Ras GTPase-activating protein binds to Akt and is required for its activation

RasGAP (Ras GTPase-activating protein) is a negative regulator as well as a downstream effector of Ras. To identify partners of RasGAP we used it as the bait in a yeast two-hybrid screen. This resulted in discovering its interaction with Akt. Overexpression of RasGAP or a mutant lacking the GTPase-activating domain (nGAP) enhanced phosphorylation and activity of Akt, which was dependent on the upstream integrin-linked kinase. Also, nGAP protected the cells against staurosporin-induced apoptosis through an Akt-dependent pathway. To determine the role of RasGAP in receptor-mediated activation of Akt, we used short hairpin RNA interference to knock out endogenous RasGAP expression. Although this procedure resulted in enhanced Ras activity, it inhibited Akt phosphorylation. Thus, we propose that Ras-GAP interacts with Akt and is necessary for its activation, possibly via integrin-linked kinase-mediated phosphorylation of Ser-473. The data suggest that this effect is independent of Ras activity.     

(T2AG3)n Telomeric Sequence Hybridization Indicating Centric Fusion Rearrangements in the Karyotype of the Rodent Oryzomys Subflavus

Chromosome preparations of 30 specimens of Oryzomys subflavus trapped in eight Brazilian localities were C-, and G-banded and analyzed by fluorescence in situ hybridization (FISH). Two karyotypes were found, 2n=50/FN=64, at three coastal localities of the Atlantic Forest domain, and 2n=58/FN=70 at two sites located in the Cerrado biome, Brazil Central. Two fluorescence in situ hybridization (FISH) patterns of the telomeric sequence (T₂AG₃)_n were observed: in both karyotypes the probes hybridized to the telomeres of all chromosomes and also a hybridization signal in the centromeric regions of two autosome pairs was seen in the 2n=50 karyotype. These results, together with the occurrence of other diploid numbers described in the literature, suggest that O. subflavus is a complex species, bearing fusion/fission rearrangements proper to the different biomes which it inhabits.     

Prevalence of hepatitis C Virus infection among hemophiliacs in Central Brazil

In order to investigate the hepatitis C virus (HCV) infection prevalence and risk factors in hemophiliacs in Central Brazil, 90 patients were interviewed and serum samples tested for HCV RNA and anti-HCV antibodies. An overall prevalence of 63.3% (CI 95%: 53.0-72.7) was found. Multivariate analysis of risk factors showed that number of blood transfusions was significantly associated with this infection. Most hemophiliacs received locally produced cryoprecipitate. All infected patients were transfused before the screening of blood units for anti-HCV. However, hemophiliacs who received exclusively screened cryoprecipitate were HCV negative. It confirms the expected decline in transfusion-acquired hepatitis C.     

The effect of growth in continuous culture at various input C:N ratios on the expression of proteins involved in the transport and metabolism of methanol and short-chain amides by Methylophilus methylotrophus

Physiological regulation of proteins involved in the transport and metabolism of methanol and short-chain amides by Methylophilus methylotrophus was investigated following growth in continuous culture at various input C:N ratios. The concentrations of the methanol porin and methanol dehydrogenase were highest during C-limited growth (C:N<4.6), but declined gradually as a function of the increasing C:N ratio and were lowest during N-limited growth (C:N>16.3). In contrast, the concentrations of the amide-urea porin, the amide-urea binding protein, formamidase and acetamidase (together with formamidase and acetamidase activities) were lowest during C-limited growth, but increased sharply as a function of the C:N ratio and were highest during dual CN-limited and N-limited growth (C:N 4.6–16.3). The results are discussed in terms of the physiological and biochemical requirements of growth at varying C:N ratios.     

The Ras-related Protein,Rap1A,Mediates Thrombin-stimulated,Integrin-dependent Glioblastoma Cell Proliferation and Tumor Growth

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β₁ integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β₁ integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β₁ integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo.     

Phylogenetic structure of Neotropical annual fish of the genus Cynopoecilus (Cyprinodontiformes: Rivulidae), with an assessment of taxonomic implications

The definition of species boundaries constitutes an important challenge in biodiversity studies. Cynopoecilus Regan, 1912 encompasses several endangered species of annual fish, occurring in temporary ponds in a restricted area of Southern Brazil and Uruguay. Divergences about the taxonomic status of Cynopoecilus species highlight the importance of species delimitation studies. Therefore, we address here the phylogenetic structure of Cynopoecilus, while assessing its taxonomic implications. For this, fragments of the mitochondrial COI and nuclear RAG1 genes were characterized and analyzed for a set of 275 and 280 specimens, respectively. DNA barcoding and phylogenetic analyses detected subdivision of these specimens in 8–10 clusters, which comprise the six previously described species, and suggest one invalid taxon and at least 3–5 putative new species. The phylogenetic structure also suggests that the Jacuí River and the Patos Lagoon historically acted as effective barriers to gene flow between populations, although some isolated dispersal events across these water bodies could be evidenced, especially for C. melanotaenia Regan, 1912. In general, the results highlight the need of independent conservation strategies within the distribution area of each of the endemic allopatric killifish clusters, while questioning several taxonomic boundaries and distribution data.     

Functional analysis of a mitochondrial phosphopantetheinyl transferase (PPTase) gene pptB in Aspergillus fumigatus

The mitochondrial phosphopantetheinyl transferase gene pptB of the opportunistic pathogen Aspergillus fumigatus has been identified and characterised. Unlike pptA, which is required for lysine biosynthesis, secondary metabolism, and iron assimilation, pptB is essential for viability. PptB is located in the mitochondria. In vitro expression of pptA and pptB has shown that PptB is specific for the mitochondrial acyl carrier protein AcpA.     

Hybrid modeling of xanthan gum bioproduction in batch bioreactor

This work is focused on hybrid modeling of xanthan gum bioproduction process by Xanthomonas campestris pv. mangiferaeindicae. Experiments were carried out to evaluate the effects of stirred speed and superficial gas velocity on the kinetics of cell growth, lactose consumption and xanthan gum production in a batch bioreactor using cheese whey as substrate. A hybrid model was employed to simulate the bio-process making use of an artificial neural network (ANN) as a kinetic parameter estimator for the phenomenological model. The hybrid modeling of the process provided a satisfactory fitting quality of the experimental data, since this approach makes possible the incorporation of the effects of operational variables on model parameters. The applicability of the validated model was investigated, using the model as a process simulator to evaluate the effects of initial cell and lactose concentration in the xanthan gum production.