Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.
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PMID:A method for the sequence analysis of dermatan sulphate. 216 67

The role of glycosphingolipids as adhesion receptors for yeasts was examined. Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, as well as Histoplasma capsulatum and Sporotrichum schenckii (in their yeast phases), bound specifically to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), as measured by overlaying glycosphingolipid chromatograms with 125I-labeled organisms. An unsubstituted galactosyl residue was required for binding, because the yeasts did not bind to glucosylceramide (Glc beta 1-1Cer) derived from lactosylceramide by treatment with beta-galactosidase or to other neutral or acidic glycosphingolipids tested that contained internal lactosyl residues. Interestingly, the yeasts preferentially bound to the upper band of the lactosylceramide doublet in human lung and bovine erythrocytes, suggesting that the ceramide structure also affects binding. Active metabolism of the yeasts was required for binding to lactosylceramide, as binding was maximal in buffer containing glucose and was almost completely abolished in nutrient-deficient medium. C. neoformans also bound to human glioma brain cells grown in monolayers, and this binding was inhibited by liposomes containing lactosylceramide but not by liposomes containing glucosylceramide. Lactosylceramide is a major glycosphingolipid in these cells and the only one to which the yeasts bound. As lactosylceramide is widely distributed in epithelial tissues, this glycosphingolipid may be the receptor for yeast colonization and disseminated disease in humans.
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PMID:Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), a possible adhesion receptor for yeasts. 219 58

An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus. The vector utilizes the Escherichia coli beta-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp 70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter. Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal. The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA. The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of Mr 46,000. Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector. Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells. This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus.
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PMID:Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector. 223 Jul 25

The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.
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PMID:The sialylated oligosaccharides of recombinant bovine lutropin modulate hormone bioactivity. 229 23

We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.
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PMID:A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity. 232 7

The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.
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PMID:A novel phosphate-regulated expression vector in Escherichia coli. 237 33

Human papillomavirus (HPV) type 6b genome contains two large open reading frames (ORFs), designated L1 and L2, in a putative late region. These ORFs are expected to code for viral structural proteins. To examine antigenic properties of a L2 gene product, we constructed two plasmids which contain N-terminal (L2-N) and internal (L2-I) regions of the HPV6b L2 ORF and then each region was expressed in Escherichia coli as a fusion protein with E. coli beta-galactosidase (beta-Gal). Both L2-N/beta-Gal fusion proteins reacted with anti-beta-Gal antibody, but did not react with the antibody prepared against bovine papillomavirus type 1 (BPV1), in contrast with a high reactivity of HPV6b L1-beta-Gal fusion protein with the anti-BPV1 antibody. Antibody raised against the L2-I/beta-Gal protein in a rabbit reacted with viral antigens in the nuclei of cells in superficial epithelium of the condyloma acuminatum tissue, but did not react with the antigens in the bovine papilloma tissue. This antibody recognized a protein from condyloma acuminata which migrates to the position of mol wt 70K-76K on an electrophoresed SDS-polyacrylamide gel. These results suggested that the L2 ORF of HPV6b codes for a capsid protein which is less cross-reactive than the L1 antigen with anti-BPV1 antibody.
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PMID:Expression of the human papillomavirus type 6b L2 open reading frame in Escherichia coli: L2-beta-galactosidase fusion proteins and their antigenic properties. 243 99

Expression of xylose isomerase was repressed in Bacillus subtilis strains W23, 168, and BR151 and could be induced in the presence of xylose. The expression was also glucose repressed in strains 168 and BR151, although this effect was not observed with W23. A xyl-cat fusion gene was constructed on a multicopy plasmid, from which the xyl promoter located on a 366-base-pair (bp) DNA fragment derived from W23 directed the expression of chloramphenicol resistance. The regulation of expression was not very pronounced in this multicopy situation. The xyl promoter is a strong signal for transcription initiation. The 5' sequence of the xyl mRNA was identified by nuclease S1 mapping. The promoter consisted of the -10 sequence TAAGAT, the -35 sequence TTGAAA spaced by 17 bp, and an upstream poly(A) block with 14 As out of 17 bp. To study the regulation, a xyl-lacZ fusion gene was constructed and integrated as a single copy into the amygene of B. subtilis 168. This strain grows blue on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) indicator plates in the presence of xylose and white in the presence of glucose. Quantitatively, the induction of beta-galactosidase by xylose was 100-fold. In the presence of xylose plus glucose, the expression of the indicator gene was repressed to 30% of the fully induced level. About 25 to 60% of the maximal lacZ expression was obtained with this strain when the 366-bp xyl DNA fragment was provided in trans on a multicopy plasmid. This result indicates that repression in the absence of xylose is mediated in trans by a soluble factor which is expressed at a low level in B. subtilis 168. The xylose effect depended on negative regulation. The estimations of mRNA amounts by dot blot analysis showed unambiguously that the induction by xylose occurs at the level of transcription. The possible molecular mechanisms are discussed with respect to the nucleotide sequence of the 366-bp xyl regulatory DNA.
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PMID:Expression of the Bacillus subtilis xyl operon is repressed at the level of transcription and is induced by xylose. 245 11

By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.
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PMID:A versatile phage lambda expression vector system for cloning in Escherichia coli. 246 28

In order to delineate clinical subtypes of GM1 gangliosidosis enzymologically, we prepared galactosyl oligosaccharides from the urine of patients, as substrates, and established the method of the galactosyl oligosaccharide beta-galactosidase assay. Galactosyl oligosaccharides beta-galactosidase activities (nmol/mg protein/20 h) in vitro, using substrates without repeating structures were; type 1, 1.0 +/- 0.5 (n = 6), type 2A, 2.1, type 2B, 3.4 +/- 0.7 (n = 5), type 3, 4.9 +/- 0.2 (n = 2). The activities in vitro using substrates with repeating structures were: type 1, 0.3 +/- 0.2 (n = 5), type 2A, 1.2, type 2B, 2.2 +/- 0.5 (n = 4), type 3, 4.2 +/- 0.3 (n = 2). The activities using substrates with and without repeating structures were affected in the fibroblasts of patients, and the residual activities in each subtype were reduced progressively with the increasing severity of the clinical features. The ratio between activities using substrates without repeating structures and activities using substrates with repeating structures indicated that beta-galactosidase activities toward Gal beta 1- of repeating structures were reduced progressively with the increasing severity of the clinical features. The activities in vivo (pmol/mg protein per 24 h) were: type 1, 11.8 +/- 1.8 (n = 2), type 2A, 24.8, type 2B, 40.0 +/- 9.7 (n = 2), type 3, 63.2. The activities in vivo were affected in the fibroblasts of patients and the residual activities were reduced in proportion to the severity of the clinical features. These differences of residual activities among each subtype make it possible to delineate clinical subtypes enzymologically.
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PMID:Diagnosis of subtypes of GM1 gangliosidosis in vitro and in vivo--using urinary oligosaccharides as substrates. 249 42


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