Role of bradykinin in gastric vasodilation caused by hypertonic saline and acid back diffusion

Protective vasodilation in response to tissue injury and acid back diffusion is associated with release of bradykinin in the rat stomach. We hypothesized that bradykinin might be involved in mechanisms behind such vasodilation via influence on mast cells and sensory neurons. Acid back diffusion after mucosal barrier disruption with hypertonic saline evoked degranulation of mast cells in the rat stomach wall. Acid back diffusion was also associated with increased luminal release of histamine and gastric blood flow in normal rats, but not in mast cell-deficient rats. Bradykinin (BK(2)) receptor blockade inhibited degranulation of submucosal mast cells in the stomach and attenuated gastric vasodilation both in response to acid back diffusion and after stimulation of sensory neurons with capsaicin. Gastric vasodilation caused by mucosal injury with hypertonic saline alone was associated with degranulation of mucosal mast cells. These events were unaffected by inhibition of prostaglandin synthesis, whereas bradykinin (BK(2)) receptor blockade was associated with abolished vasodilation and inhibition of mucosal mast cell degranulation. We conclude that bradykinin is involved in gastric vasodilation caused by hypertonic injury alone via influence on mast cells, and by acid back diffusion via influence on both sensory neurons and mast cells.     

Growth and enzyme production by three Penicillium species on monosaccharides

The growth and preference for utilisation of various sugar by the Penicillium species Penicillium pinophilum IBT 4186, Penicillium persicinum IBT 13226 and Penicillium brasilianum IBT 20888 was studied in batch cultivations using various monosaccharides as carbon source, either alone or in mixtures. P. pinophilum IBT 4186 and P. persicinum IBT 13226 had a micro(max) around 0.08-0.09 h(-1) using either glucose or xylose as carbon source. The micro(max) of P. brasilianum IBT 20888 was 0.16 and 0.14 h(-1) on glucose and xylose, respectively. Glucose was found to exert repression on the catabolism of mannose, galactose, xylose and arabinose. The three species were able to utilise all the tested monosaccharides, but arabinose was only slowly metabolised. Glucose was also found to repress the production of endoglucanases, endoxylanases and beta-xylosidases. After glucose depletion, the fungi started producing beta-glucosidase and endoglucanases. Xylose did not repress the enzyme production and it induced the production of endoxylanases and beta-xylosidases.     

Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK), which is regulated by protein stability. However, its function is unknown and no physiological substrates for ERK3 have yet been identified. Here we demonstrate a specific interaction between ERK3 and MAPK-activated protein kinase-5 (MK5). Binding results in nuclear exclusion of both ERK3 and MK5 and is accompanied by ERK3-dependent phosphorylation and activation of MK5 in vitro and in vivo. Endogenous MK5 activity is significantly reduced by siRNA-mediated knockdown of ERK3 and also in fibroblasts derived from ERK3-/- mice. Furthermore, increased levels of ERK3 protein detected during nerve growth factor-induced differentiation of PC12 cells are accompanied by an increase in MK5 activity. Conversely, MK5 depletion causes a dramatic reduction in endogenous ERK3 levels. Our data identify the first physiological protein substrate for ERK3 and suggest a functional link between these kinases in which MK5 is a downstream target of ERK3, while MK5 acts as a chaperone for ERK3. Our findings provide valuable tools to further dissect the regulation and biological roles of both ERK3 and MK5.     

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells

UDP-GlcNAc 2-epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals. Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism. This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy. For a detailed understanding of the enzyme, large amounts of the pure active protein are needed. Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli, the yeast strains Saccharomyces cerevisiae and Pichia pastoris, and insect cells. In all these cell types, the expressed enzyme displayed both epimerase and kinase activities. In E. coli, up to 2mg protein/l cell culture was expressed, in yeast cells only 0.4mg/L, while up to 100mg/L, were detected in insect cells. In all three cell systems, insoluble protein aggregates were also observed. Purification from E. coli resulted in 100microg/L pure and structurally intact protein. For insect cells, purification methods were established which resulted in up to 50mg/L pure, soluble, and active protein. In summary, expression and purification of the UDP-GlcNAc 2-epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme. However, the easier expression in E. coli and yeast provides sufficient quantities for enzymatic and kinetic characterization.     

