UNIPROT:P30536 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
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High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.
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PMID:Differential binding properties of the peripheral-type benzodiazepine ligands [3H]PK 11195 and [3H]Ro 5-4864 in trout and mouse brain membranes. 274 35

Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.
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PMID:Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells. 291 Oct 13

In order to answer the question of whether there is an optimal buffer system for the preservation and reoxygenation period in liver transplantation, sodium/potassium phosphate, HEPES, TRIS (THAM), MOPS and histidine/His-HCl buffers were investigated. The buffers were added to an "extracellular" electrolyte composition of preservation solution. The solutions were incubated with in vitro cultures of pig hepatocytes in two different models. I: during cold hypoxia (4 degrees C, PO2 < 0.1 mmHg) for 24 h, and II. during the reoxygenation period of 3 h after preservation in UW solution. Cell viability, cell detachment rate, and LDH and GOT liberation were used as parameters of cell alteration. The lowest amount of enzyme release during the preservation period and reoxygenation was obtained using sodium or potassium phosphate buffer. Rising LDH and GOT liberation rates during preservation and reoxygenation were observed with HEPES and TRIS buffer. The enzyme release induced by these three buffer systems correlated with their pKa values. Higher pH of the preservation solution resulted in higher enzyme leakage from the cells. In contrast, the Histidine/HCl buffer system with low pH led to striking cell damage during preservation as well as during reoxygenation. MOPS, a weak acid with the lowest pH in solution, led to the lowest enzyme release during the preservation period, but to high enzyme release after reoxygenation with standard medium. Incubation of the cultures with MOPS after UW preservation resulted in lower enzyme levels in comparison to the controls. In summary, PBS had the best results in our study.
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PMID:[The effect of buffers in liver preservation solutions on hepatocytes in a model of in vitro preservation and reoxygenation]. 799 Jun 20

The aim of the study was to delineate the most optimal preservation conditions for small bowel grafts. Established preservation solutions such as EuroCollins, University of Wisconsin, histidine-tryptophane-ketoglutarate-Brettschneider, phosphate-buffered sucrose (PBS 140), and 3 new solutions--extracellular fluid (ECF), lactobionate fructose, and a modified lactobionate fructose solution--were compared with saline to determine the most optimal solution for the intestine. Recipient survival, standard histology, and glutaminase activity were used to assess the degree of injury encountered after 12 hr of preservation followed by transplantation. To evaluate the various preservation conditions, ECF was used at pH 6.8 (original ECF). Grafts were preserved most optimally when a vascular washout after the cold storage period was omitted and when topical rewarming of the graft with 37 degrees C saline before reperfusion was performed. Graft survival was not significantly different after preservation with any solution tested (50-83%). Highest graft survival (83%) was achieved with lactobionate fructose and PBS140. Histologic evaluation 20 min after reperfusion revealed minor differences between most groups; a slight advantage was observed for PBS140-preserved grafts. Mucosal glutaminase activity of PBS140-preserved grafts was significantly higher 20 min after reperfusion compared with any other solution evaluated, indicating a superior graft function. These data indicate that different preservation conditions have a great impact on postoperative graft survival and that PBS140 might be preferable to any of the other preservation solutions tested.
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PMID:Evaluation of preservation conditions and various solutions for small bowel preservation. 814 Jun 26

Nucleocapsid protein NCp10 of murine leukemia virus (MuLV) is encoded by the 3' domain of gag and contains a zinc finger of the form Cys-X2-Cys-X4-His-X4-Cys flanked by basic amino acids. In the course of virus assembly, NCp10 is necessary for core formation, and the zinc finger flanked by the basic residues is required for the packaging of the genomic RNA dimer. In vitro, NCp10 exhibits strong nucleic acid binding and annealing activities that appear to be critical for virus infectivity since NCp10 promotes dimerization of the viral RNA containing the E/DLS packaging-dimerization signal and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (PBS). Recent in vitro studies have suggested that NCp10 may also play a role in proviral DNA synthesis. To investigate the function of NCp10 in proviral DNA synthesis in vivo, we developed a simple and convenient genetic packaging system consisting of two DNA constructs expressing the packaging components gag-pol and env of Friend MuLV and a Moloney MuLV-based lacZ vector with either the MuLV E+ or a rat VL30 E packaging signal. This system allowed us to examine the consequences of a set of mutations in NCp10 on a single round of recombinant virus replication. Most mutations in the N- or C-terminal domain of NCp10 do not significantly alter infectivity, while those in the zinc finger drastically impair infectivity. Analysis of the viral RNA content in virions showed that all mutations in the zinc finger decrease but do not abolish packaging of the recombinant genome. Interestingly enough, mutation of Y-28 to S (mutation Y28S) in the zinc finger results in RNA packaging at a level similar to that observed upon deletion of three prolines and three arginines in the C-terminal domain of NCp10 (mutant delta PR3). However, mutant Y28S is noninfectious while mutant delta PR3 is only threefold less infectious than the wild-type virus, which prompted us to examine the role of NCp10 protein in proviral DNA synthesis in vivo using these nucleocapsid mutants. PCR amplification was used to analyze viral DNA synthesized in newly infected cells, and results indicate that the Y28S zinc finger mutation impairs reverse transcription, thus suggesting that the nucleocapsid protein zinc finger plays a key role in proviral DNA synthesis in vivo.
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PMID:The zinc finger of nucleocapsid protein of Friend murine leukemia virus is critical for proviral DNA synthesis in vivo. 870 95

