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)

Cytochrome bd is a respiratory quinol oxidase in Escherichia coli. Besides the structural genes (cydA and cydB) encoding the oxidase complex, the cydD and cydC genes, encoding an ABC-type transporter, are required for assembly of this oxidase. Recently, cysteine has been identified as a substrate (allocrite) that is transported from the cytoplasm by CydDC, but the mechanism of cysteine export to the periplasm and its role there remain unknown. To initiate an understanding of structure-function relationships in CydDC, its membrane topography was analysed by generating protein fusions between random and selected residues in the two polypeptides with both alkaline phosphatase and beta-galactosidase. CydD and CydC are experimentally shown each to have six transmembrane segments, two major cytoplasmic loops and three minor periplasmic loops; both termini of each protein face the cytoplasm. The cydD1 allele is shown to have two point mutations (G319D, G429E) within the ATP-binding domain of CydD; either mutation alone is sufficient to cause loss or severe reduction of cytochrome bd assembly. A comparative sequence analysis prompted the targeting of residues in CydD for site-directed mutational analysis, which identified (i) the 'start' methionine residue, (ii) essential residues in the ATP-binding site (Walker sequence A) and (iii) a duplicated positively charged heptameric motif, R-G/T-L/M-X-T/V-L-R, in CydD cytoplasmic loop II. The replacement of arginines in these motifs with glycines resulted in Cyd- phenotypes; however, activity could be restored at these positions by replacing the glycine with lysine or histidine and hence returning the positive charge. The conservation of these charges in CydD-like proteins indicates functional importance. Evolutionary aspects of bacterial cyd genes are discussed.
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PMID:Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly. 1547 Jan 19

Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an N-linkage following signal peptide cleavage. Most lipoproteins are anchored in the outer membrane, facing the periplasm, but some lipoproteins remain in the plasma membrane, depending on the amino acid at position +2, immediately after the fatty-acylated cysteine. In vitro, the last step in lipoprotein maturation, N-acylation of apolipoproteins by the plasma membrane apolipoprotein N-acyltransferase (Lnt), is necessary for efficient recognition of outer membrane lipoproteins by the Lol system, which transports them from the plasma to the outer membrane (Fukuda, A., Matsuyama, S.-I., Hara, T., Nakayama, J., Nagasawa, H., and Tokuda, H. (2002) J. Biol. Chem. 277, 43512-43518). To study the role of Lnt in vivo, we constructed a conditional lnt mutant of Escherichia coli. The apo-form of peptidoglycan-anchored major lipoprotein (Lpp) and two other outer membrane lipoproteins accumulated in the plasma membrane when lnt expression was reduced. We also found that Lnt is an essential protein in E. coli and that the lethality is partially because of the retention of apoLpp in the plasma membrane. Topology mapping of Lnt with beta-galactosidase and alkaline phosphatase fusions indicated the presence of six membrane-spanning segments. The lnt gene in a mutant of Salmonella enterica displaying thermosensitive Lnt activity (Gupta, S. D., Gan, K., Schmid, M. B., and Wu, H. C. (1993) J. Biol. Chem. 268, 16551-16556) was found to carry a mutation causing a single glutamate to lysine substitution at a highly conserved position in the last predicted periplasmic loop of the protein.
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PMID:Depletion of apolipoprotein N-acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia coli. 1551 25

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine-rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor-beta (TGF-beta). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF-beta transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF-beta caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF-beta and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the proalpha2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial beta-galactosidase gene. Serial injections of CTGF after TGF-beta resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF-beta alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF-beta in comparison with the injection of TGF-beta alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF-beta-induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.
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PMID:Connective tissue growth factor causes persistent proalpha2(I) collagen gene expression induced by transforming growth factor-beta in a mouse fibrosis model. 1560 79

Beta-Galactosidase production by Bifidobacterium longum CCRC 15708, Bifidobacterium longum B6 and Bifidobacterium infantis CCRC 14633 was first examined with B. longum CCRC 15708 showing the highest production of beta-galactosidase and the highest specific activity. Further study with B. longum CCRC 15708 revealed that the highest level of beta-galactosidase was produced with lactose and yeast extract as carbon and nitrogen sources, respectively. Optimal enzyme production occurred at an initial pH of 6.5 and at 37 degrees C. Under these optimum culture conditions, a maximumbeta-galactosidase activity of 18.6 U/ml could be obtained after 16 h of fermentation in a medium contain 4% lactose, 3.5% yeast extract, 0.3% K2HPO4, 0.1% KH2PO4, 0.05% MgSO4.7H2O and 0.03% L-cysteine. The highest transgalactosylation activity was also detected in this culture after 14-16 h of fermentation.
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PMID:Production of beta-galactosidase by Bifidobacteria as influenced by various culture conditions. 1598 5

