Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus

Adaptive mutations in viral polymerase, which is composed of PB1, PB2, and PA, of avian influenza viruses are major genetic determinants of the host range. In this study, to elucidate the molecular mechanism of mammalian adaptation of avian viral polymerase, we performed cell-based vRNP reconstitution assays and biochemical analyses using purified recombinant viral polymerase complexes. We found that avian viral polymerase from A/duck/Pennsylvania/10,218/84 (DkPen) enhances the viral polymerase activity in mammalian cells by replacing the PA or PB2 gene with that from human influenza virus A/WSN/33 (WSN). Chimeric constructs between DkPen PA and WSN PA showed that the N-terminal endonuclease domain of WSN PA was essential for the mammalian adaptation of DkPen viral polymerase. We also found that the cap-snatching activity of purified DkPen viral polymerase was more than 5 times weaker than that of WSN in vitro in a PB2 Glu627-dependent manner. However, the cap-snatching activity of DkPen viral polymerase was hardly increased by replacing DkPen PA to WSN PA. These results suggest that the activity of viral genome replication may be enhanced in the DkPen reassortant containing WSN PA.     

Evaluation of five promoters for use in transformation of oil palm (Elaeis guineensis Jacq.)

The efficiency of GUS (-Glucuronidase) gene expression in embryogenic callus and young leaflets of mature and seedling palm after microprojectile bombardment with five constructs (pEmuGN, pAHC25, pAct1-F4, pGH24 and pBARGUS) was evaluated to identify the most suitable promoter(s) to use in transformation attempts in oil palm. Expression of the GUS gene driven by theEmu, Ubi1, Act1 35S orAdh1 was assayed, both histochemically and fluorometrically, from a total of 200 plates of tissues in eight independent experiments two days after bombardment. A completely randomized experimental design was used for each experiment, and the data analysed by ANOVA and Duncan's Multiple Range Test. The expression level of GUS driven by theEmu orUbi1 promoters was significantly higher than that of the Act], 35S and Adhl promoters in many experiments, and that of theAdhl was significantly lower than those of the other four promoters. Both histochemical and fluorometric data indicate that in embryogenic callus, the expression of theEmu promoter was higher than that of theUbi1 whereas in young leaflets from mature palm the Ubi1 expression was stronger. The performances of the five promoters were also tested in tobacco callus using a fluorometric GUS assay. The activity of the 35S promoter was highest, and significantly different from that of all the other promoters except theEmu, and that of theAct1 promoter was lowest. These results indicate that either theUbil orEmu promoter should facilitate the expression of desired genes in oil palm and aid in development of an efficient stable transformation system.Abbreviations GUS -Glucuronidase - EC embryogenic callus - YLMP young leaflet from mature palm - YLSP young leaflet from seedling palm - MU methyl umbelliferone - MUG 4-methyl--D-glucuronide - X-glue 5-bromo-4-chloro-3-indoyl-glucuronide - Ubil maize ubiquitin 1 - Actl rice actin 1 - Adh1 maize alcohol dehydrogenase 1 - Emu a recombinant truncated maize alcohol dehydrogenase 1 - ANOVA Analysis of variance - DMRT Duncan's Multiple Range Test Communicated by W A. Parrott     

In vitro effect of carbonic anhydrase inhibitor acetazolamide on cell viability,migration and colony formation of colorectal cancer cells

Acidification of extracellular medium in malignant tumors increases the invasive behaviors of cancer cells. In normal healthy tissues, acid production is catalyzed by carbonic anhydrases. Some of the carbonic anhydrase enzymes are overexpressed in certain types of cancer. The present study aimed to investigate the effect of acetazolamide, a potent carbonic anhydrase inhibitor, on in vitro cultivated cancer cells. Three different assays (MTT test, wound healing and clonogenic assay) were performed using human colorectal adenocarcinoma cells (SW620) to evaluate the suppressive effect of acetazolamide, on the colorectal cancer cells migration ability, colony formation and cell viability. The dose-dependent (1–1000 μM) reducing effect of acetazolamide on the cell viability was more significant within the first 48 h. This inhibitory effect of acetazolamide was found to be decreased at 72 h, and affects cells migration ability of cells at 24 and 48 h. Acetazolamide was observed to inhibit the cell viability, migration and colony formation ability of cells, depending on dose.     

