Natural thermal stress and heat-shock protein expression in Drosophila larvae and pupae

1. Whether Drosophila larvae and pupae naturally experience temperatures that can cause heat damage or death is poorly understood, but bears directly on numerous investigations of the thermal biology and heat-shock response in Drosophila . Accordingly, the temperatures of necrotic fruit, which Drosophila larvae and pupae inhabit, the temperatures of larvae and pupae outside the laboratory, and the levels of the heat-shock protein hsp 70 expressed by larvae in nature were examined.
2. When necrotic fruit was sunlit, internal temperatures rose to levels that can harm indwelling insects. Fruit size and evaporative water loss affected these temperatures. Temperatures of larvae and pupae in the field commonly exceeded 35 °C, with living larvae recorded at >44°C and pupae at >41°C. Natural mortality was evident, presumably because of heat.
3. In the laboratory, these temperatures kill larvae rapidly, with LT₅₀s (time taken for half the sample to be killed) of 30 min at 39 °C, 15 min at 40 °C and 8·5 min at 41 °C. Gradual transfer from 25°C to these temperatures resulted in no lesser mortality than did direct transfer.
4. Hsp 70 levels in lysates of whole larvae were measured by ELISA (enzyme-link immunosorbent assay) with an hsp 70-specific antibody. For larvae within necrotic apples experimentally transferred from shade to sun and within necrotic fruit in situ , hsp 70 levels equalled or exceeded levels detected in parallel laboratory studies of whole larvae or cells in culture.
5. These data provide an ecological context for studies of thermal stress and the heat-shock response in Drosophila that has heretofore been lacking.     

Insulin-induced expression of human heat-shock protein gene hsp70

In human hepatoma cell line Hep3B/T2, the human heat-shock-inducible gene hsp70 could be induced by insulin. The dose-dependent insulin effect correlates very well with the dissociation constant of the insulin receptor, indicating that the insulin effect is mediated by the insulin receptor. The expression of hsp70 gene was neither significantly induced by growth factors of epidermal and platelet-derived growth factors, nor by tumor promoter, calcium ionophore, cAMP, and glucocorticoids. These results indicate that the induction of expression of hsp70 gene by insulin is very specific and not cell cycle-related. Furthermore, the insulin-induced expression of hsp70 gene is transient, occurring specifically from 4 to 8 h after insulin addition.     

Integration of the study of natural and anthropogenic disturbances using severity gradients

Disturbance is an integral part of every ecosystem, but humans are altering disturbance regimes in fundamental ways that can alter outcomes for ecosystem structure and function. Fortunately, advances in understanding ecosystem responses to natural disturbances can address the ecological consequences of the novel suite of disturbances now created by humans. Complex interactions among both natural and anthropogenic disturbances at many overlapping spatial and temporal scales can be examined across severity gradients. The gradient approach applies ecological tools to differential conditions of stability and fertility, degrees of biological legacy and rates of successional recovery and can help address modern concerns about socio‐economic consequences of disturbance and the sustainability of ecosystem services.     

Structure and expression of a yeast gene encoding the small heat-shock protein Hsp26

The nucleotide sequence of the Saccharomyces cerevisiae gene encoding a small heat-shock protein (Hsp26) has been determined. It reveals a 213-amino acid protein (27 kDa) that contains no methionine (Met) residues. Radiolabelling studies demonstrate the N-terminal Met residue is cleaved post-translationally. The Hsp26 amino acid sequence shows significant homology with both a range of eukaryotic small Hsps and with vertebrate alpha-crystallins. Particularly highly conserved among these proteins is a hydrophobic tetrapeptide sequence Gly-Val-Leu-Thr. These findings are discussed in relation to the structure and function of small Hsps.     

Effect of heat-shock on Plasmodium falciparum viability, growth and expression of the heat-shock protein 'PFHSP70-I' gene.

Cultures of the human malaria parasite Plasmodium falciparum were subjected to heat-shock for varying times and temperatures and then tested for their viability, growth and expression of heat-shock protein. Results show that the majority of parasites remained viable after heat-shock but their growth was affected. However, the expression of the heat-shock protein 'PFHSP70-I' gene was enhanced after heat-shock. We conclude that malarial parasites are able to survive in vivo during fever probably due to the overexpression of the heat-shock protein gene.     

Induction of macrophage elastase (MMP-12) gene expression by statins

The statins (including mevastatin and lovastatin) are a widely prescribed class of serum-cholesterol lowering drugs that function by inhibiting 3-hydroxymethylglutaryl coenzyme A (HMG CoA) reductase activity and cellular sterol synthesis. Statins are also widely being appreciated for their inhibitory effects upon inflammation, primarily mediated through direct regulation of inflammatory gene expression. Here we report that statins are also capable of increasing the expression of macrophage elastase (MMP-12). The induction of MMP-12 in mouse macrophages by statins is specific for HMG CoA reductase inhibition, rescued by mevalonate and not observed after inhibition of subsequent steps in the cholesterol biosynthetic pathway. Modulation of cholesterol metabolism may lead to changes in MMP-12 expression and subsequent impacts during physiological and pathophysiological states. We conclude that statins, in addition to their previously described anti-inflammatory properties, may promote the production of some proteinases from activated macrophages.     

