Proteomic study of the yeast Rhodotorula mucilaginosa RCL-11 under copper stress

In order to understand the mechanism involved in Rhodotorula mucilaginosa RCL-11 resistance to copper a proteomic study was conducted. Atomic absorption spectroscopy showed that the copper concentration in the medium decreased from 0.5 to 0.19 mM 48 h after inoculation of the yeast. Analysis of one-dimensional gel electrophoresis of crude cell extracts revealed expression of differential bands between cells with and without copper. In order to study this difference, two-dimensional electrophoresis of R. mucilaginosa RCL-11 exposed to Cu for 16, 24, and 48 h was carried out. Identification of differentially expressed proteins was performed by MALDI-TOF/TOF. Ten of the 16 spots identified belonged to heat shock proteins. Superoxide dismutase, methionine synthase and beta-glucosidase were also found over-expressed at high copper concentrations. The results obtained in the present work show that when R. mucilaginosa RCL-11 is exposed to 0.5 mM copper, differential proteins, involved in cell resistance mechanisms, are expressed.     

Sodium valproate inhibits MDA-MB-231 breast cancer cell migration by upregulating NM23H1 expression

Breast cancer is a common cancer in women, with a highly variable course, from inoffensive to lethal. To find a more effective strategy for its treatment, sodium valproate has been tested as an anti-cancer drug; it is the only clinically available histone deacetylase inhibitor. However, data about the effects of sodium valproate on breast cancer are insufficient in both animals and humans; studies have yielded conflicting conclusions. In particular, little is known about the association between expression of the metastasis suppressor Nm23H1 gene and breast cancer. We hypothesized that sodium valproate regulates NM23H1 expression, and affects migration and/or invasion. We found that sodium valproate at concentrations of 0.8-3.2 mM inhibits migration and modulates Nm23H1 gene expression in a concentration-dependent manner. Confluent MDA-MB-231 cells were scratched by a micropipette tip after VPA treatment for 24 h; 24 h later, the scratch was almostly closed in the 0 mM VPA-treated cells, while the 3.2 mM VPA-treated cells migrated the slowest. The cell migration ratio exposed to 0.8, 1.6 and 3.2 mM VPA was about 66.67, 30.67 and 26.67% (P < 0.05). We also found evidence that sodium valproate upregulates NM23H1 expression, which is a clue to its anti-cancer mode of action. The NM23H1 gene expression was relative fold increased determined by Western blotting at 3.2 mM VPA. Collectively, these observations indicate that sodium valproate has potential for use in breast cancer treatment.     

Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection.     

Dynamics and modeling of temperature-regulated gene product expression in recombinant yeast fermentation

The dynamic response of temperature-regulated gene expression in the recombinant yeast Saccharomyces cerevisiae, strain XK1-C2 carrying plasmid pSXR125, to temperature changes during fed-batch and continuous (chemostat) cultures was studied. The production of the gene product, beta-galactosidase, in the yeast cell is sensitive to the growth temperature. Gene expression of this product was fully turned on or off by temperature shifts between 24 and 30 degrees C. However, the response for gene turn-on and turn-off in this recombinant yeast was slow, requiring from several hours to over 10 h to fully appear. The continuous reactor took 30-60 h after the temperature shift to reach a new steady state. A dynamic process model was developed to simulate the reactor and cell responses to temperature shift. A first-order model was used to account for the effect of dilution rate on the change of protein concentration in the chemostat. It was found that cell response in gene expression to temperature shift followed first-order plus dead-time dynamics. Also, the response time for gene expression to temperature shift varied with specific growth rate or dilution rate of the continuous reactor. In general, the response was slower at a higher dilution rate and for gene turn-on than for gene turn-off. (c) 1996 John Wiley & Sons, Inc.     

Optimization of medium for lipase production by Acinetobacter haemolyticus from healthy human skin

A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the "one variable at a time" and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone--1, yeast extract--0.5, sodium chloride-1, olive oil-1, Tween-80 1, manganese sulphate--5 mM, sucrose--1, pH-7. It was found that maximum production occurred in late log phase, i.e., after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3% (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.     

Fermentation kinetics of recombinant yeast in batch and fed-batch cultures

Fed-batch cultures of recombinant microorganisms have attracted attention as they can separate cell growth stage from cloned-gene expression phase during fermentations. In this work, the effect of different glucose feeding strategies on cell growth and cloned gene expression was studied during aerobic fed-batch fermentations of recombinant yeast, containing the plasmid pRB58. The plasmid contains the yeast SUC2 gene, which codes for the enzyme invertase. Some feeding policies resulted in a constant glucose concentration inside the fermentor, while others deliberately introduced a cyclic variation. The cell mass yield was found to be higher at low glucose concentrations, thus indicating a shift to the more energy-efficient respiratory pathway. The SUC2 gene expression was derepressed at glucose levels below 2 g/L. The response of specific invertase activity to changes in the medium glucose concentration was found to be almost immediate.     

