Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpin_Psscauses yeast cell death that shares features of cell death pathway with harpin_Pss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpin_Pss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpin_Pssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpin_Pss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml⁻¹catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpin_Pss-mediated cell death.     

Functional diversity of microbial communities in the mixed boreal plain forest of central Canada

This study was designed to examine whether or not specific tree species (Picea glauca, Picea mariana, Pinus banksiana, Populus tremuloides), their post-fire stand age, or their position in a successional pathway had any significant effect on the functional diversity of associated soil microbial communities in a typical mixed boreal forest ecosystem (Duck Mountain Provincial Forest, Manitoba, Canada). Multivariate analyses designed to identify significant biotic and/or abiotic variables associated with patterns of organic substrate utilization (assessed using the BIOLOG™ System) revealed the overall similarity in substrate utilization by the soil microbial communities. The five clusters identified differed mainly by their substrate-utilization value rather than by specific substrate utilization. Variability in community functional diversity was not strongly associated to tree species or post-fire stand age; however, redundancy analysis indicated a stronger association between substrate utilization and successional pathway and soil pH. For example, microbial communities associated with the relatively high pH soils of the P. tremuloides-P. glauca successional pathway, exhibited a greater degree of substrate utilization than those associated with the P. banksiana-P. mariana successional pathway and more acidic soils. Differences in functional diversity specific to tree species were not observed and this may have reflected the mixed nature of the forest stands and of their heterogeneous forest floor. In a densely treed, mixed boreal forest ecosystem, great overlap in tree and understory species occur making it difficult to assign a definitive microbial community to any particular tree species. The presence of P. tremuloides in all stand types and post fire stand ages has probably contributed to the large amount of overlap in utilization profiles among soil samples.     

Bioguided fractionation and isolation of free radical scavenging components from in vitro propagated chinese medicinal plants Dendrobium tosaense Makino and Dendrobium moniliforme SW

This study was performed to investigate the free radical scavenging active components from in vitro propagated medicinal herbs of the genus Dendrobium, namely, Dendrobium tosaense Makino and Dendrobium moniliforme SW, using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical antioxidative assay. Seeds of the capsules derived after 12 weeks of hand-pollination germinated asymbiotically (50-74%) on half-strength Murashige and Skoog's (MS) basal medium with 3% sucrose and solidified with 0.9% Difco agar. Active growth in the germinated seedlings was achieved by reculturing on full-strength MS basal medium supplemented with 8% banana homogenate, 8% potato homogenate, 8% coconut water, 1.5% sucrose, and 0.9% Difco agar. Healthy plantlets transferred to plastic trays containing moss or moss and tree fern successfully acclimatized (84-100%) in the greenhouse. Extracts were prepared from plants grown in the greenhouse for a period of 6 months. Methanolic extracts of D. tosaense and D. moniliforme scavenged DPPH at 95.9 and 83.4%, respectively, at a concentration of 0.4 mg/mL. Therefore, methanolic solubles of D. tosaense and D. moniliforme were subjected to bioguided fractionation and separation by column chromatographic methods individually. After chromatographic separation of these crude extracts, the obtained fractions (Dm 1, Dm 2, Dm 3, Dt 1, Dt 2, and Dt 3) were tested for their activity. Among them, fractions Dm 2 and Dt 1 showed significant antioxidant activity by DPPH radical antioxidative assay. Active fractions were purified further by column chromatography and resulted in identification of the antioxidant components alkyl ferulates from D. moniliforme and quercetin from D. tosaense.     

Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation

The antioxidant ferulic and caffeic acid phenolics are ubiquitous in plants and abundant in fruits and vegetables. We have synthesized a series of ferulic and caffeic acid esters and tested for tumor cell proliferation, cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation inhibitory activities in vitro. In the tumor cell proliferation assay, some of these esters showed excellent growth inhibition of colon cancer cells. Among the phenolics esters assayed, compounds 10 (C12-caffeate), 11 (C16-caffeate), 21 (C8-ferulate), and 23 (C12-ferulate) showed strong growth inhibition with IC50 values of 16.55, 13.46, 18.67, and 7.57 microg/mL in a breast cancer cell line; 9.65, 7.45, 17.05, and 4.35 microg/ mL in a lung cancer cell line; 5.78, 3.5, 4.29, and 2.46 microg/mL in a colon cancer cell line; 12.04, 12.21, 14.63, and 8.09 microg/ mL in a central nervous system cancer cell line; and 8.62, 7.76, 11.0, and 5.37 in a gastric cancer cell line. In COX enzyme inhibitory assays, ferulic and caffeic acid esters significantly inhibited both COX-1 and COX-2 enzymes. Caffeates 5-10 (C4-C12), inhibited COX-1 enzyme between 50% and 90% and COX-2 enzyme by about 70%, whereas ferulates 15-21 (C3-C8) inhibited COX-1 and COX-2 enzymes by 85-95% 25 microg/mL. Long-chain caffeates 11-14 (C16-C22) and short-chain ferulates 15-20 (C3-C5) were the most active in lipid peroxidation inhibition and showed 60-70% activity at 5 microg/mL concentration.     

