Reactive oxygen species (ROS) are a by-product of aerobic metabolism and have been implicated in cancer, arthrosclerosis, diabetes and aging. Antioxidant enzymes, such as superoxide dismutase (SOD), work to neutralize ROS and oxidative stress occurs when the antioxidant capacity of the cell is overwhelmed. Using a Drosophila mutant with defective cytoplasmic SOD function (cSODn108), we are able to study the consequences of excess ROS on gene expression. Microarray experiments indicate gene expression changes associated with immune response, heat shock, detoxification, proteolysis, carbohydrate metabolism, lipid metabolism and behaviour. Behavioural and physiological assays investigated possible phenotypes predicted by changes in gene expression. We found that cSODn108 mutants feed less yet demonstrate a remarkable resistance to starvation. In addition, cSODn108 mutants show a reduced response to sucrose, odorants and decreased locomotor activity. These phenotypes correlate with observed gene expression changes and suggest a potentially altered energy metabolism in response to chronic oxidative stress.

Reactive oxygen species (ROS) are a by-product of aerobic metabolism and have been implicated in cancer, arthrosclerosis, diabetes and aging. Antioxidant enzymes, such as superoxide dismutase (SOD), work to neutralize ROS and oxidative stress occurs when the antioxidant capacity of the cell is overwhelmed. Using a Drosophila mutant with defective cytoplasmic SOD function (cSODn108), we are able to study the consequences of excess ROS on gene expression. Microarray experiments indicate gene expression changes associated with immune response, heat shock, detoxification, proteolysis, carbohydrate metabolism, lipid metabolism and behaviour. Behavioural and physiological assays investigated possible phenotypes predicted by changes in gene expression. We found that cSODn108 mutants feed less yet demonstrate a remarkable resistance to starvation. In addition, cSODn108 mutants show a reduced response to sucrose, odorants and decreased locomotor activity. These phenotypes correlate with observed gene expression changes and suggest a potentially altered energy metabolism in response to chronic oxidative stress.

The global gene expression profiles of industrial strains of <i>Saccharomyces cerevisiae</i> responding to nitrogen deficiency and very high sugar concentrations stresses were determined by oligonucleotide microarray analysis of ~ 6200 yeast open reading frames. Genomics analysis showed that 400 genes in S. cerevisiae was differentially expressed by more than 1.5-fold compared with controls at late-logarithmic phase of fermentation, as the yeast adapted to changing nutritional, environmental and physiological conditions. The genes of many pathways are regulated in a highly coordinated manner. The repressed expression of GDH1 and up-regulation of ARO10 within the contrast of Q270/Q10 indicated high energy demanding of yeast cells under high sugar stress. Activities of G3P shuttle indicated that under very high gravity environment, sufficient assimilatory nitrogen enhances yeasts ability of redox balancing, and therefore higher stress-tolerance and higher fermentation efficiency of yeast. Under contrast W270/Q270, the up-regulation of DUR1,2 responsible for urea degradation induces the glutamate biosynthesis and the consumption of -ketoglutarate. This may indicate that higher nitrogen level would enable higher activities in the TCA cycle, and therefore generate more energy for biosynthesis and yeast cell proliferation under very high gravity fermentation conditions. Nitrogen metabolism was also stimulated by high nitrogen level when yeast was grown in very high gravity environment.

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.

Pasture feeding depresses adipose tissue development in beef cattle whereas grain feeding, enhances adipogenesis. Therefore, we hypothesized that specific fatty acids would differentially affect lipogenesis in explants of bovine subcutaneous (SC) and intramuscular (IM) adipose tissues. Angus steers were harvested at 12, 14, and 16 mo of age, and IM and SC adipose tissue explants from the 8-11th thoracic rib region were dissected and cultured in media. Media contained no supplemental fatty acids or 40 microM of five fatty acids, stearic acid (18:0), oleic acid (18:1 n-9), trans-11 vaccenic acid (18:1 trans-11), conjugated linoleic acid (CLA, 18:2 trans-10, cis-12), or alpha-linolenic acid (18:3 n-3). After 48 h of culture, lipogenesis using [U-14C]glucose and [1-14C]acetate was measured. Lipogenesis from glucose decreased between 12 and 16 mo of age in SC adipose tissue (from 8.9 to 4.0 nmol per 2 h per 100 mg; P = 0.001) and IM adipose tissue (from 4.4 to 2.7 nmol per 2 h 100 mg ; P = 0.08). Lipogenesis from acetate did not change over time in SC adipose (approximately 56 nmol per 2 h per 100 mg; P = 0.23), but increased over time in IM adipose tissue (from from 11.3 to 17.1 nmol per 2 h 100 mg; P = 0.02). Oleic acid increased lipid synthesis from glucose 125 percent (P = 0.04) in IM adipose tissue, whereas stearic acid and trans-vaccenic acid increased lipogenesis from glucose in SC adipose tissue by approximately 50 percent (P = 0.04). In SC adipose tissue only, trans-vaccenic and increased, lipogenesis from glucose (P < 0.02). Lipogenesis from acetate was depressed by CLA nearly 50 percent in SC adipose tissue. PPARγ gene expression increased between 14 and 16 mo of age in control IM and SC adipocytes. The increase in activity was also observed in AMPK gene expression. C/EBPβ and SCD gene expression did not increase in control samples until 16 mo of age. SC adipose tissue responded to stearic acid by increased GPR43 and AMPK gene expression at 12 mo of age. We conclude that fatty acids differentially affect lipid synthesis in IM and SC adipose tissues, which may account for the effects of pasture and grain feeding on adiposity.

Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death.

The global gene expression profiles of industrial strains of <i>Saccharomyces cerevisiae</i> responding to nitrogen deficiency and very high sugar concentrations stresses were determined by oligonucleotide microarray analysis of ~ 6200 yeast open reading frames. Genomics analysis showed that 400 genes in S. cerevisiae was differentially expressed by more than 1.5-fold compared with controls at late-logarithmic phase of fermentation, as the yeast adapted to changing nutritional, environmental and physiological conditions. The genes of many pathways are regulated in a highly coordinated manner. The repressed expression of GDH1 and up-regulation of ARO10 within the contrast of Q270/Q10 indicated high energy demanding of yeast cells under high sugar stress. Activities of G3P shuttle indicated that under very high gravity environment, sufficient assimilatory nitrogen enhances yeasts ability of redox balancing, and therefore higher stress-tolerance and higher fermentation efficiency of yeast. Under contrast W270/Q270, the up-regulation of DUR1,2 responsible for urea degradation induces the glutamate biosynthesis and the consumption of -ketoglutarate. This may indicate that higher nitrogen level would enable higher activities in the TCA cycle, and therefore generate more energy for biosynthesis and yeast cell proliferation under very high gravity fermentation conditions. Nitrogen metabolism was also stimulated by high nitrogen level when yeast was grown in very high gravity environment.

Cadmium (Cd) is one of the major contaminants in agricultural soil, threatening agricultural production and human health. The objectives of this research work were to understand the tolerance mechanism in rice (Oryza sativa L.) genotype with more Cd-tolerance, and the relation between changes of peroxidases activities and peroxidase gene expression profiles after Cd-treated. For more, we analysis about cis-acting elements in the rice peroxidase genes promoter sequences region, gene structure of rice peroxidase genes and phylogenic relation among 9 peroxidase genes which were blasted from 6 Arabidopsis thaliana Cd-induced based on the peroxidase genes protein sequences. We used 9 upland and 32 varieties rice seeds as materials for germination and the growth of seedlings test with 50 or 500 £gM CdCl2 application, respectively. Rice seeds germination is a complex physiological and biochemical process, and is highly affected by cadmium. The results showed that Cd inhibited both the growth of radicles and coleoptiles. At germination stage, Cd highly inhibited the growth of upland rice. Among these rice varieties, Japonica type cultivars are more tolerant to Cd, but Indica type are more sensitive to Cd. Upland rice cultivars are the most sensitive to Cd. At seedling stage, Cd highly inhibited the growth of roots, but slightly inhibited the growth of shoots. To cope with Cd-induced stresses, plants adopt different strategies and posses a variety of defense mechanisms to prevent themselves from Cd damage. Peroxidase (POXs) is an important antioxidative enzyme for defense responses against Cd oxidative stress. The results suggested that different rice variety has a specific peroxidase gene expression pattern by the pI focusing electrophoresis after Cd-treated, and the peroxidase activities are significant differences when these rice varieties faced Cd stress. In this study, we searched the rice databases (japonica type) and cloned the promoter sequences of the indica type of the rice peroxidase genes, pI 4.5 POX and pI 5.1 POX genes. According to the search results about cis-acting elements from PLACE and PlantCARE databases, these cis-acting elements can be divided into three classes, showing as follow¡G1. Transcriptional related; 2. Light regulated related, and 3. Plant hormones- and stress-related. Based on all reported cis-acting elements, there are many types of transcription factors (TFs) involved in regulation of rice pI 4.5 POX and pI 5.1 POX genes expression. These TFs, which can recognize the specific cis-acting elements to regulate the gene expression, and will be induced by stresses and defense-related plant hormones. We found a cis-acting element (CURECORECR) which response to copper in both rice peroxidase genes promoter regions. There is a number of difference between Japonica type and Indica type peroxidase genes, japonica type peroxidase has more cis-acting elements than indica type. Plant class III peroxidases are present in all land plants. All land plant peroxidase genes are with the same putative ancestor of peroxidase genes and are orthologous genes, but they have specific functions of individual perxidase genes owing to their promoter sequences are very divergent. We used 6 Arabidopsis Cd-related peroxidases protein sequences as a starting point for rice peroxidase datas mining. We found 9 rice peroxidase genes have a closely relation among them. For more details about these rice peroxidase genes, searching each one of these rice peroxidases its gene structure on the Rice Genome Annotation Project (RGAP), comparison and their relation. The expressions of these peroxidase genes are very different among them after Cd treatment, and we also found the same cis-acting element (CURECORECR) which response to copper in all 9 rice peroxidase genes promoter regions. There is a number of difference among them.

