Polymorphism of the angiotensin II type 1 receptor in patients with essential hypertension in Ukrainian population

The distribution of polymorphism of the angiotensin II type 1 receptor in Ukrainian population was investigated. Healthy persons had genotypes AA (51%), AC (34%), CC (15%) and alleles A (68%), C (32%). We suppose the prevalence of allele C and genotypes CC in health persons in Ukrainian population. The frequencies of genotypes and alleles in patients with essential hypertension were AA (22,85%), AC (51,9%), CC (25,3%) and A (48,7%), C (51,3%). Thus the development of essential hypertension was associated with the presence of allele C and its homozygote variant. Moreover the severity and complications of hypertension depended on the presence of this allele and genotype. We concluded that Ukrainian population has specific distribution of polymorphism of the angiotensin II type 1 receptor with prevalence of allele C1166 and genotype CC. The presence of these genetic variants is a risk factor for essential hypertension.     

Changes in serum leptin levels in strenuous exercise and its relation to zinc deficiency in rats

This study aimed to investigate the possible changes in serum leptin concentration caused by acute exercise and the effects of zinc deficiency on these changes. Forty male rats were divided into control-control, control-elercise, zinc-deficient-control, and zinc-deficient-exercise groups (10 rats in each). Control-exercise and zinc-deficient-exercise groups performed exercisse at 6 m/min speed on a rodent treadmill for 60 min or until exhaustion. All rats were decapitated 48h after the exercise, and blood samples were collected to determine serum leptin and zinc levels. Serum leptin levels in the zinc-deficient-control group were lower than in the control-control group. The mean exercise time of control-exercise group was significantly longer than the zinc-deficient-exercise group. We conclude that serum leptin levels significantly decrease both 48 h after strenuous exercise and in the zinc-deficient rats, and there is a further decrease in leptin levels when rats fed on a zinc-deficient diet performed exercise.     

Virulotypes of Bacillus anthracis strains isolated in Poland

Bacillus anthracis--the causative agent of anthrax--possesses several virulence genes located in the chromosome as well as in two B. anthracis virulence plasmids: pXO1 and pXO2. In the presented study, we determined occurrence of six virulence markers located in the virulence plasmids (capA, capB, capC, pagA, lef and cya) for capsule and toxin production together with virulence-associated gene gerXA and chromosomal gene sap, which are responsible for germination and S-layer biosynthesis respectively. Fourteen strains of B. anthracis isolated in Poland and belonging to five different MLVA genotypes were analyzed by PCR for presence of the aforementioned genes. Two virulotypes were found in tested strains. The only variation was absence of capA, capB and capC due to a lack of pXO2.     

Donuts, scratches and blanks: robust model-based segmentation of microarray images

MOTIVATION: Inner holes, artifacts and blank spots are common in microarray images, but current image analysis methods do not pay them enough attention. We propose a new robust model-based method for processing microarray images so as to estimate foreground and background intensities. The method starts with a very simple but effective automatic gridding method, and then proceeds in two steps. The first step applies model-based clustering to the distribution of pixel intensities, using the Bayesian Information Criterion (BIC) to choose the number of groups up to a maximum of three. The second step is spatial, finding the large spatially connected components in each cluster of pixels. The method thus combines the strengths of the histogram-based and spatial approaches. It deals effectively with inner holes in spots and with artifacts. It also provides a formal inferential basis for deciding when the spot is blank, namely when the BIC favors one group over two or three. RESULTS: We apply our methods for gridding and segmentation to cDNA microarray images from an HIV infection experiment. In these experiments, our method had better stability across replicates than a fixed-circle segmentation method or the seeded region growing method in the SPOT software, without introducing noticeable bias when estimating the intensities of differentially expressed genes. AVAILABILITY: spotSegmentation, an R language package implementing both the gridding and segmentation methods is available through the Bioconductor project (http://www.bioconductor.org). The segmentation method requires the contributed R package MCLUST for model-based clustering (http://cran.us.r-project.org). CONTACT: fraley@stat.washington.edu.     

Organelle proteomics of rat synaptic proteins: correlation-profiling by isotope-coded affinity tagging in conjunction with liquid chromatography-tandem mass spectrometry to reveal post-synaptic density specific proteins

Organelle proteomics is the method of choice for global analysis of cellular proteins. However, it is difficult to isolate organelles to homogeneity. Recently, correlation-profiling has been used to filter off the contaminants ad hoc and to disclose the genuine organelle-specific proteins. In the present study, we further extend the method to include subcellular compartments that contain proteins shared by multiple distinct subcellular domains. We performed correlation profiling of proteins contained in synaptic membrane and postsynaptic density (PSD) fractions isolated from rat brain. Proteins were labeled with isotope-coded affinity-tag reagents, digested with trypsin, and resulting peptides were resolved by cation exchange chromatography followed by reversed phase chromatography. Peptides were then subjected to mass spectrometry for quantification and identification. We confirm that the core PSD proteins were enriched in the PSD preparation. Other functional protein groups such as cytoskeleton-associated proteins, protein kinases and phosphatases, signaling components and regulators, as well as proteins involved in energy production partitioned to multiple organelles. When analyzed as groups, they were shown to accumulate to a lesser extent. Mitochondrial proteins and transporters were generally strongly depleted from the PSD fraction confirming that they were contaminants of the PSD preparation. Finally, immunoelectron microscopy was performed on selected proteins to validate the proteomics results, and confirm that synaptophysin that was highly depleted in the PSD preparation is localized in the presynaptic compartment, whereas LASP-1 that was slightly enriched in the PSD preparation is present in the PSD as well as other subdomains within the synapse.     

