The present invention provides mutant genes encoding variant galactose oxidase enzymes, termed vGOs; constructs and recombinant host cells incorporating the genes; the vGO polypeptides encoded by the genes; antibodies to the polypeptides; and methods of making and using all of the foregoing.

The present invention relates generally to the fields of genetics, cellular and molecular biology, and enzymology. More particularly, the invention relates to novel galactose oxidase enzymes, polynucleotides, and polypeptides.
The enzyme galactose oxidase (GO), encoded by the galactose oxidase gene (go) of Dactylium dendroides and other organisms, catalyzes a reaction in which a primary alcohol such as the C6 hydroxyl group of galactose is oxidized to an aldehyde with concomitant reduction of molecular oxygen to hydrogen peroxide, as shown generally in Formula I.
RCH2OH+O2xe2x86x92RCHO+H2O2xe2x80x83xe2x80x83(I)
GO enzymes may be readily used to oxidize the D-galactose (and other hydroxyl-containing) side-chains of many complex compounds, including, but not limited to molecules comprising a D-galactose moiety that is not sterically hindered or blocked at the C6 hydroxyl, and molecules comprising a moiety such as dihydroxyacetone, glycerol, or similar short-chain alcohols in which a primary hydroxyl functional group is present. Of particular interest is the oxidation of guar gum. When guar gum is oxidized by GO, the resulting compound, called oxidized guar, can be used for many purposes, including use in paper manufacturing to add strength to paper products via the formation of acetal, hemiacetal, and other crosslinks with cellulose fibers (see, e.g., 1996; Aldehyde cationic derivatives of galactose containing polysaccharides used as paper strength additives; U.S. Pat. No. 5,554,745 (Chiu et al.); U.S. Pat. No. 5,502,091 (Dasgupta).
Galactose oxidases have been isolated from several species. For example, U.S. Pat. No. 6,090,604 discloses a genomic DNA sequence and deduced amino acid sequence for the GO enzyme from Fusarium venenatum.
Wildtype GO enzymes, however, are relatively inefficient oxidizers of guar gum and other compounds. Thus, there exists a need in the art for superior oxidizers of such compounds. Additionally, one obstacle to the development of variant GO""s is the high viscosity of guar (e.g., the cationic guar used in this work has a viscosity of 1000 cps in 1% aqueous solution), a high molecular weight polymeric substrate. Indeed, many natural or synthetic polymers are insoluble or highly viscous when in solution, and are consequently difficult to pipette by hand or robotic means. Therefore, various methods of high-throughput screening used to evaluate such variant enzymes useful in adding functionality to viscous or insoluble polymers are needed by the art. Such methods enable those of skill in the art to create mutant GO enzymes capable of more efficient oxidative enzymatic reactions.
Although GO does display significant activity towards guar, the present inventors improved its specific activity via in vitro evolution of the enzyme. Using selected methods of mutagenesis, the present inventors created mutant galactose oxidase genes (mgo""s) which encode variant galactose oxidase enzymes (vGO""s), which variants are superior to wildtype GO in terms of efficiency of oxidizing guar and other compounds, as well as in conferring improved thermostability.
Error prone PCR (EPP) was used to generate mutant go genes, encoding variant GO enzymatic proteins. One of skill in the art will appreciate that any method capable of generating mutant genes would be suitable for practicing the present invention. In order to evaluate the efficiency of oxidation of the variants, certain recently developed methods of high throughput screening were used. However, one of skill in the art may choose a method of screening suitable to the substrate of interest. One aspect of the particular screening method used for the evolution of vGO""s by the present inventors is the use of a proxy, i.e., a substrate that represents an adequate substitute for a problematic compound. Particularly, in order to evaluate vGO""s oxidation of guar, the proxy methyl-xcex1-D-galactose (methyl galactose) was used. The variant GO""s of the present invention are demonstrably superior to wildtype GO in terms of their ability to oxidize guar and other complex compounds having hydroxyl-containing sidechains.
In one aspect, the present invention provides polynucleotides comprising mutant go genes. In another aspect, the invention provides polypeptides encoded by such polynucleotides. In another aspect, the invention provides variant GO enzymes having superior enzymatic activity (on methyl galactose or other substrates), and/or thermostability (i.e., resistance to heat inactivation), and which differ from wildtype GO by having at least one substituted amino acid. In another aspect, the present invention provides vectors comprising the polynucleotides. In another aspect, the present invention provides cells transfected or transformed with such vectors. In still another aspect, the present invention provides antibodies specific to the variant GOs. In another aspect, the present invention provides methods of using these molecules and constructs.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) encoding variant GO enzymes which differ from wildtype GO by at least one amino acid, and which exhibit superior enzymatic activity (on methyl galactose or other substrates) and/or thermostability. DNA polynucleotides of the invention include genomic DNA, cDNA, and DNA that has been chemically synthesized in whole or in part. The present invention also provides vectors comprising such polynucleotides, and cells transfected with such vectors. The present invention also provides the proteins encoded by such polynucleotides (i.e., variant GO enzymes), and methods of using the polynucleotides and polypeptides. The present invention also provides antibodies specific to vGOs capable of binding specifically to the variants while remaining unbound to wildtype GO.
Various definitions are made throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as are typically understood by those skilled in the art.
xe2x80x9cSynthesizedxe2x80x9d as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. xe2x80x9cWhollyxe2x80x9d synthesized DNA sequences are therefore produced entirely by chemical means, and xe2x80x9cpartiallyxe2x80x9d synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. By the term xe2x80x9cregionxe2x80x9d is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. The term xe2x80x9cdomainxe2x80x9d is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.
As used herein, the term xe2x80x9cactivityxe2x80x9d refers to a variety of measurable indicia suggesting or revealing the extent of a catalytic reaction peformed by an enzyme, binding, either direct or indirect; affecting a response, i.e. having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event. The term xe2x80x9cbetterxe2x80x9d activity, or xe2x80x9cincreasedxe2x80x9d activity, or xe2x80x9csuperiorxe2x80x9d activity, or the like, means an activity compared to the wildtype (and measured under the same conditions) that is at least about 10% higher, preferably at least about 25% higher, more preferably at least about 50% higher, more preferably at least about 75% higher, more preferably at least about 100% higher, more preferably at least about 1.35-fold higher, more preferably at least about 2-fold higher, more preferably at least about 3-fold higher, and most preferably at least about 4-fold higher. Such better, increased, or superior activity may be exhibited at particular desirable conditions of temperature, pressure, solution contents, and the like. Enzymatic activity of a GO enzyme may be measured using Vmax/Km, as described below in Example 1. A variant GO enzyme of the present invention has a Vmax/Km greater than 0.005 xcex94OD405/min mM.
As used herein, the abbreviation in italicized lower case (go) refers to a gene, cDNA, RNA or nucleic acid sequence while the upper case version (GO) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence.
As used herein, the term xe2x80x9cantibodyxe2x80x9d is meant to refer to complete, intact antibodies, and Fab, Fabxe2x80x2, F(ab)2, and other fragments thereof. Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, polyclonal antibodies, chimeric antibodies, and humanized antibodies.
As used herein, the term xe2x80x9cbindingxe2x80x9d means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, Hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.
As used herein, the term xe2x80x9ccompoundxe2x80x9d means any identifiable chemical or molecule, including, but not limited to a small molecule, peptide, protein, sugar, nucleotide, or nucleic acid, and such compound may be natural or synthetic.
As used herein, the term xe2x80x9ccomplementaryxe2x80x9d refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.
As used herein, the term xe2x80x9ccontactingxe2x80x9d means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention. The polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc. Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoded by the nucleic acid, or fragment thereof.