Identification and expression analysis of a heat-shock protein 70 gene in Polycelis sp.

Heat-shock protein 70 (HSP70) is ubiquitously found in a variety of organisms and plays an important role in cytoprotection, environmental monitoring, and disease resistance. In this study, the full-length complementary DNA (cDNA) of hsp70 from planarian Polycelis sp. was first cloned using rapid amplification of cDNA ends (RACE). The expression levels of Pyhsp70 were analyzed in the presence of various stressors by real-time PCR, and its temporal-spatial expression patterns were also examined in both intact and regenerative animals by whole-mount in situ hybridization. The results show that (1) the deduced amino acid sequence of Pyhsp70 includes three typical HSP70 family signature motifs and is highly conserved during evolution; (2) Pyhsp70 expression is induced by prolonged starvation, tissue damage, and ionic liquid but inhibited by high or low temperatures; and (3) Pyhsp70 mRNA is mainly expressed in the head peripheral region and in the regenerating blastema during regeneration. These results suggest that the highly expressed Pyhsp70 gene may contribute to enhance cytoprotection and tolerance against stress-induced molecular damage, and the migration of neoblasts to the wound, which might also be involved in the proliferation and differentiation of neoblasts. Our work provides basic data for the study of stress responses and regenerative mechanism in freshwater planarians.     

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to invesitgate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidiumiodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.     

Induction of Salmonella enterotoxin (stn) gene expression by epithelial cells (IEC-6)

Prevalence of Salmonella enterotoxin (stn) gene among Salmonella enterica and S. bongori was investigated by polymerase chain reaction (PCR) and gene probe and its status of phenotypic expression was examined on chinese hamster ovary cells by cultivating the strains with conventional method for enterotoxin production and by cultivating the organisms in contact with intestinal epithelial cells of rats (IEC-6). All the 19 strains and serovars of S. enterica such as Typhimurium, Enteritidis, Newport, Weltevreden, Indiana, Gallinarum and Kentucky were found to carry stn gene as examined by PCR and gene probe but only a limited number of strains (13 out of 19) expressed phenotypically the enterotoxin when cultured by conventional method. Cultivation of organisms in contact with epithelial cells induced expression of stn gene phenotypically in all the 19 strains. In contrast to S. enterica, strains of S. bongori were found neither genotypically (stn) nor phenotypically (Stn) positive.     

Induction of a proteinase by heat-shock in yeast

Abstract In Saccharomyces cerevisiae heat-shock induces an increase in proteinase activity. The induction is probably due to newly synthesized enzyme molecules, since the increase in proteinase activity can be inhibited by cycloheximide. Degradation of endogenous proteins is enhanced by EDTA, while the azocasein assay is not affected by MnCl₂, MgCl₂, or EDTA. The proteinase has a pH optimum of 8, and phenylmethylsulfonyl fluoride (PMSF) as well as chymostatin are strong inhibitors. We infer that the induced proteinase is probably identical with proteinase B of yeast.     

Analysis of stress-induced hepatic gene expression in rainbow trout (Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.     

Introgressive hybridization in Rorippa (Brassicaceae): gene flow and its consequences in natural and anthropogenic habitats

Introgressive hybridization between three Rorippa species (R. amphibia, R. palustris and R. sylvestris) in northern Germany has been studied using isozymes and noncoding chloroplast DNA (trnL/F spacer). Our results provide substantial evidence for different patterns of gene flow in natural and in anthropogenic environments. Hybridization and bi-directional introgression (chloroplast DNA and allozymes) between R. amphibia and R. sylvestris were detected at the river Elbe, which is one of the last rivers in Central Europe showing a natural dynamic of erosion and sedimentation. The natural dynamic of the Elbe leads to periodic habitat disturbance and the temporal breakdown of ecological isolation barriers between R. amphibia and R. sylvestris. However, the high dynamic does not provide the opportunity for persistence of the morphologically intermediate hybrid R. x anceps (R. amphibia x R. sylvestris). We did not find hybrid zones between R. amphibia and R. sylvestris in the more anthropogenic landscape of northwest Germany. However, contact zones between R. amphibia and R. palustris were detected in drainage ditches in northwest Germany. We found substantial evidence for unidirectional introgression of R. palustris markers (chloroplast DNA and allozymes) into R. amphibia in the man-made habitats. The R. amphibia introgressants in the drainage ditches often showed strongly serrate upper cauline leaves instead of the entire upper cauline leaves typical for R. amphibia. We argue that landscape melioration in northwest Germany, particularly the creation of drainage ditches, favoured both hybrid-zone formation and ecotypic differentiation within R. amphibia.