Differential gene expression of P. aeruginosa in a rhizosphere biofilm on poplar tree roots was examined in order to identify new virulence factors from this human pathogen. Changes in gene expression for poplar trees contacted with P. aeruginosa was examined as well to identify the response of poplar roots to P. aeruginosa infection. This is the first study of the whole-transcriptome analysis of P. aeruginosa on a plant tree root. The 20 most highly-induced genes of P. aeruginosa were examined for their role in biofilm formation, rhizosphere colonization, barley germination, and poplar tree killing assays. Seven previously uncharacterized virulence genes (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150, and PA4295) were identified. The role of PA2663, a hypothetical protein discovered in the microarrays of P. aeruginosa while killing poplar trees, was examined in further detail. Expression of PA2663 protein increases biofilm formation in P. aeruginosa PAO1 drastically. By complementing the PA2663 mutation in trans and by studying with DNA microarrays and RT-PCR the PA2663 mutant vs. the wild-type strain, PA2663 was confirmed to be related to biofilm formation and was found that it is the first protein to control the psl operon in P. aeruginosa PAO1. Furthermore, PA2663 protein increases pyoverdine synthesis and quorum sensing (QS)- regulated phenotypes. A biofilm formation-related hypothetical protein, PA0939, was identified in this study. The effects of indole and 7-hydroxyindole on P. aeruginosa virulence factors were also examined for the first time. Indole and 7HI repressed expression of mexGHI-opmD multidrug efflux pump genes and genes involved in synthesis of QS-regulated virulence factors (pyocyanin, rhamnolipid, PQS, and pyoverdine production). In addition, the effects of an anti-cancer uracil analog, 5-fluorouracil (5-FU) on P. aeruginosa virulence factors and E. coli K-12 biofilm formation were examined. 5-FU repressed biofilm formation, abolished quorum-sensing phenotypes, and reduced virulence in P. aeruginosa. DNA microarray and biofilm studies with 5-FU in E. coli revealed that 5-FU controls biofilm formation through the AriR protein in E. coli K-12 strain. The effects of lsrR and lsrK mutations on E. coli biofilm formation were also examined by flow cell experiments.

Infectious agents such as bacteria or viruses can grow rapidly. If a microorganism invades a host, it must be recognized rapidly and destroyed before it overwhelms the immune system. Limiting infection to a minimum in the early stage is critical for the outcome and the recovery from infection. The innate immune system has evolved to recognize a few highly conserved, constitutive structures present only in microorganisms, such as bacterial lipopolysaccharide (LPS), called pathogen-associated molecular patterns (PAMP). Toll-like receptors are the host receptors that recognize PAMP, ultimately activating a variety of transcription factors to induce expression of a wide spectrum of immune related genes, e.g. defensins. Defensins are antimicrobial peptides that play an important role in innate defense against microorganisms in plants and animals. Beta-defensins are the largest family of antimicrobial peptides, which can directly kill microorganisms and have regulatory effects on the immune system. Thirteen beta-defensins have been identified; however, the regulation of these genes has not been well-investigated in the chicken. The objective of this research was to understand constitutive and inducible gene expression of beta-defensins in chicken white blood cells. Real-time RT-PCR was used to quantify gene expression level before and after LPS stimulation. Transcription factor binding sites in the genes were identified to understand the gene expression regulation. From the expression profile results, most chicken beta-defensins had induced gene expression by LPS stimulation in the early phase (0- to 3-hour) and reduced gene expression in the late phase (3- to 8-hour). As for the level of gene expression, the results show that the induced gene expression in the early phase corresponded to the higher levels of expression at 3-hours after LPS stimulation, and the reduced gene expression in the late phase corresponded to the lower levels of gene expression at 8-hours after LPS stimulation.