Adaptation of the yeast Yarrowia lipolytica to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45 degrees C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. The 60-min preincubation of cells at 37 degrees C or pretreatment with low concentrations of H2O2 (0.5 mM) and menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Short-term regulation of adiponectin secretion in rat adipocytes

Adiponectin belongs to the group of biologically active substances secreted by adipocytes and referred to as adipokines. Disturbances in its secretion and/or action are thought to be involved in the pathogenesis of some metabolic diseases. However, regulation of adiponectin secretion is poorly elucidated. In the present study, short-term regulation of adiponectin secretion in primary rat adipocytes was investigated. Isolated rat adipocytes were incubated in Krebs-Ringer buffer containing 5 mM glucose and insulin alone or in the combination with epinephrine, dibutyryl-cAMP, adenosine A(1) receptor antagonist (DPCPX), palmitate, 2-bromopalmitate or inhibitor of mitochondrial electron transport (rotenone). Adipocyte exposure for 2 h to insulin (1-100 nM) significantly increased secretion of adiponectin compared with secretion observed without insulin. Furthermore, secretion of adiponectin from adipocytes incubated with glucose and insulin was reduced by 1 and 2 microM epinephrine, but not by 0.25 and 0.5 microM epinephrine. Under similar conditions, 1 and 2 mM dibutyryl-cAMP substantially diminished secretion of adiponectin, whereas 0.5 mM dibutyryl-cAMP was ineffective. Secretion of adiponectin was found to be effectively decreased by DPCPX. Moreover, adipocyte exposure to rotenone also resulted in a substantial diminution of secretory response of adipocytes incubated for 2 h with glucose and insulin. It was also demonstrated that palmitate and 2-bromopalmitate (0.06-0.5 mM) failed to affect secretion of leptin. The obtained results indicated that in short-term regulation of adiponectin secretion, insulin and epinephrine exert the opposite effects. These effects appeared as early as after 2 h of exposure. Moreover, deprivation of energy or blockade of adenosine action substantially decreased secretion of adiponectin.     

Inducible types of cyclooxygenase and nitric oxide synthase in adaptive cytoprotection in rat stomachs.

Roles of cyclooxygenases (COX-1 and COX-2) and nitric oxide (NO) synthases (nNOS and iNOS) in adaptive cytoprotection induced by 20 mM taurocholate dissolved in 50 mM HCl (TC) were investigated in rat stomachs. Intragastric administration of 0.6 N HCl caused haemorrhagic damage in the stomach. These lesions were prevented by pretreatment of the animals with TC p.o. 0.5 h before 0.6 N HCl, and a significant protection persisted for more than 5 h. The protection afforded by TC given 0.5 h before HCl was almost totally reversed by indomethacin and slightly mitigated by N(G)-nitro-L-arginine methyl ester (L-NAME) but not affected by NS-398 or aminoguanidine. By contrast, the mucosal protective action of TC given 5 h before HCl was significantly reversed by NS-398, L-NAME and aminoguanidine as well as indomethacin. Mucosal prostaglandin E2 (PGE2) contents were significantly increased for over 5 h after TC, while luminal NOx output tended to elevate at 0.5 h and be significantly increased at 5 h after TC. The increased PGE2 generation observed 0.5 h after TC was attenuated only by indomethacin, while that observed 5 h after TC was inhibited by NS-398 as well as indomethacin. On the other hand, the NOx output determined at 5 h after TC was significantly reduced by both L-NAME and aminoguanidine. The expression of mRNA for both COX-2 and iNOS was apparently detected in the stomach from 3 h after TC treatment. These results suggest that TC induced adaptive cytoprotection in the rat stomach against 0.6 N HCl, the effect lasting for over 5 h, and the underlying mechanism differs depending on the period after the irritation. The early phase is mediated mainly by COX-1/PGs, while the later phase is mediated by iNOS/NO, in addition to prostaglandins (PGs) produced by both COX-1 and COX-2.     