As used herein, the phrase xe2x80x9chomologous nucleotide sequence,xe2x80x9d or xe2x80x9chomologous amino acid sequence,xe2x80x9d or variations thereof, refers to sequences characterised by a homology, at the nucleotide level or amino acid level, of at least the specified percentage. Homologous nucleotide sequences include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding other known wildtype go genes. Homologous amino acid sequences include those amino acid sequences which encode conservative amino acid substitutions, as well as polypeptides having neuropeptide binding and/or signalling activity. A homologous amino acid sequence does not, however, include the amino acid sequence encoding other known wildtype GOs. Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein by reference in its entirety).
As used herein, the term xe2x80x9cisolatedxe2x80x9d nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
As used herein, the terms xe2x80x9cmodulatesxe2x80x9d or xe2x80x9cmodifiesxe2x80x9d means an increase or decrease in the amount, quality, or effect of a particular activity or protein.
As used herein, the term xe2x80x9coligonucleotidexe2x80x9d refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may be used as probes.
As used herein, the term xe2x80x9cprobexe2x80x9d refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.
By xe2x80x9camplificationxe2x80x9d it is meant increased numbers of DNA or RNA in a cell compared with normal cells. xe2x80x9cAmplificationxe2x80x9d as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA. In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, compared to the basal level.
As used herein, the phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d or xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes, primers or oligonucleotides (e.g. 10 to 50 nucleotides) and at least about 60xc2x0 C. for longer probes, primers or oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
The amino acid sequences are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. The nucleotide sequences are presented by a single strand only, in the 5xe2x80x2 to 3xe2x80x2 direction, from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letters code, or by the accepted single letter code.
xe2x80x9cVariantxe2x80x9d GO (or vGO) enzymes are those which have a substitution of at least one amino acid, and have superior enzymatic activity and/or thermostability when compared with the wildtype enzyme. The enzymatic activity may be improved with regard to the substrate methyl galactose, but alternatively may be improved with regard to other substrates. The particular substitutions are described by indicating the one letter code for the wildtype amino acid, followed by the amino acid position, followed by the substituted amino acid in the variant. The position number is determined counting the first amino acid of the mature sequence, the mature sequence being defined by the crystal structure as number 1, with each consecutive amino acid numbered consecutively. The pre and prosequences are not included in the mature sequence. For example, the V494A variant describes a variant GO having the wildtype""s valine at position 494 substituted with alanine. In cases where the wildtype""s amino acid may be replaced with one of two or more amino acids, the variants are described by adding the additional single letter codes preceded by a slash (/); e.g., Y436N/H indicates a valine at position 494 substituted by either asparagine or histidine. Thus two distinct variants are described in this example, namely Y436N and Y436H.
A polynucleotide of the invention must have a mutation which renders at least one codon xe2x80x9cnondegeneratexe2x80x9d, i.e., the codon must encode a different amino acid than the unmutated codon.
Genomic DNA of the invention comprises the protein coding region for a polypeptide of the invention and is also intended to include allelic variants thereof. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or xe2x80x9cspliced out.xe2x80x9d RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a GO polypeptide, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts. Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation). While allelic variants of wildtype GO are not encompassed by the present invention, those allelic variants which have been mutated and which encode variant GOs of the invention are within the scope of the invention.
The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding vGO (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA).
The wildtype GO nucleotide sequence is set forth in the following SEQ ID NO: I (gaoA, Genbank Accession No. M86819):
The corresponding amino acid sequence for the mature wildtype GO is set forth in the following SEQ ID NO: 2.
The sequence for the precursor protein, which includes the signal sequence and prosequence, may be found in Genbank Accession No. Q01745.
Several preferred DNA sequences of a mgo encoding a vGO polypeptide of the invention having superior enzymatic activity compared to wildtype are those which encode a variant GO having a substitution at any of positions C383, Y436, V494, and Q63. More preferably, the preferred polynucleotide encodes a variant GO having more than one of these preferred substitutions. More preferably, the polynucleotide encodes a variant GO having at least one of the substitutions selected from the group consisting of C383S, V494A, Q63K, and either Y436N or Y436H, more preferably at least two of those substitutions, more preferably at least three of those substitutions. A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the xe2x80x9cnon-coding strandxe2x80x9d or xe2x80x9ccomplementxe2x80x9d) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA. These preferred examples are illustrative; the invention embraces other variants which have additional substitutions, and the polynucleotides that encode such variants, provided such variants have superior activity.
Several preferred DNA sequences of a mgo encoding a vGO polypeptide of the invention having superior enzymatic activity compared to wildtype are those which encode a variant GO having a substitution at any of positions G195, S553, G6, Q238, K342, N427, and Q63. More preferably, the preferred polynucleotide encodes a variant GO having more than one of these preferred substitutions. More preferably, the polynucleotide encodes a variant GO having at least one of the substitutions selected from the group consisting of S553C, G6R, Q238L, K342E, N427T, Q63K, and either G195A or G195E, more preferably at least two of those substitutions, more preferably at least three of those substitutions. In a preferred embodiment of the invention, the polynucleotide encodes a variant GO having at least one substitution conferring superior enzymatic activity and at least one substitution conferring superior thermostability. A preferred DNA of the invention comprises a double stranded molecule along with the complementary molecule (the xe2x80x9cnon-coding strandxe2x80x9d or xe2x80x9ccomplementxe2x80x9d) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA. These preferred examples are illustrative; the invention embraces other variants which have additional substitutions, and the polynucleotides that encode such variants, provided such variants have superior thermostability. Preferred polypeptides include all those encoded by the preferred DNA sequences as described above.
The invention further embraces species homologs of the mgo DNA. For example, species homologs have been found in Gibberella fujikuroi, Polyporus circinatus, Arabidopsis thaliana (CAB65567), and Streptomyces coelicolor A3(2) (CAB41193), among others. Species homologs, sometimes referred to as xe2x80x9corthologs,xe2x80x9d in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with DNA of the invention. Generally, percent sequence xe2x80x9chomologyxe2x80x9d with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the preferred polynucleotides discussed above, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related vGO polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR). Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to vGO and structurally related polypeptides sharing one or more biological, enzymatic, immunological, and/or physical properties of vGO. Non-human species genes encoding proteins homologous to vGO can also be identified by Southern and/or PCR analysis. Knowledge of the sequence of a human mgo DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding vGO expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express vGO.
The disclosure herein of a full-length polynucleotide encoding a vGO polypeptide makes readily available to the worker of ordinary skill in the art every possible fragment of the full length polynucleotide which contains a mutation encoding the variant amino acid. The invention therefore provides fragments of vGO-encoding polynucleotides comprising at least 14, and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding vGO, provided that the fragment contains the mutated nucleotide codon. Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also may include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides (e.g., the wildtype), and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.
