Usage of primary cells to delineate IFN-gamma-responsive DNA elements in the HLA-DRA promoter and to identify a novel IFN-gamma-enhanced nuclear factor.

The IFN-gamma regulation of the HLA-DRA gene was examined in a primary cell type, the astrocyte. Site-specific mutagenesis of the DRA promoter reveals that three known sequences, S, X, and Y, are required for an optimal IFN-gamma response. Specifically for the X sequence, the X1, but not the X2, site is involved in IFN-gamma regulation of HLA-DRA in the astrocyte. Most interesting, a novel IFN-gamma-enhanced protein (IFNEX) with specificity for the X element has consistently been observed in nuclear extracts made from primary astrocytes. The correlation of the functional importance of X1 in IFN-gamma-regulated DRA expression and the enhancement of IFNEX by IFN-gamma strongly suggests that IFNEX may play a crucial role in IFN-gamma-regulated class II MHC gene expression.     

腐蹄病致病因子节瘤拟杆菌蛋白酶在大肠杆菌...

Bacteroides nodosus is the essential causative agent of ovine footrot. It produces extracellular proteases which involved in pathogenesis of footrot. In this paper, we report the subcloning of Bacteroides nodosus protease, its overexpression in E. coli and its N-terminal polypeptide sequence. The subclone library was constructed in E. coli using SphI digested original clone (15 kb) and plasmid PTZ18R and screened using immunological assay. The expression was observed using SDS-PAGE. The subcloned DNA fragment was then cut with Sau3AI, cloned into pKK232-8 vector to perform promoter isolation and analysis. The promoter strength was determined using spectrophotometric assay.     

ATP-dependent DNA aggregation is a novel function of rat serum albumin

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.     

Biological activity of nasally administered insulin in normal subjects

Nasally administered (IN) insulin has been advocated as a potentially useful alternative to subcutaneously administered regular insulin because of its more rapid onset and time to peak action and its shorter duration of action. This study further defines the pharmacodynamics of IN insulin by using a euglycemic clamp technique to determine the bioavailability of IN insulin as compared with intravenous (IV) insulin, and to ascertain whether multiple sequentially administered doses of IN insulin alter pharmacodynamics. Eight normal volunteers received 2 control doses of IV insulin (0.05 U/kg), and 3 high doses (0.7 U/kg) and 3 low doses (0.35 U/kg) of IN insulin with an absorption enhancer (tauro-24,25 dihydrofusidate) given sequentially over a 2 day period. A euglycemic clamp was performed with a Biostator (Ames) that infused dextrose to keep the subject's blood glucose at his fasting level. Analysis of dextrose infusion curves for the low and high doses of IN insulin revealed an onset of action of 9.4 +/- 0.4 and 10.5 +/- 0.3 minutes, time to peak action of 20.6 +/- 5.6 and 23.7 +/- 4.4 minutes and duration of action of 82.1 +/- 5.2 and 95 +/- 5.7 minutes respectively. Both the onset of action and time to peak action were slightly longer (P less than .05) for the high as compared with the low dose IN insulin, although this should not represent a clinically significant difference. The total dextrose requirement was 21.9 +/- 2.3 g for the low dose IN insulin and 34.1 +/- 3.3 g for the high dose IN insulin, the latter value being significantly greater (P less than .01) than the former.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis by DNA polymerase I on bleomycin-treated deoxyribonucleic acid: a requirement for exonuclease III

phi X174 RFI DNA treated with bleomycin (BLM) under conditions permitting nicking does not serve as a template-primer for Escherichia coli DNA polymerase I. Purified exonuclease III from E. coli and extracts from wild-type E. coli strains are able to convert the BLM-treated DNA to suitable template-primer, but extracts from exonuclease III deficient strains are not. Brief digestion by exonuclease III is enough to create the template-primer, suggesting that the exonuclease III is converting the BLM-treated DNA by a modification of 3' termini. The exonucleolytic rather than the phosphatase activity of exonuclease III appears to be involved in the conversion. Comparative studies with micrococcal nuclease indicate that BLM-created nicks do not have a simple 3'-P structure. Bacterial alkaline phosphatase does not convert BLM-treated DNA to template-primer. The endonuclease VI activity associated with exonuclease III does not incise DNA treated with BLM under conditions not allowing nicking, in contrast to DNA with apurinic sites made by acid treatment, arguing that conversion does not require the endonuclease VI action on uncleaved sites.     

Isolation of synaptonemal complexes from hamster spermatocytes

Nuclei from Chinese hamster testicular cells in suspension were prepared in a sucrose gradient. Following the basic procedure of Blobel and co-workers for separating a fibrous lamina-nuclear pore complex, synaptonemal complexes (SCs) from spermatocytes were isolated free of other nuclear structures, except for fibrillar tufts at the attachment plaques in which annuli were observed. All the major morphological components of the SC appeared to be intact, showing that the structure could survive the procedure and was not dispersed by the removal of DNA with DNase and solubilization of membranes and some proteins with Triton X-100. Isolated sex bodies were also well preserved, as were various structures from other cell types in the mixed cell suspension, such as spermatid manchettes, acrosomal ‘ghosts’, axonemes, etc. While no nuclear matrix was found associated with autosomal SCs, a residual material was present in the sex body, in which the X and Y axes were embedded. The results indicate the feasibility of isolating and fractionating SCs from testicular cell suspensions enriched for pachytene spermatocytes. The association between SC attachment plaques and annuli that is seen in spreads of whole nuclei persists through the isolation procedure and implies an integrated structural relationship.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Immunological cross-reactivity of multiplication-stimulating activity polypeptides

The immunoreactivity of the multiple species of multiplication-stimulating activity (MSA) purified from medium conditioned by a rat liver cell line (BRL-3A) has been examined. Antibodies were raised in rabbits following immunization with MSA II polypeptides. Subpopulations of antibodies were purified from one antiserum using DEAE-cellulose chromatography. One antibody subpopulation recognized common antigenic determinants on MSA I and MSA II polypeptides; whereas a second antibody subpopulation recognized common determinants on MSA I, II, and III polypeptides. In a radioimmunoassay utilizing 125I-MSA III-2 and a purified antibody subpopulation, the human somatomedins (somatomedin A, insulin-like growth factor I and II) showed weak, but significant cross-reactivity: insulin-like growth factor II was 10% as potent as MSA II. By contrast, somatomedin partially purified from rat serum, insulin, growth hormone, epidermal growth factor, nerve factor, and fibroblast growth factor, showed no reactivity in the radioimmunoassay.     

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.