Evaluation of commercial soy sauce <Emphasis Type="Italic">koji</Emphasis> strains of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for γ-aminobutyric acid (GABA) production

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P < 0.05) among themselves. Based on the A. oryzae strains, highest GABA concentration was obtained from NSK (194 mg/L) followed by NSZ (63 mg/L), NSJ (51.53 mg/L) and NST (31.66 mg/L). Therefore, A. oryzae NSK was characterized and the sequence was found to be similar to A. oryzae and A. flavus with 99 % similarity. The evolutionary distance (K _nuc) between sequences of identical fungal species was calculated and a phylogenetic tree prepared from the K _nuc data showed that the isolate belonged to the A. oryzae species. This finding may allow the development of GABA-rich ingredients using A. oryzae NSK as a starter culture for soy sauce production.     

Optimization of immersion frequency and medium substitution on microtuberization of Chlorophytum borivilianum in RITA system on production of saponins

In this study, different immersion times were compared for optimization of Chlorophytum borivilianum microtuber production in a RITA (Récipient à Immersion Temporaire Automatique) system. Significantly higher mean number and length of microtubers and growth index were obtained (16, 52 mm and 16, respectively) with 15 min immersion in microtuberization medium (MM) for every 60 min rest period, and could eliminate the occurrence of hyperhydricity. The substitution of MM with liquid hormone-free MS (MSO) medium on week 6 of culture differed significantly in terms of mean number and length of microtubers and growth index compared to earlier substitution at week 3 with MSO but did not differ significantly in comparison to cultures maintained throughout the 9 weeks in MM. Comparison of total saponin of TIS-derived microtubers with tubers of field grown mother plant showed a 1.3-fold increase of saponin (≈21% diosgenin) in the microtubers. The above results hold a promising application of TIS for large scale production of microtubers and accumulated saponin in C. borivilianum.     

Selenium and Vitamin E Modulates Radiation-Induced Liver Toxicity in Pregnant and Nonpregnant Rat: Effects of Colemanite and Hematite Shielding

The levels of liver lipid peroxidation, glutathione peroxidase, reduced glutathione, and vitamins A and E were used to follow the level of oxidative damage caused by ionizing radiation in pregnant rats. The possible protective effects of selenium and vitamin E supplemented to rats housed in concrete-protected cages using hematite and colemanite were tested and compared to untreated controls. Ninety-six rats were randomly divided into four main equal groups namely control (A), normal concrete (B), concrete containing colemanite (C), and concrete containing hematite (D). Except group A, all groups exposed to 7 Gy radiation. The four main groups were divided into four subgroups each as follows: subgroups 1 (n?=?6): nonpregnant control rats. Subgroups 2 (n?=?6): selenium and vitamin E combination was intraperitoneally (i.p.) given to the nonpregnant rats for 20 days. Subgroups 3 (n?=?6): pregnant control rats. Subgroups 4 (n?=?6): selenium and vitamin E combination was i.p. given to the pregnant rats for concessive 20 days. Lactate dehydrogenate, alkaline phosphates, and lipid peroxidation values were higher in subgroups 1 and 3 than in no radiation group although glutathione peroxidase and vitamin E levels in liver were lower in radiation group than in no radiation group. Lactate dehydrogenate activity and lipid peroxidation levels were found to be decreased in subgroups 2 and 4 protected with concrete containing hematite and colemanite when compared to subgroup 1 and 3 with normal concrete. The radiation doses in rats housed by concrete without colemanite and hematite exposed radiation clearly showed liver degeneration. In conclusion, selenium and vitamin E supplementations and housing by concrete with colemanite was found to offer protection against gamma-irradiation-induced liver damage and oxidative stress in rats, probably by exerting a protective effect against liver necrosis via its free radical scavenging and membrane stabilizing. Protective effects of colemanite in the liver seem to be more important than in hematite.     

