Temporal and spatial archaeal colonization of hydrothermal vent deposits

Thermocouple arrays were deployed on two deep-sea hydrothermal vents at Guaymas Basin (27 degrees 0.5'N, 111 degrees 24.5'W) in order to measure in situ temperatures at which microorganisms colonize the associated mineral deposits. Intact sections of three structures that formed around the arrays were collected after 4 and 72 day deployments (named BM4, BM72 and TS72). Archaeal diversity associated with discreet subsamples collected across each deposit was determined by polymerase chain reaction amplification of 16S rRNA genes. Spatial differences in archaeal diversity were observed in all deposits and appeared related to in situ temperature. In BM4, no 16S rRNA genes were detected beyond about 1.5 cm within the sample (> 200 degrees C). Phylotypes detected on the outside of this deposit belong to taxonomic groups containing mesophiles and (hyper)thermophiles, whereas only putative hyperthermophiles were detected 1.5 cm inside the structure (approximately 110 degrees C). In contrast, the more moderate thermal gradient recorded across TS72 was associated with a deeper colonization (2-3 cm inside the deposit) of putative hyperthermophilic phylotypes. Although our study does not provide a precise assessment of the highest temperature for the existence of microbial habitats inside the deposits, archaeal 16S rRNA genes were detected directly next to thermocouples that measured 110 degrees C (Methanocaldococcus spp. in BM4) and 116 degrees C (Desulfurococcaceae in TS72). The successive array deployments conducted at the Broken Mushroom (BM) site also revealed compositional differences in archaeal communities associated with immature (BM4) and mature chimneys (BM72) formed by the same fluids. These differences suggest a temporal transition in the primary carbon sources used by the archaeal communities, with potential CO(2)/H(2) methanogens prevalent in BM4 being replaced by possible methylotroph or acetoclastic methanogens and heterotrophs in BM72. This study is the first direct assessment of in situ conditions experienced by microorganisms inhabiting actively forming hydrothermal deposits at different stages of structure development.     

Rac and phosphatidylinositol 3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3 mast cells

FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.     

Somatostatin inhibition of insulin release from freshly isolated and organ cultured rat islets of langerhans in vitro

1×10^?6M somatostatin causes a 37–44% inhibition of glucose induced insulin release from freshly isolated rat islets of Langerhans. A 81 to 95% inhibition is observed when the isolated islets are maintained in organ culture for 2 days prior to the somatostatin treatment. The dose curve of somatostatin on cultured islets shows an apparent K_I of 1.4×10^?9. The tetradecapeptide also causes a reversible inhibition of the stimulation of insulin release by 5 mM theophylline and 23 mM K⁺.     

Internal ribosome entry site structural motifs conserved among mammalian fibroblast growth factor 1 alternatively spliced mRNAs

Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.     

Severe abnormalities in the oral mucosa induced by suprabasal expression of epidermal keratin K10 in transgenic mice

Previous studies have demonstrated that keratin K10 plays an important role in mediating cell signaling processes, since the ectopic expression of this keratin induces cell cycle arrest in proliferating cells in vitro and in vivo. However, apart from its well known function of providing epithelial cells with resilience to mechanical trauma, little is known about its possible roles in nondividing cells. To investigate what these might be, transgenic mice were generated in which the expression of K10 was driven by bovine K6beta gene control elements (bK6(beta)hK10). The transgenic mice displayed severe abnormalities in the tongue and palate but not in other K6-expressing cells such as those of the esophagus, nails, and hair follicles. The lesions in the tongue and palate included the cytolysis of epithelial suprabasal cells associated with an acute inflammatory response and lymphocyte infiltration. The alterations in the oral mucosa caused the death of transgenic pups soon after birth, probably because suckling was impaired. These anomalies, together with others found in the teeth, are reminiscent of the lesions observed in some patients with pachyonychia congenita, an inherited epithelial fragility associated with mutations in keratins K6 and K16. Although no epithelial fragility was observed in the bK6(beta)hK10 oral epithelia of the experimental mice, necrotic processes were seen. Collectively, these data show that the carefully regulated tissue- and differentiation-specific patterns displayed by the keratin genes have dramatic consequences on the biological behavior of epithelial cells and that changes in the specific composition of the keratin intermediate filament cytoskeleton can affect their physiology, in particular those of the oral mucosa.     

Parasitism in Trialeurodes variabilis (Quaintance) (Hemiptera: Aleyrodidae) by Encarsia hispida De Santis (Hymenoptera: Aphelinidae), in papaya, in Brazil

Infestation of Trialeurodes variabilis (Quaintance) was observed in October 2004, in papaya plants of cultivar Sunrise Solo, under screenhouse conditions, in Cruz das Almas, State of Bahia, Brazil. In infested leaves, around 20% of parasitism on nymphs was verified. Leaves with parasitized nymphs were kept in laboratory until emergence of the parasitoid, identified as Encarsia hispida De Santis. This is the first time that this parasitoid was detected on T. variabilis nymphs in Brazil.     

Effect of Cattle on Salmonella Carriage,Diversity and Antimicrobial Resistance in Free-Ranging Wild Boar (Sus scrofa) in Northeastern Spain

Salmonella is distributed worldwide and is a pathogen of economic and public health importance. As a multi-host pathogen with a long environmental persistence, it is a suitable model for the study of wildlife-livestock interactions. In this work, we aim to explore the spill-over of Salmonella between free-ranging wild boar and livestock in a protected natural area in NE Spain and the presence of antimicrobial resistance. Salmonella prevalence, serotypes and diversity were compared between wild boars, sympatric cattle and wild boars from cattle-free areas. The effect of age, sex, cattle presence and cattle herd size on Salmonella probability of infection in wild boars was explored by means of Generalized Linear Models and a model selection based on the Akaike’s Information Criterion. Prevalence was higher in wild boars co-habiting with cattle (35.67%, CI 95% 28.19–43.70) than in wild boar from cattle-free areas (17.54%, CI 95% 8.74–29.91). Probability of a wild boar being a Salmonella carrier increased with cattle herd size but decreased with the host age. Serotypes Meleagridis, Anatum and Othmarschen were isolated concurrently from cattle and sympatric wild boars. Apart from serotypes shared with cattle, wild boars appear to have their own serotypes, which are also found in wild boars from cattle-free areas (Enteritidis, Mikawasima, 4:b:- and 35:r:z35). Serotype richness (diversity) was higher in wild boars co-habiting with cattle, but evenness was not altered by the introduction of serotypes from cattle. The finding of a S. Mbandaka strain resistant to sulfamethoxazole, streptomycin and chloramphenicol and a S. Enteritidis strain resistant to ciprofloxacin and nalidixic acid in wild boars is cause for public health concern.