Reactive oxygen species (ROS) were generated in all oxygen-utilizing organisms. Peroxiredoxin II (Prx II) as one of antioxidant enzymes may play a protective role against the oxidative damage caused by ROS. In order to define the role of Prx II in organismal aging, we evaluated cellular senescence in Prx II(-/-) mouse embryonic fibroblast (MEF). As compared to wild type MEF, cellular senescence was accelerated in Prx II(-/-) MEF. Senescence-associated (SA)-beta-galactosidase (Gal)-positive cell formation was about 30% higher in Prx II(-/-) MEF. N-Acetyl-l-cysteine (NAC) treatment attenuated SA-beta-Gal-positive cell formation. Prx II(-/-) MEF exhibited the higher G2/M (41%) and lower S (1.6%) phase cells as compared to 24% and 7.3% [corrected] in wild type MEF, respectively. A high increase in the p16 and a slight increase in the p21 and p53 levels were detected in PrxII(-/-) MEF cells. The cellular senescence of Prx II(-/-) MEF was correlated with the organismal aging of Prx II(-/-) mouse skin. While extracellular signal-regulated kinase (ERK) and p38 activation was detected in Prx II(-/-) MEF, ERK and c-Jun N-terminal kinase (JNK) activation was detected in Prx II(-/-) skin. These results suggest that Prx II may function as an enzymatic antioxidant to prevent cellular senescence and skin aging.
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PMID:Inhibitory role of peroxiredoxin II (Prx II) on cellular senescence. 1610 12

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P(AdhC) (or P(CopA)) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.
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PMID:NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family. 1613 33

Spodoptera frugiperda Sf-9 insect cells were infected with recombinant Autographa californica nuclear polyhedrosis virus at a low multiplicity of infection (MOI) (0.1), and the effect of dissolved oxygen (DO) on the production of a polyhedrin promoter-driven recombinant protein (beta-galactosidase), intrinsic proteases (carboxyl and cysteine proteases), and the virus was determined. The DO concentrations used in the present study were 45%, 25%, 5%, and 1.3% of air saturation. At 5% DO the cell growth following viral infection was greatest and beta-galactosidase was about 5-fold increased in volumetric yield compared to that at 45% and 25% DO, whereas the growth at 1.3% DO was extremely poor. The virus titer in the medium at 4-8 d post-infection (dpi) was also highest at 5% DO, but the titer was significantly decreased by further increasing the culture time. This was in part attributed to the fact that baculovirus is susceptible to oxidative inactivation under aerobic conditions. The DO dependency of the specific oxygen consumption rate of virus-infected and uninfected Sf-9 cells was expressed by a Monod-type equation. A critical DO, above which the rate of oxygen utilization is not limited by DO, was estimated to be 3.5% of air saturation for virus-infected Sf-9 cells. These results indicated that for a baculovirus-infected Sf-9 insect cell culture of low MOI, the optimal DO was likely to be approximately 5% of air saturation, which is above the critical DO for the infected Sf-9 cells but sufficiently low to reduce the possibility of the oxidative inactivation of virus. For the production of carboxyl and cysteine proteases, the accumulation behavior and concentrations did not significantly vary with DO, except that a peak of cysteine protease activity was observed intracellularly only at 5% DO, coinciding with beta-galactosidase production.
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PMID:Significant increase in recombinant protein production of a virus-infected Sf-9 insect cell culture of low MOI under low dissolved oxygen conditions. 1623 29