Fragment polynucleotides are particularly useful as probes for detection of full-length or fragment mgo polynucleotides. One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding vGO, or used to detect variations in a polynucleotide sequence encoding vGO.
The invention also embraces DNAs encoding vGO polypeptides that hybridize under moderately stringent or high stringency conditions to the non-coding strand, or complement, of the preferred polynucleotides set forth above.
Exemplary highly stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60xc2x0 C. in a wash solution comprising 0.1xc3x97 SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley and Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.
Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided. Expression constructs wherein vGO-encoding polynucleotides are operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided. Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and are typically selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Preferred constructs of the invention also include sequences necessary for replication in a host cell.
Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify a vGO-encoding polynucleotide sequence.
According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner which permits expression of the encoded vGO polypeptide. Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector. Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.
Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with a vGO of the present invention. Host cells of the invention are also useful in methods for the large-scale production of vGO polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like. Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
Knowledge of mgo DNA sequences allows for modification of cells to permit, or increase, expression of endogenous vGO. Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring mgo promoter with all or part of a heterologous promoter so that the cells express vGO at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous vGO encoding sequences. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the mgo coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the mgo coding sequences in the cells.
The invention also provides purified and isolated vGO polypeptides encoded by a polynucleotide of the invention. Presently preferred are vGO polypeptides comprising at least one substitution at any of the positions S553, G6, Q238, K342, N427, Q63, and G195. More preferably, the vGO enzyme has a combination of substitutions, and more preferably the substitutions are selected from the group consisting of S553C, G6R, Q238L, K342E, N427T, Q63K, and either G195A or G195E.
The invention also embraces polypeptides that have at least 99%,at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% identity and/or homology to the preferred polypeptide of the invention, provided that the sequence has at least the amino acid variations present in any of the preferred polypeptides set forth above. Percent amino acid sequence xe2x80x9cidentityxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the vGO sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence xe2x80x9chomologyxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the vGO sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.
In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment (Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference).
Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of vGO polypeptides are embraced by the invention.
The invention also embraces variant (or analog) vGO polypeptides. In one example, insertion variants are provided wherein one or more amino acid residues supplement a vGO amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the vGO amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.
Insertion variants include vGO polypeptides wherein one or more amino acid residues are added to a vGO amino acid sequence, or to an enzymatically active fragment thereof.
Other products of the invention also include mature vGO products, i.e., vGO products wherein leader or signal sequences are removed, with additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins. vGO products with an additional methionine residue at position xe2x88x921 (Metxe2x88x921-vGO) are contemplated, as are variants with additional methionine and lysine residues at positions xe2x88x922 and xe2x88x921 (Metxe2x88x922-Lysxe2x88x921-vGO). Variants of vGO with additional Met, Met-Lys, Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.
The invention also embraces vGO variants having additional amino acid residues which result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position xe2x88x921 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated.
Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of vGO is/are fused to another polypeptide.
In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a vGO polypeptide are removed. Deletions can be effected at one or both termini of the vGO polypeptide, or with removal of one or more non-terminal amino acid residues of vGO. Deletion variants, therefore, include all fragments of a vGO polypeptide.
The invention thus embraces polypeptide fragments of the preferred vGO polypeptides set forth above, wherein the fragments maintain enzymatic properties of a vGO polypeptide, provided the fragments contain the substituted amino acid(s) distinguishing the vGO polypeptide from the wildtype. Such fragments comprising at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of are comprehended by the invention. Preferred polypeptide fragments display antigenic properties unique to, or specific for, human vGO and its allelic and species homologs. Fragments of the invention having the desired enzymatic properties can be prepared by any of the methods well known and routinely practiced in the art.
In still another embodiment, the invention provides substitution variants of vGO polypeptides. Substitution variants include those polypeptides wherein one or more amino acid residues of a vGO polypeptide are removed and replaced with alternative residues, provided the substitution variants contain the variant amino acid(s) distinguishing the vGO polypeptide from the wildtype. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables 1, 2, or 3 below.
Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 1 (from WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.
Alternatively, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77) as set out in Table 2, immediately below.
As still another alternative, exemplary conservative substitutions are set out in Table 3, below.
It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells. Similarly, the invention further embraces vGO polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. Variants that display enzymatic properties of vGO and are expressed at higher levels are also contemplated.
In a related embodiment, the present invention provides compositions comprising purified polypeptides of the invention. Preferred compositions comprise, in addition to the polypeptide of the invention, an acceptable liquid, semisolid, or solid diluent that serves as a vehicle, excipient, or medium. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.
With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode vGOs from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., xe2x80x9cMolecular cloning: a laboratory manualxe2x80x9d, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference in its entirety.
A nucleic acid molecule comprising any of the vGO nucleotide sequences described above may be obtained using any means of mutagenesis performed on the wildtype go sequence. Such methods include error prone PCR, use of mutagenic strains such as the XL1-Red mutator strain of E. coli (Stratagene Inc.), use of random mutagenesis methods involving mutagenic chemicals such as ethyl-methyl sulfonate (EMS) or involving irradiation by UV light or other radiations of higher or lower energy, combinatorial cassette mutagenesis (Delagrave, et al., (1993), Bio/Technology, 10, 1548-52), site-directed mutagenesis, mutagenesis by PCR involving the incorporation of one or more primers encoding mutations, mutagenesis by DNA shuffling (e.g., Stemmer, 1994, Nature, 370:389, and other closely related methods), and mutagenesis by any PCR method.
A nucleic acid molecule comprising any of the vGO nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein. See U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis. PCR provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotides probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art. Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger), which is incorporated herein by reference in its entirety.
Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above. Vectors are used herein either to amplify DNA or RNA encoding vGO and/or to express DNA which encodes vGO. Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses. Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Other expression vectors include, but are not limited to, pSPORT vectors, pGEM vectors (Promega), pPROEXvectors (LTI, Bethesda, Md.), Bluescript vectors (Stratagene), pQE vectors (Qiagen), pSE420 (Invitrogen), and pYES2 (Invitrogen).
Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding vGO is operably linked or connected to suitable control sequences capable of effecting the expression of the vGO in a suitable host. DNA regions are operably linked or connected when they are functionally related to each other. For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence. Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding, and sequences which control the termination of transcription and translation.
Preferred vectors preferably contain a promoter that is recognised by the host organism. The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1980), which is incorporated herein by reference in its entirety); the trp, recA, heat shock, and lacz promoters of E. coli and the SV40 early promoter (Benoist, et al. Nature, 1981, 290, 304-310, which is incorporated herein by reference in its entirety). Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.
Additional regulatory sequences can also be included in preferred vectors. Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgamo of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda. The Shine-Dalgarno sequence may be directly followed by DNA encoding vGO and result in the expression of the mature vGO protein.
Moreover, suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells. The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.
An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and vGO DNA. An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat. No. 4,399,216).
Nucleotide sequences encoding vGO may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al., Mol. Cell. Biol., 1983, 3, 280, Cosman et al., Mol. Immunol., 1986, 23, 935, Cosman et al., Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.
Another embodiment of the present invention is directed to transformed host cells having an expression vector comprising any of the nucleic acid molecules described above. Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell. Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.
If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence. Preferably, eukaryotic cells are cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973), which is incorporated herein by reference in its entirety).
In addition, a yeast host may be employed as a host cell. Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluyveromyces. Preferred yeast hosts are S. cerevisiae and P. pastoris. Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Shuttle vectors for replication in both yeast and E. coli are also included herein.
Alternatively, insect cells may be used as host cells. In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O""Rielly et al. (Eds.), W. H. Freeman and Company, New York, 1992, and U.S. Pat. No. 4,879,236, each of which is incorporated herein by reference in its entirety). In addition, the MAXBAC(trademark) complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells.
Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for vGO or fragments thereof. Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fabxe2x80x2, F(abxe2x80x2)2, and FV, are also provided by the invention. The term xe2x80x9cspecific for,xe2x80x9d when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind vGO polypeptides exclusively (i.e., are able to distinguish vGO polypeptides from other known vGO polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between vGO and such polypeptides). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the vGO polypeptides of the invention are also contemplated, provided that the antibodies are specific for vGO polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
Non-human antibodies may be humanized by any of the methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity. Antibodies of the invention are useful for, e.g., detecting or quantifying vGO, as well as purifying vGO. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is imnuunospecific.
In other embodiments of the invention, the polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins. Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypetides are used to capture interacting molecules. For example, molecules are incubated with the labeled polypeptides, washed to removed unbound polypeptides, and the polypeptide complex is quantified. Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.
Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors of GO, such as 6-thio-methyl-galactose identified as an inhibitor by Wachter et al., 1996, Biochemistry 35:14425 -14435, and others. In one embodiment of affinity purification, a polypeptide is covalently coupled to a chromatography column. Cells and their membranes are extracted, and various cellular subcomponents are passed over the column. Molecules bind to the column by virtue of their affinity to the polypeptide. The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library.
The vGO enzyme may be used to oxidize compounds, such as, for example, hydroxyl containing compounds, provided that the group to be oxidized is available (e.g., sterically) for the enzyme to carry out its oxidation function. A group available to the enzyme in this regard is referred to herein as free of steric hindrance. Steric factors involved in the GO enzyme""s action are discussed generally in Cathmann et al., 1981, Biochem. Biophys. Res. Commun. 103:68-76, the disclosure of which is hereby incorporated by reference in its entirety. Examples of compounds for which vGO may be used to perform oxidation, and wherein the vGO may have improved enzymatic activity, include, but are not limited to, galactose, lactose, raffinose, dihydroxyacetone, diethylene glycol, ethanol and other primary alcohols, and guaran gums, D-galacto-hexodialdose, dihydroxyacetone, 3-hydroxy-2-oxo-propionaldehyde, glycerol, S(xe2x88x92)-glyceraldehyde, 6xe2x80x3-carboxyraffinose, methyl-alpha-D-galactopyranose, methyl-beta-D-galactopyranose, major glycolipid of human red cells, D-talose, 3-halo-1,2-propane-diols, GM1 ganglioside, D-galactosamine, melibiose, stachyose, desialyated glycoproteins (e.g., fetuin, mucin), N-acetyl-D-galactosamine, isopropyl-beta-D-thiogalactosylpyranoside, beta-thiodigalactoside, melibiitol, melibionic acid, 1,5-anhydrogalactitol, planteose, 2-glycerol-alpha-D-galactopyranoside, galactobiose, beta-D-galactopyranosyl, beta-D-galactopyranosyl, D-glucose, methyl-beta-D-thiogalactosylpyranoside, and the like. The GO enzyme has also been characterized as possessing activity as a superoxide dismutase (Cleveland et al., 1974, Biochim. Biophys. Acta 341:517-523).
For example, the oxidation of guar may be performed on a 1% guar mixture in pH 7 phosphate buffer containing galactose oxidase, catalase, and horse radish peroxidase. The reaction may be run at 26xc2x0 C. with constant mixing and sparging with air for approximately 3 hours. Suitable reaction conditions are set forth, for example, in U.S. Pat. No. 6,022,717, the disclosure of which is hereby incorporated herein by reference in its entirety.
The oxidized compounds resulting from interaction with the variant GO enzymes of the invention are useful in a wide variety of applications and chemical processes that will be readily apparent to those of skill in the art. Oxidized guar, for example, may be used in papermaking processes, such as those described generally in Smook, Handbook for Pulp and Paper Technologists (Canadian Pulp and Paper Assn. 1982), which is hereby incorporated by reference in its entirety.
Using the variants of the invention, one of skill in the art may more efficiently oxidize guar gum for use in, for example, a method of making paper. The variant enzyme is isolated in sufficient quantity, then used to oxidize guar gum, and the oxidized guar is added to the paper pulp during the paper-making process. More detailed descriptions of such papermaking processes may be found in U.S. Pat. No. 6,022,717.
There are many other possible uses of the variants of the invention, including, but not limited to, generation of H2O2 in situ; enzymatic synthesis of other aldehydes; pulp biobleaching; the use of galactose oxidase-Schiff""s reagent for early detection and prognosis in human colorectal adenocarcinoma (e.g., Carter et al., Clin Cancer Res 1997 September; 3(9):1479-89); and the use of galactose oxidase-glucan binding domain fusion proteins as targeting inhibitors of dental plaque bacteria (Lis and Kurimitsu. Antimicrob Agents Chemother 1997 May; 41(5):999-1003). Those of skill in the art will readily appreciate the many uses to which a galactose oxidase enzyme may be put.
Additional features of the invention will be apparent from the following Examples. Example 1 is actual, while the remaining Examples are prophetic.

