Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP-RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 X 10(-5) s(-1), considerably slower than free RhoA. Hydrolysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP-bound G14V-RhoA/RhoGDI complex (but not the GDP form) into serum-starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These results show that GTP-triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.     

Lipid phase separation correlates with activation in platelets during chilling

When human platelets are chilled below 22 degrees C, they spontaneously activate, a phenomenon that severely limits their storage life. It has previously been proposed that there is a correlation between cold-induced platelet activation and passage of the membranes through a liquid-crystalline to gel phase transition. Because animal models are essential for developing methods for cold storage of platelets, it is necessary to investigate such a correlation in animal platelets. In this work, horse platelets were used as a model, and it was found that cold-induced morphological activation is related to the lipid phase transition. Using fluorescence microscopy with the lipophilic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-C18), and Fourier transform infrared spectroscopy (FTIR), it was found that lipid phase separation occurs during cooling and low temperature storage. Furthermore, removal of cholesterol from the plasma membrane also induced a phase separation, possibly between specific phospholipid classes. Steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-DPH (TMA-DPH) were compared in cells and multilamellar vesicles (MLV) composed of platelet lipids. Cholesterol depletion led to a decrease in the fluorescence anisotropy of the two probes, which can be explained by changes in the order of the phospholipid molecules. In addition, the lipid composition and fatty acid profile of the cellular phospholipids were determined. Based of the similarities between horse and human platelets, it is suggested that horse platelets may be used as a model for studying cold-stored platelets. The results are discussed in relation to the possible role of phase separation during cell signalling.     

Nitric oxide-dependent pulmonary vasodilation in polycythemic rats

Polycythemia causes increased vascular production of nitric oxide (NO), most likely secondary to an effect of elevated vascular shear stress to enhance expression of endothelial nitric oxide synthase (eNOS). Because both polycythemia and increased eNOS expression are associated with chronic hypoxia-induced pulmonary hypertension, experiments were performed to test the hypothesis that increased hematocrit leads to upregulation of pulmonary eNOS and enhanced vascular production of NO independent of hypoxia. Rats were administered human recombinant erythropoietin (rEpo; 48 U/day) or vehicle for 2 wk. At the time of study, hematocrit was significantly greater in the rEpo-treated group than in the vehicle group (65.8 +/- 0.7% vs. 45.1 +/- 0.5%), although mean pulmonary artery pressure did not differ between treatments. Experiments on isolated, saline-perfused lungs demonstrated similar vasodilatory responses to the endothelium-derived NO-dependent agonist ionomycin in each group. Additional experiments showed that the vasoconstrictor response to the thromboxane mimetic U-46619 was diminished at lower doses in lungs from the rEpo group compared with the vehicle group. However, perfusate nitrite/nitrate concentration after 90 min of perfusion in isolated lungs was not different between groups. Additionally, no difference was detected between groups in lung eNOS levels by Western blot. We conclude that the predicted increase in shear stress associated with polycythemia does not result in altered pulmonary eNOS expression.     

Hanging on to your homolog: the roles of pairing, synapsis and recombination in the maintenance of homolog adhesion

Homologous chromosomes initially undergo weak alignments that bring homologous sequences into register during meiosis. These alignments can be facilitated by two types of mechanisms: interstitial homology searches and telomere-telomere alignments. As prophase (and chromatin compaction) proceeds, these initial pairings or alignments need to be stabilized. In at least some organisms, such as Saccharomyces cerevisiae and S. pombe, these pairings can apparently be maintained by the creation of recombination intermediates. In contrast, synapsis during zygotene may be able to facilitate and/or maintain chromosome pairing even in the absence of exchange in several higher organisms. It thus seems possible that the synaptonemal complex plays a role both in maintaining homolog adhesion during meiotic prophase and, more speculatively, in facilitating meiotic exchange. Received: 15 November 1999; in revised form: 17 January 2000 / Accepted: 18 January 2000     

De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B₁4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing ¹⁵NH₄Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.     

Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1

 Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors, and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix. In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete distance from the lesion not exceeding 350 μm from the inflammatory cells. Staining was associated with mesenchymal cells with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the regulation of extracellular matrix accumulation. Accepted: 19 December 1998     

Pleistocene phylogeographic effects on avian populations and the speciation process.

Pleistocene biogeographic events have traditionally been ascribed a major role in promoting speciations and in sculpting the present-day diversity and distributions of vertebrate taxa. However, this paradigm has recently come under challenge from a review of interspecific mtDNA genetic distances in birds: most sister-species separations dated to the Pliocene. Here we summarize the literature on intraspecific mtDNA phylogeographic patterns in birds and reinterpret the molecular evidence bearing on Pleistocene influences. At least 37 of the 63 avian species surveyed (59%) are sundered into recognizable phylogeographic units, and 28 of these separations (76%) trace to the Pleistocene. Furthermore, use of phylogroup separation times within species as minimum estimates of ''speciation durations'' also indicates that many protracted speciations, considered individually, probably extended through time from Pliocene origins to Pleistocene completions. When avian speciation is viewed properly as an extended temporal process rather than as a point event, Pleistocene conditions appear to have played an active role both in initiating major phylogeographic separations within species, and in completing speciations that had been inaugurated earlier. Whether the Pleistocene was exceptional in these regards compared with other geological times remains to be determined.     

Isolation of isoproturon-degrading bacteria from treated soil via three different routes

Three different isolation routes (flask enrichment/flask degradation assay, flask enrichment/microplate degradation assay, MPN assay/microplate degradation assay) were used to obtain pure cultures of bacteria which degraded isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) as sole carbon and nitrogen source in a mineral salts medium from a field soil treated with isoproturon in the laboratory. All three isolation routes were successful, but the microplate assay of degradation was more successful than the flask assay. Characterization of 36 isolates indicated that they formed 16 distinct phenotypes (10 Gram-positive phenotypes, six Gram-negative phenotypes) which are likely to represent distinct species. Low concentrations of the degradation product 3-(4-isopropylphenyl)-1-methylurea (IPPMU) were occasionally found in the culture solutions. When provided as the sole source of carbon and nitrogen, the monomethyl degradation product was itself rapidly degraded by several of the isolates. Some isolates were also able to use the demethylated degradation product 3-(4-isopropylphenyl)-urea (IPPU) as sole source of carbon and nitrogen, although there was occasionally an extended lag-phase before rapid degradation commenced. One isolate was particularly active and degraded isoproturon, the monomethyl and demethylated degradation products of isoproturon, and demethylated the related phenylureas diuron and linuron.