Cell cycle regulation of NF-YC nuclear localization

NF-Y is a trimeric activator with histone fold, HFM, subunits that binds to the CCAAT-box and is required for a majority of cell cycle promoters, often in conjunction with E2Fs. In vivo binding of NF-Y is dynamic during the cell cycle and correlates with gene activation. We performed immunofluorescence studies on endogenous, GFP- and Flag-tagged overexpressed NF-Y subunits. NF-YA, NF-YB are nuclear proteins. Unexpectedly, NF-YC localizes both in cytoplamatic and nuclear compartments and its nuclear localization is determined by the interaction with its heterodimerization partner NF-YB. Most importantly, compartmentalization is regulated during the cell cycle of serum restimulated NIH3T3 cells, accumulating in the nucleus at the onset of S phase. These data point to the control of HFM heterodimerization as an important layer of NF-Y regulation during cell cycle progression.     

Modulation of endothelin-A receptor, Galpha subunit, and RGS2 expression during H9c2 cardiomyoblast differentiation

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.     

Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.     

Mechanosensing role of caveolae and caveolar constituents in human endothelial cells

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2.     

Outbreak of hind limb paralysis in young CFW Swiss Webster mice

An outbreak of paralysis among 16- to 20-week-old CFW Swiss Webster sentinel mice developed in one of our barrier facilities. Two months after arrival and over a period of four weeks, six of 400 mice purchased from an approved vendor, developed progressive hind limb paralysis without other clinical signs of disease. On the basis of the histopathologic changes and negative serologic test results, lymphoblastic lymphoma causing compression of the spinal cord was diagnosed. There were two leading features to this outbreak: its unusual epidemiologic presentation, and the localization of the lesions principally in the lumbar muscles. A presumptive diagnosis of retroviral infection with Abelson's murine leukemia virus (A-MuLV) was established on the basis of histopathologic and immunohistochemical findings. Little is known about retroviral status in many commercial colonies, and few users report presence of spontaneous lymphomas. This report points out complications derived from commercially available animals that carry endogenous retroviruses. It also emphasizes the need of diagnosing and reporting clusters of hind limb paralysis or lymphomas in mice to assess the prevalence and relevance of retroviral infections in commercial colonies.     

Patterns of polymorphism detected in the chloroplast and nuclear genomes of barley landraces sampled from Syria and Jordan

In order to examine how molecular polymorphism in barley landraces, sampled from five different ecogeographical regions of Syria and Jordan, is organised and partitioned, genetic variability at 21 nuclear and 10 chloroplast microsatellite loci were examined. Chloroplast polymorphism was detected, with most variation being ascribed to differences between the five regions (F_st 0.45) and to within sites within each region (F_st 0.44). Moreover, the distribution of chloroplast polymorphism is structured and not distributed randomly across the barley landraces sampled. From a total of 125 landrace accessions (five lines from each of five sites from each of five regions) genotyped with 21 SSRs a total of 244 alleles were detected, of which 38 were common to the five regions sampled. Most nuclear variation was detected within sites. Significant differentiation between sites (F_st 0.29) was detected with nuclear SSRs and this partially mirrored polymorphism in the chloroplast genome. Strong statistical associations/interaction was also detected between the chloroplast and nuclear SSRs, together with non-random association (linkage disequilibrium) of alleles at both linked and unlinked SSR loci. These results are discussed in the context of adaptation of landraces to the extreme environment, the concept of 'adapted gene complexes' and the exploitation of landraces in breeding programmes.Communicated by P. Langridge     

Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase

Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.     

Unusual glycoconjugates in the oesophagus of a tilapine polyhybrid

The aim of this work is to elucidate the glycoconjugate composition of the secretory products of the oesophageal mucous cells in a tilapine polyhybrid. Lectin histochemistry gave evidence of the presence of β-galactose, α-N-acetylgalactosamine and sialic acid residues in the terminal position. The majority of sialic acid belongs to short side chains; a few sialic acid residues are acetylated at the C₇ and/or C₈ and/or C₉ level. The heterogeneity of the carbohydrate chains may mask potential receptor sites for micro-organisms and hamper the formation of multiple bonds.