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)

The effect of amino acid substitution at the P(3) and P(4) subsites on the cleavage activity of a recombinant secretory-type Kex2 protease (Kex2-660) was investigated using recombinant fusion proteins. They were constructed from a truncated Escherichia coli beta-galactosidase (beta G-117S) followed by human parathyroid hormone amino acids 1-34 [hPTH(1-34)] with a junction designed to allow cleavage with Kex2-660. Kex2-660 preferentially cleaved the fusion protein with Arg-His at the P(3)-P(4) subsite, whereas it poorly cleaved the fusion proteins with Val-Asp, Gly-His, Cys-His and Leu-His compared with the original sequence, Val-Lys. As for Asp and Glu at P(4), they precluded the cleavage almost completely. Some of the substitutions were investigated kinetically using the fusion proteins and peptide-substrate mimics corresponding to the 15 amino acids contained in the junction. The substitution Val-Lys to Arg-His resulted in a 2-fold increase in k(cat) and the introduction of Asp at P(4) of the peptide substrate resulted in a large increase in K(m) and a change in the optimum pH. The ordered cleavage of the fusion protein was attained by introducing the two dibasic sites, Arg-His and Asp-His, at P(4)-P(3). The fusion protein was cleaved with Kex2 only at the former site in 3.0 M urea at pH 8.0 and the liberated peptides containing the latter site could be cleaved further with Kex2 at pH 6.5 once purified in urea-free solution. Thus Kex2 exhibited extended substrate specificity beyond P(2)-P(1) in the cleavage of the fusion proteins, although it depended on the reaction conditions.
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PMID:Effect of amino acid substitution at the P(3) and P(4) subsites of fusion proteins on kex2 protease activity. 1091 38

The genus Proteus belongs to the tribe of Proteae in the family of Enterobacteriaceae, and consists of five species: P. mirabilis, P. vulgaris, P. morganii, P. penneri and P. myxofaciens. They are distinguished from the rest of Enterobacteriaceae by their ability to deaminate phenylalanine and tryptophane. They hydrolyze urea and gelatin and fail to ferment lactose, mannose, dulcitol and malonate; and do not form lysine and arginine decarboxylase or beta-galactosidase [1]. Colonies produce distinct "burned chocolate" odor and frequently show the characteristics of swarming motility on solid media. P. mirabilis, P. vulgaris and P. morganii are widely recognized human pathogens. They have been isolated from urinary tract infections, wounds, ear, and nosocomial bacteremic infections, often in immuncompromised patients [2-6]. P. myxofaciens has no clinical interest to this time. P. penneri as species nova was nominated by the recommendation of Hickman and co-workers [7]. Formerly it was recognized as P. vulgaris biogroup 1 or indole negative P. vulgaris [8, 9]. Although it has been less commonly isolated from clinical samples than the other three human pathogenic Proteus species, it has nevertheless been connected with infections of the urinary tract, wounds and has been isolated from the feces of both healthy and diarrheic individuals [10-12]. Potential virulence factors responsible for virulence of Proteae are: IgA protease, urease, type3 fimbriae associated with MR/K haemagglutinins of at least two antigenic types, endotoxin, swarming motility and HlyA and/or HpmA type hemolysins [for review see ref. 13]. In the followings we give a survey of accumulated concepts about the position and characteristics of HlyA type alpha-hemolysins both in general and with emphasis on virulence functions in the tribe of Proteae.
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PMID:Proteus virulence: involvement of the pore forming alpha-hemolysin (a short review). 1105 65

Because arginase hydrolyzes arginine to produce ornithine and urea, it has the potential to regulate nitric oxide (NO) and polyamine synthesis. We tested whether expression of the cytosolic isoform of arginase (arginase I) was limiting for NO or polyamine production by activated RAW 264.7 macrophage cells. RAW 264.7 cells, stably transfected to overexpress arginase I or beta-galactosidase, were treated with interferon-gamma to induce type 2 NO synthase or with lipopolysaccharide or 8-bromo-cAMP (8-BrcAMP) to induce ornithine decarboxylase. Overexpression of arginase I had no effect on NO synthesis. In contrast, cells overexpressing arginase I produced twice as much putrescine after activation than did cells expressing beta-galactosidase. Cells overexpressing arginase I also produced more spermidine after treatment with 8-BrcAMP than did cells expressing beta-galactosidase. Thus endogenous levels of arginase I are limiting for polyamine synthesis, but not for NO synthesis, by activated macrophage cells. This study also demonstrates that it is possible to alter arginase I levels sufficiently to affect polyamine synthesis without affecting induced NO synthesis.
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PMID:Arginase I: a limiting factor for nitric oxide and polyamine synthesis by activated macrophages? 1108 91