Yeast lacking the SRO7/SOP1-encoded tumor suppressor homologue show increased susceptibility to apoptosis-like cell death on exposure to NaCl stress

Yeast cells deleted for the SRO7/SOP1 encoded tumor suppressor homologue show increased sensitivity to NaCl stress. On exposure to growth-inhibiting NaCl concentrations, sro7Delta mutants display a rapid loss in viability that is associated with markers of apoptosis: accumulation of reactive oxygen species, DNA breakage, and nuclear fragmentation. Additional deletion of the yeast metacaspase gene YCA1 prevents the primary fast drop in viability and diminishes nuclear fragmentation and DNA breakage. We also observed that NaCl induced loss in viability of wild-type cells is Yca1p dependent. However, a yeast strain deleted for both SRO7 and its homologue SRO77 exhibits NaCl-induced cell death that is independent on YCA1. Likewise, sro77Delta single mutants do not survive better after additional deletion of the YCA1 gene, and both sro77Delta and sro77Deltayca1Delta mutants display apoptotic characteristics when exposed to growth-inhibiting salinity, suggesting that yeast possesses Yca1p-independent pathway(s) for apoptosis-like cell death. The activity of Yca1p increases with increasing NaCl stress and sro7Delta mutants achieve levels that are higher than in wild-type cells. However, mutants lacking SRO77 do not enhance caspase activity when subject to NaCl stress, suggesting that Sro7p and Sro77p exert opposing effects on the cellular activity of Yca1p.     

Alternative mRNAs arising from trans-splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin Glutathione reductase

Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.     

Base-pair dynamics in an antiparallel DNA triplex measured by catalyzed imino proton exchange monitored via 1H NMR spectroscopy

Using (1)H NMR spectroscopy, the base-pair opening dynamics of an antiparallel foldback DNA triplex and the corresponding duplex has been characterized via catalyzed imino proton exchange. The triplex system was found to be in an equilibrium between a duplex and a triplex form. The exchange rate between the two forms (i.e., the on/off-rate of the third strand) was measured to be 5 s(-1) at 1 degrees C, and the base-pair dynamics of both forms were investigated separately. Both Watson-Crick and reverse Hoogsteen base pairs were found to have base-pair lifetimes in the order of milliseconds. The stability of the Watson-Crick base pairs was, however, substantially increased in the presence of the third strand. In the DNA triplex, the opening dynamics of the reverse Hoogsteen base pairs was significantly faster than the dynamics of the Watson-Crick pairs. We were able to conclude that, for both Watson-Crick and reverse Hoogsteen base pairs, spontaneous and individual opening from within the closed base triplet is the dominating opening pathway.     

How well can blood pressure be controlled? Progress report on the Systolic Hypertension in Europe Follow-Up Study (Syst-Eur 2)

Background

The randomised, double-blind, placebo-controlled Systolic Hypertension in Europe trial (Syst-Eur 1) proved that blood pressure (BP) lowering therapy starting with nitrendipine reduces the risk of cardiovascular complications in elderly patients with isolated systolic hypertension. In an attempt to confirm the safety of long-term antihypertensive therapy based on a dihydropyridine, the Syst-Eur patients remained in open follow-up after the end of Syst-Eur 1. This paper presents the second progress report of this follow-up study (Syst-Eur 2). It describes BP control and adherence to study medications.

Methods

After the end of Syst-Eur 1 all patients, treated either actively or with placebo, were invited either to continue or to start antihypertensive treatment with the same drugs as previously used in the active treatment arm. In order to reach the target BP (sitting SBP <150 mmHg), the first line agent, nitrendipine, could be associated with enalapril and/or hydrochlorothiazide.

Results

Of the 3787 eligible patients, 3516 (93%) entered Syst-Eur 2. At the last available visit, 72% of the patients were taking nitrendipine. SBP/DBP at entry in Syst-Eur 2 averaged 160/83 mmHg in the former placebo group and 151/80 mmHg in the former active-treatment group. At the last follow-up visit SBP/DBP in the patients previously randomised to placebo or active treatment had decreased by 16/5 mmHg and 7/5 mmHg, respectively. The target BP was reached by 74% of the patients.

Conclusion

Substantial reductions in systolic BP may be achieved in older patients with isolated systolic hypertension with a treatment strategy starting with the dihydropyridine calcium-channel blocker, nitrendipine, with the possible addition of enalapril and/or hydrochlorothiazide.     

ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the 'universal' agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.