pH dependence of extracellular calcium sensing receptor activity determined by a novel technique

BACKGROUND: Increasing evidence points to the role of the extracellular Calcium Sensing Receptor (CaSR) as a multimodal receptor responding to diverse physiologic stimuli, such as extracellular divalent and polyvalent cations, amino acids, and ionic strength. Within the kidney, these stimuli converge on the CaSR to coordinate systemic calcium and water homeostasis. In this process, the impact of urinary pH changes on the activity of the CaSR has not yet been defined. We therefore performed the present study to analyze the pH sensitivity of the CaSR. METHODS: To assess the activation state of the CaSR, we developed a new method based on the functional coupling between CaSR activity and gating of calcium sensitive potassium currents mediated by SK4 potassium channels. Two-electrode voltage clamping was used to determine whole cell currents in Xenopus oocytes heterologously expressing rat CaSR and rat SK4 potassium channels. RESULTS: Coexpression of CaSR and SK4 gave rise to potassium currents that were dependent on CaSR-mediated intracellular calcium release, and thereby corresponded to the activation state of the CaSR. In presence of extracellular calcium, ambient alkalinization above pH 7.5 increased CaSR activity. Evaluation of the CaSR calcium sensitivity at various ambient proton concentrations revealed that this effect was due to a sensitization of the CaSR towards extracellular calcium. CONCLUSION: Coexpression with SK4 potassium channels provides a fast and sensitive approach to evaluate CaSR activity in Xenopus oocytes. As disclosed by this novel technique, CaSR activity is regulated by extracellular pH.     

Glutathione and glutathione peroxidase activities in blood of patients in early stages following kidney transplantation

This study focuses on glutathione (GSH) level in red blood cells, as well as on glutathione peroxidases (GSH-Px) activities in red blood cells and in plasma of chronic renal failure (CRF) patients following renal transplantation. We want to focus our main attention on plasma GSH-Px, the selenoenzyme that is synthesized primarily in the kidney. In CRF patients, activity of this enzyme is significantly reduced, and the reduction decreases with the progress of the disease, reaching in the end-stage 20% to 30% of the activity of healthy patients. We have shown that following renal transplantation the activity of plasma GSH-Px is restored very rapidly, and 2 weeks after surgery it reached the value of the control group. Red blood cell GSH level is significantly higher in CRF patients, and following transplantation, no significant changes were observed. Red blood cell GSH-Px activity before transplantation was the same as in healthy patients and did not change significantly after surgery.     

Lung cancer and indoor pollution from heating and cooking with solid fuels: the IARC international multicentre case-control study in Eastern/Central Europe and the United Kingdom

Exposure to fuel from cooking and heating has not been studied in Europe, where lung cancer rates are high and many residents have had a long tradition of burning coal and unprocessed biomass. Study subjects included 2,861 cases and 3,118 controls recruited during 1998-2002 in the Czech Republic, Hungary, Poland, Romania, Russia, Slovakia, and the United Kingdom. The odds ratio of lung cancer associated with solid fuel use was 1.22 (95% confidence interval (CI): 1.04, 1.44) for cooking or heating, 1.37 (95% CI: 0.90, 2.09) for solid fuel only for cooking, and 1.24 (95% CI: 1.05, 1.47) for solid fuels used for both cooking and heating. Risk increased relative to the percentage of time that solid fuel was used for cooking (p(trend) < 0.0001), while no risk increase was detected for solid fuel used for heating. The odds ratio of lung cancer in whole-life users of solid cooking fuel was 1.80 (95% CI: 1.35, 2.40). Switching to nonsolid fuels resulted in a decrease in risk. The odds ratio for the longest duration of time since switching was 0.76 (95% CI: 0.63, 0.92). The data suggest a modest increased risk of lung cancer related to solid-fuel use for cooking rather than heating.     

Plant genotoxicity: a molecular cytogenetic approach in plant bioassays

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).     

Molecular basis of the low activity of antitumor anthracenediones, mitoxantrone and ametantrone, in oxygen radical generation catalyzed by NADH dehydrogenase. Enzymatic and molecular modelling studies

Synthetic antitumor anthracenedione drugs, in contrast to anthracycline antibiotics, are ineffective in free radical formation in NADH dehydrogenase system. Our results have indicated that neither the reduction potential nor the side chain conformation and the energies of border orbitals (HOMO and LUMO) determine the ability of anthracenediones to stimulate reactive oxygen species formation in NADH dehydrogenase system. It was shown that the distribution of the molecular electrostatic potential (MEP), around the quinone system was crucial for this ability. We have found for non-stimulating anthracenediones that the clouds of positive MEP cover the quinone carbon atoms while for agents effective in stimulating reactive oxygen species formation the clouds of negative MEP cover continuously the aromatic core together with the quinone system.     

