic DNA, or endotoxin, but detection methods and limits were not described. This process has numerous scalability issues. DEAE resins typically have relatively low capacity for plasmid. Furthermore, using isopropanol precipitation and centrifugation for product concentration and desalting is not feasible at large scale.
U.S. Pat. No. 6,197,553, issued to Lee and Sagar, describes an integrated process involving cell wall digestion with lysozyme, lysis by passing through a flow-through heat exchanger to heat the cell suspension to about 80° C., clarification by centrifugation, diafiltration, treatment with RNase, diafiltration, anion exchange column chromatography on POROS® PI/M (Applied Biosystems, Foster City, Calif.) with NaCl gradient elution, reverse phase chromatography on POROS® R2/M with isopropanol gradient elution, and UF/DF. Final product contained 2.9% genomic DNA, <1% protein, <1% RNA, and endotoxin levels of 2.8 endotoxin units (EU) per milligram of plasmid. However, this process suffers from the use of two enzymes (lysozyme and RNase), gradient-based anion exchange chromatography, and gradient-based reverse phase chromatography using isopropanol. These present substantial scalability and/or regulatory issues.
U.S. Pat. No. 6,410,274, issued to Bhikhabhai, describes a process involving alkaline lysis, filtration, precipitation of RNA and genomic DNA with CaCl2, centrifugation, filtration, anion exchange column chromatography on Q Sepharose™ XL (Amersham Biosciences Corp., Piscataway, N.J.) with step elution, and further anion exchange column chromatography on Source™ 15Q (Amersham Biosciences Corp., Piscataway, N.J.) with step elution. Final product was reported to contain 0.6% genomic DNA (by PCR), 100% supercoiled plasmid (by anion exchange high performance liquid chromatography, "HPLC"), and no detectable RNA (by reverse phase HPLC), protein (by Micro BCA™ assay, Pierce Biotechnology, Rockford, Ill.), or endotoxin (by limulus amebocyte lysate, "LAL"). The use of two successive anion exchange steps is an obvious inefficiency of this process. Furthermore, the process relies on column chromatographic techniques, which involve expensive hardware and resins.
WO 00/05358, submitted by Bridenbaugh et al., describes a process where plasmid-containing cells are resuspended in the presence of RNase. A continuous lysis procedure is described, where the resuspended cells and an alkaline lysis solution are simultaneously pumped through a static mixer to achieve lysis. The lysate is then mixed with potassium acetate precipitation solution via a second static mixer. The precipitated lysate then passes into a continuous centrifuge to remove the flocculent precipitate, resulting in a clarified lysate. Clarified lysate is filtered to remove fine particulates and purified by anion exchange column chromatography using Fractogel® TMAE-650M (Merck KGaA, Darmstadt, Germany). The anion exchange eluate is then passed through glass and nylon filters, which are claimed to help remove endotoxin and genomic DNA. Purified plasmid was then concentrated and desalted by UF/DF, and sterilized by filtration. Final endotoxin levels were 16.2 EU/mg. Residual RNA, protein, and genomic DNA were said to routinely be <2%, <0.1%, and <1%, respectively. Use of continuous centrifugation is a significant drawback of this process, due to high shear rates and subsequent release of excess genomic DNA into solution, as well as the high cost of such equipment. Use of RNase is a further drawback of this process from a regulatory standpoint.
U.S. Patent Application No. 2001/0034435, submitted by Nochumson et al., describes a process where plasmid-containing cells are lysed with alkali and SDS in a continuous process using static mixers. The lysate is neutralized by continuous addition (via a second set of static mixers) of a neutralization/precipitation solution. The neutralized lysate is held for 6–12 hours at 4° C. to precipitate the majority of the RNA. The flocculent precipitate and the precipitated RNA are removed by centrifugation and/or filtration, and the plasmid-containing solution is subjected to anion exchange column chromatography using Fractogel® TMAE-650S (Merck KGaA, Darmstadt, Germany). Plasmid is then eluted and subjected to hydrophobic interaction chromatography ("HIC"), also in column format, using Octyl Sepharose™ 4FF (Amersham Biosciences Corp., Piscataway, N.J.). Under appropriate conditions, genomic DNA, RNA, and endotoxin bind to the resin, while plasmid passes through. After HIC, the product is concentrated and desalted by UF/DF, and sterile filtered. Detailed information on yields and purity were not described in this application. However, plasmid binding capacities for the resins are relatively low (1–3 mg/mL for the anion exchange, and <1 mg/mL for the HIC if used in binding mode), and again, there is a reliance on column chromatography.
