Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
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The amino acid metabolism of 23 different Brucella strains was investigated for differentiation purposes. The results were evaluated by thin layer chromatography, after enzymatic incubation. The organisms (Tab. 1) were grown on Tryptose blood agar at 37 degree C for 24 or 48h. Two mg wet weight of bacteria in 0.2 ml PBS, 0.01 M, were incubated with 12.5 microng (0.025 ml) amino acid in small tubes for 16h at 37 degree C, and centrifuged for 15 min at 7500 X g. For controls, bacterial suspensions were heated for 15 min at 100 degree C to destroy enzymatic activity, and also contrifuged for 15 min at 7500 X g. Usually 4 micronl of the supernatant fluids (6 micronl for L-asparagine, and 10 micronl for L-proline) were pipetted on the thin layer plate. The tests were run in n-butanol acetic acid water, 20:5:5, with a distance of 8 cm. Amino acids were stained with ninhydrine. The tests were repeated 3-5 times with identical results. Amino acid metabolism was indicated by different staining intesities (+ to ++) in comparison to control preparations. All species could be exactly differentiated from each other, with the exception of B. suis, biotype 2, and B canis, which could not be differentiated by their amino acid metabolism. Biotyps of the same species were mostly identical. The results of these investigations could be reproduced qualitatively as well as quantitatively. The method described is recommended for routine investigations.
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PMID:[Investigations of amino acid metabolism by thin layer chromatography for differentiation of Brucella (author's transl)]. 86 72

The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.
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PMID:Biochemical characterization of a rat oncofetal colonic antigen defined by a monoclonal antibody raised against gastric surface epithelium. 768 17

Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 1x PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.
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PMID:Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation. 1115 May 48

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of Abeta protofibrils. Library screening yielded several molecules that stimulate Abeta aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts Abeta(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37 degrees C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-Abeta aggregates exhibit a low thioflavin T response. Like Abeta fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-Abeta aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous Abeta fibril formation reaction: approximately 12 of the 39 Abeta(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the Abeta molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in Abeta conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester Abeta in a form and/or location that cannot engage the toxic pathway.
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PMID:Structural properties of Abeta protofibrils stabilized by a small molecule. 1588 77

Cryopreservation is a pivotal process in cellular engineering for creating a continuous source of generated functional cell lines and for the convenience of various medical treatments that involve cell culture. FBS (fetal bovine serum) supplemented with 10% (v/v) DMSO is extensively used as a freezing medium for mammalian cells using conventional methods. However, FBS should ideally be avoided, owing to serious concerns regarding bovine spongiform encephalopathy and other infections such as viruses, and an alternative to FBS is eagerly awaited. Furthermore, bio-medicines and cells for transplantation should not be infectious. The present study aimed to develop a novel serum-free freezing medium. For this purpose, we focused on using the silk protein sericin as a cryoprotectant for storage and developed a novel serum-free freezing medium consisting of PBS, 1% (v/w) sericin, 0.5% (v/w) maltose, 0.3% (v/w) proline, 0.3% (v/w) glutamine and 10% DMSO. This novel freezing medium was compared with the conventional FBS supplemented with DMSO and also with three purchased freezing media with respect to cryopreservation of the P 3 U1 myeloma cell line and Chinese-hamster ovary cells. As a result, the constructed medium containing sericin successfully cryopreserved both cell types as efficiently as the conventional medium of FBS containing 10% DMSO and was superior to all three of the purchased media. The constructed medium containing sericin also cryopreserved normal human dermal fibroblasts, human epidermal keratinocytes, the rat phaeochromocytoma cell line PC12 and insect (Spodoptera frugiperda) cell line S f 9 as effectively as the conventional medium of FBS and DMSO.
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PMID:Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin. 1594 83

The role of free radicals in protein modification and the importance of anti-4-hydroxy-2-nonenal (HNE) antibodies as marker of HNE-mediated cell toxicity has been well documented. Proteins modified by HNE in vitro, prior to immobilization on ELISA plates, have served as substrates for assaying these antibodies. We found preferential binding of HNE-modified versus unmodified proteins to ELISA plates and this prompted us to seek a more reliable assay. We report a method to HNE-modify any cysteine/histidine/lysine-containing protein or multiple antigenic peptide (MAP) following their immobilization on an ELISA plate. To a set of wells, HNE (200 microM) dissolved in PBS is added and incubated for 4 h, followed by regular ELISA. Since HNE was supplied dissolved in ethanol, PBS with appropriate amount of ethanol added was used as control. For inhibition experiments, HNE is incubated with or without inhibitors and then added to the wells. The commercial anti-HNE serum bound only to HNE-modified antigens. Sera from rabbits and mice immunized with HNE-modified 60 kDa Ro autoantigen preferentially bound the modified antigens. Modification of solid phase antigens in this manner makes assaying anti-HNE antibodies unambiguous. Lengthy dialysis procedures or the use of spin columns that lead to antigen loss becomes unnecessary for the separation of free HNE. We were able to HNE-modify various antigens (BSA, the autoantigens Ro, La and Sm/nRNP, 60 kDa Ro and Sm MAPs) using this procedure. Using MAPs, we confirmed the importance of histidine, lysine and cysteine residues in HNE modification. In addition, this method allowed identification of inhibitors of HNE-modification. We obtained 61%, 70% and 74% inhibition of HNE-modification of solid phase Ro MAP 166 substrate using BSA, Ro MAP 482 and Ro MAP 166, respectively. Glycyl-proline dipeptide and a MAP from the Sm autoantigen (PPPGMRPP) showed 0% inhibition of HNE-modification.
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PMID:In vitro modification of solid phase multiple antigenic peptides/autoantigens with 4-hydroxy-2-nonenal (HNE) provide ideal substrates for detection of anti-HNE antibodies and peptide antioxidants. 1605 45