脂肪细胞分化相关基因在大鼠再生肝中表达变化

肝脏由多种细胞构成,肝再生与细胞分化密切相关,细胞分化受基因转录水平调控。为在基因转录水平了解脂肪细胞分化基因在大鼠肝再生中作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因,用Rat Genome2302.0芯片检测它们在大鼠肝再生(liver regeneration,LR)中表达情况,将三次检验结果相同或相似、在肝再生中表达变化2倍以上、真手术组和假手术组相比差异显著的基因视为肝再生相关基因。初步证实上述基因中75个基因与肝再生相关。肝再生启动(PH后0.5-4h)、G0/G1过渡(PH后4-6h)、细胞增殖(PH后6-66h)、细胞分化和组织结构功能重建(PH后72-168h)等四个阶段起始表达的基因数为44、13、30和1;基因的总表达次数为88、58、302和90。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共表达上调313次、下调167次,分为43种表达方式。表明肝再生中脂肪细胞发生和分化相关基因活动多样和复杂。根据本文研究结果推测,上述基因不仅调节脂肪细胞分化,而且参与肝再生的生理生化活动。     

Effects of hexavalent chromium on the survival and cell cycle distribution of DNA repair-deficient S. cerevisiae

A broad spectrum of genetic damage results from exposure to hexavalent chromium. These lesions can result in DNA and RNA polymerase arrest, chromosomal aberrations, point mutations and deletions. Because of the complexity of Cr genotoxicity, the repair of Cr(VI)-induced DNA damage is poorly understood. Therefore, our aim was to investigate the sensitivities of DNA repair-deficient Saccharomyces cerevisiae strains to Cr(VI)-induced growth inhibition and lethality. Wild-type, translesion synthesis (rev3) and excision repair (apn1, ntg1, ntg2, rad1) mutants exhibited similar survival following Cr(VI) treatment (0-50mM) and underwent at least one population doubling within 2-4h post-treatment. The simultaneous loss of several excision repair genes (apn1 rad1 ntg1 ntg2) led to slower growth after Cr(VI) exposure (10mM) manifested as an initial delay in S phase progression. Higher concentrations of Cr(VI) (25mM) resulted in a prolonged transit through S phase in every strain tested. A G(2)/M arrest was evident within 1-2h after Cr(VI) treatment (10mM) in all strains and cells subsequently divided after this transient delay. In contrast to all other strains, only recombination-deficient (rad52, rad52 rev3) yeast were markedly hypersensitive towards Cr(VI) lethality. RAD52 mutant strains (rad52, rad52 rev3) also exhibited a significant delay (>6h) in the resumption of replication after Cr(VI) exposure which was related to the immediate and apparently terminal arrest of these yeast in G(2)/M after Cr(VI) treatment. These results, taken together with the recombinogenic effects of Cr(VI) in yeast containing a functional RAD52 gene, suggest that RAD52-mediated recombination is critical for the normal processing of lethal Cr-induced genetic lesions and exit from G(2) arrest. Furthermore, only the combined inactivation of multiple excision repair genes affects cell growth after Cr(VI) treatment.     

Toxic effects caused by heavy metals in the yeast Saccharomyces cerevisiae: a comparative study

The decreasing order of toxicity of select heavy metals on the yeast Saccharomyces cerevisiae, in 10 mM MES (2-(N-morpholino)ethanesulfonic acid) pH buffer at pH 6.0, was found to be copper, lead, and nickel. Heavy metal (200 microM) induced a decrease in the number of viable cells by about 50% in the first 5 min for copper and in 4 h for lead, while nickel was not toxic up to a 200 microM concentration over a period of 48 h. Glucose (25 mM) strongly enhanced the toxic effect of 50 microM copper but had little or no effect on the toxicity of 200 microM lead or nickel. Copper, lead, and nickel induced the leakage of UV260-absorbing compounds from cells with different kinetics. The addition of 0.5 mM calcium, before addition of 200 microM copper, showed a protective action against cell death and decreased the release of UV-absorbing compounds, while no effect was observed against lead or nickel toxic effects. Copper complexation capacities of the filtrates of cells exposed for 2 h in 200 microM copper and 24 h in 200 microM lead were 51 and 14 microM, respectively. The implication of the complexation shown by these soluble compounds in the bioavailability of heavy metals is discussed.     

Wound- and systemin-inducible calmodulin gene expression in tomato leaves

Using a calmodulin (CaM) cDNA as a probe in northern analyses, transgenic tomato plants that overexpress the prosystemin gene were found to express increased levels of CaM mRNA and protein in leaves compared to wild-type plants. These transgenic plants have been reported previously to express several wound-inducible defense-related genes in the absence of wounding. Calmodulin mRNA and protein levels were found to increase in leaves of young wild-type tomato plants after wounding, or treatment with systemin, methyl jasmonate, or linolenic acid. CaM mRNA appeared within 0.5 h after wounding or supplying young tomato plants with systemin, and peaked at 1 h. The timing of CaM gene expression is similar to the expression of the wound- or systemin-induced lipoxygenase and prosystemin genes, signal pathway genes whose expression have been reported to begin at 0.5–1 h after wounding and 1–2 h earlier than the genes coding for defensive proteinase inhibitor genes. The similarities in timing between the synthesis of CaM mRNA and the mRNAs for signal pathway components suggests that CaM gene expression may be associated with the signaling cascade that activates defensive genes in response to wounding.