Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antibody responses and health parameters were compared in rabbits immunized with a synthetic polypeptide antigen, [L-Tyr,L-Glu,DL-Ala]-poly-L-lysine ((TG)-AL), in Freund's (FA) or Ribi (RA) adjuvants. Rabbits, 12 weeks old, of both sexes, were inoculated with 0.5 ml divided between two intramuscular (i.m.) sites. Eight received FA and antigen (50 micrograms); eight RA and antigen, eight PBS and antigen; four FA and PBS; four RA and PBS, and four PBS. Identical booster inoculations were made 21 days later, except that incomplete FA was substituted for complete FA. Rabbits were monitored until euthanasia and necropsy 7 weeks after the primary inoculation. Sera, obtained weekly, were analyzed for immunoglobulins using an enzyme immunoassay. Only rabbits given antigen with adjuvant produced high titered antibodies. Mean optical density values for immunoglobulin (Ig)M were greater the week after the booster in the group given FA. IgG values were similar for both adjuvant/antigen groups the week after the booster, but thereafter decreased in rabbits given RA. Antisera from rabbits given antigen with FA had greater avidity for the antigen than that from rabbits given antigen with RA, however, the difference was not significant (p greater than 0.05). Rabbits inoculated with FA and antigen had high serum creatinine kinase levels the day after inoculation, showed evidence of discomfort, and extensive granulomatous inflammation at the inoculation sites. Lesions were minimal to mild in rabbits given antigen with RA and PBS with either adjuvant. While RA did not result in adverse side effects, the IgG response to (TG)-AL with RA was transient compared to FA.
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PMID:Comparison of Freund's and Ribi adjuvants for inducing antibodies to the synthetic antigen (TG)-AL in rabbits. 164 Jan 5

Rat cerebral astrocytes from confluent primary cultures were grown for two weeks in medium made hyperosmotic with additional NaCl. At the time the cells were harvested (four weeks in culture), the medium osmolality of experimental cultures was approximately 600 mOsm. Amino acid, protein, and potassium contents and the cell volume were measured. Compared to cells maintained in control medium (approximately 300 mOsm), cells grown in hyperosmotic conditions had over two times the content of taurine and five times the content of glutamine. Alanine, aspartate, glutamate, glycine, and tyrosine contents also were elevated in these hyperosmotic-treated cells, while asparagine contents were unchanged relative to control cells. Cell volume and potassium content were decreased to approximately 50% of control levels by the hyperosmotic treatment while total protein content per cell was unchanged relative to cells from control cultures. Seven min after hyperosmotic-exposed cells were rapidly diluted into PBS with osmolality equal to about 330 mOsm, cell contents of alanine, asparagine, glutamine, glutamate, glycine, taurine, and tyrosine fell toward control levels. The data indicate that significant alterations in intracellular osmolytes occur in astrocytes adapted to hyperosmotic conditions. We suggest that a loss of intracellular potassium is at least partially compensated by accumulation of taurine, glutamine, and perhaps other amino acids acting as intracellular osmolytes.
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PMID:Amino acid content of rat cerebral astrocytes adapted to hyperosmotic medium in vitro. 225 66

Mutants have been isolated which correspond to every step concerned with the biosynthesis of the aromatic amino acids in Bacillus subtilis. Each mutant has been characterized, and the lesion it bore was analyzed by deoxyribonucleic acid transformation and PBS-1 mediated transduction. The biochemical analysis revealed that each of the mutations appears to have affected a single enzyme, except for two groups of pleiotropic mutations. All aroF mutants (chorismic acid synthetase) lack dehydroquinic acid synthetase (aroB) activity. The gene that specifies aroB is closely linked to the gene coding for the aroF enzyme. Both genes are a part of the aro cluster. Mutants lacking chorismate mutase activity also lack d-arabino-heptulosonic acid-7-phosphate synthetase and shikimate kinase activity, presumably as a result of these three activities forming a multi-enzyme complex. Another mutant, previously undescribed, had been isolated. The affected gene codes for the tyrosine and phenylalanine aminotransferase activity. All of the mutations have been located on the B. subtilis genome except those in the genes specifying shikimate kinase activity and tyrosine-phenylalanine aminotransferase activity.
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PMID:Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis. 420 Aug 44

