Calcium/Calmodulin-Stimulated Protein Kinase II Is Present in Primary Cultures of Cerebral Endothelial Cells

Abstract: Calcium/calmodulin-stimulated protein kinase II (CaMPK II). a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the α-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that α-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that α-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.     

Immunophenotyping of mitotic cells from long-term cultures of chorionic villi

Chromosomal mosaicism in chorionic villus samples (CVS) may arise from different sources, such as clonal diversity within the chorionic tissue or contamination with maternal cells. To determine the origin of karyotyped cells, we compared the immunocytochemical features of mitotic cells in CVS long-term cultures with histological sections of their tissue of origin, i.e. chorionic villi. Immunolabelling of intermediate filaments specific for epithelial cells (cytokeratin) and mesenchymal cells (vimentin) established that mitoses yielded from CVS long-term cultures indeed stem from independently growing clones derived from both the epithelial and mesenchymal parts of the chorionic villi. Thus, mosaicism in CVS cultures may reflect true genetic heterogeneity within the biopsy. However, epithelial chorionic cells undergo in vitro metaplasia leading to co-expression of cytokeratins and vimentin. Fetal-specific immune markers (-HCG and SP1-glycoprotein) are not reliably expressed in CVS cell culture.     

Climate change disrupts core habitats of marine species

Driven by climate change, marine biodiversity is undergoing a phase of rapid change that has proven to be even faster than changes observed in terrestrial ecosystems. Understanding how these changes in species composition will affect future marine life is crucial for conservation management, especially due to increasing demands for marine natural resources. Here, we analyse predictions of a multiparameter habitat suitability model covering the global projected ranges of >33,500 marine species from climate model projections under three CO₂ emission scenarios (RCP2.6, RCP4.5, RCP8.5) up to the year 2100. Our results show that the core habitat area will decline for many species, resulting in a net loss of 50% of the core habitat area for almost half of all marine species in 2100 under the high-emission scenario RCP8.5. As an additional consequence of the continuing distributional reorganization of marine life, gaps around the equator will appear for 8% (RCP2.6), 24% (RCP4.5), and 88% (RCP8.5) of marine species with cross-equatorial ranges. For many more species, continuous distributional ranges will be disrupted, thus reducing effective population size. In addition, high invasion rates in higher latitudes and polar regions will lead to substantial changes in the ecosystem and food web structure, particularly regarding the introduction of new predators. Overall, our study highlights that the degree of spatial and structural reorganization of marine life with ensued consequences for ecosystem functionality and conservation efforts will critically depend on the realized greenhouse gas emission pathway.     

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.     

Gene transfer by electroporation into intact scutellum cells of wheat embryos

Summary Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-F capacitor and with 100 g DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MES 2-N-morpholinoethane sulfonic acid     

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.     

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (k_cat/k_m) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono-layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.     

Enantioselective lipase-catalyzed ester hydrolysis: Effects on rates and enantioselectivity from a variation of the ester structure

In order to make a preliminary study of substituent effects on the rate and enantioselectivity obtained in esterolytic reactions catalyzed by a lipase from Candida rugosa, a series of racemic esters, derived from some α-alkyl and α-halo phenylacetic acids, were prepared. The reactions were studied at pH 6.0 and 50°C under which conditions uncatalyzed hydrolysis was relatively slow. Reaction samples were studied at different points of time by means of analytical chiral reversed-phase liquid chromatography, which permitted the simultaneous determination of product enantiomeric excess and of the degree of total ester hydrolysis. These data were then used to calculate initial rates as well as enantioselectivity. An increase of the steric bulk of the α-substituent was found to highly decrease the rate of the reaction. On the other hand, rates were higher for the p-nitrophenyl esters than for the corresponding 2-chloroethyl esters. Consistently, the enantioselectivity was found to be higher for the latter type of ester. The esters of the α-halo (bromo and chloro) phenylacetic acids gave mandelic acid as the final product. This was caused by a rapid solvolysis of the α-halo phenylacetic acid initially formed. © 1993 Wiley-Liss, Inc.     

Phylogeny of Saxifraga section Saxifraga subsection Arachnoideae (Saxifragaceae) and the origin of low elevation shade-dwelling species

Saxifraga section Saxifraga subsection Arachnoideae is a lineage of 12 species distributed mainly in the European Alps. It is unusual in terms of ecological diversification by containing both high elevation species from exposed alpine habitats and low elevation species from shady habitats such as overhanging rocks and cave entrances. Our aims are to explore which of these habitat types is ancestral, and to identify the possible drivers of this remarkable ecological diversification. Using a Hybseq DNA-sequencing approach and a complete species sample we reconstructed and dated the phylogeny of subsection Arachnoideae. Using Landolt indicator values, this phylogenetic tree was used for the reconstruction of the evolution of temperature, light and soil pH requirements in this lineage. Diversification of subsection Arachnoideae started in the late Pliocene and continued through the Pleistocene. Both diversification among and within clades was largely allopatric, and species from shady habitats with low light requirements are distributed in well-known refugia. We hypothesize that low light requirements evolved when species persisting in cold-stage refugia were forced into marginal habitats by more competitive warm-stage vegetation. While we do not claim that such competition resulted in speciation, it very likely resulted in adaptive evolution.