Poly(L-valine-L-proline-L-alanine-L-valine-L-glycine) (VPAVG) is a new kind of proteinaceous polymer belonging to the Elastin-like family. These polymers are based on the recurrence of certain short peptide monomers that are considered as "building blocks" in the natural elastin. This smart thermoresponsive polymer has the ability to self-associate at physiological temperature to form aggregates with about 60% in water. This ability can be harnessed to prepare microparticles loaded with an active substance. The aim of this report is to evaluate, from the results of the experiment conducted, the biocompatibility of microparticles prepared from poly(VPAVG). We have studied the cytotoxic effects of microparticles, edema formation after subcutaneous injection (1 and 2.5 mg) in rats (n = 6), and also intraocular tolerance after the intravitreal injection of 2.5 mg of poly(VPAVG) microparticles into pigmented rabbits (n = 12). The polymer did not induce any cytotoxicity or nonspecific depression of cellular respiration on macrophages under the range of polymer concentrations investigated in this study (20, 30, 40, and 60 mg/mL). We observed no inflammatory response to microparticles after subcutaneous injection in the hind-paw of rats, with no significant differences between the control group (PBS) and experimental groups. Anterior and posterior segment signs were evaluated after intraocular injection of poly(VPAVG) microparticles. Only a few eyes (2/11) of the experimental group presented inflammation signs at day 28 postinjection. Nevertheless, 45% (5/11) of the eyes receiving microparticles showed tractional retinal detachment. The results observed in this work suggested certain fibroblastic activity induced by poly(VPAVG) microparticles after their intraocular injection.
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PMID:Biocompatibility of elastin-like polymer poly(VPAVG) microparticles: in vitro and in vivo studies. 1664 66

The objective of this study was to test the activity of microbicides against herpes simplex virus type 2 (HSV-2) introduced in seminal plasma. We found that seminal plasma interfered with the activity of PRO 2000 and of cellulose sulfate, increasing by 100-fold the concentration of drug required to inhibit 90% of viral plaque formation. Seminal plasma competitively inhibited binding of the microbicides to the HSV-2 envelope. Most of the interference was found in a high molecular-weight fraction; tandem mass spectrometry identified the proteins as fibronectin-1 and lactoferrin. In a murine model, the interference translated in vivo into a loss in protection. We found that 2% PRO 2000 gel protected 100% of mice challenged intravaginally with HSV-2 introduced in PBS, whereas only 55% of mice were protected if virus was introduced in seminal plasma (P=.0007, log rank test). If these findings are reflective of what occurs in humans, modifications to microbicides to ensure that they retain activity in the presence of seminal plasma are indicated.
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PMID:Seminal plasma reduces the effectiveness of topical polyanionic microbicides. 1792 91

The viridins like demethoxyviridin (Dmv) and wortmannin (Wm) are nanomolar inhibitors of the PI3 kinases, a family of enzymes that play key roles in a host of regulatory processes. Central to the use of these compounds to investigate the role of PI3 kinase in biological systems, or as scaffolds for drug development, are the interrelated issues of stability, chemical reactivity, and bioactivity as inhibitors of PI3 kinase. We found that Dmv was an even more potent inhibitor of PI3 kinase than Wm. However, Dmv was notably less stable than Wm in PBS, with a half-life of 26min versus Wm's half-life of 3470min. Dmv, like Wm, disappeared in culture media with a half-life of less than 1min. To overcome Dmv's instability, it was esterified at the C1 position, and then reacted with glycine at the C20 position. The resulting Dmv derivative, termed SA-DmvC20-Gly had a half-life of 218min in PBS and 64min in culture media. SA-DmvC20-Gly underwent an exchange reaction at the C20 position with N-acetyl lysine in a manner similar to a WmC20 derivative, WmC20-Proline. SA-DmvC20-Gly inhibited PI3 kinase with an IC(50) of 44nM, compared to Wm's IC(50) of 12nM. These results indicate that the stability of Dmv can be manipulated by reactions at the C1 and C20 positions, while substantially maintaining its ability to inhibit PI3 kinase. Our results indicate it may be possible to obtain stabilized Dmv derivatives for use as PI3 kinase inhibitors in biological systems.
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PMID:A stabilized demethoxyviridin derivative inhibits PI3 kinase. 1952 25

In this research work, proline ester prodrug of acetaminophen (Pro-APAP) was synthesized and evaluated for its stability in PBS buffer at various pH and Caco-2 cell homogenate. The Pro-APAP is more stable at lower pH than higher pH, with half-life of 120min in PBS buffer at pH 2.0, half-life of 65min at pH 5.0, and half life of 3.5min at pH 7.4, respectively. The half-life of Pro-APAP in Caco-2 cell homogenate is about 1min, much shorter than the half-life in PBS buffer at pH 7.4, indicating enzymes in the cell homogenate contribute to the hydrolysis of the ester bond. Carboxypeptidase A was incubated with Pro-APAP at pH 7.4 with half-life of 3.8min which is very close to the half life in buffer itself. This clearly indicates carboxypeptidase A is not one of the enzymes contributing to the hydrolysis of the prodrug. Physicochemical characteristics such as melting point and stability of newly synthesized prodrug were determined by MDSC technique.
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PMID:Development of acetaminophen proline prodrug. 2062 61


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