Fungichromin: a substance from Streptomyces padanus with inhibitory effects on Rhizoctonia solani

Streptomyces padanus strain PMS-702 is an antagonist of Rhizoctonia solani AG-4, the causal agent of damping-off of cabbage. Treatment of cabbage seeds with the culture filtrate of S. padanus strain PMS-702 was effective in reducing the incidence of damping-off of cabbage. The major active ingredient from the culture filtrate of S. padanus strain PMS-702 was purified by silica gel column chromatography and identified as the polyene macrolide, fungichromin, by NMR and mass spectral data. Bioassay studies showed that fungichromin had a strong antifungal activity against R. solani AG-4, and its minimum inhibitory concentration (over 90% inhibition) was found to be 72 microg/mL. This is the first report of fungichromin from S. padanus as an active ingredient for the control of Rhizoctonia damping-off of cabbage.     

Cultivars of apple fruits that are not marketed with potential for anthocyanin production

The red coloration of apple skin is mainly due to anthocyanins that are reported to possess health benefits. The aim of the present study was to determine the anthocyanin content in three underutilized Malus pumila Mill cultivars, Cranberry, Kerr, and Niedzwetzkyana, and confirm their anti-inflammatory and antioxidant activities. Our analysis revealed that the three cultivars studied contained primarily cyanidin-3-O-glucosyl rutinoside (1) at >99%. The anthocyanin was purified by C-18 medium pressure liquid chromatography and characterized by NMR spectral methods. The quantification of anthocyanins in M. pumila cultivars revealed that Cranberry, Kerr, and Niedzwetzkyana contained 1.12, 0.55, and 0.36 mg/g of fresh weight of 1, respectively. The lipid peroxidation (LPO) and cyclooxygenase enzyme (COX) inhibitory activities of 1 in water were compared with the activities of cyanidin-3-O-rutinoside (2) and cyanidin-3-O-glucoside (3) found in cherries and berries. There is a significant increase in LPO and COX enzyme-inhibitory activities of anthocyanin when tested in water compared to using dimethylsulfoxide as the carrier. The LPO inhibition of anthocyanins 1, 2, and 3 were 53.3, 68.3, and 87.9, respectively, at a 0.25 microM concentration. They inhibited the COX-1 enzyme by 42.7, 45.2, and 50.4 and COX-2 by 52.7, 61.5, and 68.5, respectively, at 5 microM. The LPO inhibitory values for commercial standards, BHA, BHT, and TBHQ, were 85, 89, and 94%, respectively at 1 microM. Similarly, positive controls aspirin, celecoxib, and robecoxib inhibited COX-1 and -2 enzymes by 68.6, 40.7, and 0% and 26.6, 72.2, and 92.4%, respectively, at 60, 26, and 32 nM.     

A study on the biochemical and cytogenetic status in the blood of glioma patients

Malignant gliomas, the most common subtype of primary brain tumors, are aggressive, highly invasive and neurologically destructive tumors, considered being the deadliest of human cancers. As an attempt to understand the biology of glial tumor, a study on macromolecules like proteins, matrix metalloproteinases, lipids antioxidants and Deoxyribonucleic acid, in the blood of glioma patients was made. Biochemical assessment of significant pathophysiological enzymes, antioxidants and marker enzymes was performed. MMP expression was determined using gelatin zymography. Karyotyping analysis was done to determine chromosomal aberrations. A marked rise was observed in the proteins and lipids of glioma patients as compared to the normal cases. The antioxidant status of the patients was found to be lowered. Karyotypic analysis of the peripheral blood chromosomes presented various chromosomal aberrations in glioma patients. The biochemical parameters were significantly increased in the patient population (p<0.01, p<0.001) when compared to those of normal. Zymographic analysis showed the presence of MMP-2 and MMP-9 in the patient sample. Karyotypic investigation showed alterations in the chromosomal pattern of the glioma patients. The study provides baseline information on the biochemical alterations in the blood of glioma patients which can be further exploited for detailed investigations.