Hyperacute xenogeneic rejection is partly initiated by the binding of performed natural antibodies on the recipient's vascular endothelium. In this study, isotypic suppression of IgM in the recipient was conducted in the guinea pig-to-rat combination. Anti-IgM HIS 40 and 56 monoclonal antibodies were injected intraperitoneally in rats (n = 3) (Group 1). Control animals were rats (n = 13) treated by PBS (Group 2). Guinea pig hearts were implanted heterotopically in all rats. Depletion of circulating IgM in the recipient was assessed by ELISA. The circulating and splenic B-lymphocyte population was scanned by FACS analysis. Isotypic suppression was very effective for the depletion of circulating IgM in Group 1 rats (alpha < 0.01) as assessed by ELISA. Similarly, the rate of circulating and splenic B-lymphocytes was significantly decreased in treated animals (alpha < 0.01). However, the graft survival in Group 1 (14 +/- 6.9 min) was not different from that observed in Group 2 (15.4 +/- 5 min). Isotypic suppression of IgM in the recipient did not delay hyperacute xenogenic rejection in the guinea pig-to-rat combination. The prevention of hyperacute rejection must therefore be based on the various mechanisms of this phenomenon.
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PMID:[Attempt at preventing hyperacute xenogenic rejection by isotopic suppression of IgM in the recipient]. 903 21

The accumulation of LDL in the arterial intima is considered a key event in atherogenesis. We investigated the binding of oxidized LDL (ox-LDL) to microtiter plates coated with type I or II collagen, laminin, fibronectin, or poly-D-lysine. Oxidation of LDL, 125I-LDL, or Eu(3+)-LDL was performed with CuCl2, varying the time of oxidation. Bound lipoprotein was assessed by counting radioactivity or fluorescence in the wells. Binding of highly ox-LDL in PBS followed the order: type I collagen > poly-D-lysine > type II collagen > laminin > fibronectin. Comparing various collagen types, the binding of ox-LDL followed the order: type I > type V and, type III > type IV > type II collagen. Binding of ox-LDL in PBS was dependent on an increase in negative charge of ox-LDL. Testing certain amino acids as competitors for binding of highly ox-LDL to type I collagen put lysine first, followed by arginine and histidine. On laminin, histidine competed most, followed by lysine and arginine. When studying the influence of Na+, K+, Ca2+, Mg2+ (equivalent to their concentrations in the interstitial fluid), native LDL, moderately ox-LDL, and highly ox-LDL showed the same affinity to type I collagen. However, a fivefold dilution of the buffer increased the affinity of moderately and highly ox-LDL 3.9- and 10-fold compared with native LDL. Application of the F(ab')2 from a monoclonal antibody to ox-LDL revealed a strong competition of the binding of highly ox-LDL to type II collagen (60%), laminin (35%), type I collagen (20%), and poly-D-lysine (15%), whereas the binding to fibronectin was not affected.
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PMID:In vitro interactions of oxidatively modified LDL with type I, II, III, IV, and V collagen, laminin, fibronectin, and poly-D-lysine. 940 48

The ultrasonically-induced in vitro cell damaging effect of fluorine-containing anthracycline derivative (FAD104) was investigated. Sarcoma 180 cells suspended in air-saturated PBS were exposed to ultrasound for up to 60 s in the presence and absence of FAD104. The rate of inducing cell damage with ultrasound was doubled with 80 microM FAD104, while no cell damage was observed with FAD104 alone. This enhancement was significantly inhibited by histidine, which may suggest a sonochemical mechanism.
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PMID:Sonodynamically-induced cell damage with fluorinated anthracycline derivative, FAD104. 956 17

