Pressure response in the yeast Saccharomyces cerevisiae: from cellular to molecular approaches.

Yeasts are unicellular organisms that are exposed to a highly variable environment, concerning the availability of nutrients, temperature, pH, radiation, access to oxygen and, specially, water activity. Evolution has selected yeasts to tolerate, to a certain extent, these environmental stresses. High hydrostatic pressure (HHP) exerts a broad effect upon yeast cells, interfering with the cell membranes, cellular architecture and in processes ofpolymerisation and denaturation of proteins. Gene expression patterns in response to HHP revealed a stress response profile. The majority of the upregulated genes were involved in stress defence and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. In addition, in the present work it was seen that mild pressure induced cell cycle arrest and protection against severe stresses, such as high temperature, high pressure and ultra cold shock. Nevertheless, this protection was only significant if the cells were incubated at atmospheric pressure after the HHP treatment. Expression of genes that were upregulated by HHP and are related to resistance to this stresses were also analyzed, and, for the majority of them, higher induction was attained after 15 min post-pressurization. Taken together, the results imply an interconnection among stresses.     

水稻氮饥饿诱导基因的克隆与表达分析

为了解水稻(Oryza sativa L.)对氮饥饿反应的分子与基因背景,利用RaSH策略构建了水稻氮(N)饥饿诱导cDNA文库.通过反向Northern筛选该文库,获得氮饥饿诱导的18个功能已知基因和2个功能未知基因.这些已知基因涉及碳代谢、次生代谢产物合成、蛋白质分解代谢、激素代谢、信号转导、生长调控过程及转录因子.这些基因表现出不同的时空表达模式.研究结果表明了植物对氮饥饿反应涉及互相关联的多种生理与分子机理,提供了相关的一些基因信息.     

水稻氮饥饿诱导基因的克隆与表达分析

为了解水稀(Oryza sativa L.)对氮饥饿反应的分子与基因背景,利用RaSH策略构建了水稻氮(N)饥饿诱导cDNA文库。通过反向Northern筛选该文库,获得氮饥饿诱导的18个功能已知基因和2个功能未知基因。这些已知基因涉及碳代谢、次生代谢产物合成、蛋白质分解代谢、激素代谢、信号转导、生长调控过程及转录因子。这些基因表现出不同的时空表达模式。研究结果表明了植物对氮饥饿反应涉及互相关联的多种生理与分子机理,提供了相关的一些基因信息。     

Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure

Gene expression patterns in response to hydrostatic pressure were determined by whole genome microarray hybridization. Functional classification of the 274 genes affected by pressure treatment of 200 MPa for 30 min revealed a stress response expression profile. The majority of the >2-fold upregulated genes were involved in stress defense and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. Furthermore, uncharacterized genes were among the 10 highest expressed sequences and represented 45% of the total upregulated genes. The results of this study revealed a hydrostatic pressure-specific stress response pattern and suggested interesting information about the mechanisms involved in adaptation of cells to a high-pressure environment.     

Transcriptomic profiles of Japanese medaka (Oryzias latipes) in response to alkalinity stress

Oryzias latipes (Adrianichthyidae), known as Japanese medaka or Japanese killifish, is a small 2-4 cm long fish common in rice paddies in coastal Southeast Asia and is also a popular aquarium fish. It has been widely used as a research model because of its small size and because it is very easy to rear. Alkalinity stress is considered to be one of the major stressors on fish in saline-alkaline water. As very little is known about molecular genetic responses of aquatic organisms to alkalinity stress, we examined genome-wide gene expression profiles of Japanese medaka in response to carbonate alkalinity stress. Adult fish were exposed to freshwater and high carbonate alkaline water in the laboratory. We designed a microarray containing 26,429 genes for measuring gene expression change in the gills of the fish exposed to high carbonate alkalinity stress. Among these genes, 512 were up-regulated and 501 were down-regulated in the gills. These differentially expressed genes can be divided into gene groups using gene ontology, including biological processes, cellular components and molecular function. These gene groups are related to acid-base and ion regulation, cellular stress response, metabolism, immune response, and reproduction processes. Biological pathways including amino sugar and nucleotide sugar metabolism, porphyrin and chlorophyll metabolism, metabolism of xenobiotics by cytochrome P450, drug metabolism, aminoacyl-tRNA biosynthesis, glycine, serine and threonine metabolism, ascorbate and aldarate metabolism, pentose and glucuronate interconversions, glutathione metabolism, and fructose and mannose metabolism were significantly up-regulated. Alkalinity stress stimulates the energy and ion regulation pathways, and it also slows down the pathways related to the immune system and reproduction.     

Induction of baroresistance by hydrogen peroxide, ethanol and cold-shock in Saccharomyces cerevisiae

The acquisition of tolerance to high hydrostatic pressure of 220 MPa (HHP) in response to a 0.4 mM hydrogen peroxide, 6% ethanol and cold-shock (10 degrees C) pretreatment for different lengths of times was studied in the yeast Saccharomyces cerevisiae. The protection conferred by these different treatments was similar ( approximately 3 log cycles) and time-dependent. Analysis of the induction of the most pressure up-regulated genes under these conditions was investigated by RT-PCR. Our results revealed that the cell stress response to HHP shares common features with hydrogen peroxide and ethanol stresses, but differs in some way to cold-shock.     