Variant galactose oxidase, nucleic acid encoding same, and methods of using same


A wired line connecting an existing fixed station and a wired telephone network is divided into a plurality of channels on a time division multiplex basis for a digital signal, at least one of the plurality of channels is allocated for a public mobile station, a spatial radio link established between the fixed station and a specific mobile station exclusively connected only to the fixed station is divided into a plurality of time division slots, and at least one of the time slots is used for a public portable radio telephone set.

1. Field of the Invention
The present invention relates to a radio communication system, and more particularly, to a realization of a multiplicity of fixed stations having small spatial radio zones for radio connection of mobile stations.
2. Description of the Related Art
As one of the prior art radio communication systems of the type referred to, there is a portable radio telephone system of a cellular type which arrangement is schematically shown in FIG. 18.
In FIG. 18, a portable telephone control station 201 is connected at its one side to a wired telephone network 202 and also connected at the other side to radio base stations 203-1 to 203-3 as master stations through respective wired lines 206-1 to 206-3. These radio base stations 203-1 to 203-3 have respective spatial radio zones 204-1 to 204-3. When a portable radio telephone set 205 as a mobile station is located within the spatial radio zone 204-2, the portable radio telephone set 205 can communicate with the radio base station 203-2 through the spatial radio circuit and further can communicate with the wired telephone network 202 through the portable telephone control station 201.
If the portable radio telephone set 205 issues a calling signal, then the calling signal issued from the portable radio telephone set 205 is received at any of the respective radio base stations 203-1 to 203-3 even when the portable radio telephone set 205 is located within any of the spatial radio zones 204-1 to 204-3, so that interconnection control between the portable radio telephone set 205 and the wired telephone network 202 is carried out and a communication is started.
FIG. 19 is a block diagram of an internal arrangement of the portable radio telephone set 205, and FIG. 20 is a sequential chart for explaining the brief operation of the portable radio telephone system when a calling signal is issued from the portable radio telephone set.
Explanation will be made as to the case where a calling signal is issued from the portable radio telephone set 205.
First of all, when a hook switch (not shown) of the portable radio telephone set 205 is turned ON, a controller 259 of the portable radio telephone set 205 detects the off-hook state, sets a synthesizer 258 at a frequency of a control channel, and applies to a transmitter 252 a calling signal A including an identification number of the portable radio telephone set 205. And a modulation output of the transmitter 252 is amplified at a power amplifier 253 and then applied through an antenna duplexer 254 to an antenna 255, whereby the calling signal A is transmitted from the antenna.
The radio base station 203-2, when receiving the calling signal A from the portable radio telephone set 205, transmits a calling signal B including identification numbers of the radio base station 203-2 and the portable radio telephone set 205 through the wired line 206-2 to the portable telephone control station 201.
The portable telephone control station 201, when determining that the received identification numbers are for use in its own portable radio telephone system, returns to the radio base station 203-2 a calling answer signal C including the designation of a speech channel while the control station 201 transmits a calling signal D to the wired telephone network 202.
The radio base station 203-2, when receiving the calling answer signal C, transmits a calling answer signal E to the portable radio telephone set 205 through the control channel.
In the portable radio telephone set 205, an input received at the antenna 255 is demodulated at a receiver 256 to form the calling answer signal E which is then applied to the controller 259. This causes a display (not shown) of the portable radio telephone set 205 to indicate thereon a message telling the user of the portable radio telephone set 205 that the user can dial.
Under such a condition, when the user of the portable radio telephone set 205 depresses a dial key (not shown) mounted thereon to enter a desired dial number, this causes the controller 259 to apply a dialing signal F indicative of the dial number to the transmitter 252 to transmit the dialing signal F from the antenna 255.
The radio base station 203-2, when receiving the dialing signal F, transmits a dialing signal G indicative of the dial number to the wired telephone network 202 through the wired line 206-2 and the portable telephone control station 201. As a result, a party telephone set as speech party corresponding to the dial number is called so that, when the speech party answers the calling, the speech can be started. A down voice signal from the wired telephone network 202 is transmitted through the portable telephone control station 201, radio base station 203-2 and a spatial down voice circuit to the portable radio telephone set 205. On the other hand, an up voice signal transmitted from the portable radio telephone set 205 is transmitted through a spatial up voice circuit, the radio base station 203-2 and the portable telephone control station 201 to the wired telephone network 202.
In such a portable radio telephone system, the major problem is the capacity for accommodating the portable radio telephone sets within radio zones. In other words, with the prior art cellular type portable radio telephone system, since the transmission output of the portable radio telephone set is as large as several watts, it has been necessary to set a large distance among each of the radio base stations in order to avoid any interference between the portable radio telephone set and the radio base station using the same frequency. Thus, the number of portable radio telephone sets to be accommodated within the unit area per the entire frequency band allocated to the portable radio telephone system must inevitably be small. This has led disadvantageously to a poor frequency use efficiency.
Two methods for improving the capacity for accommodating portable radio telephone sets have been recently proposed. One method is to set small the transmission outputs of the radio base station and the portable radio telephone set; while the other method is to digitize a voice signal to employ a time division multiple access (TDMA) communication system. When the transmission outputs of the radio base station and portable radio telephone set are set small, that is, when it is desired to secure the same service area as the portable radio telephone system having a large spatial radio zone, this requires the number of radio base stations to be increased, involving an economical problem. Further, unless such a large number of radio base stations are not installed, there occurs such a problem that an unserviceable area is created. In addition, when the number of radio base stations is thoughtlessly increased to solve the aforementioned problems, this also involves other problems such as a scene appearance problem, locations for installation of the system, and in particular, the necessity of an AC power source.
When a voice signal is digitized to employ the time division multiplex access communication system, the frequency band is divided into a predetermined number of time slots as shown in FIG. 21, for example, and speech channels and the time slots are managed in a centralized manner by the portable telephone control station as previously explained above.
In the example of FIG. 21, one frame is divided into 6 time slots (SLOT 1 to SLOT 6) to be repetitively used. Since the frequency band per radio channel can be made to be the same, a frequency effective utilization can be 6 times increased. In other words, 6 times the number of portable radio telephone sets used in the prior art can be used.
In this case, the aforementioned calling answer signals C and E each contains the speech channel number and the number of any one of the 6 time slots and the speech is carried out with use of the designated time slot in the designated speech channel.
Meanwhile, the portable radio telephone system based on the time division multiplex access communication system can digitize a voice signal and can increase relatively easily the number of portable radio telephone sets to be used, but has a limit of increasing the number of portable radio telephone sets. That is, when the number of divided time slots is increased, this requires high-speed data transmission and thus a wide frequency band, which results in that the frequency use efficiency cannot be increased.
On the other hand, as the transmission output or power is made smaller, the capacity for accommodating portable radio telephone sets increases, the consumption current of the portable radio telephone set also decreases, its battery can last longer, and thus the operating time of the portable radio telephone set can be made longer. However, it is difficult to install an increased number of radio base stations efficiently and economically.


