Chemical identification of<Emphasis Type="Italic"> N</Emphasis>-acyl homoserine lactone quorum-sensing signals produced by<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> strains in defined medium

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.     

Single-chain Tet transregulators

We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse ‘tet-on’ phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.     

The Proteome Analysis database: a tool for the in silico analysis of whole proteomes

The Proteome Analysis database (http://www.ebi.ac.uk/proteome/) has been developed by the Sequence Database Group at EBI utilizing existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archeae and eukaryotes. Three main projects are used, InterPro, CluSTr and GO Slim, to give an overview on families, domains, sites, and functions of the proteins from each of the complete genomes. Complete proteome analysis is available for a total of 89 proteome sets. A specifically designed application enables InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.     

Metabolic Changes in <Emphasis Type="Italic">Clostridium absonum</Emphasis> ATCC 27555 Accompanying Induction of Epimerization of a Primary Bile Acid

Some parameters of fermentation have been determined for Clostridium absonum in a chemostat by using a chemically defined medium with glucose as the sole source of carbon and energy. Steady-state continuous cultures were achieved at two dilution rates (D). Trends of the carbon flow were determined by comparison of ratios between the specific rates of formation of the three products of metabolism (lactate, acetate, butyrate). Chenodeoxycholate induced the 7- and 7-hydroxysteroid dehydrogenases of C. absonum. In the presence of this inducer, the growth yield and the carbon recovery decreased, the carbon flow distribution was altered favoring acetate production, and a deficit in the reoxydation of nucleotidic cofactors was observed. In the presence of chenodeoxycholate, C. absonum would favor the production of energy at the expense of the reoxidation of nucleotidic cofactors so as to ensure its growth, and the epimerization of chenodeoxycholate to ursodeoxycholate.     

Genetic studies in south Balkan populations

Within a study of the genetics of Balkan populations, four DNA-STR systems and 19 classical markers were examined in seven samples: Romanians (two groups), Albanians, Greeks and Aromuns (three groups). The results for the DNA-STR systems have been compared with data from the literature. The results show four clear separated groups: sub-Saharan black populations, North-African, Japanese and European populations. The large Balkan populations, except the Greek sample, are genetically more homogenous than the Aromun populations. A second Neighbor-joining tree based on all 23 analyzed systems, show a particular trend of the Aromun groups, which indicates a particular genetic structure.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.     

Food hoarding is increased by food deprivation and decreased by leptin treatment in Syrian hamsters

Compensatory increases in food intake are commonly observed after a period of food deprivation in many species, including laboratory rats and mice. Thus it is interesting that Syrian hamsters fail to increase food intake after a period of food deprivation, despite a fall in plasma leptin concentrations similar to those seen in food-deprived rats and mice. In previous laboratory studies, food-deprived Syrian hamsters increased the amount of food hoarded. We hypothesized that leptin treatment during food deprivation would attenuate food-deprivation-induced increases in hoarding. Baseline levels of hoarding were bimodally distributed, with no hamsters showing intermediate levels of hoarding. Both high (HH) and low hoarding (LH) hamsters were included in each experimental group. Fifty-six male hamsters were either food deprived or given ad libitum access to food for 48 h. One-half of each group received intraperitoneal injections of leptin (4 mg/kg) or vehicle every 12 h during the food-deprivation period. Within the HH group, the hoarding score increased significantly in food-deprived but not fed hamsters (P < 0.05). Leptin treatment significantly decreased hoarding in the food-deprived HH hamsters (P < 0.05). The LH hamsters did not increase hoarding regardless of whether they were food deprived or had ad libitum access to food. These results are consistent with the idea that HH hamsters respond to energetic challenges at least in part by changing their hoarding behavior and that leptin might be one factor that mediates this response.     

Remodeling of the adventitia during coronary arteriogenesis

We studied the role of the adventitia in adaptive arteriogenesis during the phase of active growth of coronary collateral vessels (CV) induced by chronic occlusion of the left circumflex coronary artery in canine hearts. We used electron microscopy and immunoconfocal (IF) labeling for bFGF, matrix metalloproteinase (MMP)-2, MMP-9, tissue-type plasminogen activator (tPA), its inhibitor (PAI-1), fibronectin (FN), and Ki-67. Proliferation of smooth muscle cells and adventitial fibroblasts was evident. Quantitative IF showed that adventitial MMP-2, MMP-9, and FN were 9.2-, 7.5-, and 8.6-fold, bFGF was 5.1-fold, and PAI-1 was 3.4-fold higher in CV than in normal vessels (NV). The number of fibroblasts was 5-fold elevated in CV, but the elastic fiber content was 25-fold greater in NV than in CV. Perivascular myocyte damage and induction of endothelial nitric oxide synthase in peri-CV capillaries indicate expansion of CV. It was concluded that adventitial activation is associated with the development of CV through cell proliferation, production of growth factors, and induction of extracellular proteolysis thereby contributing to remodeling during adaptive arteriogenesis.     

Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.