Genome‐wide mosaicism in divergence between zoonotic malaria parasite subpopulations with separate sympatric transmission cycles

Plasmodium knowlesi is a significant cause of human malaria transmitted as a zoonosis from macaque reservoir hosts in South‐East Asia. Microsatellite genotyping has indicated that human infections in Malaysian Borneo are an admixture of two highly divergent sympatric parasite subpopulations that are, respectively, associated with long‐tailed macaques (Cluster 1) and pig‐tailed macaques (Cluster 2). Whole‐genome sequences of clinical isolates subsequently confirmed the separate clusters, although fewer of the less common Cluster 2 type were sequenced. Here, to analyse population structure and genomic divergence in subpopulation samples of comparable depth, genome sequences were generated from 21 new clinical infections identified as Cluster 2 by microsatellite analysis, yielding a cumulative sample size for this subpopulation similar to that for Cluster 1. Profound heterogeneity in the level of intercluster divergence was distributed across the genome, with long contiguous chromosomal blocks having high or low divergence. Different mitochondrial genome clades were associated with the two major subpopulations, but limited exchange of haplotypes from one to the other was evident, as was also the case for the maternally inherited apicoplast genome. These findings indicate deep divergence of the two sympatric P. knowlesi subpopulations, with introgression likely to have occurred recently. There is no evidence yet of specific adaptation at any introgressed locus, but the recombinant mosaic types offer enhanced diversity on which selection may operate in a currently changing landscape and human environment. Loci responsible for maintaining genetic isolation of the sympatric subpopulations need to be identified in the chromosomal regions showing fixed differences.     

Preferential CEBP binding to T:G mismatches and increased C-to-T human somatic mutations

DNA cytosine methylation in mammals modulates gene expression and chromatin accessibility. It also impacts mutation rates, via spontaneous oxidative deamination of 5-methylcytosine (5mC) to thymine. In most cases the resulting T:G mismatches are repaired, following T excision by one of the thymine DNA glycosylases, TDG or MBD4. We found that C-to-T mutations are enriched in the binding sites of CCAAT/enhancer binding proteins (CEBP). Within a CEBP site, the presence of a T:G mismatch increased CEBPβ binding affinity by a factor of >60 relative to the normal C:G base pair. This enhanced binding to a mismatch inhibits its repair by both TDG and MBD4 in vitro. Furthermore, repair of the deamination product of unmethylated cytosine, which yields a U:G DNA mismatch that is normally repaired via uracil DNA glycosylase, is also inhibited by CEBPβ binding. Passage of a replication fork over either a T:G or U:G mismatch, before repair can occur, results in a C-to-T mutation in one of the daughter duplexes. Our study thus provides a plausible mechanism for accumulation of C-to-T human somatic mutations.     

Diet-induced obesity suppresses ghrelin in rat gastrointestinal tract and serum

The aims of the present study were to examine ghrelin expression in serum and gastrointestinal tract (GIT) tissues, and to measure tissue ghrelin levels and obesity-related alterations in some serum biochemical variables in rats with diet-induced obesity (DIO). The study included 12 male rats, 60 days old. The rats were randomly allocated to two groups (n = 6). Rats in the DIO group were fed a cafeteria-style diet to induce obesity, while those in the control group were fed on standard rat pellets. After a 12 week diet program including an adaptation period all rats were decapitated, tissues were individually fixed, ghrelin expression was examined by immunohistochemistry , and tissue and serum ghrelin levels were measured by radioimmunoassay. Serum biochemical variables were measured using an autoanalyzer. When the baseline and week 12 body mass index and GIT ghrelin expression were compared between DIO and control rats, BMI had increased and ghrelin expression decreased due to obesity. The RIA results were consistent with these findings. Serum glucose, LDL cholesterol, and total cholesterol levels were elevated and HDL cholesterol significantly decreased in the DIO group. A comparison of GIT tissues between the control and obese groups demonstrated that ghrelin was decreased in all tissues of the latter. This decrease was brought about a decline in the circulating ghrelin pool. This suggests that rather than being associated with a change in a single tissue, obesity is a pathological condition in which ghrelin expression is changed in all tissues.