Techniques for implementing series-fed antenna arrays with a variable dielectric waveguide. In one implementation, coupling elements with optional controlled phase shifters are placed adjacent each radiating element of the array. To avoid frequency sensitivity of the resulting array, one or more waveguides have a variable propagation constant. The variable waveguide may use certain materials exhibiting this phenomenon, or may have configurable gaps between layers. Plated-through holes and pins can control the gaps; and/or a 2-D circular or a rectangular travelling wave array of scattering elements can be used as well.

1. Technical Field
This patent relates to series-fed phased array antennas and in particular to a coupler disposed between the radiating antenna elements of the array and a waveguide having an adjustable wave propagation constant.
2. Background Art
Phased array antennas have many applications in radio broadcast, military, space, radar, sonar, weather satellite, optical and other communication systems. A phased array is an array of radiating elements where the relative phases of respective signals feeding the elements may be varied. As a result, the radiation pattern of the array can be reinforced in a desired direction and suppressed in undesired directions. The relative amplitudes of the signals radiated by the individual elements, through constructive and destructive interference effects, determines the effective radiation pattern. A phased array may be designed to point continuously in a fixed direction, or to scan rapidly in azimuth or elevation.
There are several different ways to feed the elements of a phased array. In a series-fed arrangement, the radiating elements are placed in series, progressively farther and farther away from a feed point. Series-fed arrays are thus simpler to construct than parallel arrays. On the other hand, parallel arrays typically require one feed for each element and a power dividing/combining arrangement.
However, series fed arrays are typically frequency sensitive therefore leading to bandwidth constraints. This is because when the operational frequency is changed, the phase between the radiating elements changes proportionally to the length of the feedline section. As a result the beam in a standard series-fed array tilts in a nonlinear manner.


Disclosed is a method and apparatus for reducing communication system downtime when enabling cryptographic operation of a cryptographic system of the communication system where the cryptographic system includes a first cryptographic device operatively coupled to a plurality of second cryptographic devices via a communication network of the communication system. The method includes causing a pass-through mode of the second cryptographic devices to be suspended, sequentially determining a state of each of the second cryptographic devices, causing the second cryptographic devices and the first cryptographic device to substantially simultaneously operate in a secure mode if each of the second cryptographic devices is determined to have a first state, and causing the second cryptographic devices and the first cryptographic device to operate in the pass-through mode if at least one of the plurality of second cryptographic devices is determined to have a second state.

The present invention generally relates to cryptographic systems, and more specifically, to a method and apparatus for reducing communication system downtime when configuring a cryptographic system of the communication system.
Supervisory Control and Data Acquisition (SCADA) systems are used in virtually every industry, but especially in utility industries such as gas delivery, electric power, sewage treatment, water supply, transportation, etc. A SCADA system is one type of communication system and includes, among other things, a control center having an operator display/control panel and a SCADA master operatively coupled to the operator display/control panel. A typical SCADA system is configured to measure key operating aspects of a process or system, and then to transmit via a SCADA communication network, the associated measurement data to a central control center. Operators, either human or machine at the control center, make decisions based on the measurement data. The SCADA system is also configured to transmit commands from the operators to the process or system via the SCADA communication network.
As is known, the SCADA communication network may include one of any number of suitable communication network links, depending on the process or system monitored by the SCADA system. For example, the SCADA communication network may include radio, analog and/or digital microwave links, fiber optic links, analog or digital modems on utility owned or leased telephone circuit links, or even the public switched telephone network.
In the electric utility industry a SCADA system may be configured to measure the voltages associated with a power system substation bus (i.e., bus voltages), to measure the current coming into the bus from a power transmission line (i.e., a line current), and to measure the status or position of numerous switches in the substation. The status measurements may include indications of circuit breaker positions and electrical power routing switch positions (e.g., open position, closed position). The SCADA system may also be configured to transmit the current, voltage and switch position measurements to a central control center (CC) via a SCADA communication network for review by an operator. The operator can then make decisions such as closing a circuit breaker to enable additional electric power to a particular load. In that case, a command from the operator delivered via the SCADA communication network results in closure of the circuit breaker.
"Intelligent" devices of the SCADA system that measure the current, voltage and switch positions, and that cause actions to be taken based on commands from the operator are often referred to as intelligent electronic devices (IEDs). The IEDs may include an electric power meter, a programmable controller, a Remote Terminal Unit (RTU), a communications processor, a protective relay, or any number of other suitable intelligent devices configured to take measurements, transmit those measurements over the SCADA communication network to the SCADA master and the operator display/control panel, and respond to commands sent via the operator display/control panel over the SCADA communication network.
As previously mentioned, the SCADA communication network may include any one of a number of suitable communication network links. A typical SCADA communication network may also span long distances of many miles. Unfortunately, due to their sheer expanse, SCADA communication networks are vulnerable to electronic intrusions thereby putting associated IEDs and other SCADA system components at risk for compromise by an eavesdropper (e.g., adversary, attacker, interceptor, interloper, intruder, opponent, enemy). For example, if an eavesdropper gains access to a telephone circuit used to transmit switch position measurements from a power system substation to a CC and used to transmit circuit breaker control commands from the CC to the substation, the eavesdropper could launch numerous attacks on the power system. Such an attack may include altering settings on a protective relay thereby rendering the relay useless in the event of a short circuit, IED damage, operator confusion causing unnecessary power system blackouts, etc. Further, for systems other than power systems, the eavesdropper may cause havoc to any SCADA-monitored critical infrastructure including natural gas delivery systems, transportation or communications systems, waste water treatment and fresh water delivery, etc. The US government has recognized this growing threat. In a report created by the U.S. Department of Energy titled "21 Steps to Improve Cyber Security of SCADA communication networks" published jointly by the President's Critical Infrastructure Protection Board, and the Office of Energy Assurance, the authors concluded:                "Supervisory control and data acquisition (SCADA) networks contain computers and applications that perform key functions in providing essential services and commodities (e.g., electricity, natural gas, gasoline, water, waste treatment, transportation) to all Americans. As such, they are part of the nation's critical infrastructure and require protection from a variety of threats that exists in cyber space today. By allowing the collection and analysis of data and control of equipment, such as pumps and valves from remote locations, SCADA communication networks provide great efficiency and are widely used. However, they also present a security risk: SCADA communication networks were initially designed to maximize functionality, with little attention paid to security. As a result, performance, reliability, flexibility and safety of distributed control/SCADA systems are robust, while the security of these systems is often weak. This makes some SCADA communication networks potentially vulnerable to disruption of service, process redirection or manipulation of operational data that could result in public safety concerns and/or serious disruptions to the nation's critical infrastructure. Action is required by all organizations, government or commercial, to secure their SCADA communication networks as part of the effort to adequately protect the nation's critical infrastructure".
To address the SCADA communication network security issue, numerous types of cryptographic devices are used to encrypt, decrypt and authenticate the data transmitted by a SCADA communication network. Unfortunately, current cryptographic devices require manual installation, manual cryptographic setting changes and manual commissioning; a time consuming effort for a typical SCADA communication network spanning many miles. As a result, IEDs in need of cryptographic protection remain unprotected for unacceptable time periods until all of the cryptographic devices associated with the individual IEDs and the SCADA master have been installed, commissioned or had settings changed. As each cryptographic device is installed, the SCADA master loses communication with the IED's connected to the SCADA communication network segment associated with that cryptographic device. As installation of new cryptographic devices progress, the SCADA master loses communication with more of the IEDs until all cryptographic devices have been installed, including the cryptographic device for the SCADA master. Such a lack of complete SCADA communications may continue for days or even weeks, depending on how long it takes an operator(s) to visit all of the sites of the SCADA system 10 requiring cryptographic device installation.
Moreover, if the cryptographic device is installed on the SCADA master first, then the SCADA master will lose communications with all equipment on the SCADA communication network until cryptographic devices are installed at the various IEDs. The best a cryptographic device installer can do is to install cryptographic devices at about half of the IEDs, and then install the cryptographic device at the SCADA master. The SCADA master will lose communications with the half of the equipment not connected to cryptographic devices until the installer completes installing cryptographic devices at all of the intended sites.
When installed, each of the cryptographic devices may be manually placed in a "pass-through mode", making the cryptographic device transparent to the SCADA communication network. Unlike encryption/decryption operation or "secure mode operation", a cryptographic device in the pass-though mode performs no encryption, decryption, or authentication functions for data transmitted via the SCADA communication network. Unfortunately, cryptographic devices are placed in and removed from pass-through mode via either a hardware switch or button, or via an electronic command received via a maintenance interface of the cryptographic device. As a result, an installer has to travel from cryptographic device location to cryptographic device location to place the cryptographic devices in, or remove them from, pass-through mode. During that time, the SCADA system remains unprotected from eavesdropper activity.
Similarly, as each installed cryptographic device is undergoing a parameter value change (e.g., an encryption key change, an initialization vector size change, a synchronization mode change, a network architecture parameter change, a max allowable frame length parameter change), the SCADA master loses communication with the SCADA communication with the IEDs connected to the network segment associated with that cryptographic device. As each of the installed cryptographic devices are visited by an operator to change parameter values, the SCADA master loses communication with more of the IEDs until all cryptographic devices have undergone parameter value changes. Again, the lack of complete SCADA communications may continue for days or even weeks, depending on how long it takes an operator(s) to visit all of the sites of the SCADA system 10 requiring parameter value updates or changes. Moreover, if the parameter value(s) of the cryptographic device associated with the SCADA master are updated first, then the SCADA master will lose communications with all equipment on the SCADA communication network until all of the parameters values of the remaining cryptographic devices are similarly updated.

