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Osteotesticular phosphatase (OST-PTPase) is a class III receptor-type tyrosine phosphatase (RPTPase). It has 10 tandem fibronectin III-like (FN-III) repeats in the extracellular region and two phosphatase domains in the intracellular region. The expression of the rat OST-PTPase gene, Esp, is restricted to osteoblasts and Sertoli cells, and the transcript level in osteoblasts is highly up-regulated by parathyroid hormone and cAMP treatment. We report here the cloning and characterization of the rat Esp gene, including a 2.9-kb 5' flanking region sequence. Two potential binding sites for Osf2/Cbfa1, an osteoblast-specific transcription factor, are present in the promoter. Esp is composed of 35 exons, but spans merely 20 kb, making it the most compact RPTPase gene identified. Each FN-III repeat is encoded by a single exon flanked with phase 1 introns. Two phosphatase domains are encoded by 16 exons in a genomic organization similar to those in RPRPalpha, RPTPgamma, and Ptprc genes. Esp was mapped by fluorescence in situ hybridization to rat chromosome 13q1. These results represent the first genomic structure of a mammalian class III RPTPase gene.

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Bioassay-guided fractionation of the marine alga Dictyochloris fragrans led to the isolation and identification of sulfonoquinovosyl dipalmitoyl glyceride (1). The structure of 1 was determined by a combination of spectroscopic methods. On the basis of P-selectin inhibition assays (i.e., P-selectin-IgG ELISA, cell binding assay of receptor globulin, and platelet:HL60 adhesion, it was demonstrated that 1 selectively blocks the P-selectin-ligand interaction in vitro and could be considered a lead compound for synthetic modification in order to design more potent inhibitors of cell adhesion processes that play important roles in development of inflammatory-mediated disease states.

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The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

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P-Selectin (CD62/GMP140/PADGEM) is an inducible cell-surface glycoprotein expressed by endothelial cells and platelets following stimulation by inflammatory mediators such as thrombin, histamine, or peroxides. P-Selectin mediates the binding of leukocytes to activated vascular endothelium at sites of inflammation and plays a role in mediating the binding of activated platelets to leukocytes and the vascular cell wall. The adhesive function of P-selectin is mediated by its calcium-dependent (or C-type) lectin domain, which is known to bind to carbohydrate ligands including fucosyl-N-acetyllactosamine (Lex, CD15), sialyl-Lex, and 3-sulfated galactosylceramides (sulfatides). Sulfatides can efficiently block P-selectin/myeloid cell binding in vitro and are excreted at high levels by activated granulocytes. These observations led to the hypothesis that sulfatide may play a role in facilitating the disengagement of CD62, allowing the efficient exit of granulocytes from the blood stream at sites of inflammation. In this report, we extend our previous mutagenesis analysis of the P-selectin binding site [Hollenbaugh, D., Bajorath, J., Stenkamp, R., & Aruffo, A. (1993) Biochemistry 32, 2960] and show that replacement of Tyr48 with Ser or Lys113 with Arg results in P-selectin mutants that, although correctly folded, do not bind to HL60 cells. These results suggest that the conservation of charged and hydrogen-bonding site chains is not sufficient to maintain the P-selectin function and that the exact stereochemistry provided by the side chains of residues lining the P-selectin binding pocket is critical for P-selectin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

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A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86% of the Vmax and was used as the standard concentration for quantitation of enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red has an extinction coefficient that is 21-fold higher than o-nitrophenol; these factors make CPRG about 10-fold greater in sensitivity for the quantitation of enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as substrate permitted the rapid detection of low levels of enzyme production from transfected human cells that could not be detected using ONPG.

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The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1-cytochrome c and iso-2-cytochrome c, encoded by the genes CYC1 and CYC7, respectively. The amino acid sequences of these two isozymes are approximately 80% identical. Cyc3- mutants lack both holocytochromes c, because of a deficiency of cytochrome c heme lyase, the enzyme catalyzing covalent attachment of the heme group to apocytochrome c. A deficiency of heme lyase also prevents import into mitochondria. Surprisingly, apo-iso-1-cytochrome c is absent in cyc3- strains, although apo-iso-2-cytochrome c is present at approximately the same level at which holo-iso-2-cytochrome c is found in CYC3+ strains. The lack of apo-iso-1-cytochrome c is not due to a deficiency of either transcription or translation, but to rapid degradation of the protein. Apocytochromes c encoded by composite cytochrome c genes composed of the central portion of iso-2-cytochrome c flanked by amino and carboxyl regions of iso-1-cytochrome c exhibit increased stability compared with apo-iso-1-cytochrome c. A region encompassing no more than four amino acid differences between iso-1- and iso-2-cytochromes c is sufficient to partially stabilize the protein. In contrast to what is observed in vivo with the apo forms, the holo forms of the composite isocytochromes c are even less stable to thermal denaturation than iso-1-cytochrome c or iso-2-cytochrome c. Either a small region of the sequence of apo-iso-1-cytochrome c is involved in degradation of the protein, or the corresponding region in apo-iso-2-cytochrome c is preventing degradation. The differential stability of the two isocytochromes c may be part of a regulatory process that increases the proportion of iso-2-cytochrome c under certain physiological conditions.

