TRPC channels and diacylglycerol dependent calcium signaling in rat sensory neurons

Transient receptor potential (TRP) channels of the TRPV, TRPA, and TRPM subfamilies play important roles in somatosensation including nociception. While particularly the Thermo TRPs have been extensively investigated in sensory neurons, the relevance of the subclass of "canonical" TRPC channels in primary afferents is yet elusive. In the present study, we investigated the presence and contribution to Ca(2+) transients of TRPC channels in dorsal root ganglion neurons. We found that six of the seven known TRPC subtypes were expressed in lumbar DRG, with TRPC1, C3, and C6 being the most abundant. Microfluorimetric calcium measurements showed Ca(2+) influx induced by oleylacylglycerol (OAG), an activator of the TRPC3/C6/C7 subgroup. Furthermore, OAG induced rises in [Ca(2+)](i) were inhibited by SKF96365, an inhibitor of receptor and store operated calcium channel. OAG induced calcium transients were also inhibited by blockers of diacylglycerol (DAG) lipase, lipoxygenase or cyclooxygenase and, intriguingly, by inhibitors of the capsaicin receptor TRPV1. Notably, SKF96365 did not affect capsaicin-induced calcium transients. Taken together, our findings suggest that TRPC are functionally expressed in subpopulations of DRG neurons. These channels, along with TRPV1, contribute to calcium homeostasis in rat sensory neurons.     

Dynamics of oligomer formation by denatured carbonic anhydrase II

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.     

Ecological Aspects of Circadian Rhythms in Six Species of Omni-Seasonal Beetles (Coleoptera, Tenebrionidae) Inhabiting Kara Kum Desert (Turkmenistan)

Circadian rhythms of activity were compared in ground-dwelling Tenebrionid beetles from the Kara Kum sand desert: Trigonoscelis gigas Reitter, Trigonoscelis sublaevicollis Reitter, Pisterotarsa gigantea Fish.-W., Sternodes caspicus Pall, Blaps faustii Seidlitz, and Ocnera imbricata Fish.-W. For the observations, artificial pens (enclosures) 60 × 60 cm and 80 cm deep were arranged in field conditions and filled with various kinds of sand to simulate natural habitats as closely as possible. The activity of individually marked beetles was assessed visually and recorded every 30 min. The beetle species studied had different types of behavior, from strictly crepuscular to strictly nocturnal. Closely related species sharing one habitat differed in activity pattern — they occupied different time niches; hence their circadian rhythms contribute to reproductive isolation. In contrast, non-closely related species could have similar circadian patterns as a result of convergent evolution. In experiments with T. gigas, we failed to stimulate individual variability among beetles in circadian pattern by applying additional light and heat at night. Therefore, the circadian rhythms in field conditions were found to be remarkably stable, which can be explained by our model of the beetle circadian system consisting of two strongly coupled oscillators. However, in laboratory conditions, we previously observed a wide range of individual variability in free-running circadian rhythms. The strong coupling between two putative endogenous circadian oscillators is a crucial tool for beetles to survive in a harsh arid environment. Such organization of the circadian system ensures synchronous activity of beetles despite individual differences, thus preserving the variability of circadian clock properties in a population, which increases chances for survival of a population in a changing environment.     

Impact of Weight Reduction on Early Carotid Atherosclerosis in Obese Premenopausal Women

Objective: To investigate the extent of carotid atherosclerosis and the effect of weight loss on carotid intima‐media thickness (IMT) in obese premenopausal women. Research Methods and Procedures: In 43 obese premenopausal women who participated in a 3‐month weight reduction program with a hypocaloric diet, IMT was measured by B‐mode high‐resolution ultrasound at entry and after 5 months of follow‐up. Blood samples were analyzed at entry, after intervention, and after 5 months of follow‐up. Nineteen lean women served as control subjects. Results: At entry, common carotid IMT (0.72 vs. 0.59 mm), carotid bulb IMT (0.90 vs. 0.71 mm), and overall mean IMT (0.81 vs. 0.65 mm) were greater in obese women than in lean women (all p < 0.01). After dietary intervention decreases in blood pressure, low density lipoprotein to high density lipoprotein cholesterol ratio, triglycerides, fibrinogen, plasminogen activator inhibitor‐1, and an increase in tissue‐type plasminogen activator activity levels were observed. These effects persisted after follow‐up in 14 women who maintained reduced weight. Reduction in carotid bulb IMT (to 0.81 mm, p < 0.01) and overall mean IMT (to 0.79 mm, p < 0.05) was observed in this subgroup. No significant change of carotid IMT was detected in eight women who regained weight. Changes in IMT were associated independently and significantly with changes in body mass index, low density lipoprotein to high density lipoprotein cholesterol ratio, and plasminogen activator inhibitor‐1 antigen. Discussion: Obese premenopausal women had greater IMT than did age‐matched lean controls. It seems that this early atherosclerotic changes may be reversed by normalization of body weight.     

Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin.

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn-binding site of PAI-1 appear to reside between amino acids 110-147, which includes alpha helix E (hE, amino acids 109-118). Ten different PAI-1 variants (mostly harboring modifications in hE) as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli containing a His(6) tag and purified by affinity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114-118, in which all amino-acids at positions 114-118 of PAI-1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild-type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114-118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn.     

Blood Collection Procedure of Laboratory Primates: A Neglected Variable in Biomedical Research

A survey of 75 biomedical articles dealing with stress-dependent blood parameters in caged primates revealed that the conditions under which blood collection occurred were in most cases described either not at all or so haphazardly that it would be impossible to determine if humane handling procedures were used and basic principles of scientific methodology applied. These findings were unexpected because there is ample scientific evidence not only that stress-sensitive research data are influenced by traditional blood sampling procedures, but also that those data-biasing effects can be avoided. If dependent variables of the blood collection procedure are not controlled, data variability will increase, automatically increasing the number of animals needed for statistical analysis. For ethical and scientific reasons, it was recommended that editors of biomedical journals require authors to provide sufficient information of the blood collection--and, when applicable, the sedative injection--procedure to ensure that the experiment was done with the smallest number of animals possible to achieve statistical significance and that the investigation can be replicated reliably in another laboratory and the research data interpreted with reasonable accuracy.     

Cardiotrophin-1 review

Background: Cardiotrophin-1 is newly discovered chemokin with a lot of functions. Aim of our work was to describe most important of them. Methods: systematically scan of available scientific resources. Results: Cardiotrophin-1 stimulates the proliferation of cardiomyocytes. Cardiotrophin-1 expression and plasma values are elevated in individuals with heart failure and have high diagnostic efficacy for the heart failure. Plasma values are also an independent prognostic factor. Preliminary findings suggest that the determination of plasma cardiotrophin-1 may be useful for the follow-up of hypertensive heart disease in routine clinical practice. Cardiotrophin-1 also plays an important cardioprotective effect on myocardial damage, is a potent regulator of signaling in adipocytes in vitro and in vivo and potentiates the elevation the acute-phase proteins. Cardiotrophin-1 may play also an important protective role in other organ systems (such as hematopoietic, neuronal, developmental). Conclusion: Cardiotrophin is a newly discovered chemokin with a lot of system effects and is stable in system circulation hence permitting its development in the routine clinical investigation.