Studies were undertaken to identify intracellular mediators of prolactin inhibition of glucocorticoid-induced apoptosis in Nb2 lymphoma cells. A short-term assay was implemented that quantitates fragmented DNA released from the genome by reaction with diphenylamine. Induction and inhibition of internucleosomal DNA cleavage (indicative of apoptosis) was verified by agarose gel electrophoresis of extracted cellular DNA. Synchronized Nb2 cells (G0/G1) exhibited increased DNA fragmentation after 4-hr incubation with dexamethasone (DEX) (25-100 nM) which was inhibited by ovine prolactin (oPRL) (0.1-1 ng/ml), the glucocorticoid receptor antagonist, RU486 (500 nM), and the nuclease inhibitor, aurintricarboxylic acid (100 microM). Signals previously implicated in prolactin induction of mitogenesis in Nb2 cells were investigated for their role in prolactin inhibition of apoptosis including: protein kinase C activation, arachidonic acid metabolism, polyamine production, tyrosine phosphorylation, and extracellular calcium. Protein kinase C agonists, phorbol-12-myristate-13-acetate, and 1,2-dioctanoyl-sn-glycerol, +/- the calcium ionophore, A23187 (200 nM), did not mimic oPRL inhibition of DEX-induced DNA fragmentation. Protein kinase C inhibitors, gossypol and quercetin, did not block prolactin action. Arachidonic acid did not mimic prolactin protection against DEX-induced DNA fragmentation. Inhibitors of arachidonic acid metabolism, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and indomethacin did not block prolactin action. The polyamine, spermine, inhibited DEX-induced DNA fragmentation at 1.5 to 2.5 mM. However, inhibition of polyamine synthesis with alpha-difluoromethyl ornithine or methylglyoxal bis(guanylhydrazone) did not inhibit prolactin action. Prolactin action was not blocked by inhibitors of tyrosine kinase activation, genistein and tyrphostin-47. On the other hand, pervanadate, a potent tyrosine phosphatase inhibitor, consistently inhibited DEX-induced DNA fragmentation. Prolactin action and DEX-induced apoptosis both occurred in calcium-free PBS. In summary, protein kinase C activation and eicosanoid production do not appear to mediate this prolactin action. Although spermine could block DNA fragmentation, blockade of the polyamine cascade did not inhibit prolactin action, suggesting that polyamines do not mediate this prolactin effect. While inhibitors of tyrosine kinase activation did not block prolactin action, tyrosine phosphatase inhibition in the presence of basal tyrosine kinase activity mimicked prolactin action, suggesting tyrosine phosphorylation participation in the anti-apoptotic effect. Extra-cellular calcium was not required for prolactin or DEX action.
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PMID:Investigation of intracellular signals mediating the anti-apoptotic action of prolactin in Nb2 lymphoma cells. 777 88

The literature reported DPP-IV substrate specificity includes oligopeptides with a penultimate Pro, Hyp or Ala residue. Bovine GRF is a substrate for DPP-IV and is rapidly degraded by the enzyme via removal of its N-terminal Tyr-Ala. Incubation of selected GRF analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series with a porcine-kidney-derived DPP-IV in PBS (pH 7.4) resulted in cleavage at the X2-Asp3 bond. The extent of enzymatic hydrolysis varied with X2 as reflected in the following relative cleavage rates: Ala2 (100%), Ser2 (4%), Thr2 (2.5%), Val2 (0.53%), Ile2 (0%). These cleavages were sequestered when similar experiments were performed in the presence of the DPP-IV-specific inhibitor N-epsilon-(p-NO2-benzyloxycarbonyl)-Lys-Pro-OH. A side reaction, buffer-induced deamidation of Asn8, contributed less than 5% of the total substrate degradation. Although our finding qualitatively extends the DPP-IV substrate specificity to also include N-terminal X-Ser, X-Thr and X-Val sequences, quantitatively, relatively fast cleavages of the GRFs with Ala2 make the latter preferred substrates for DPP-IV. The data presented here indicates that the observed GRF(3-29) fragment formation upon incubation of Ser2- and Thr2-substituted bGRF analogs in bovine plasma could have been DPP-IV-related.
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PMID:Dipeptidyl peptidase IV (DPP-IV) from pig kidney cleaves analogs of bovine growth hormone-releasing factor (bGRF) modified at position 2 with Ser, Thr or Val. Extended DPP-IV substrate specificity? 810 71