Genes up- and down-regulated by dermcidin in breast cancer: a microarray analysis

Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, which is co-amplified with multiple oncogenes with a well-established role in the growth, survival and progression of breast cancers. Here, we present a summary of a DNA microarray-based study that identified the genes that are up- and down-regulated in a human MDA-361 pLKO control clone and three clones expressing short hairpin RNA against three different regions of DCD mRNA. A list of 235 genes was differentially expressed among independent clones (> 3-fold change and p < 0.005). The gene expression of 208 was reduced and of 27 was increased in the three DCD-RNAi clones compared to pLKO control clone. The expression of 77 genes (37%) encoding for enzymes involved in amino acid metabolism, glucose metabolism and oxidoreductase activity and several genes required for cell survival and DNA repair were decreased. The expression of EGFR/ErbB-1 gene, an important predictor of outcome in breast cancer, was reduced together with the genes for betacellulin and amphiregulin, two known ligands of EGFR/ErbB receptors. Many of the 27 genes up-regulated by DCD-RNAi expression have not yet been fully characterized; among those with known function, we identified the calcium-calmodulin-dependent protein kinase-II delta and calcineurin A alpha. We compared 132 up-regulated and 12 down-regulated genes in our dataset with those genes up- and down-regulated by inhibitors targeting various signaling pathway components. The analysis showed that the genes in the DCD pathway are aligned with those functionally influenced by the drugs sirolimus, LY-294002 and wortmannin. Therefore, DCD may exert its function by activating the PI3K/AKT/mTOR signaling pathway. Together, these bioinformatic approaches suggest the involvement of DCD in the regulation of genes for breast cancer cell metabolism, proliferation and survival.     

Hydrostatic pressure induced death of mammalian cells engages pathways related to apoptosis or necrosis.

We investigated the response to high hydrostatic pressure (HHP) of mammalian cells, since HHP is proposed to be suitable to inactivate mammalian cells in biopharmaceutics and patient's material. We observed that cells were not restricted in their viability by pressures up to 100 MPa. Mammalian cells die when treated with pressures of 200 MPa or more. But the effects of 200, 300 or 400 MPa do not follow the same pattem. At 200 MPa, cells die in a way that is related to apoptosis. Some apoptotic characteristics like phosphatidylserine (PS) exposure and morphological alterations appear very fast. Other features like a higher exposure of intracellular NPn ligands and pronounced degradation of DNA and lectin ligands are unique features of HHP induced apoptosis. Cells treated with 300 and 400 MPa die immediately following a unique necrotic pathway, since treated cells harbour high DNA and glycoprotein degrading activities.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

Large-scale EST sequencing in rice

Large-scale cDNA analysis provides several great advantages for genome investigations in rice. Isolated and partially characterized cDNA clones have contributed not only to the construction of an RFLP linkage map and physical maps of the chromosomes but also to investigations of the mechanisms of expression of various isozymes and family genes. The ultimate aim of our large-scale cDNA analysis is to catalogue all the expressed genes of this important cereal, including tissue-specific, developmental stage-specific, and stress-specific genes. As of August 1996, the Rice Genome Research Program (RGP) has isolated and partially sequenced more than 29000 cDNA clones from various tissues and calluses in rice (Nipponbare, a japonica variety). The sequence data were translated into amino acid sequences for the 3 possible reading frames, and the similarity of these amino acid sequences to known proteins registered in PIR were examined. About 25% of the clones had significant similarities to known proteins. Some of the hit clones showed library-specific distributions, indicating that the composition of the clones in each library reflects, to some extent, the regulation of gene expression specific to differentiation, growth condition, or environmental stress. To further characterize the cDNA clones, including unknown clones, nucleotide sequence similarities of 24728 clones were analyzed and the clones were classified into around 10000 independent groups, suggesting that around a half or one third of expressed genes in rice have already been captured. These results obtained from our large-scale cDNA analysis provide useful information related to gene expression and regulation in rice.     

Modeling conductive heat transfer and process uniformity during batch high-pressure processing of foods

A numerical model for predicting conductive heat transfer during batch high hydrostatic pressure (HHP) processing of foods was developed and tested for a food simulator (agar gel). For a comprehensive evaluation of the proposed method, both "conventional" HHP processes, HHP processes with gradual, step-by-step pressure buildup and pressure release, and pressure cycling HHP processes were included. In all cases, good agreement between experimental and predicted temperature profiles was observed. The model provides a very useful tool to evaluate batch HHP processes in terms of uniformity of any heat- and/or pressure-related effect. This is illustrated for inactivation of Bacillus subtilis alpha-amylase, an enzymatic model system with known pressure-temperature degradation kinetics.