The generation of free radicals by Ni(2+) and Co(2+) was studied at physiological pH in H(2)O(2)-containing solutions in the absence and presence of various radical-mediating ligands and in human peripheral blood mononuclear cell (PBMC) cultures. With ESR spectroscopy, free radical species were identified and quantitated by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Co(2+) generated hydroxyl radicals from H(2)O(2) in PBS solutions containing glutathione (GSH) or histidine (His). Omission of GSH or His from the reaction mixture significantly reduced the ESR-signal, indicating the importance of metal-chelation in free radical generation. Carnosine did not significantly enhance the reactivity of Co(2+) toward H(2)O(2), whereas cysteine (Cys) and N-acetylcysteine (NAC) suppressed free radical generation. Under identical reaction conditions, Ni(2+) was markedly less reactive toward H(2)O(2) in comparison with Co(2+). GSH, His, Cys and NAC did not enhance free radical generation of Ni(2+) from H(2)O(2). However, in the presence of carnosine weak but significantly enhanced ESR intensities were found. Incubation of PBMC cultures from healthy subjects with Co(2+) (10-50 microM) yielded the DMPO-.OH adduct, suggesting Co(2+)-mediated hydroxyl radical generation. In contrast, incubation of PBMC cultures with Ni(2+) (10-50 microM) did not produce a detectable ESR-signal. Ascorbic acid efficiently inhibited Co(2+)-mediated free radical generation in PBS solutions and PBMC cultures. The observed difference in free radical generating capacity between Ni(2+) and Co(2+) is of interest with respect to the absence of cross-reactivity between the two metal-ions in experimental allergic contact dermatitis.
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PMID:Free radicals as potential mediators of metal-allergy: Ni2+- and Co2+-mediated free radical generation. 979 82

Functionalization of biologically relevant molecules for the labeling with the novel fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) precursor has gained considerable attention recently. Therefore, we tested seven different tridentate (histidine L(1)(), iminodiacetic acid L(2)(), N-2-picolylamineacetic acid L(3)(), N, N-2-picolylaminediacetic acid L(4)()) and bidentate (histamine L(5)(), 2-picolinic acid L(6)(), 2,4-dipicolinic acid L(7)()) ligand systems, with the potential to be bifunctionalized and attached to a biomolecule. The ligands allowed mild radiolabeling conditions with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) (30 min, 75 degrees C). The ligand concentrations necessary to obtain yields of >95% of the corresponding organometallic complexes 1-7 ranged from 10(-)(6) to 10(-)(4) M. Complexes of the general formula "fac-[(99m)TcL(CO)(3)]" (L = tridentate ligand) and "fac-[(99m)Tc(OH(2))L'(CO)(3)]" (L' = bidentate ligand), respectively, were produced. Challenge studies with cysteine and histidine revealed significant displacement of the ligands in complexes 5-7 but only little exchange with complexes 1-4 after 24 h at 37 degrees C in PBS buffer. However, no decomposition to (99m)TcO(4)(-) was observed under these conditions. All complexes showed a hydrophilic character (log P(o/w) values ranging from -2.12 to 0.32). Time-dependent FPLC analyses of compounds 1-7 incubated in human plasma at 37 degrees C showed again no decomposition to (99m)TcO(4)(-) after 24 h at 37 degrees C. However, the complexes with bidentate ligands (5-7) became almost completely protein bound after 60 min, whereas the complexes with tridentate coordinated ligands (1-4) showed no reaction with serum proteins. The compounds were tested for their in vivo stability and the biodistribution characteristics in BALB/c mice. The complexes with tridentate coordinated ligand systems (1-4) revealed generally a good and fast clearance from all organs and tissues. On the other hand, the complexes with only bidentate coordinated ligands (5-7) showed a significantly higher retention of activity in the liver, the kidneys, and the blood pool. Detailed radiometric analyses of murine plasma samples, 30 min p.i. of complex fac-[(99m)TcL(1)(CO)(3)], 1, revealed almost no reaction of the radioactive complex with the plasma proteins. By contrast, in plasma samples of mice, which were injected with complex fac-[(99m)Tc(OH(2))L(5)(CO)(3)](+), 5, the entire radioactivity coeluded with the proteins. On the basis of these in vitro and in vivo experiments, it appears that functionalization of biomolecules with tridentate-chelating ligand systems is preferable for the labeling with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+), since this will presumably result in radioactive bioconjugates with better pharmacokinetic profiles.
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PMID:Influence of the denticity of ligand systems on the in vitro and in vivo behavior of (99m)Tc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules. 1082 50

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