A method to attach a fluorochrome sequence-specifically to supercoiled plasmid DNA (pDNA) without perturbing transgene expression would provide an invaluable aid in a variety of applications requiring probes for the intracellular tracking of transfected pDNA. Here we report a method to couple commercially available fluorochromes covalently and sequence-specifically to pDNA using a peptide nucleic acid (PNA) as a linker molecule. The terminal cysteine thiol group on the PNA peptide backbone is reacted with a maleimide moiety on the fluorochrome to produce a fluorescent conjugate which is in turn hybridized to a plasmid expression vector containing an 11-bp target sequence. Spectroscopic evaluation and an electrophoretic mobility shift assay showed that the pDNA hybridized to one PNA-fluorochrome conjugate molecule. The fluorescence signal comigrated with pDNA on acrylamide gels, confirming the stable attachment of the fluorescent conjugate to the pDNA. The utility of one of the conjugates, PNA-Oregon green 488/pCMVbeta-DTS, to probe pDNA transport across the nuclear envelope, a significant barrier to gene transfer, was undertaken using a digitonin-permeabilized HeLa cell assay. The PNA-Oregon green 488/pCMVbeta-DTS conjugate is able to efficiently traverse the nuclear membrane of the permeabilized cells, accumulating in the nuclei within 30 min and reaching maximal levels by 1h. When transfected into HeLa cells, the PNA-Oregon green 488/pCMVbeta-DTS conjugate retained 55% of the native plasmid's biological activity, as determined by a beta-galactosidase assay. Thus, this method allows for the sequence-specific coupling of commercially available fluorochromes to DNA expression vectors while retaining biological function.
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PMID:Nondisruptive, sequence-specific coupling of fluorochromes to plasmid DNA. 1657 51

Two combinatorial glycopeptide libraries were synthesized on solid support via the "split-and-mix" method combined with the ladder synthesis strategy. The O-glycopeptide library contained Gal(beta1-O)Thr, whereas the S-,N-glycopeptide library contained both Gal(beta1-S)Cys and Gal(beta1-N)Asn. In this model study, the two libraries were screened against the fluorescently labeled lectin Ricinus communis agglutinin (RCA120). The screening results showed that both O- and S- or S-,N-glycopeptides were recognized by the lectin with similar amino acid recognition patterns. Surface plasmon resonance interaction studies demonstrated that both the selected S- or S-,N-glycopeptide hits and the O-glycopeptides bound to the lectin with a similar affinity. Structure 19, containing two glycosylated cysteine residues, bound to the receptor with the highest affinity (KA = 3.81 x 10(4) M(-1)), which is comparable to N-acetyllactosamine. Competition assays, in which some selected glycopeptides and methyl beta-d-galactopyranoside competed for the binding site of immobilized RCA120, showed that the glycopeptide-lectin interaction was carbohydrate-specific. Incubation of the O- and S-,N-glycopeptides with beta-galactosidase demonstrated the complete stability of S-,N-glycopeptides toward enzymatic degradation, whereas O-glycopeptides were not completely stable.
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PMID:Affinity determination of ricinus communis agglutinin ligands identified from combinatorial O- and S-,N-glycopeptide libraries. 1709 69

In this study, S-allyl cysteine sulfoxide (SACS) was used to evaluate its preventive effect in isoproterenol (ISO)-induced myocardial ischemia in male Wistar rats. Rats were pretreated with SACS (40 and 80 mg kg(-1)) orally for 5 weeks. After the treatment period, ISO (150 mg kg(-1)) was administered subcutaneously to rats at an interval of 24 h for 2 days. The activities of beta-D-N-acetyl-glucosaminidase, beta-galactosidase, beta-glucosidase, and acid phosphatase increased in serum and heart in ISO-induced rats. In addition, these rats showed a significant (p < 0.05) increase in the activities of beta-glucuronidase and cathepsin-D in serum and heart and a significant (p < 0.05) decrease in their activities in lysosomal fraction of the heart. The activity of Na(+)K(+)-ATPase declined, while those of Ca(2+)- and Mg(2+)-ATPases significantly (p < 0.05) elevated in the heart of ISO-induced rats. Pretreatment with SACS (40 and 80 mg kg(-1)) showed a significant (p < 0.05) effect in all the biochemical parameters studied. The effect at a dose of 80 mg kg(-1) body weight was more effective than that at 40 mg kg(-1) body weight and brought back all the biochemical parameters to near normal levels. Hereby, our study shows the membrane-stabilizing as well as antioxidant effects of SACS in ISO-induced rats.
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PMID:Preventive effect of S-allyl cysteine sulfoxide (alliin) on lysosomal hydrolases and membrane-bound ATPases in isoproterenol-induced myocardial infarction in Wistar rats. 1762 87


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