Method and apparatus for reducing communication system downtime when configuring a cryptographic system of the communication system

Schweitzer Engineering Laboratories, Inc.


A canine-derived hemoglobin blood substitute comprising an extract of dog erythrocyte cytoplasm in a pharmaceutically acceptable carrier, the process for preparing the same and uses thereof. The process comprises utilizing membrane molecular phase transitions to induce and reverse a nonporous-porous behavior of erythrocytes, conserving all membrane and cytoskeletal material as well as all cytoplasmic enzymes including methemoglobin-reducing enzymes, yielding large quantities that do not require dialysis to remove potassium, and steps to easily prepare and use the product without extensive processing or chromatographic separation. In contrast to the prior art, the canine-derived blood substitute does not exhibit hypertension, vasoconstriction or loss of blood volume. The homologous product is intended for treatment of diseases or medical conditions of dogs requiring an increase, restoration or supplement in oxygen perfusion without hemodynamic or immunologic restriction.

There are many veterinary medical conditions, for example hemorrhagic hypotension and anaphylactic shock, in which significant blood loss and/or hypotension (abnormally low blood pressure) occur leading to tissue hypoxia. For subjects with such medical conditions, it is desirable and often critical for their survival to stabilize their blood pressure and to increase the amount of oxygen provided to body tissues by their circulatory systems.
Considerable effort has therefore been expended in developing substances which may be used as resuscitation fluids and/or blood plasma expanders for stabilizing blood pressure and which are capable of carrying and delivering oxygen to bodily tissues. While transfusion of whole blood or commercially prepared red blood cells (packed) may stabilize blood pressure and provide the needed oxygen for survival, there are many costs (blood-typing; testing for antigens/antibodies, viruses and bacteria; processing expense, etc.) and risks (adverse effects on hematology or immunology; pathological organisms, etc.) associated with these therapies. Therefore, the research and development of an alternate oxygen-carrying substance is being pursued in many countries for applications in the transfusion marketplace as well as for novel therapies for many disease conditions.
Hemoglobin, the natural respiratory protein of erythrocytes which carries oxygen to body tissues from the lungs and carbon dioxide from the tissues to the lungs, is a potential alternate oxygen-carrying substance. Erythrocytes contain approximately 34 grams of hemoglobin per 100 ml of cells.
Since native hemoglobin is readily oxidized in air, commercially available preparations may comprise up to 75% methemoglobin, with the balance being primarily oxyhemoglobin (Sigma Chemical Company, St. Louis, Mo., 1996). The high methemoglobin content limits the usefulness of these preparations for use in a blood substitute or organ preservative as well as for use as a diagnostic or biochemical reagent simply because of the cost and labor required to convert or maintain methemoglobin at acceptable minimum concentrations.
Various methods have been developed to isolate hemoglobin from erythrocytes. Bonsen et al., U.S. Pat. No. 4,001,401 (issued Jan. 4, 1977) describes purifying hemoglobin by lysing red blood cells with toluene and then filtering the lysate through diatomaceous earth filters followed by dialysis. Simmonds et al., U.S. Pat. No. 4,401,652 (issued Aug. 30, 1983) and Tye et al., U.S. Pat. No. 4,473,494 (issued Sep. 25, 1984) disclose purification methods involving selective precipitation of hemoglobin with ions followed by ultrafiltration or dialysis. Hsia, U.S. Pat. No. 4,925,574 (issued May 15, 1990) describes purification of hemoglobin by affinity chromatography. Sheffield et al., Biotechnology and Applied Biochemistry, 9, 230-238 (1987) describe the dialysis of red blood cells using a hypotonic solution followed by ultrafiltration through hollow fibers. Finally, Rausch et al, U.S. Pat. No. 5,084,558 (issued Jan. 28, 1992) disclose the use of high performance liquid chromatography to isolate hemoglobin from erythrocytes.
The foregoing methods all have shortcomings in that they are time-consuming and labor-intensive, requiring multiple extraction, dialysis or filtration steps, but most importantly all require a costly postpurification reconcentration step to obtain the resulting hemoglobin solution. Additionally, the foregoing methods eliminate redox enzymes responsible in vivo for maintaining the hemoglobin in a "useful" state. In the absence of these enzymes, hemoglobin is oxidized to methemoglobin which is not capable of binding oxygen. Furthermore, the above inventions do not take advantage of dog red cell physiology which, because of the relative absence of intracellular potassium, allows for quicker and cleaner preparation of a blood substitute because no dialysis step is required to remove a potentially toxic potassium ion. In addition, this dialysis step is required in all prior art and, because dialysis removes numerous small molecular weight compounds, will thereby introduce an alteration of the natural components of the cytoplasmic extract. Another advantage of deriving a canine blood substitute from dog erythrocytes is the homologous basis of the product, i.e., there will be less risk of immunological incompatibility with proteins derived from the same species, especially when these proteins are planned for intravenous infusion in large quantities as part of a blood substitute. Immunological effects of heterologous hemoglobin blood substitutes are a central concern because of the inherent antigenicity of hemoglobin (Yoshioka et al., Biochem. J., 234, 441-447 (1986); Garver et al., Biochem. Genet., 13, 743-757 (1975). Furthermore, the prior art has demonstrated amplified antigenicity with heterologous hemoglobin that has been polymerized or cross linked (Chang et al., Biomat. Artif. Cells Immobilization Biotechnol., 20, 611-618 (1992); Viazova et al., Biull Exsp. Biol. Med., 106, 446-448 (1988); Hertzman et al., Int. J. Artif. Organs, 9, 179-182 (1986). Finally, an adverse side effect of hemoglobin blood substitutes is vasoconstriction or hypertension which may be related to a specific impurity or contaminant of the formulation or may be caused by some unique