Association analysis of alcohol metabolizing enzymes <Emphasis Type="Italic">ADH1B,ADH7, CYP2E1</Emphasis> gene polymorphism with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5′-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B ^* G (+MslI) allele, CYP2E1 ^* C2 (+PstI) allele and CYP2E1 ^* C (-DraI) allele were 8.48 ± 1.86, 1.20 ± 0.69, and 10.00 ± 1.90%, respectively. The ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7 ^* C (-StyI) allele was 44.58 ± 3.21%. A significantly higher frequency of CYP2E1 PstI C2 allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03–20.01). The tendency to significant effect of A1A2 genotype in ADH1B MslI polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0.042) were also revealed. Association of A1A2 genotype in ADH1B MslI polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.     

Differential cytotoxic effects ofAnnona squamosa seed extracts on human tumour cell lines: Role of reactive oxygen species and glutathione

Annonaceous acetogenins are a new class of compounds that have been reported to have potent pesticidal, parasiticidal, anti-microbial, cell growth inhibitory activities. In this study, organic and aqueous extracts from the defatted seeds ofAnnona squamosa (custard apple) were tested on different human tumour cell lines for antitumoural activity. While organic and aqueous extracts induced apoptosis in MCF-7 and K-562 cells, they failed to do so in COLO-205 cells. Treatment of MCF-7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA fragmentation, induction of reactive oxygen species (ROS) generation and reduced intracellular glutathione levels. In addition downregulation of Bcl-2 and PS externalization by Annexin-V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts through oxidative stress. On the contrary, COLO-205 cells showed only PS externalization but no change in ROS and glutathione levels. These observations suggest that the induction of apoptosis byA. squamosa extracts can be selective for certain types of cancerous cells     

Common Cuckoo Cuculus canorus and host behaviour at Reed Warbler Acrocephalus scirpaceus nests

This study is based on continuous video recordings made at 53 Reed Warbler Acrocephalus scirpaceus nests each day during the laying period. Egg-laying by the Common Cuckoo Cuculus canorus was recorded in 14 (26.4%) of these nests. By analysing the activity of the host birds around and at the nest, we found that this is probably not the only cue used by the Common Cuckoo when locating suitable nests to parasitize. Furthermore, in most cases there was no significant difference between the length of time the host birds spent at the nest in the morning and afternoon, thus providing little support for the hypothesis that the Common Cuckoo lays in the afternoon because it is less likely to be seen by the nest owners then. Parasitized Reed Warblers rejected the Common Cuckoo egg more frequently when they observed the parasite at their nests. However, contrary to what should be expected, most Common Cuckoos laid their eggs in the presence of the host(s), and in general their egg-laying behaviour (for example duration of stay at the nest) was less secretive than described earlier. When partially depredating host clutches, Cuckoos showed the same behavioural pattern at parasitized and unparasitized nests, indicating that the latter may act as a potential reserve for egg-laying.     

Analysis of the p53 gene alterations in mouse embryo fibroblast cell line Balb 3T12 and its derivative 312

We have analysed the status of the p53 gene in the mouse embryo fibroblast cell line Balb 3T12 (TD₅₀=10⁶) and its transformed clonal derivative 312 (TD₅₀=10⁴) with an aim to determine whether there exists a correlation between increased tumorigenicity and clonal expansion of cells bearing a mutation in the p53 gene. While Southern hybridizations did not show any obvious changes in the p53 gene organization in 3T12 and 312 cells, sequencing the p53 cDNA revealed that 3T12 is mutated at the amino acid residue 233 (Tyr→ Asp) whereas 312 is mutated at the residue 132 (Cys→Trp). Exploiting the altered RFLP pattern due to mutations, we identified that 3T12 contains p53 alleles that are different from the already identified mutant p53. On the basis of these observations, we conclude that 3T12 and 312 have evolved independently.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Curcumin mediated apoptosis in AK-5 tumor cells involves the production of reactive oxygen intermediates.

Curcumin, the active ingredient of the rhizome of Curcuma longa has anti-inflammatory, antioxidant and antiproliferative activities. Although its precise mode of action remains elusive, studies have shown that chemopreventive action of curcumin might be due to its ability to induce apoptosis in cancer cells. Curcumin was shown to be responsible for the inhibition of AK-5 tumor (a rat histiocytoma) growth by inducing apoptosis in AK-5 tumor cells via caspase activation. This study was designed to investigate the mechanism leading to the induction of apoptosis in AK-5 tumor cells. Curcumin treatment resulted in the hyperproduction of reactive oxygen species (ROS), loss of mitochondrial membrane potential (delta psi(m)) and cytochrome c release to the cytosol, with the concomitant exposure of phosphatidylserine (PS) residues on the cell surface. This study suggests redox signalling and caspase activation as the mechanisms responsible for the induction of curcumin mediated apoptosis in AK-5 tumor cells.     

Induction of nitric oxide production by natural killer cells: its role in tumor cell death.

It has been recognized that natural killer (NK) cells destroy AK-5 tumor cells, largely by cytolysis and apoptosis. The objective of this study was to elucidate the existence and the role of nitric oxide (NO) during this killing. The target cell killing ability of NK cells was associated with an increased production of NO with higher expression of inducible nitric oxide synthase. In part, the production of NO was confirmed by significant increase in cell lysis in the presence of l-arginine and attenuation of cell lysis, DNA fragmentation, and apoptosis by N(omega)-nitro-l-arginine methyl ester (L-NAME). An increased oxidation of intracellularly trapped dichlorofluorescein was observed in NK cells, which was effectively prevented by L-NAME. Exposure of AK-5 cells to chemically generated NO also induced DNA fragmentation in AK-5 cells. Further evidence for the involvement of NO in apoptosis was provided by the inhibition of specific cleavage of PARP and activation of CPP32 by L-NAME. Increased production of NO with simultaneous enhancement of the cytotoxic activity of NK cells from sc tumor-transplanted animals has been implicated in tumor regression when compared to the ip tumor-bearing animals. Overall, these observations suggest an important role for NO during NK cell-mediated apoptosis and lysis of AK-5 cells.     

Fast Docking on Graphics Processing Units via Ray-Casting

Docking Approach using Ray Casting (DARC) is structure-based computational method for carrying out virtual screening by docking small-molecules into protein surface pockets. In a complementary study we find that DARC can be used to identify known inhibitors from large sets of decoy compounds, and can identify new compounds that are active in biochemical assays. Here, we describe our adaptation of DARC for use on Graphics Processing Units (GPUs), leading to a speedup of approximately 27-fold in typical-use cases over the corresponding calculations carried out using a CPU alone. This dramatic speedup of DARC will enable screening larger compound libraries, screening with more conformations of each compound, and including multiple receptor conformations when screening. We anticipate that all three of these enhanced approaches, which now become tractable, will lead to improved screening results.