Lymphocyte subpopulations in the surface secretion on the adenoid

The significance and protective efficacy of surface secretions on mucosal membranes in the upper airways are well recognized. The aim of our study was to reveal the role of the adenoids as a source of cellular components in the mucosal secretion. The adenoid removed because of its hypertrophy and the samples of surface secretions taken by "imprint method" described by Ebenfelt et al. from the group of 38 children were examined. By flow cytometry the lymphocyte subsets with following antigens: CD3, CD4, CD8, CD19, CD23, CD16+56, CD45RA, CD45RO, HLA-DR were analyzed. The results were compared between the groups of younger and older children and in the group of concomitant otitis media with effusion (OMS) and pure adenoid hypertrophy. The percentage of lymphocyte subsets in adenoid and secretion were similar, except for lymphocytes Th with memory phenotype which were more numerous in the adenoid and lymphocytes B CD23+ which were more numerous in the secretion. In the adenoid the percentage of T and Th lymphocytes increased with age and the percentage of B lymphocytes decreased. In the secretion the percentage of lymphocytes Th and B CD23+ was higher among older children. In cases of otitis media with effusion higher percentage of lymphocytes B (CD19+ and CD19+CD23+) and lower of lymphocytes Ts and Th with naive phenotype were observed in the secretion. In adenoid however lower percentage of lymphocytes B and higher of lymphocytes Ts was observed in cases of OMS.     

The protective action of alpha-tocopherol on the white matter lipids during moderate hypoxia in rats

Hypoxia and ischemia acting on the brain cause alterations of the level of lipids and sterols. Famile 3.0-3.5-month-old rats were used for the experiment. They were given alpha-tocopherol in the dose of 11.43 mg/kg of body weight through seven days, then underwent hypoxia (7% of oxygen in the breathing mixture) and myelin was isolated in four times after experiment: 4, 24 hours, 14 days and 2 months after experiment. Three lipids groups were isolated that are neutral lipids, galactolipids and phospholipids. They were quantitatively analyzed with spectrophotocolorimetry. The obtained results indicate that vitamin E administration to animals does not cause significant changes of brain lipids levels. However, alpha-tocopherol administred before moderate hypoxia balances the concentrations of lisophosphatidylcholine and phosphatidylinositide and cerebrosides with control level 2 months after experiment. Vitamin E changes in concentration of the myelin neutral lipids. Vitamin E administered before experimental moderate hypoxia stabilizes some membrane lipids and could be used in brain hypoxia.     

Thromboembolic and haemorrhagic complications in patients treated with anticoagulants after artificial heart valve replacement. Risk factors dependent on quality of anticoagulant treatment

Thromboembolic (TE) and hemorrhagic (H) complications are the most significant causes of morbidity and mortality in patients treated with oral anticoagulants (OA) after implantation of artificial valvular prostheses. The risk of complications is dependent on recommended target INR ranges and quality of anticoagulation treatment. We studied 358 patients with a mean age 56.9 +/- 10.3 (who underwent 144 mitral, 172 aortic, 42 both artificial heart valve replacement). The follow up was averaged 36.7 +/- 12.1 month. Linearized incidents of TE events was 2.7%/pty, H events--4.15%/pty. The quality of anticoagulant treatment based on following parameters: averaged INR +/- SD, percentage measurements of INR within the target range 2.5-3.5, within the broad range 2-4 and under 2 and over 5. Average frequency of tests, SD/averaged INR X 100%--was similar in patients with TE and H complications and event free patients. Significant differences were found when a shorter period of time-3 months preceding the TE or H incident was analized compared with the average results of measurements of event free patients. The patients with TE and H complications have statistically significant higher fluctuation ratio than event free patients (respectively: 1.09 -/+ 0.71 and 1.71 -/+ 1.06 vs 0.5 -/+ 0.32, p = 0.04 and 0.02 ) CONCLUSIONS: 1. The risk of TE and H complications was dependent on high fluctuation of INR values in the period of 3 months before the event. 2. The worse control of measurements of INR in the period of 3 months before the event in comparison with yearly values within the recommended target ranges show, that TE and H complications are directly dependent on period of poor INR control.     

Receptors for VIP and PACAP in guinea pig cerebral cortex

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of ¹²⁵I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of ¹²⁵I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a Bmax of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of ¹²⁵I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) ≥ PACP38 ∼ PACAP27 ∼ guinea pig VIP (gpVIP) ≥ mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of ¹²⁵I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific ¹²⁵I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [³H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 μM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 μM, produced net increases in cAMP production in the range of 8–9% conversion, whereas 5 μM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.