The calmodulin inhibitor R24571 induces a short-term Ca2+ entry and a pulse-like secretion of ATP in Ehrlich ascites tumor cells

The properties of the Ca2+ channel induced by a calmodulin inhibitor in Ehrlich ascites tumor cells were investigated using fluorescent indicators Indo-1 and chlortetracycline. The inhibitor of calmodulin calmidazolium (R24571) in concentrations of 1-2 microM induces a short-term Ca2+ entry and a pulse-like ATP secretion. Repeated addition of R24571 also causes a transient Ca2+ signal. Ca2+ channels induced by R24571 are permeable for Mn2+. Ca2+ entry does not depend on endoplasmic reticulum depletion by thapsigargin, ATP, or ionomycin and is suppressed by nordihydroguaretic acid (EC50 = 6.7 microM), quercetin (EC50 = 1.5 microM), dihydroquercetin (EC50 = 17 microM), arachidonic acid (AA) (EC50 = 8.6 microM), and suramin (EC50 = 0.25 +/- 0.05 MM), and weakly depends on temperature in the range of 18 - 37 degrees C. The apparent activation constant for R24571 and the Hill coefficient are 2.5 +/- 0.2 and 4 +/- 0.3 microM, respectively. The products of arachidonic acid oxidation are neither activators nor inhibitors of these channels. The inhibitory effect of nordihydroguaretic acid is indirect and is conceivably caused by the accumulation of arachidonic acid due to suppression of its lipoxygenase-catalyzed oxidation at phospholipase A2 activation. The maximal level of about 1.3 microM in the dependence of Ca2+ signal amplitude on R24571 concentration points to possible inhibition of the channel by increased Ca2+ concentration in the cytosol. The weak dependence on temperature implies that the channel is highly permeable, the chain of enzymic processes is not involved in Ca2+ entry activation, and the mutual compensation of processes with opposite contributions is possible. Using chlortetracycline fluorescence, we have shown in model experiments on calmodulin solution that Ca2+ induces cooperatively a conformational transition of calmodulin with the exposure of a hydrophobic chlortetracycline-Ca(2+)-binding site. The interaction of R24571 with the CaM-Ca2+ complex results in quenching of fluorescence to its level in water, which is interpreted as the elimination of the availability of calmodulin hydrophobic site for chlortetracycline-Ca+. Nordihydroguaretic acid, quercetin, and dihydroquercetin, but not suramin, also interact with calmodulin, but this does not result in the complete closing of its hydrophobic site. It is supposed that the activation of the Ca2+ channel occurs owing to the activation of calmodulin-dependent phospholipase A2 by R24571, which leads to the formation of a low-molecular short-lived secondary messenger, or because of the interaction of R24571 with calmodulin, which directly inhibits the channel. The termination of Ca2+ entry is probably due to the inhibition of phospholipase A2 and/or of the channel at increased concentrations of arachidonic acid and Ca2+.     

Adenovirus-mediated expression of the C-terminal domain of SARS-CoV spike protein is sufficient to induce apoptosis in Vero E6 cells

The pro-apoptotic properties of severe acute respiratory syndrome coronavirus (SARS-CoV) structural proteins were studied in vitro. By monitoring apoptosis indicators including chromatin condensation, cellular DNA fragmentation and cell membrane asymmetry, we demonstrated that the adenovirus-mediated over-expression of SARS-CoV spike (S) protein and its C-terminal domain (S2) induce apoptosis in Vero E6 cells in a time- and dosage-dependent manner, whereas the expression of its N-terminal domain (S1) and other structural proteins, including envelope (E), membrane (M) and nucleocapsid (N) protein do not. These findings suggest a possible role of S and S2 protein in SARS-CoV induced apoptosis and the molecular pathogenesis of SARS.     

Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.     

Glutathione S-transferase pi as a target for tricyclic antidepressants in human brain

GST pi, the main glutathione S-transferase isoform present in the human brain, was isolated from various regions of the brain and the in vitro effect of tricyclic antidepressants on its activity was studied. The results indicated that amitripyline and doxepin - derivatives of dibenzcycloheptadiene, as well as imipramine and clomipramine - derivatives of dibenzazepine, inhibit the activity of GST pi from frontal and parietal cortex, hippocampus and brain stem. All these tricyclics are noncompetitive inhibitors of the enzyme with respect to reduced glutathione and noncompetitive (amitripyline, doxepin) or uncompetitive (imipramine, clomipramine) with respect to the electrophilic substrate. Their inhibitory effect is reversible and it depends on the chemical structure of the tricyclic antidepressants rather than on the brain localization of the enzyme. We conclude that the interaction between GST pi and the drugs may reduce their availability in the brain and thus affect their therapeutic activity. On the other hand, tricyclic antidepressants may decrease the efficiency of the enzymatic barrier formed by GST and increase the exposure of brain to toxic electrophiles. Reactive electrophiles not inactivated by GST may contribute in adverse effects caused by these drugs.     

A simple method for selection of trypsin chromogenic substrates using combinatorial chemistry approach

A tetrapeptide combinatorial library, considered as chromogenic substrates of bovine beta-trypsin, was synthesized by the solid phase method. The peptides contain an analog of p-nitroanilide, obtained by attaching 5-amino-2-nitrobenzoic acid (Anb(5,2)) to the C-termini. Deconvolution of the peptide library, performed in solution using an iterative method, yielded four efficient trypsin substrates. The most active one, Phe-Val-Pro-Arg-Anb(5,2)-NH(2), appeared to be 125-fold more active than Bz-D,L-Arg-pNA (BAPNA) used as a reference compound. The reported method of designing trypsin chromogenic substrate libraries is straightforward. Such p-nitroanilides may be useful for the investigation of any protease substrate specificity.