Calcium control of Saccharomyces cerevisiae actin assembly.

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.     

Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol. II. Purification of mitochondrial and microsomal DT-diaphorase from 3-methylcholanthrene-treated rats

Liver mitochondrial and microsomal DT-diaphorase have been purified from 3-methylcholanthrene-treated rats. A 1150-fold and 3500-fold purification of mitochondrial and microsomal DT-diaphorase, respectively, is achieved after solubilization of the membranes with deoxycholate followed by affinity chromatography on azodicoumarol Sepharose 6B and subsequent gel filtration on Sephadex G-100. From this purification procedure, 65–70% of mitochondrial DT-diaphorase is recovered and the purified enzyme has a specific activity comparable to that of cytosolic DT-diaphorase; i.e., 50.4 kat/kg protein. Microsomal DT-diaphorase is obtained with a yield of 45% and a specific activity of 15.5 kat/kg protein.Purified mitochondrial DT-diaphorase exhibits an absorption spectrum characteristic of a flavoprotein and very similar to that of the cytosolic enzyme. Purification of both mitochondrial and microsomal DT-diaphorase results in fractions enriched in a polypeptide with a molecular weight of 28,000 which comigrates with purified cytosolic DT-diaphorase on SDS-polyacrylamide gel electrophoresis. Employing antiserum raised against cytosolic DT-diaphorase, immunological identity between DT-diaphorase isolated from the three cell fractions is observed with both the Ouchterlony immunodiffusion technique and fused rocket immunoelectrophoresis. The latter method also reveals that DT-diaphorase isolated from mitochondria and microsomes contains several antigenic forms identical to those observed in purified cytosolic DT-diaphorase. Furthermore, this antiserum inhibits DT-diaphorase to about the same extent whether the enzyme is isolated from mitochondria, microsomes, or cytosol. In addition, this antiserum efficiently inhibits membrane-bound microsomal DT-diaphorase.     

Inheritance of enzymes and blood proteins in the leopard frog,Rana pipiens: Three linkage groups established

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.The project was supported by Grant No. RR-00572 from the Division of Research Resources, National Institutes of Health. This paper is contribution No. C-87 from the Amphibian Facility, George W. Nace, Director.     

Enzymatic basis for the selective inhibition of varicella-zoster virus by 5-halogenated analogues of deoxycytidine.

5-Bromodeoxycytidine (BrdC) and 5-iododeoxycytidine, at a concentration of 100 mug/ml, effectively inhibit the replication of varicella-zoster (VZ) virus in tissue culture. No toxicity could be demonstrated in uninfected cells under the same conditions. Studies on the enzymatic basis for this selective inhibition were undertaken. Infection of human embryonic lung cell monolayers with VZ virus-infected cells results in the induction of thymidine (dT), deoxycytidine (dC), and BrdC kinase activities (which are increased 10-, 40-, and 60-fold, respectively) and in a 70-fold stimulation in the incorporation of 3H nucleotide (5-bromodeoxyuridylate) derived from BrdC into DNA. The thermal stability of the VZ virus-induced activities differs significantly from the activities induced by herpes simplex virus type 1 and herpes simplex virus type 2 and those present in uninfected human embryonic lung cells. The VZ virus-induced dT, dC, and BrdC kinase are similarly affected by temperature and cofractionate upon Sephadex gel filtration, findings consistent with the hypothesis that these activities are the function of a single enzyme: a pyrimidine deoxyribonucleoside kinase. The molecular weight, calculated on the basis of the elution pattern on Sephadex G-150, is 70,000. Kinetic studies, demonstrating that dT and dC competively inhibit the phosphorylation of BrdC, are consistent with the phosphorylation of these substrates at a common active site. Kinetic parameters include: KidT = 0.6 MUM; KidC = 60 muM; KmBrdC = 8.5 muM. In contrast to its relatively high affinity for the VZ virus-induced kinase, BrdC is a relatively poor substrate for the host kinases. Therefore, the basis for the selective inhibition of VZ virus by 5-halogenated analogues of dC is reflected in the induction of a pyrimidine deoxyribonucleoside kinase with a high affinity for BrdC.     

Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharide from Escherichia coli O 75

The structure of the O-specific side-chains of the cell-wall lipopolysaccharide of Escherichia coli O 75 has been investigated, using methylation analysis and Smith degradation as the principal methods. The O-specific side-chain was found to be composed of a tetrasaccharide repeating-unit of the following structure:

     

Vaginal microbiome-hormonal contraceptive interactions associate with the mucosal proteome and HIV acquisition

Alterations to the mucosal environment of the female genital tract, such as genital inflammation, have been associated with increased HIV acquisition in women. As the microbiome and hormonal contraceptives can affect vaginal mucosal immunity, we hypothesized these components may interact in the context of HIV susceptibility. Using previously published microbiome data from 685 women in the CAPRISA-004 trial, we compared relative risk of HIV acquisition in this cohort who were using injectable depot medroxyprogesterone acetate (DMPA), norethisterone enanthate (NET-EN), and combined oral contraceptives (COC). In women who were Lactobacillus-dominant, HIV acquisition was 3-fold higher in women using DMPA relative to women using NET-EN or COC (OR: 3.27; 95% CI: 1.24–11.24, P = 0.0305). This was not observed in non-Lactobacillus-dominant women (OR: 0.95, 95% CI: 0.44–2.15, P = 0.895) (interaction P = 0.0686). Higher serum MPA levels associated with increased molecular pathways of inflammation in the vaginal mucosal fluid of Lactobacillus-dominant women, but no differences were seen in non-Lactobacillus dominant women. This study provides data suggesting an interaction between the microbiome, hormonal contraceptives, and HIV susceptibility.     

A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

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Highlights

• •Liver Mallory-Denk-Body inducers elicited an IκBα-loss and NF-κB-activation.
• •IκBα-loss was due to its sequestration into insoluble cytoplasmic aggregates.
• •Four proteomic approaches identified 10 IκBα-interacting/aggregating proteins.
• •Nup153/RanBP2-aggregation prevented IκBα nuclear entry for ending NF-κB-activation.