Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle.

The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.     

Sex chromosomes,micronuclei and aging in women

Several studies on aneuploidy and aging have shown a significant increase in the loss of chromosomes in both males and females with age. Others have observed a significant increase in micronucleus formation in lymphocytes with age. The objectives of this investigation were to determine the relationship between sex chromosome loss and increased micronucleus frequencies with age, to establish sex chromosome loss frequencies unbiased by cellular survival factors or slide preparation, and to determine the effect of smoking on sex chromosome loss. Blood samples were obtained from 8 newborn females and 38 adult females ranging in age from 19 to 77. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Two thousand binucleated cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. Slides were then hybridized with a 2.0 kb centromeric X chromosome-specific probe labeled with biotinylated dUTP, and detected with fluorescein-conjugated avidin. All micronucleated cells were relocated and their X chromosome status was determined. We found the X chromosome to be present in 72.2% of the micronuclei scored; additionally our results show a significant increase with age in the number of micronuclei containing an X chromosome.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Karyotype diversity and interspecific 4C DNA variation inBupleurum

Investigation on karyotype, 4C nuclear DNA amount and interphase nuclear volume (INV) of different HimalayanBupleurum species belonging toUmbelliferae revealed genetic differentiation. Numerical and structural alternation of chromosomes in interspecific level were manifested in their statistically significant altered species specific 4C nuclear DNA content. Somatic chromosome number ranged between 2n = 14 and 2n = 16.B. himalayense was reported for the first time having 2n = 16 chromosomes. Correlation coefficient among the various chromosomal and nuclear parameters showed no significant progressive or regressive interdependence except in between INV and nuclear DNA amount. Critical differences between 4C DNA content showed interspecific variation.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

Possible causes of the physiological decline in soybean nitrogen fixation in the presence of nitrate

Nodulated soybean plants (Glycine max (L.) Merr. cv. Clarke)were supplied with 10 mol m^-3 nitrate at the vegetative stage.This treatment caused a rapid decline in nitrogen fixation (acetylenereduction) activity and a consequent decline in ureides in thexylem sap. However, there was virtually no effect on the nitrogenasecomplex, according to Western blots against components 1 and2. The effect on nitrogen fixation was matched by a decreasein nitrogenase-linked respiration and increases in nodule oxygendiffusion resistance and the carbon cost of nitrogen fixation.The addition of nitrate had little effect on protein contentfrom either nodule plant or bacteroid fractions. Activitiesof nitrate reductase (NR) and nitrite reductase (NiR) from eitherthe plant fraction or the bacteroids were affected in differentways during 8 d of supply. Nodule plant NR and bacteroid NiR were not affected. However,nodule plant NiR increased 5-fold within 2 d of supplying Bacteroid NR only increased after6 d. These results could be interpreted in terms of a restrictednitrate access into the infected region of nodules. However,denitrification was detected within 2 d of nitrate supply insoybean nodules. The results are discussed in relation to possiblecauses of the nitrate-induced decline in nitrogenase activity. Key words: Glycine max, nitrate, nitrogen fixation, nodules     

Additional evidence implicating Triticum searsii as the B-genome donor to wheat

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid wheat species ancestral to the B genome of Triticum turgidum. ³H-T. turgidum DNA was hybridized to the unlabeled DNAs of T. urartu, T. speltoides, T. sharonensis, T. bicorne, T. longissimum, and T. searsii. ³H-Labeled DNAs of T. monococcum and a synthetic tetraploid AADD were hybridized with unlabeled DNAs of T. urartu and T. searsii to determine the relationship of the A genome of polyploid wheat and T. urartu. The heteroduplex thermal stabilities indicated that T. searsii was most closely related to the B genome of T. turgidum (AB) and that the genome of T. urartu and the A genome have a great deal of base-sequence homology. Thus, it appears that T. searsii is the B-genome donor to polyploid wheat or a major chromosome donor if the B genome is polyphyletic in origin.Published with the approval of the Director of The West Virginia Agricultural Experiment Station as Scientific Paper No. 1837.