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The 3-aryl-2-oxooxazolidinones are a new class of synthetic antibacterial agents that potently inhibit protein synthesis. An automated pulse labelling method with [3H]-lysine was developed with Bacillus subtilis to obtain additional quantitative activity data for structure-activity relationship studies with the oxazolidinones. Inhibition constants were calculated after a Logit fit of the data into the formula: % of control = 100/(1 + e[-B(X - A)]), where B is the slope of the model, X is the natural log of the inhibitor concentration and A is the natural log of the inhibitor concentration required to inhibit protein synthesis by 50% (ln IC50). When substituents at the 5-methyl position of the heterocyclic ring (B-substituent) were NHCOCH3, OH or Cl, the correlation coefficient was 0.87 between the MIC and IC50 values (for all compounds with MICs less than or equal to 16 micrograms/ml). The D-isomers of DuP 721 (A-substituent = CH3CO) and DuP 105 (A-substituent = CH3SO) gave MICs of 128 micrograms/ml and IC50s of greater than or equal to 50 micrograms/ml for protein synthesis, showing that only the L-isomers were active. By MIC testing, oxazolidinones with the B-substituent of NHCOCH3 and the A-substituent of CH3CO, NO2, CH3S, CH3SO2 or (CH3)2CH had comparable antibacterial potency; however, pulse labelling analysis showed that compounds with an A-substituent of CH3CO or NO2 were more potent inhibitors of protein synthesis.

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The synthesis and structure/activity studies of the effect of varying the "B" group in a series of oxazolidinone antibacterials (I) are described. Two synthetic routes were used: (1) alkylation of aniline with glycidol followed by dialkyl carbonate heterocyclization to afford I (A = H, B = OH), whose arene ring was further elaborated by using electrophilic aromatic substitution methodology; (2) cycloaddition of substituted aryl isocyanates with epoxides to give A and B with a variety of values. I with B = OH or Br were converted to other "B" functionalities by using SN2 methodology. Antibacterial evaluation of compounds I with A = acetyl, isopropyl, methylthio, methylsulfinyl, methylsulfonyl, and sulfonamido and a variety of different "B" groups against Staphylococcus aureus and Enterococcus faecalis concluded that the compounds with B = aminoacyl, and particularly acetamido, were the most active of those examined in each A series, possessing MICs in the range of 0.5-4 micrograms/mL for the most active compounds described.

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The mode of action of DuP 721 was investigated. This compound was active primarily against gram-positive bacteria, including multiply resistant strains of staphylococci. Although inactive against wild-type Escherichia coli, DuP 721 did inhibit E. coli when the outer membrane was perturbed by genetic or chemical means. Pulse-labeling studies with E. coli PLB-3252, a membrane-defective strain, showed that DuP 721 inhibited amino acid incorporation into proteins. The 50% inhibitory concentration of DuP 721 for protein synthesis was 3.8 micrograms/ml, but it was greater than 64 micrograms/ml for RNA and DNA syntheses. The direct addition of DuP 721 to cell-free systems did not inhibit any of the reactions of protein synthesis from chain initiation through chain elongation with either synthetic or natural mRNA as template. However, cell extracts prepared from DuP 721 growth-arrested cells were defective in initiation-dependent polypeptide synthesis directed by MS2 bacteriophage RNA. These cell-free extracts were not defective in polypeptide elongation or in fMet-tRNA(fMet)-dependent polypeptide synthesis stimulated by poly(G.U). We conclude, therefore, that DuP 721 exerts its primary action at a step preceding the interaction of fMet-tRNA(fMet) and 30S ribosomal subunits with the initiator codon.

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Pulse labeling studies with Bacillus subtilis showed that DuP 721 inhibited protein synthesis. The IC50 of DuP 721 for protein synthesis was 0.25 micrograms/ml but it was greater than 32 micrograms/ml for RNA and DNA synthesis. In cell-free systems, DuP 721 concentrations up to 100 microM did not inhibit peptide chain elongation reactions under conditions where chloramphenicol, tetracycline and hygromycin B inhibited these reactions. Furthermore, Dup 721 did not cause phenotypic suppression of nonsense mutations suggesting that DuP 721 did not inhibit peptide chain termination. Thus, the mechanism of action of DuP 721 is at a target preceeding chain elongation.

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One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.

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The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

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The CYC1-239-O mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu- replacement of the normal -Ala-Gly- sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-1-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYC1+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYC1-239-O mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYC1 mRNA diminishes translation by inhibiting elongation.

