Ethanol-response genes and their regulation analyzed by a microarray and comparative genomic approach in the nematode Caenorhabditis elegans

The nematode shows responses to acute ethanol exposure that are similar to those observed in humans, mice, and Drosophila, namely hyperactivity followed by uncoordination and sedation. We used in this report the nematode Caenorhabditis elegans as a model system to identify and characterize the genes that are affected by ethanol exposure and to link those genes functionally into an ethanol-induced gene network. By analyzing the expression profiles of all C. elegans ORFs using microarrays, we identified 230 genes affected by ethanol. While the ethanol response of some of the identified genes was significant at early time points, that of the majority was at late time points, indicating that the genes in the latter case might represent the physiological consequence of the ethanol exposure. We further characterized the early response genes that may represent those involved directly in the ethanol response. These genes included many heat shock protein genes, indicating that high concentration of ethanol acts as a strong stress to the animal. Interestingly, we identified two non-heat-shock protein genes that were specifically responsive to ethanol. glr-2 was the only glutamate receptor gene to be induced by ethanol. T28C12.4, which encodes a protein with limited homology to human neuroligin, was also specific to ethanol stress. Finally, by analyzing the promoter regions of the early response genes, we identified a regulatory element, TCTGCGTCTCT, that was necessary for the expression of subsets of ethanol response genes.     

Developmental control of stress stimulons in Streptomyces coelicolor revealed by statistical analyses of global gene expression patterns

Stress-induced regulatory networks coordinated with a procaryotic developmental program were revealed by two-dimensional gel analyses of global gene expression. Four developmental stages were identified by their distinctive protein synthesis patterns using principal component analysis. Statistical analyses focused on five stress stimulons (induced by heat, cold, salt, ethanol, or antibiotic shock) and their synthesis during development. Unlike other bacteria, for which various stresses induce expression of similar sets of protein spots, in Streptomyces coelicolor heat, salt, and ethanol stimulons were composed of independent sets of proteins. This suggested independent control by different physiological stress signals and their corresponding regulatory systems. These stress proteins were also under developmental control. Cluster analysis of stress protein synthesis profiles identified 10 different developmental patterns or "synexpression groups." Proteins induced by cold, heat, or salt shock were enriched in three developmental synexpression groups. In addition, certain proteins belonging to the heat and salt shock stimulons were coregulated during development. Thus, stress regulatory systems controlling these stimulons were implicated as integral parts of the developmental program. This correlation suggested that thermal shock and salt shock stress response regulatory systems either allow the cell to adapt to stresses associated with development or directly control the developmental program.     

Heat shock induces intestinal-type alkaline phosphatase in rat IEC-18 cells

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.     

Protein response of insect cells to bioreactor environmental stresses

Protein expression of Spodoptera frugiperda (Sf9) insect cells was characterized upon exposure to environmental stresses typically present in bioreactors including heat shock, oxygen deprivation, shear stress, change of pH, and salinity or ethanol shock. This study fills the void in knowledge as to how bioreactor hydrodynamics, anoxia, small changes in pH as well as salinity alterations due to pH control or exposure to ethanol used in asepsis treatments affect protein expression in Sf9 cells. Heat shock at 43 degrees C induced proteins at 83 kDa, 68-78 kDa and six small heat shock proteins (hsps) at 23-15.5 kDa. Anaerobic conditions in CO2 atmosphere reduced significantly the normal protein synthesis and induced a small subset of heat shock proteins at 70 kDa. Oxygen deprivation in nitrogen atmosphere transiently induces the 70 kDa proteins and had minor effects on the normal protein synthesis. Exposure to increased salinity or ethanol concentration failed to trigger the stress response, but may extensively inhibit the induction of normal proteins even though there was a negligible change in cell viability. Shear stress that had a major reducing effect on cell viability did not change the protein synthesis profile of Sf9 cells. Both long and short term exposures to small pH changes had negligible effects on protein synthesis.     

The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response

The concentrations of two structurally distinct membrane fluidizers, the local anesthetic benzyl alcohol (BA) and heptanol (HE), were used at concentrations so that their addition to K562 cells caused identical increases in the level of plasma membrane fluidity as tested by 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy. The level of membrane fluidization induced by the chemical agents on isolated membranes at such concentrations corresponded to the membrane fluidity increase seen during a thermal shift up to 42 degrees C. The formation of isofluid membrane states in response to the administration of BA or HE resulted in almost identical downshifts in the temperature thresholds of the heat shock response, accompanied by increases in the expression of genes for stress proteins such as heat shock protein (HSP)-70 at the physiological temperature. Similarly to thermal stress, the exposure of the cells to these membrane fluidizers elicited nearly identical increases of cytosolic Ca2+ concentration in both Ca2+-containing and Ca2+-free media and also closely similar extents of increase in mitochondrial hyperpolarization. We obtained no evidence that the activation of heat shock protein expression by membrane fluidizers is induced by a protein-unfolding signal. We suggest, that the increase of fluidity in specific membrane domains, together with subsequent alterations in key cellular events are converted into signal(s) leading to activation of heat shock genes.     

Continuous heat shock enhances translational initiation directed by internal ribosomal entry site

Many cellular mRNAs contain internal ribosomal entry sites (IRES) that become functional under conditions of cellular stress, when the rate of protein synthesis for most cellular mRNA is reduced. Internal ribosomal entry increases in response to hypoxia, cell differentiation, apoptosis, gamma irradiation, and heat shock. Heat shock is the principal cellular stress in which general cap-dependent translation is inhibited. On the other hand, heat shock induces the preferential translation of a small class of mRNA, called heat shock protein (HSP) mRNAs, which probably occurs because little or no eIF4F activity is required for their translation. In this study, we found that continuous heat stress enhances expression of the heat shock protein BiP at the level of translation. Interestingly, heat stress also enhanced the viral IRES-dependent translation of encephalomyocarditis virus and hepatitis C virus but not poliovirus. Although several BiP inducers increased BiP protein expression, BiP IRES-dependent translation was enhanced only during heat shock, suggesting that heat shock is a specific inducer for BiP IRES-dependent translation. Taken together, these results indicate that the mechanism of IRES-dependent translation can be used during heat shock and suggest that this translational mechanism may be critical to the survival and proliferation of cells under stress.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

Chronic Ethanol Exposure Increases Cytochrome P‐450 and Decreases Activated in Blocked Unfolded Protein Response Gene Family Transcripts in Caenorhabditis elegans

Ethanol is a widely consumed and rapidly absorbed toxin. While the physiological effects of ethanol consumption are well known, the underlying biochemical and molecular changes at the gene expression level in whole animals remain obscure. We exposed the model organism Caenorhabditis elegans to 0.2 M ethanol from the embryo to L4 larva stage and assayed gene expression changes in whole animals using RNA‐Seq and quantitative real‐time PCR. We observed gene expression changes in 1122 genes (411 up, 711 down). Cytochrome P‐450 (CYP) gene family members (12 of 78) were upregulated, whereas activated in blocked unfolded protein response (ABU) (7 of 15) were downregulated. Other detoxification gene family members were also regulated including four glutathione‐S‐transferases and three flavin monooxygenases. The results presented show specific gene expression changes following chronic ethanol exposure in C. elegans that indicate both persistent upregulation of detoxification response genes and downregulation of endoplasmic reticulum stress pathway genes.