pharmacology or structure of the hemoglobin in the formulation. Whether the hypertension or vasoconstriction is caused by, or is amplified by, heterologous hemoglobin is as yet uncertain. However, recent studies with a bovine hemoglobin blood substitute in dogs, cats and humans all produced similar vasoconstriction or hypertension (Standl et al., Intensive Care Med., 23, 865-872 (1997); Ulatowski et al., Crit. Care Med., 24, 558-565 (1996); Standl et al., Anaesthetist, 46, 763-770 (1996). In view of the above presentation, it becomes immediately apparent that several needs exist in the development of a canine-derived hemoglobin blood substitute: a method of preparation that poses less risk for contamination either by membrane, membrane-associated cytoskeletal material or by extra processing steps currently used in the art, a method that preserves the endogenous or natural enzymes of the erythrocyte including methemoglobin-reducing substances, a method that, in general, has a large yield with less steps and less cost, a method that produces a canine-derived hemoglobin blood substitute that is immunologically safer with less risk of incompatibility and a method that produces a canine-derived hemoglobin blood substitute that is more efficacious without adverse restriction on blood flow and oxygen delivery that will occur with vasoconstriction or hypertension.

Canine-derived hemoglobin blood substitute, the process for preparing same and uses thereof


In a metadata collection system comprising a plurality of content management servers each capable of managing content and content metadata as local content and managing content metadata stored in another content management server as reference content and a metadata collection apparatus connected with the plurality of content management servers to collect metadata from the plurality of content management servers, the metadata collection apparatus collects metadata from the individual content management servers based upon server information held at the content management servers and category identifiers appended to tree structures generated at the content management servers by individually referencing the plurality of content management servers.

1. Field of the Invention
The present invention relates to a metadata collection system, a content management server, a metadata collection apparatus, a metadata collection method and a program.
2. Description of the Related Art
An aspect of the advent of data communication networks in recent years is evident in data exchange among home appliances, computers and other peripheral devices connected through so-called home networks in private homes. The use of a home network, which assures high levels of convenience and comfort to users by allowing various devices to share a data processing function and content to be exchanged among the devices, is expected to become even more prevalent in the future.
The technologies developed to improve the ease of content management by allowing various devices to share content, with identifiers (IDs), each assigned to a specific set of content among a plurality of sets of content that may be exchanged among the devices for identification purposes, which remain unchanged even when the content is transmitted from one device to another, are of particular interest (e.g., see Japanese Patent Application Publication JP-A-2004-234158).

Metadata collection system, content management server, metadata collection apparatus, metadata collection method and program


An upstanding garment hanger comprises a transversely extending support member and a succession of garment support elements depending downwardly from the support member to free ends, at least first and second adjacent ones of the garment support elements defining transversely extending, vertically spaced projections upwardly of the free ends thereof.

U.S. Pat. No. 4,703,878 ("the '878 patent") shows a hanger for underwear which has a transversely extending support member with a hook extending upwardly of the support member for engaging a support rail to suspend the support member. A plurality of garment interior support elements depend downwardly of the support member arcuately to the vertical and have hook formations on ends thereof to entrap an undergarment thereabove. At ends of the support member, the hanger includes downwardly and outwardly extending single garment support elements, i.e., exterior garment support elements, with projections extending outwardly of the exterior garment support elements. A notch or detent is formed in the support member above each of the exterior support elements. Such hanger exterior garment support elements are shown in U.S. Pat. No. Des. 202,240.
The hanger of the '878 patent has facility for the hanging of underwear (briefs) of various waistband sizes. Underwear is hung by applying one end of its waistband to an interior garment support element above the hooked end thereof, drawing the waistband about one of the exterior garment support elements and then applying the other end of the waistband about the other of the exterior garment support elements and then applying the other end of the waistband to another of the interior support elements. Different interior garment support elements are used for differently-sized underwear.
South Africa Patent Application No. 85/7516, which is noted in the '878 patent as the first-filed application counterpart to the application for the '878 patent, describes a similar hanger structure wherein, however, the interior garment support elements depend straightwise from the support member, i.e., the elements are not arcuate.
By way of further background to the subject invention, note is taken of a commercially-known hanger for briefs bearing the legend "PLASTI-FORM-1-800-HANGER-4". This hanger also has garment support elements which extend downwardly and straightwise from the hanger support member, i. e., not arcuately to the vertical. Contrary to the hangers of the '878 patent and the above-noted South African patent application, each of which has hook formations at the lower ends of the garment support elements, the commercially-known hanger has a spring-like member extending continuously with the ends of the garment support elements vertically upwardly toward the support member.
Common to above-discussed hangers is to provide the support member in a downwardly arcuate configuration such that the support member is not seen when the hangers support underwear.