Normal human fibroblasts have been shown to undergo a p16(Ink4a)-associated senescence-like growth arrest in response to sustained activation of the Ras/Raf/MEK/ERK pathway. We noted a similar p16(Ink4a)-associated, senescence-like arrest in normal human astrocytes in response to expression of a conditional form of Raf-1. While HPV16 E7-mediated functional inactivation of the p16(Ink4a)/pRb pathway in astrocytes blocked the p16(Ink4a)-associated growth arrest in response to activation of Raf-1, it also revealed a second p21(Cip1)-associated, senescence-associated, beta-galactosidase-independent growth arrest pathway. Importantly, the p21(Cip1)-associated pathway was present not only in normal astrocytes but also in p53-, p14(ARF)-, and p16(Ink4a)/pRb-deficient high grade glioma cells that lacked the p16(Ink4a)-dependent arrest mechanism. These results suggest that normal human cells have redundant arrest pathways, which can be activated by Raf-1, and that even tumors that have dismantled p16(Ink4a)-dependent growth arrest pathways are potentially regulated by a second p21(Cip1)-dependent growth arrest pathway.
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PMID:Dual growth arrest pathways in astrocytes and astrocytic tumors in response to Raf-1 activation. 1127 20

Canestrato Pugliese cheeses from ewe milk were produced according to a traditional protocol and by adding 7.0 log10 cfu of fresh cells per gram of Bifidobacterium bifidum Bb02, Bifidobacterium longum Bb46, or both species. The traditional technology was modified slightly to favor the survival of probiotic microorganisms. After 56 d of ripening, the survival of B. bifidum Bb02 and B. longum Bb46 was 6.0 and 5.0 log10 cfu/g, respectively. After 19 d cheeses contained ca. 7.0 log10 cfu/g of bifidobacteria. Compared to traditional cheese, the addition of bifidobacteria seemed to support the growth and survival of mesophilic lactobacilli and Streptococcus thermophilus, used as starter, during ripening. No significant differences were observed in the main chemical composition, and only a slightly higher concentration of acetic acid was found in cheeses with bifidobacteria added. On the contrary, alpha- and beta-galactosidase activities were markedly more pronounced in the presence of bifidobacteria, especially with B. bifidum Bb02. In contrast with traditional cheese, the lactose was completely hydrolyzed in cheeses made with bifidobacteria. Urea-PAGE electrophoresis of the pH 4.6-soluble and pH 4.6-insoluble N fractions did not show appreciable variations. Only the reversed-phase-HPLC analysis of the pH 4.6-soluble N showed a slightly more complex profile in the presence of bifidobacteria. This finding was in agreement with the higher value of the pH 4.6-soluble N/total N ratio and with the more pronounced amino-, imino-, and dipeptidase activities found in all the cheeses with the bifidobacteria added, especially B. bifidum Bb02. No differences were found in the free amino acid and free fatty acid contents. The amino acids glutamic acid, valine, proline, leucine, and lysine and the fatty acids butyric, caproic, capric, and oleic acids were found at the highest concentrations. The sensory evaluation did not show significant differences, and Canestrato Pugliese cheeses were characterized by small and uniformly distributed eyes, were pale yellow, had an elastic consistency and a Pecorino-like smell, were very salty, and tended to be moderately piquant.
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PMID:Microbiological and biochemical properties of canestrato pugliese hard cheese supplemented with bifidobacteria. 1128 6

Arginase, which exists as the isoforms arginase I and II, catalyzes the hydrolysis of arginine to ornithine and urea. Ornithine is the principal precursor for production of polyamines, which are required for cell proliferation. Rat aortic smooth muscle cells (RASMC) contain constitutive arginase I, and arginase inhibitors cause inhibition of cell proliferation. The objective of this study was to determine whether the elevated expression of arginase I in RASMC causes increased cell proliferation. RASMC were stably transfected with either rat arginase I cDNA or a beta-galactosidase control expression plasmid. Western blots and arginase enzymatic assays revealed high-level expression of cytosolic arginase I in arginase I-transfected RASMC. Moreover, this observation was associated with the increased production of urea and polyamines and higher rates of RASMC proliferation. The two selective inhibitors of arginase, N(G)-hydroxy-l-arginine and S-(2-boronoethyl)-l-cysteine, inhibited arginase and decreased the production of urea and polyamines in arginase I-transfected RASMC, all of which were associated with the inhibition of cell proliferation. This study demonstrates that elevated arginase I expression increases RASMC proliferation by mechanisms involving increased production of polyamines. These observations suggest that arginase I plays a potentially important role in controlling RASMC proliferation.
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PMID:Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation. 1147 Sep 19

