Attila Miseta

Relationship between cellular ATP, potassium, sodium and magnesium concentrations in mammalian and avian erythrocytes

Intracellular K+/Na+ ratios of erythrocytes of various mammalian species are known to differ markedly. We have measured ATP, K+, Na+, Mg2+, H2O contents of erythrocytes of twelve mammalian and three avian species. Our results indicate that the intracellular ATP concentration in erythrocytes of different species is in close positive correlation with the K+/Na+ ratios (linear correlation coefficient, r = 0.852). Furthermore, ATP levels in erythrocytes of individual sheep with different potassium concentrations correspond with their K+/Na+ ratios (r = 0.747). Intracellular magnesium concentrations also correlate with ATP concentrations in erythrocytes of different species (r = 0.629) and in different sheep (r = 0.549).

Extracellular Ca2+ sensing contributes to excess Ca2+ accumulation and vacuolar fragmentation in a pmr1Δ mutant of S. cerevisiae

Previous studies have suggested that yeast strains lacking the Ca2+-ATPase Pmr1p are unable to maintain an adequate level of Ca2+ within the Golgi apparatus. It is thought that this compartmental store depletion induces a signal that causes an increased rate of Ca2+ uptake and accumulation in a manner similar to the capacitative Ca2+ entry (CCE) response in non-excitable mammalian cells. To explore this model further, we examined cellular Ca2+ uptake and accumulation in a pmr1Δ strain grown in the presence of a reduced level of divalent cations. We found that the level of Ca2+ uptake and accumulation in a pmr1Δ strain increased as the concentration of divalent cations in the growth medium decreased. These results are inconsistent with a model in which cellular Ca2+ uptake and accumulation are determined solely by the depletion of Ca2+ in an intracellular compartment. Instead, our results suggest that a second regulatory mechanism couples cellular Ca2+ uptake to the availability of Ca2+ in the extracellular environment. Furthermore, we found that various conditions that increase the level of cytosolic Ca2+ correlate with vacuolar fragmentation in wild-type (WT), pmr1Δ and pmr1Δ/pmc1Δ yeast strains. This suggests that vacuolar fragmentation might function as a normal physiological response to Ca2+ stress that increases the vacuolar surface/volume ratio, thereby maximizing the sequestration of this important signaling molecule.

Impact of genetic variants on post-clopidogrel platelet reactivity in patients after elective percutaneous coronary intervention.

AIM To determine the effect of various SNPs on post-clopidogrel platelet reactivity and clinical outcome. MATERIALS & METHODS Cytochrome 2C19 (CYP2C19) loss-of-function (LOF; *2, *3) and gain-of-function (GOF; *17) allelic variants, together with ABCB1 (3435 C→T and 2677 G→T/A) and paraoxonase-1 (PON-1; 192 Q→R) SNPs were analyzed in 189 patients after elective stent implantation who participated in a randomized, placebo-controlled trial (NCT00638326). Platelet reactivity was determined with light transmission aggregometry and vasodilator stimulated phosphoprotein phosphorylation (VASP-PRI) 12-24 h after 600 mg clopidogrel. High on-treatment platelet reactivity (HTPR) was defined according to the consensus definition (ADP 5 µM >46%; VASP-PRI>50%). RESULTS In the case of CYP2C19 genotypes, a gene-dose effect was observed in ADP reactivity with the lowest values in GOF homozygotes and the highest degree in patients carrying two LOF alleles. The odds for HTPR also increased with the number of LOF alleles. There were no significant differences in platelet reactivity according to PON-1 or ABCB1 genotypes. In multivariate analysis, the presence of a CYP2C19 LOF allele turned out to be the independent determinant of HTPR. Although the study was not powered to clinical outcome (not LOF heterozygotes), only patients with two LOF alleles had a significantly higher risk for cardiovascular death, myocardial infarction or unplanned target vessel revascularization at 1 year compared with non-LOF carriers. CONCLUSION Genetic variants in CYP2C19 have a gene-dose effect on post-clopidogrel platelet reactivity, with homozygote LOF carriers having the highest risk for HTPR and for adverse ischemic events. Neither ABCB1 nor PON-1 genotypes significantly influenced platelet reactivity or outcome.

Chronic stress reduces the number of GABAergic interneurons in the adult rat hippocampus, dorsal-ventral and region-specific differences

Major depressive disorder is a common and complex mental disorder with unknown etiology. GABAergic dysfunction is likely to contribute to the pathophysiology since disrupted GABAergic systems are well documented in depressed patients. Here we studied structural changes in the hippocampal GABAergic network using the chronic mild stress (CMS) model, as one of the best validated animal models for depression. Rats were subjected to 9 weeks of daily stress and behaviorally characterized using the sucrose consumption test into anhedonic and resilient animals based on their response to stress. Different subtypes of GABAergic interneurons were visualized by immunohistochemistry using antibodies for parvalbumin (PV), calretinin (CR), calbindin (CB), cholecystokinin (CCK), somatostatin (SOM), and neuropeptide Y (NPY). We used an unbiased quantification method to systematically count labeled cells in different subareas of the dorsal and ventral hippocampus. Chronic stress reduced the number of specific interneurons in distinct hippocampal subregions significantly. PV+ and CR+ neurons were reduced in all dorsal subareas, whereas in the ventral part only the CA1 was affected. Stress had the most pronounced effect on the NPY+ and SOM+ cells and reduced their number in almost all dorsal and ventral subareas. Stress had no effect on the CCK+ and CB+ interneurons. In most cases the effect of stress was irrespective to the behavioral phenotype. However, in a few specific areas the number of SOM+, NPY+, and CR+ neurons were significantly reduced in anhedonic animals compared to the resilient group. Overall, these data clearly demonstrate that chronic stress affects the structural integrity of specific GABAergic neuronal subpopulations and this should also affect the functioning of these hippocampal GABAergic networks.

Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to environ‐mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose‐induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high‐affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc‐1‐P (or a related glucose metabolite) in yeast.

Augmented Water Binding and Low Cellular Water Content in Erythrocytes of Camel and Camelids

We investigated a link between hemoglobin primary structure, hemoglobin hydrophobicity-hydrophilicity, and erythrocyte water content in various mammalian species. Some hemoglobin molecules, particularly those of the camel and camelids, contain more charged amino acid residues and are more hydrophilic than the hemoglobins of human and a number of other mammalian species. To test the in vivo significance of these alterations of hemoglobin primary structure, we determined the osmotically unresponsive erythrocyte water fractions in mannit solutions of various osmolarities at 4 degreesC. Among the species investigated, the size of the osmotically unresponsive erythrocyte water fraction relates in a positive linear way to hemoglobin hydrophilicity. The extreme low total erythrocyte water content of camel erythrocytes (1.1-1.3 g water/g dry mass) may be explained by a comparatively high osmotically unresponsive erythrocyte water fraction. It is proposed that alterations of hemoglobin sequences of camel and camelids may be the part of a natural selection process aimed at protecting these animals against osmotic dehydration in arid environments.

Lithium induces phosphoglucomutase activity in various tissues of rats and in bipolar patients

Phosphoglucomutase catalyses the reversible conversion of glucose-6-P and glucose-1-P. Lithium is a potent inhibitor of phosphoglucomutase in vitro, however, it is not known if phosphoglucomutase was significantly inhibited by Li+ in Li+-treated bipolar patients. Here, we demonstrate that phosphoglucomutase inhibition by chronic Li+ treatment causes alterations of glucose-phosphate levels in various tissues of rats. Also, phosphoglucomutase inhibition results in compensatory elevation of phosphoglucomutase activity in rat tissues and in leukocytes isolated from Li+-treated bipolar patients. The increase of uninhibited phosphoglucomutase activity in leukocytes of Li+-treated bipolar patients is due to the increased expression of the PGM1 gene.

Pilot study for the characterization of pharmacogenetically relevant CYP2D6, CYP2C19 and ABCB1 gene polymorphisms in the Hungarian population

Polymorphisms of CYP450 metabolizer enzymes and transport proteins play crucial roles in the inter‐individual variability of drug efficiency. The aim of our study was to predict the frequency of functional variants of CYP2D6, CYP2C19 and ABCB1 genes in the Hungarian population. One hundred twelve unrelated healthy subjects donated DNA sample in the study. ABCB1 C3435T and G2677T/A single‐nucleotide polymorphisms (SNPs) were determined by LightCycler polymerase chain reaction. Because only limited amount of data is available on the rare allelic variants of CYP2D6 in the European populations, our study applied an expanded set of CYP2D6 and CYP2C19 alleles by using AmpliChip test. Our results show that the CYP2D6 phenotypes were 1.9% ultra‐rapid metabolizer, 6.5% intermediate metabolizer (IM), 8.3% poor metabolizer (PM) and 83.3% extensive metabolizer (EM), and the CYP2C19 phenotypes were 1.8% PM, 31.2% IM and 67% EM. The prevalence of the commonly observed CYP2D6 and CYP2C19 alleles in our study corresponds with that of other European populations. Nevertheless, our study confirms that extending the CYP2D6 allele set with loss‐of‐function variants such as CYP2D6*7, *9, *41 is worth considering. Frequency of the wild type ABCB1 3435C was 42.8% whereas the prevelance of 2677 G was 50.4%. Although frequency data of G2677T/A SNP in the European area are limited, some discrepancies with other studies were found. Copyright

Cardioprotective action of urocortin in early pre- and postconditioning

Abstract:  Pre‐ and postconditioning are powerful endogenous adaptive phenomenon of the organism whereby different stimuli enhance the tolerance against various types of stress. Urocortin (Ucn), member of the corticotropin‐releasing factor (CRF) family has potent effects on the cardiovascular system. The aim of this article was to investigate the action of Ucn on cultured cardiomyocytes in the process of pre‐ and postconditioning. Isolated neonatal rat ventricular myocytes were preconditioned with adenosine, simulated ischemia, and Ucn (10‐min treatment followed by 10‐min reperfusion/recovery). For detecting the effect of alternative types of preconditioning, necrosis enzyme (lactate dehydrogenase [LDH]) release, vital staining (trypan blue), and ratio of apoptosis/necrosis were examined after cardiac cells were exposed to 3‐h sustained ischemia and 2‐h reperfusion. Same parameters were measured in the postconditioned groups (30‐ or 60‐min ischemia followed by postconditioning with 10‐min ischemic stimulus or Ucn and 2‐h reperfusion). Cells exposed to 3‐h ischemia followed by 2‐h reperfusion were shown as control. Our results show that LDH release a number of trypan blue‐stained dead cells and the ratio of apoptotized and necrotized cells was decreased in all preconditioned groups compared with control group. In postconditioned groups LDH content of culture medium, trypan blue‐positive cardiomyocytes, and the rate of apoptotic/necrotic cells was reduced contrasted with non‐postconditioned group. We can conclude that preconditioning with Ucn induced such a powerful cell protective effect as adenosine and ischemia. Furthermore, postconditioning with Ucn after 60‐min ischemia was more cardioprotective than ischemic postconditioning.

A Saccharomyces cerevisiae Mutant Unable To Convert Glucose to Glucose-6-Phosphate Accumulates Excessive Glucose in the Endoplasmic Reticulum due to Core Oligosaccharide Trimming

ABSTRACT d-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu3Man9GlcNac2 effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Δ or alg6Δ, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [14C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca2+ uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.