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Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 microM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin- and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C] lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.

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A homologous in vitro method was developed from Tetrahymena for ribosomal A-site binding of aminoacyl-tRNA to poly(uridylic acid)-programmed ribosomes with very low error frequency. The reaction mixture pH was the crucial factor in the stable A-site association of aminoacyl-tRNA with high fidelity. At a pH greater than 7.1, endogenous activity translocated A-site-bound aminoacyl-tRNA to the P site. If translocation was allowed to occur, a near-cognate amino-acyl-tRNA, Leu-tRNA, could stably bind to the ribosome by translocation to the ribosomal P site. Near-cognate aminoacyl-tRNA did not stably bind to either site when translocation was blocked. Misreading antibiotics stimulated the stable association of near-cognate aminoacyl-tRNA to the ribosomal A site, thereby increasing the error frequency by several orders of magnitude. Ribosome binding of total aminoacyl-tRNA near equilibrium was not inhibited by misreading antibiotics; however, initial rate kinetics of the binding reaction were dramatically altered such that a 6-fold rate increase was observed with paromomycin or hygromycin B. The rate increase was evident with both cognate and near-cognate aminoacyl-tRNAs. Several antibiotics were tested for misreading potency by the ribosome binding method. We found gentamicin G418 greater than paromomycin greater than neomycin greater than hygromycin B greater than streptomycin in the potentiation of misreading. Tetracycline group antibiotics effectively inhibited A-site aminoacyl-tRNA binding without promoting misreading.

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Selenomethionine and methionine were compared as substrates for in vitro aminoacylation, ribosome binding, and peptide bond formation with preparations from wheat germ. Selenomethionine paralleled methionine in all steps of the translation process except peptide bond formation. Peptide bond formation with the initiating species of tRNA(Met) demonstrated that selenomethionyl-tRNA(Met) was less effective as a substrate than was methionyl-tRNA(f) (Met). Participation of selenomethionine in the initiation process of translation could be expected to reduce the overall rate of protein synthesis and might aid in explaining selenium toxicity in selenium-sensitive plants.

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Selenium-accumulator plants synthesize selenium compounds that differ from those produced by nonaccumulators. To determine if there are any subcellular differences between accumulators and nonaccumulators in the use of selenomethionine in vitro, polysomes from Astragalus crotalariae (accumulator) and Astragalus lentiginosis (nonaccumulator) were translated in the presence of selenomethionine. Polysomes from both species efficiently used selenomethionine in vitro during the translation process. Inasmuch as no differences in the incorporation of selenomethionine into protein were observed between polysomes from the two types of Astragalus, it can be inferred that in accumulators there exists a mechanism that either prevents synthesis of selenomethionine or modifies this selenocompound to a derivative that cannot be incorporated into protein.

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Vigna radiata polysomes efficiently incorporated [(75)Se]selenomethionine, [(14)C]methionine, and [(14)C]leucine in vitro. The optimal conditions for translation were determined to be 4.8 millimolar Mg(2+), 182 millimolar K(+), and pH 7.4. The rates of incorporation of [(75)Se]selenomethionine and [(14)C]methionine were similar when measured separately, but [(75)Se]selenomethionine incorporation was 35% less than [(14)C]methionine incorporation when both amino acids were present in equal molar concentrations. Polyacrylamide gel electrophoresis of the hot trichloroacetic acid precipitable translation products demonstrated synthesis of high molecular weight labeled proteins in the presence of [(75)Se]selenomethionine or [(35)S]methionine. No major differences in molecular weights could be detected in the electrophoretic profiles. Utilization of selenomethionine during translation by Vigna radiata polysomes establishes a route for the assimilation of selenomethionine by plants susceptible to selenium toxicity.

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Pigmentation mutants of Pseudomonas aeruginosa, selected by observed visual differences in coloration from the wild-type strain, were examined for altered patterns of phenazine synthesis. Three classes of mutants that were incapable of pyocyanine production were identified. Pigmentation patterns that were found to characterize the various mutant classes implicated precursor-product relationships, and a biochemical scheme covering the terminal reactions of pyocyanine biosynthesis is proposed. Among compounds tested as inhibitors of pigmentation, two effectively inhibited pyocyanine production production while allowing cell growth. p-Aminobenzoate inhibited total pigmentation; i.e., no other phenazine accumulated. m-Aminobenzoate inhibited a presumptive methylation step in pyocyanine biosynthesis, abolishing the formation of pyocyanine and aeruginosin pigments but increasing the yields of phenazine 1-carboxylic acid and oxychlororaphin. D-[2,3,4,5(n)-14C]shikimate was most efficiently incorporated into phenazines in the middle to late exponential phase of growth. Label was incorporated predominantly into pyocyanine in the absence of inhibitors and into phenazine 1-carboxylic acid when the organism was grown in the presence of m-aminobenzoate.

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