Tyr183 is a constituent of the highly conserved YXDD motif common to all retroviral reverse transcriptases. The two aspartates in this motif are the crucial members of the catalytic carboxylate triad while residue X, which in the case of HIV-1 RT is Met184, is implicated in dNTP substrate recognition and fidelity of DNA synthesis. In an attempt to understand the function of Tyr183 in the catalytic mechanism, we generated mutants of this residue (Y183F and Y183A) and subjected them to in-depth analysis. The efficiency of reverse transcription of natural U5-PBS HIV-1 RNA template was severely impaired by both the conservative and nonconservative substitutions. The major defect identified was at the level of dNTP binding as determined by a 20-80-fold increase in the Km for the dNTP substrate on both homopolymeric and heteropolymeric RNA and DNA templates. A significant reduction in processivity of DNA synthesis by these mutants was also noted. However, the fidelity of DNA synthesis by the Y183F and Y183A mutants was increased significantly compared to the wild-type enzyme. Interestingly, the reduction in the polymerase activity due to single substitution of Tyr to Phe in the YMDD motif is compensated by a second substitution of Met to Val in the same motif, herein referred to as the FVDD. The loss of dNTP binding as well as decreased processivity of DNA synthesis exhibited by the Y183F mutant was also compensated by mutation at the second site. Curiously, the double mutant did not exhibit any synergistic effect in regard to fidelity of DNA synthesis as might be expected since both the single mutations (Y183F, M184V) exhibited enhanced fidelity compared to the wild-type enzyme. These data implicate Tyr183 and Met184 as important constituents of the dNTP-binding pocket. We propose a model which suggests that subtle structural changes due to mutation in the flexible beta9-beta10 loop region at the active site of the molecule influence the enzyme activity and substrate recognition.
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PMID:Loss of polymerase activity due to Tyr to Phe substitution in the YMDD motif of human immunodeficiency virus type-1 reverse transcriptase is compensated by Met to Val substitution within the same motif. 965 75

Electrolyzed anodic NaCl solutions [EW+], prepared by the electrolysis of 0.1% NaCl, have been shown to instantly inactivate most pathogens that cause food-borne disease. Elimination of food-borne pathogens does not necessarily guarantee food safety because enterotoxins produced by pathogens may remain active. We have tested whether EW+ can inactivate Staphylococcal enterotoxin A (SEA), one of the major enterotoxins responsible for food poisoning. Fixed quantities of SEA were mixed with increasing molar ratios of EW+, and SEA was evaluated by reversed-phase passive latex agglutination (RPLA) test, immunoassay, native polyacrylamide gel electrophoresis (PAGE), and amino acid analysis after 30 min incubations. Exposure of 70 ng, or 2.6 pmol, of SEA in 25 microL of PBS to a 10-fold volume of EW+, or ca. 64.6 x 10(3)-fold molar excess of HOCl in EW+, caused a loss of immuno-reactivity between SEA and a specific anti-SEA antibody. Native PAGE indicated that EW+ caused fragmentation of SEA, and amino acid analysis indicated a loss in amino acid content, in particular Met, Tyr, Ile, Asn, and Asp. Staphylococcal enterotoxin-A excreted into culture broth was also inactivated by exposure to an excess molar ratio of EW+. Thus, EW+ may be a useful management tool to ensure food hygiene by food processing industries.
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PMID:Inactivation of staphylococcal enterotoxin-A with an electrolyzed anodic solution. 1175 73

