The β3‐integrin endothelial adhesome regulates microtubule‐dependent cell migration

Integrin β3 is seen as a key anti‐angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro‐ or anti‐angiogenic depends on the context in which it is expressed. To understand precisely β3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the β3‐dependent adhesome. We show that depletion of β3‐integrin in this cell type leads to changes in microtubule behaviour that control cell migration. β3‐integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2‐driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when β3‐integrin levels are reduced.     

Simultaneous increase in cellular content and volumetric concentration of lipids in <Emphasis Type="Italic">Bracteacoccus bullatus</Emphasis> cultivated at reduced nitrogen and phosphorus concentrations

Manipulation of the nutrient concentration is an inexpensive and efficient method for increasing lipid and TAG accumulation in algal cells. However, high volumetric production requires finding a proper balance between the decrease of biomass production and the increase in the total lipid content. We isolated a strain of green microalga Bracteacoccus bullatus and increased its lipid content from 17 to 59% of biomass dry weight by manipulating of nitrogen and phosphorus content in the medium. The 10-fold reduction of the nitrogen and phosphorus concentration in the medium was the most efficient method of the lipid induction compared to nutrient deplete and high nutrient conditions. The oleic (48–64% mass of total fatty acids) and linoleic (14–24% mass of total fatty acids) acids dominated in the fatty acid profile, thus making this strain a suitable candidate for biodiesel production.     

Laminin α1 Chain Synthesis in the Mouse Developing Lung: Requirement for Epithelial–Mesenchymal Contact and Possible Role in Bronchial Smooth muscle Development

Laminins, the main components of basement membranes, are heterotrimers consisting of α, β, and γ polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of β1 and γ1 chains and differ from each other on their α chain, which is α1 and α2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial–mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin α1 chain. Synthesis of laminin α1 chain however returns upon re-establishment of epithelial–mesenchymal contact. Cell–cell contact is critical, since laminin α1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial–mesenchymal cocultures in which heterotypic cell–cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin α1 chain upon heterotypic cell– cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin α1, α2, and β/γ chains. Lung explants exposed to monoclonal antibodies to laminin α1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle α actin and desmin. Taken together, our studies suggest that laminin α1 chain synthesis is regulated by epithelial–mesenchymal interaction and may play a role in airway smooth muscle development.     

Laminin α4 and Integrin α6 Are Upregulated in Regenerating dy/dy Skeletal Muscle: Comparative Expression of Laminin and Integrin Isoforms in Muscles Regenerating after Crush Injury

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin α2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and “preactivated” myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin α1 was not detectable in skeletal muscle. Laminin α2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin α2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin α chains in early myogenic differentiation, such as laminin α4 and α5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin β1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin α3 was not expressed in uninjured or regenerating muscle, while integrin α6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin α6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin α6 occurred on some newly formed myotubes. Integrin α7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin α7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin α7 negative. No marked difference was observed in integrin α7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin α4 and integrin α6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin α4 and integrin α6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin α2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.     

Heterochromatin restricts the mobility of nuclear bodies

Nuclear bodies are relatively immobile organelles. Here, we investigated the mechanisms underlying their movement using experimentally induced interphase prenucleolar bodies (iPNBs). Most iPNBs demonstrated constrained diffusion, exhibiting infrequent fusions with other iPNBs and nucleoli. Fusion events were actin-independent and appeared to be the consequence of stochastic collisions between iPNBs. Most iPNBs were surrounded by condensed chromatin, while fusing iPNBs were usually found in a single heterochromatin-delimited compartment (“cage”). The experimentally induced over-condensation of chromatin significantly decreased the frequency of iPNB fusion. Thus, the data obtained indicate that the mobility of nuclear bodies is restricted by heterochromatin.     

Analysis of the distribution of the nucleotide sequence and electrostatic potential of the Escherichia coli genome

The oligonucleotide composition of the E. coli genome and its sigma70-specific promoters has been analyzed. The promoter DNA was shown to contain mainly AT-rich hexanucleotides having functionally important physical properties such as the ability to form easily melting sites and induce the bending of the double helix. A comparative analysis of the electrostatic characteristics of hexanucleotides within the whole sequence of the E. coli genome and its promoter regions was made. Hexanucleotides possessing a more electronegative surrounding were found to predominate in the nucleotide sequence of the promoter DNA.     

Bacterial diversity and activity along a salinity gradient in soda lakes of the Kulunda Steppe (Altai, Russia)

Here we describe the diversity and activity of sulfate reducing bacteria along a salinity gradient in four different soda lakes from the Kulunda Steppe (South East Siberia, Russia). For this purpose, a combination of culture-dependent and independent techniques was applied. The general bacterial and SRB diversity were analyzed by denaturing gradient gel electrophoresis (DGGE) targeting the 16S rDNA gene. DNA was used to detect the microbial populations that were present in the soda lake sediments, whereas ribosomal RNA was used as a template to obtain information on those that were active. Individual DGGE bands were sequenced and a phylogenetic analysis was performed. In addition, the overall activity of SRB was obtained by measuring the sulfate reduction rates (SRR) and their abundance was estimated by serial dilution. Our results showed the presence of minor, but highly active microbial populations, mostly represented by members of the Proteobacteria. Remarkably high SRR were measured at hypersaline conditions (200 g L⁻¹). A relatively high viable count indicated that sulfate reducing bacteria could be highly active in hypersaline soda lakes. Furthermore, the increase of sodium carbonate/bicarbonate seemed to affect the composition of the microbial community in soda lakes, but not the rate of sulfate reduction.     

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples

Background  

Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C₄(RP)-LC protein separation) followed by C₁₈(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.