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)

In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.
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PMID:Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene. 766 13

Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a reporter of gene expression.
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PMID:Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli. 815 97

Transcription of the manganese-superoxide dismutase gene (sodA) in Escherichia coli was shown to be activated by manganese. Addition of MnCl2 increased the expression of beta-galactosidase from a sodA::lacZ protein fusion and increased the concentration of mRNA transcribed from sodA+ and sodA::lacZ constructs. The stimulatory affect of manganese on the expression of sodA::lacZ was greatly reduced (i.e., > 90%) in a strain harboring a fur mutation. We also found that manganese was capable of altering DNA topology. These results show that Mn2+ causes activation of sodA transcription.
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PMID:Transcriptional activation of Mn-superoxide dismutase gene (sodA) of Escherichia coli by MnCl2. 824 Dec 58

The biosynthesis of Mn-containing superoxide dismutase is regulated in response to stimuli that affect the redox potential of the cell. To further investigate the mode of regulation of the gene (sodA) encoding this enzyme, cis-acting regulatory mutations in a strain containing a sodA::lacZ gene fusion were studied. The mutant strains expressed beta-galactosidase under anaerobic conditions, whereas the wild-type did not. Furthermore, the mutants were not induced in response to the presence of iron chelator, 2,2'-dipyridyl, or to the redox cycling compound, paraquat. The wild-type, however, did respond to these effectors. In vivo cloning was used to isolate the cis-acting regulatory elements from the mutants (NC4 and NC5). Replacement of the wild-type 5'-regulatory region with either of the mutants' cis-acting regulatory element resulted in the anaerobic expression of active Mn-superoxide dismutase. Sequence and restriction analysis revealed the presence of an IS2 insertion element in the promoter region of one of the mutants (NC5). This insertion caused the displacement of the 5'-regulatory region of sodA and the formation of a functional hybrid promoter consisting of the resident-10 region from sodA and -35 from IS2. The second mutation (from NC4) was similarly analyzed, and an IS5 element was identified. The insertion site of IS5 (in NC4) was 6 bp (5'-TTAATT-3') upstream from the IS2 site (in NC5). Anaerobic expression of sodA in NC4 was lower than in NC5. This difference was almost eliminated in an arc- background, suggesting that the sequence 5'-TTAATT-3' might be essential for negative regulation by ArcA.
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PMID:Characterization of cis-acting regulatory mutations causing anaerobic expression of the sodA gene in Escherichia coli. 838 43

The AMT1 metalloregulatory trans-acting factor from Candida glabrata was found to functionally mimic the ACE1 metalloregulatory trans-acting factor from Saccharomyces cerevisiae in the copper-induced expression of the chromosomal S. cerevisiae metallothionein gene. Plasmid constructs with promoters of various metal-inducible genes fused to the bacterial beta-galactosidase (lacZ) reporter gene were used in S. cerevisiae to evaluate the roles of ACE1 and AMT1 in mediating metal-stimulated expression. Promoters from the S. cerevisiae CUP1 gene and Cu,Zn-superoxide dismutase (SOD1) and from the C. glabrata MT genes MTI, MTIIa, and MTIIb were used. The ACE1 factor was effective in the metalloregulation of the two S. cerevisiae promoters, CUP1 and SOD1, but of only one C. glabrata promoter, MTI. AMT1 was found to be effective in the metalloregulation of all three C. glabrata MT promoters and the two S. cerevisiae promoters tested. The regulation mediated by both ACE1 and AMT1 was copper-dependent and copper-specific. Episomally expressed SWI5, a distinct trans-acting factor of S. cerevisiae, enhanced only the basal expression from promoters. The SWI5 enhancement was not metal dependent. In conclusion, AMT1 and ACE1 are functionally homologous in metal-specific regulation, AMT1 appears to be more promiscuous than ACE1 in this function.
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PMID:Regulation of metallothionein genes by the ACE1 and AMT1 transcription factors. 850 91

Staurosporine (0.03-0.5 microM) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 microM) or the cell cycle inhibitor mimosine (100 microM). To investigate the role of Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D28K, a Ca2+ binding protein, and Cu/ Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a beta-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage-sensitive Ca2+ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1-10 microM) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 microM) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1-10 microM) and N-acetylcysteine (100 microM) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca2+ and reactive oxygen species in staurosprine-induced neuronal apoptosis.
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PMID:Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis. 908 41

