Counter-immunoelectrophoresis for the detection of Brucella antigen and antibodies in the diagnosis of brucellosis in buffaloes

Counter-immunoelectrophoresis was employed for the detection of Brucella antigen in stomach contents of aborted buffalo fetuses and antibody in aborted as well as apparently healthy in contact buffaloes. Five of 16 aborted cases were serologically positive for brucellosis but isolation of Brucella abortus was successful in only two cases. By counter-immunoelectrophoresis, Brucella antigen was detected in the fetal stomach contents of four serologically positive cases. Of the 68 serum samples from in contact healthy buffaloes, 10 were positive with counter-immunoelectrophoresis: more than were detected by tube agglutination, Rose Bengal plate agglutination, complement fixation and agar gel precipitation test.     

Dynamic state of site-specific DNA methylation concurrent to altered prolactin and growth hormone gene expression in the pituitary gland of pregnant and lactating rats

The regulatory mechanism for the hormonal control of prolactin (PRL) and growth hormone (GH) gene expression in rat pituitary gland during gestation and lactation is verified in this study. The level of PRL-specific mRNA (mRNAPRL) sequences in the pituitary gland is elevated in the later part of gestation and more prominently so in lactation. In contrast the expression of GH gene is inhibited in the same tissue during gestation and lactation resulting in the dramatic decrease in the level of GH-specific mRNA (mRNAGH) sequences. We now demonstrate the influence of a tissue-specific altered DNA methylation pattern on the temporal modulation of expression of PRL and GH genes in the pituitary gland during alternate physiological states. An altered methylation pattern of specific "-C-" residues only in the coding region of PRL and GH gene can be detected concurrently with the altered level of expression of these genes in the pituitary gland during gestation and lactation. These results also demonstrate the dynamic state of methylation of specific -C- residues during the transition of these two genes from one state of expression to another in the same tissue. A correlation between site-specific DNA methylation and tissue-specific expression of PRL and GH gene in pituitary gland is reported. Thus a role of DNA methylation in the hormonal control of PRL and GH gene expression in physiological states such as pregnancy and lactation is proposed.     

Reconstituted U1 small nuclear ribonucleoprotein complex restores 5' splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.     

Rapid isolation of lysyl-tRNA synthetase from rat liver

The high molecular weight aminoacyl-tRNA synthetase complex (the 24S complex) was isolated from rat liver by ultracentrifugation. The lysyl-tRNA synthetase (E.C. 6.1.1.6) was selectively dissociated by hydrophobic interaction chromatography on 1,6 diaminohexyl agarose followed by hydroxylapatite chromatography and DEAE chromatography. The lysyl-tRNA synthetase dissociated from the 24S synthetase complex was purified approximately to 2700 fold with 14% yield.     

Asymmetry of lysophosphatidylcholine/cholesterol vesicles is sensitive to cholesterol modulation

Sonication of lysophosphatidylcholine (lysoPC; 20 mumol/mL) and cholesterol (chol) in aqueous medium produces lamellar structures over a wide range of concentrations. From 25 to 47 mol % cholesterol, electron microscopy (EM) after negative staining showed extended stacklike lamellae about 40 A thick. From 50 to 60 mol % chol, freeze-fracture EM showed homogeneous populations of small unilamellar vesicles averaging 260-310 A in diameter. Phosphorus-31 nuclear magnetic resonance was used to characterize the stacklike lamellae and to measure the distribution of the lysophospholipid between the outer and inner leaflet of the vesicles as a function of sterol concentration. We found that in lysoPC/chol dispersions containing less than equimolar amounts of cholesterol (25-47 mol %), the entire phosphorus signal (40.5 ppm) was shifted downfield by 10.5 ppm upon addition of Pr3+ (2.4 mM), consistent with the stacklike lamellar structures in which all lysoPC head groups are accessible to the ions. By contrast, addition of Pr3+ to lysoPC/chol vesicles containing equimolar or higher amounts of cholesterol (up to 60 mol %) gave rise to two phosphorus peaks. The more intense downfield signal (51.0 ppm) responsive to paramagnetic ions was assigned to lysoPC located in the outer vesicle leaflet. The upfield signal (40.5 ppm), which was not affected by the ions, was assigned to inside lysoPC. For lysoPC/chol (1:1) vesicles, an outside to inside lysophospholipid ratio (Ro/i) of 6.5 was determined. Essentially the same Ro/i value (6.7) was obtained on lysoPC/chol (1:1) vesicles which after dialysis contained only entrapped Pr3+.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage

A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.     