Varley et al. (1999, Bioseparation 8, 209–217) describe a process consisting of optimized alkaline lysis with RNase treatment, bag depth filtration, expanded bed anion exchange chromatography, ultrafiltration, and size exclusion chromatography. Similar processes are disclosed in U.S. Pat. No. 5,981,735 by Thatcher et al. In these processes, the pH during alkaline lysis was carefully controlled at a point just below the empirically determined level that leads to permanent plasmid denaturation. The investigators claim that this allows extended incubation in alkali, presumably to maximize lysis and/or to degrade RNA without damaging plasmid. Impurities were reported to be <2% genomic DNA (by PCR), 0.2% RNA (by HPLC), <0.1% protein, and 2.5 EU/mg endotoxin. However, the process contains several undesirable elements, including use of RNase, bag depth filtration, column-based anion exchange, and size exclusion chromatography. Performing the controlled alkaline lysis requires carefully determining the ideal pH for a given combination of host, plasmid, and growth conditions, suggesting that this step may not be very robust.
As the above examples suggest, column chromatography is often a preferred element in plasmid purification. Anion exchange chromatography is well suited for separating plasmids from certain impurities such as proteins, because plasmids, like all nucleic acids, have a high negative charge density. Thus, many known plasmid purification processes include an anion exchange step. However, anion exchange chromatography is less suited for separating plasmids from other nucleic acids with similar negative charge densities, such as genomic DNA or RNA. Thus, anion exchange chromatography is frequently combined with another chromatographic step to achieve sufficiently pure plasmid. As discussed above, these may include size exclusion chromatography, reverse phase chromatography, hydrophobic interaction chromatography, and even additional anion exchange chromatography. Other chromatographic techniques are also known. For example, Wils and Ollivier, in WO 97/35002, disclose methods for purifying plasmids with ceramic hydroxyapatite. Comparable methods are disclosed by Yamamoto in U.S. Pat. No. 5,843,731. Ion-pair or matched ion chromatography may be used, as disclosed, for example, by Gjerde et al. in U.S. Pat. No. 5,986,085. Silica, glass beads, or glass fibers may also be used, as disclosed, for example, by Padhye et al. in U.S. Pat. No. 5,808,041, by Woodard et al. in U.S. Pat. No. 5,650,506, and by Woodard et al. in U.S. Pat. No. 5,693,785. Alternatively, magnetic beads or particles may be used, as disclosed, for example, by Reeve and Robinson in U.S. Pat. No. 5,665,554, and by Hawkins in U.S. Pat. No. 5,898,071. Affinity methods are also known, with examples being disclosed by Ji and Smith in U.S. Pat. No. 5,591,841, and by Cantor et al. in U.S. Pat. No. 5,482,836.
Despite the frequent use of column chromatography, there remain substantial limitations to this general technique. Chromatography resins are often expensive, and must be carefully packed into specially designed column hardware. Reproducibly packing large-scale chromatography columns is a significant challenge, as discussed by Rathore et al. (2003, Biopharm International, March, 30–40). Furthermore, in regards to plasmids, traditional chromatography resins typically offer relatively low binding capacities. For example, Levy et al. (2000, Trends Biotechnol. 18, 296–305) examined a variety of commercially available anion exchange resins and found that all exhibited plasmid binding capacities of about 5 mg/mL or less, with most exhibiting capacities of about 2 mg/mL or less. Moreover, accessibility to binding sites for large molecules like plasmids is mostly by diffusion and resins have a limited pressure drop resulting in low throughput, making these steps time consuming, costly and impractical.