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a newly assigned member of the Ig-immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. In this study, we hypothesized that PECAM-1 plays an essential in vivo role as a counterregulator of immediate hypersensitivity reactions. We found that PECAM-1 was highly expressed on the surface of immature bone marrow mast cells and at a lower density on mature peritoneal mast cells. Examination of skin biopsies from PECAM-1(+/+) and PECAM-1(-/-) mice revealed that absence of PECAM-1 did not affect mast cell development or the capacity of mast cells to populate tissues. To examine whether the absence of PECAM-1 would influence immediate hypersensitivity reactions, PECAM-1(+/+) and PECAM-1(-/-) mice were presensitized with anti-DNP mouse IgE and then challenged 20 h later with DNP-BSA or PBS. PECAM-1(-/-) mice exhibited elevated serum histamine concentrations after Ag stimulation compared with PECAM-1(+/+) mice, indicating an increased severity of systemic IgE-mediated anaphylaxis. PECAM-1(-/-) mice have increased sensitivity to local cutaneous IgE-dependent anaphylaxis compared with PECAM-1(+/+) mice, as assessed by greater tissue swelling of their ears and mast cell degranulation in situ. PECAM-1(-/-) bone marrow mast cells showed enhanced dense granule serotonin release after Fc epsilon RI cross-linking in vitro. These results suggest that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity and negatively modulates mast cell activation.
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PMID:Absence of platelet endothelial cell adhesion molecule-1 (CD31) leads to increased severity of local and systemic IgE-mediated anaphylaxis and modulation of mast cell activation. 1205 65

Neurotrophic factors regulate a variety of cellular processes, including neuronal survival during development and after injury. For instance, brain-derived neurotrophic factor (BDNF) can prevent the death of dopaminergic substantia nigra neurons in rats. Most neurotrophic factor receptors, such as TrkB for BDNF, are tyrosine kinases whose signaling is terminated by protein tyrosine phosphatases (PTPs). We tested the idea that inhibition of PTPs, and thus potentially enhancement of the efficiency of endogenous trophic factors and their receptors, would lead to increased neuronal survival. After a 2-week infusion of the small PTP inhibitor molecule peroxovanadium (pVa, pervanadate) close to the substantia nigra of adult rats, up to 66% of axotomized substantia nigra neurons had survived, compared to only 33% in control rats infused with PBS. PVa most likely affected TrkB and/or downstream signaling molecules, as ineffective doses of BDNF and pVa had a synergistic effect when given simultaneously, rescuing 82% of the neurons. PVa stimulated tyrosine hydroxylase (TH) expression in the noninjured substantia nigra but did not prevent axotomy-induced loss of TH. These results raise the possibility that PTP inhibition can prevent neuronal death by enhancing neurotrophic factor signaling pathways in the adult mammalian nervous system, identifies an important role for PTPs in neuronal functioning, and points to a novel small molecule treatment approach for neurologic disorders
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PMID:Tyrosine phosphatase inhibition enhances neurotrophin potency and rescues nigrostriatal neurons in adult rats. 1250 84

The proteasome, a multienzymatic protease complex is present in human sperm. Here we present evidence indicating that the proteasome has an extracellular localization, on the plasma membrane of the sperm head. Motile sperm (>90%) in PBS were incubated with the proteasome inhibitors clasto-lactacystin beta-lactone or epoxomicin. Then, the substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC) was added and the enzyme activity evaluated in a spectrofluorometer. Other aliquots were resuspended in Tyrode's medium and incubated at different concentrations for various times with or without inhibitors in the presence of 0.4% azocasein. Hydrolysis of azocasein was evaluated at 440 nm. In addition, sperm membrane proteins were obtained incubating the sperm with Triton X-114 or with 0.5 M KCl plus Triton X-100 and removing insoluble material by centrifugation at 5,000g for 40 min. Proteasomal activity was evaluated with SLLVY-AMC and its presence corroborated by Western blotting. Formaldehyde fixed, unpermeabilized sperm were incubated with anti-proteasome monoclonal antibodies and evaluated using indirect immunofluorescence. The effect of proteasome inhibitors upon the progesterone-induced acrosome reaction was also evaluated. Results indicated that (a) whole, intact sperm were able to hydrolyze the proteasome substrates SLLVY-AMC and azocasein; this activity was inhibited by proteasome inhibitors; (b) proteasomal activity was detected in soluble sperm membrane protein preparations and Western blotting revealed the presence of the proteasome in these fractions; (c) indirect immunofluorescence revealed staining of the head region, particularly of the post acrosomal region; and (d) the proteasome plays an important role during the acrosome reaction.
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PMID:Extracellular localization of proteasomes in human sperm. 1503 55


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