Digitalis-like pregnanes. Cardiac and renal effects of a glycoside of 14 beta-hydroxyprogesterone

The synthesis of the glucoside, 3 beta-[(beta-D-glucopyranosyl)oxy]-14-hydroxy-14 beta-pregn-4-en-20-one, a 14 beta-hydroxyprogesterone glucoside (14 beta-OHP-glu), is described. This compound has an IC50 of 1 microM in a [3H]ouabain binding assay, and is about 10 times more potent than the aglycone. Like 14 beta-hydroxyprogesterone, the glucoside enhances contractility of isolated cardiac muscle. 14 beta-OHP-glu or ouabain, when infused at comparable doses into the renal artery of the anesthetized rat, markedly increases urine volume. Whereas ouabain significantly enhances urinary potassium excretion with little or no effect on sodium excretion, 14 beta-OHP-glu promotes a marked natriuresis with no significant effect on potassium excretion.     

Methane recovery from water hyacinth through anaerobic activated sludge process

The concepts of phase separation, anaerobic activated sludge process, and alkali pretreatment have been incorporated in this investigation with the objective of developing rational and cost-effective designs of diphasic anaerobic activated sludge systems, with and without alkali treatment, for methane recovery from water hyacinth (WH). Evaluation of process kinetics and optimization analyses of laboratory data reveal that a diphasic system with alkali treatment could be designed with an alkali pretreatment step (3.6% Na(2)CO(3) + 2.5% Ca(OH)(2) (w/w) of WH, 24 h duration) followed by an open acid phase (2.1 days HRT) and closed methane reactor with sludge recycle (5.7 days HRT, 7.7 days MCRT) for gas yield of 50 L/kg WH/d at 35-37 degrees C. Likewise, a diphasic system without alkali treatment could be designed with an open acid phase (2 days HRT) followed by closed methane reactor with sludge recycle (3.2 days HRT, 6 days MCRT) for gas yield of 32.5 L/kg WH/d at 35-37 degrees C. Detailed economic analyses bring forth greater cost-efficacy of the diphasic system without alkali treatment and reveal that the advantage accrued in terms of higher gas yield is overshadowed by the cost of chemicals in the diphasic system with alkali treatment.     

Expression of 93D heat shock puff of Drosophila melanogaster in deficiency genotypes and its influence on activity of the 87C puff

Using the overlapping deficiencies Df(3R)GC14 and Df(3R)e ^Gp 4 of the 93D region of Drosophila melanogaster, the benzamide (BM)-inducible site in polytene chromsomes was localised to the 93D6-7 region, which had earlier been identified as heat inducible. The normal developmental and BM-induced 93D6-7 puff was found to be dosage compensated since in larvae heterozygotus for a deficiency, with one dose of 93D6-7, the rate of ³H-uridine incorporation in this puff was the same as in the wild type with two doses. Curiously, however, heat shock (37° C) caused regression of the 93D6-7 puff on the normal chromosome in heterozygotes. In agreement with earlier results from our laboratory, the non-inducibility of the single-dose 93D locus by heat shock in the heterozygotes, caused the 87C puff to be nearly half as active as the 87A puff at 37° C. However, in e ^Gp 4/GC14 larvae, lacking the 93D6-7 locus on both homologues, the 87C puff was less active than 87A in some heat-shocked larvae but in other larvae 87A and 87C were equally active. Possible reasons for this inter-larval variability are discussed.     

Phosphorylation of lipocortins in vitro by protein kinase C

Protein kinase C catalyzes the incorporation of about 1.1, 0.7 and 0.4 mole of phosphate per mole of Lipocortin-I (P35), Lipocortin-II (P36) and Lipocortin-85 (P36 oligomer) respectively. The phosphorylation is specific for protein kinase C and is dependent on the presence of both calcium and phospholipids. While Lipocortin-I is phosphorylated on threonine residues, Lipocortin-II and Lipocortin-85 are phosphorylated on serine residues. The substoichiometric phosphorylation of Lipocortin-85 appears to preclude the potential regulation of this protein by protein kinase C. The phosphorylation of Lipocortin-I on threonine residues and Lipocortin-II on serine residues suggests these proteins may be regulated by distinct phosphorylation-dephosphorylation reactions.