Thus, it is desirable to develop a purification process that retains the advantages of column chromatography while avoiding its drawbacks. Use of membrane chromatography offers a potential solution. Membrane-based techniques typically offer substantially higher binding capacities, as well as very high flow rates. Expensive large-scale column hardware is not required. In addition, the difficulties associated with column packing are avoided, as well as the need for costly cleaning validation studies.
Certain previous investigators have disclosed membrane-based methods for purifying plasmids. For instance, Nieuwkerk et al., in U.S. Pat. No. 5,438,128, describe the use of an assembly containing a plurality of stacked microporous anion exchange membranes for purifying nucleic acids, including plasmids. However, their method is described for relatively small-scale purification of up to several hundred micrograms of plasmid. Furthermore, although the purified plasmid was stated to be RNA and protein free, there was no disclosure that the provided methods could substantially eliminate genomic DNA or endotoxin. Demmer and Nussbaumer, in U.S. Pat. No. 6,235,892, disclose a method of purifying nucleic acids, including plasmids, from a solution containing endotoxin, using a microporous weakly basic anion exchange membrane. Similarly, in WO 01/94573, Yang et al. claim a process involving two (or more) separate membranes, wherein one binds plasmid and the second binds endotoxin. The investigators state that their methods provide plasmid that is suitable for use in many pharmaceutical applications, but no data is provided to support this statement.
Thus, none of the disclosed membrane-based purification processes is demonstrably adequate for preparing substantially pure plasmid that is acceptable for pharmaceutical, veterinary, or agricultural applications. There is therefore a need for a purification process that employs membrane-based chromatographic separations, avoids column chromatography, and provides substantially pure plasmids or other biologically active molecules of interest.

Devices and methods for biomaterial production


In order to provide pages relating to a prescribed page to a user in a precise manner over the internet, a site page processor collects pages included at various sites, determines parent-child relationships between the pages, and stores the results of such determinations in a site page data storage section. A related page data processor calculates the relevance between pages using page feature extraction values given weightings taking into consideration at least one of a sibling relationship and a co-parent relationship between pages using data stored in the site page data storage section. Through this page feature extraction, words used in common on pages in link relationships are processed so as not to have a substantial influence on the calculation of relevance. The system can be applied to a server on the Internet for searching for pages relating to a prescribed page.

1. Field of the Invention
The present invention relates to an information processing device and method, a recording medium, and a program, and particularly relates to an information processing device and method, recording medium, and program appropriate for use in searching for homepages etc. set up in a network.
2. Description of Related Art
In recent years, the number of homepages set up on the Internet has increased with the proliferation of the Internet itself. Homepages are not just being set up by businesses but also by individual users, meaning that the number of homepages is colossal. Searching out homepages carrying information desired by a user from this enormous number of homepages is therefore very troublesome.
In order to alleviate such troublesomeness, homepages commonly referred to as "search engines" such as, for example, "Yahoo" (trademark), "goo" (trademark), "Excite" (trademark), "Google" (trademark) and "Netscape" (trademark) have started to provide services to enable desired homepages to be searched simply by inputting keywords, etc.
These search engines are suited to looking for homepages containing keywords inputted by the user that have similar features to those of the keywords, but it is often the case that there are pages, other than those successfully found, that the user would probably have liked to have seen.
As a result, several search engines have started related page search engine services referred to as "related page searches," etc. For example, there exist patent document 1 and a related page search for pages returned by Google search results, a related page search button in the Google Toolbar, and a related site search button displayed in browsers such as Netscape Navigator.
[Patent Document 1]
Japanese Patent Application Publication No. 2002-149698 (pages 4 to 7).
Searches employing a related page search engine search for pages related to a page a user is currently browsing or for prescribed pages included in search results returned by the search engine. This search takes into consideration the WWW (World Wide Web) link structure, but it is not necessarily the case that searches for related pages are carried out with high precision.

Information processing device and method, recording medium, and program