Intrastriatal grafting of embryonic dopamine-containing neurons is a promising approach for treating clinical and experimental Parkinson's disease. However, neuropathological analyses of grafted patients and transplanted rats have demonstrated that the survival of grafted dopamine neurons is relatively poor. In the present study, we pursued a strategy of transferring a potentially neuroprotective gene into rat embryonic mesencephalic rat cells in vitro, before grafting them into the denervated striatum of 6-hydroxydopamine-lesioned rats. We performed intrastriatal grafts of embryonic day 14 mesencephalic cells infected with replication-defective adenoviruses bearing either the human copper-zinc superoxide dismutase gene or, as a control, the E. coli lac Z marker gene. The transgenes were expressed in the grafts four days after transplantation and the expression persisted for at least five weeks thereafter. After five weeks postgrafting, there was more extensive functional recovery in the superoxide dismutase group as compared to the control (uninfected cells) and beta-galactosidase groups. The functional recovery was significantly correlated with the number of tyrosine hydroxylase-positive cells in the grafts, although the clear trend to increased survival of the dopamine neurons in the superoxide dismutase grafts did not reach statistical significance. Only a moderate inflammatory reaction was revealed by OX-42 immunostaining in all groups, suggesting that ex vivo gene transfer using adenoviral vectors is a promising method for delivering functional proteins into brain grafts.
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PMID:Intrastriatal grafts of embryonic mesencephalic rat neurons genetically modified using an adenovirus encoding human Cu/Zn superoxide dismutase. 915 52

Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp., the bvg locus. A risA'-'lacZ translational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of beta-galactosidase activity under different environmental conditions suggested that ris is regulated independently of bvg and is optimally expressed at 37 degrees C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetella spp., is required for the expression of acid phosphatase by B. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive and bvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.
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PMID:A second two-component regulatory system of Bordetella bronchiseptica required for bacterial resistance to oxidative stress, production of acid phosphatase, and in vivo persistence. 974 60

Overexpression of the proto-oncogene bcl-2 has been shown to protect a variety of cell types from oxidative and non-oxidative injury, blocking apoptotic and necrotic types of cell death. Retroviral vectors were used to stably overexpress bcl-2 in primary murine astrocyte cultures with more than 95% efficiency. Compared to beta-galactosidase-expressing and uninfected control cells, bcl-2 overexpressing astrocytes suffered < 40% injury after 24 h glucose deprivation, while controls were essentially completely injured. After exposure to 0.2 mM hydrogen peroxide, the bcl-2 overexpressing astrocytes suffered < 40% the injury seen in controls. In contrast, when the cultures were injured by combined oxygen-glucose deprivation, no difference in the extent or time course of injury was found between cells overexpressing bcl-2 and those expressing beta-galactosidase. To investigate one possible mechanism of bcl-2 protection, we measured the levels of glutathione and three antioxidant enzymes. Astrocytes overexpressing bcl-2 had elevated glutathione levels (130-200%), increased superoxide dismutase (170%) and glutathione peroxidase (140%) activities compared with beta-galactosidase-expressing controls. Bcl-2 overexpressing astrocytes suffered less lipid peroxidation after glucose deprivation, as assessed by cis-parinaric acid fluorescence, and demonstrated more rapid removal of hydrogen peroxide from the medium. When glutathione levels were decreased 80% by pretreatment with buthionine sulfoximine, the extent of protection from glucose deprivation of bcl-2 overexpressing cells was decreased by about half. Increased antioxidant defence contributes to protection from glucose deprivation in bcl-2 overexpressing astrocytes.
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PMID:Potentiation of murine astrocyte antioxidant defence by bcl-2: protection in part reflects elevated glutathione levels. 974 79

Parotid and mandibular saliva was obtained from red kangaroos by concurrent acetylcholine isoprenaline stimulation. Salivary proteins were separated by horizontal electrophoresis on either cellulose acetate or starch gels and assessed by specific staining techniques for 23 enzymes commonly found in mammalian tissues and body fluids. Parotid saliva was positive for acid phosphatase, alpha-amylase, carbonic anhydrase, glucose-6-phosphate dehydrogenase, sorbitol dehydrogenase and superoxide dismutase activities. Mandibular saliva was positive for alcohol dehydrogenase in addition to the above six enzymes. The kangaroo salivas lacked activity for alkaline phosphatase, beta-galactosidase and non-specific esterase which occur in saliva from some mammalian species.
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PMID:Enzyme activity in parotid and mandibular saliva from red kangaroos, Macropus rufus. 978 23


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