Growth of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in a long spiral form does not alter expression of immunodominant proteins

Background  

We have previously reported that altered culture conditions (a broth media with shaking) could induce a strain of Helicobacter pylori to assume a long spiral morphology resembling that described for Helicobacter heilmannii. The present study was initiated to determine if other strains of H. pylori could be induced to assume that morphology and if doing so would alter the expression of immunodominant proteins.     

Adaptive haemoglobin gene control in Daphnia pulex at different oxygen and temperature conditions

Hypoxia-induced haemoglobin (Hb) expression is a central regulatory mechanism in Daphnia in response to environmental hypoxia or warm temperatures. Changes in Hb concentration as well as Hb subunit composition, which modulate Hb oxygen affinity, guarantee the oxygen supply of tissues under these environmental conditions. Based on the sequenced D. pulex genome, Hb genes were related to the properties of haemolymph Hb, which included its concentration and oxygen affinity (both measured by spectrophotometry) as well as the Hb subunit composition (determined by 2-D gel electrophoresis and ESI-MS analysis). Permanent cultures of D. pulex acclimated to different oxygen conditions (normoxia and hypoxia) and temperatures (10°C, 20°C, and 24°C), showed characteristic changes in Hb concentration, subunit composition and oxygen affinity. Several subunits (Hb4, Hb7, Hb8, and Hb10) were obviously responsible for changes in oxygen affinity including those, which carry a number of hypoxia-responsive elements (HREs) upstream of the respective gene (hb4 and hb10). Analysing the effects of different oxygen- or temperature-acclimations on Hb subunit expression in D. pulex and D. magna on a common basis (Hb concentration or oxygen affinity) revealed a general pattern of oxygen and temperature effects on Hb, which implies that Hb quantity and quality are mostly influenced by the degree of tissue hypoxia. Differences between both species in the onset of hypoxia-induced differential Hb expression and Hb oxygen affinity, which are probably related to different HRE patterns and functionally important differences in the amino acid sequence of only a few subunits, cause a reduced ability of D. pulex to adjust Hb function to temperature changes in comparison to D. magna.     

Cadina-4,11-diene from Viguiera oblongifolia

Viguiera oblongifolia afforded two known furanoheliangolides and a new cadinane derivative whose structure was established by spectroscopic methods. From V. lanceolata 17,18-dihydrobudlein A was isolated.     

GABA transporter currents activated by protein kinase A excite midbrain neurons during opioid withdrawal

Adaptations in neurons of the midbrain periaqueductal gray (PAG) induced by chronic morphine treatment mediate expression of many signs of opioid withdrawal. The abnormally elevated action potential rate of opioid-sensitive PAG neurons is a likely cellular mechanism for withdrawal expression. We report here that opioid withdrawal in vitro induced an opioid-sensitive cation current that was mediated by the GABA transporter-1 (GAT-1) and required activation of protein kinase A (PKA) for its expression. Inhibition of GAT-1 or PKA also prevented withdrawal-induced hyperexcitation of PAG neurons. Our findings indicate that GAT-1 currents can directly increase the action potential rates of neurons and that GAT-1 may be a target for therapy to alleviate opioid-withdrawal symptoms.     

Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs.     

The annexin 2-S100A10 complex and its association with TRPV6 is regulated by cAMP/PKA/CnA in airway and gut epithelia

The formation of a heterotetrameric complex between annexin 2 (anx 2) and S100A10 plays an important role in regulating the cellular distribution and biochemical properties of anx 2. A major distinction between the anx 2-S100A10 complex and other annexin-S100 complexes is that S100A10 binding to anx 2 occurs independently of calcium. Here we describe a cyclic 3',5'-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA, EC 2.7.1.37)-dependent mechanism regulating anx 2-S100A10 complex formation and its interaction with the transient receptor potential vanilloid type 6 channel (TRPV6) in airway and gut epithelia. In both 16HBE14o- and Caco-2 cells, forskolin (FSK) stimulated increased anx 2-S100A10 complex formation, which was attenuated by either PKA inhibitors or calcineurin A (CnA) inhibitors. The anx 2-S100A10 complex association with TRPV6 was dependent on FSK-induced CnA-dependent dephosphorylation of anx 2. Analysis of the significance of the cAMP/PKA/CnA pathway on calcium influx showed that both PKA and CnA inhibitors attenuated Ca(45) uptake in Caco-2, but not 16HBE14o-, cells. Thus, the cAMP/PKA/CnA-induced anx 2-S100A10/TRPV6 complex may require additional factors for calcium influx or play a role independent of calcium influx in airway epithelia. In conclusion, our data demonstrates that cAMP/PKA/CnA signalling is important for anx 2-S100A10 complex formation and interaction with target molecules in both absorptive and secretory epithelia.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.