The activity of the promoter regions of the cell division genes ftsZ, ftsE, minC, minD and minE from Neisseria gonorrhoeae (Ng) was studied under different environmental conditions using lacZ translational fusions. The promoters of the minNg genes have not been previously determined and we identified promoter regions upstream of each gene (minCp, minDp and minEp). We determined that minDp had the strongest activity. Expression of the promoter regions of ftSZ(Ng) and ftsE(Ng), which we had previously identified, as well as minD(Ng), were then studied under conditions reflecting the environment of the genitourinary tract. These conditions included anaerobiosis, presence of isoleucine or urea (3 mM and 400 mM, respectively) and acidity of pH 6. Both beta-galactosidase expression and northern blot analysis indicated that all three genes were upregulated under anaerobiosis. The addition of isoleucine as well as media at pH 6 did not have any significant effects on the promoter activity of these genes while the presence of urea significantly decreased ftsZ(Ng) promoter activity. The expression of the minD(Ng) promoter region was analyzed during different growth phases and shown to follow the growth behavior of the culture. By contrast, the ftSZ(Ng) promoter activity continued to rise after the onset of the stationary phase. When gonococcal ftsZ promoter 1, (Pz1) was altered by site-directed mutagenesis, a significant decrease in the expression of ftsZ(Ng) was observed under both aerobic and anaerobic conditions. These data infer that gonococci regulate their cell division in response to different environments.
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PMID:Expression of Neisseria gonorrhoeae cell division genes ftsZ, ftsE and minD is influenced by environmental conditions. 1176 38

A beta-galactosidase isoenzyme, beta-Gall, from Bifidobacterium infantis HL96, was expressed in Escherichia coli and purified to homogeneity. The molecular mass of the beta-Gall subunit was estimated to be 115 kDa by SDS-PAGE. The enzyme appeared to be a tetramer, with a molecular weight of about 470 kDa by native PAGE. The optimum temperature and pH for o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose were 60 degrees C, pH 7.5, and 50 degrees C, pH 7.5, respectively. The enzyme was stable over a pH range of 5.0-8.5, and remained active for more than 80 min at pH 7.0, 50 degrees C. The enzyme activity was significantly increased by reducing agents. Maximum activity required the presence of both Na+ and K+, at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, divalent metal cations, and Cr3+, and to a lesser extent by EDTA and urea. The hydrolytic activity using lactose as a substrate was significantly inhibited by galactose. The Km, and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. beta-Gall possesses strong transgalactosylation activity. The production rate of galactooligosaccharides from 20% lactose at 30 and 60 degrees C was 120 mg/ml, and this rate increased to 190 mg/ml when 30% lactose was used.
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PMID:Purification and characterization of a recombinant beta-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96. 1195 89

Survival of Helicobacter pylori in acid depends on intrabacterial urease. This urease is a Ni(2+)-containing oligomeric heterodimer. Regulation of its activity and assembly is important for gastric habitation by this neutralophile. The gene complex encodes catalytic subunits (ureA/B), an acid-gated urea channel (ureI), and accessory assembly proteins (ureE-H). With the use of yeast two-hybrid analysis for determining protein-protein interactions, UreF as bait identified four interacting sequences encoding UreH, whereas UreG as bait detected five UreE sequences. These results were confirmed by coimmunoprecipitation and beta-galactosidase assays. Native PAGE immunoblotting of H. pylori inner membranes showed interaction of UreA/B with UreI, whereas UreI deletion mutants lacked this protein interaction. Deletion of ureE-H did not affect this interaction with UreI. Hence, the accessory proteins UreE/G and UreF/H form dimeric complexes and UreA/B form a membrane complex with UreI, perhaps enabling assembly of the urease apoenzyme at the membrane surface and immediate urea access to intrabacterial urease to allow rapid periplasmic neutralization.
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PMID:Interactions among the seven Helicobacter pylori proteins encoded by the urease gene cluster. 1238 7

Urea, at concentrations routinely observed in the renal inner medulla during antidiuresis in many mammals, is a potent protein destabilizing agent that reduces the activity of many enzymes. The molecular chaperone heat shock protein 72 (HSP72) is expressed at about 5 ng/ micro g protein in the renal papilla and is thus 40 times more abundant than in the isosmotic cortex and may counteract the deleterious effects of high urea concentrations in the inner medulla. To test this hypothesis, we examined the effect of recombinant HSP72 on lactate dehydrogenase activity in the presence of 0.8 M urea in a cell-free system. The urea-induced increase in K(m) was reduced by 85% in the presence of 1 microM HSP72 but only by 6% by 100 mM betaine, a "counteracting" trimethylamine osmolyte. Conversely, the decrease in V(max) with 0.8 M urea was not affected by HSP72 but was attenuated by 42% in the presence of betaine. The protective effect of HSP72 was confirmed by the attenuation of the urea-induced decrease in the activity of another model enzyme, beta-galactosidase, by lysate of HSP72-overexpressing MDCK cells. Hence, in addition to the trimethylamine osmolytes, HSP72 may participate in counteracting urea-mediated effects on protein function in the renal papilla.
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PMID:Heat shock protein 72, a chaperone abundant in renal papilla, counteracts urea-mediated inhibition of enzymes. 1239 89


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