Transient expression of the GUS gene in a unicellular marine green alga,<Emphasis Type="Italic">Chlorella</Emphasis> sp.<Emphasis Type="Italic">MACC/C95</Emphasis>, via electroporation

A transient expression system for a unicellular marine green alga,Chlorella sp.MACC/C95, was developed using a reporter GUS gene coded for by plasmid pBI121. The results demonstrated a high transformation efficiency could be achieved by using electroporation to deliver DNA into intact cells and the CaMV35S promoter to drive the foreign gene expression inChlorella sp.MACC/C95. The use of a carrier DNA coupled with osmosis treatment improved the transformation efficiency, while linearization of the plasmid had minor effects. Investigation of the effects of DNA concentration and growth phases ofChlorella sp.MACC/C95 on transformation efficiency indicated that the highest level of transient expression was observed when 6 μg mL⁻¹ of plasmid DNA and cells 2–6 days old were used.     

HSP25 in isolated perfused rat hearts: Localization and response to hyperthermia

Recent investigations concentrate on the correlation between the myocardial expression of the inducible 70-kDa heat shock protein (HSP70i) by different stress conditions and its possible protective effects. Only few studies have focused on the involvement of small heat shock proteins in this process. We analyzed the location of the small heat shock protein HSP25 in isolated cardiomyocytes as well as its location and induction in isolated perfused hearts of rats. By immunofluorescence microscopy HSP25 was found to colocalize with actin in the I-band of myofibrils in cardiomyocytes of isolated perfused hearts as well as in isolated neonatal and adult cardiomyocytes. Hyperthermic perfusion of isolated hearts for 45 min resulted in modulation of different parameters of heart function and in induction of HSP25 and HSP70i. Temperatures higher than 43°C (44–46°C) were lethal with respect to the contractile function of the hearts. Compared to control hearts perfused at 37°C, significant increases during hyperthermic perfusion at 42°C and 43°C were obtained for heart rate, contraction velocity and relaxation velocity. In response to hyperthermia at 43°C and after subsequent normothermic perfusion for 135 min at 37°C, left ventricular pressure, contraction velocity and relaxation velocity remained significantly elevated. However, heart rate returned to control values immediately after the period of heat treatment. HSP25 is constitutively expressed even in normothermic perfused hearts as shown by Western blotting. Hyperthermia increased the content of HSP25 only in the left ventricular tissue. In contrast, HSP70i was strongly induced in all analyzed parts of the myocardium (left ventricle, right ventricle, septum). Our findings suggest a differential regulation of HSP25 and HSP70i expression in response to hyperthermia in isolated perfused hearts. The constitutively expressed HSP25 seems to be located adjacent to the myofibrils which implies a specific role of this protein even under unstressed conditions for the contractile function of the myocardium.     

Transient expression of GUS and anthocyanin constructs in intact maize immature embryos following electroporation

Electroporation was used for the delivery and subsequent expression of GUS and anthocyanin reporter genes into intact maize immature embryos. The optimal conditions consisted of culturing immature embryos for 4 days on N6 1-100-25-Ag medium prior to electroporation (375 V/cm; 960 µF capacitance) in EPR buffer containing DNA and 0.07 M sodium glutamate at room temperature (22°C) after a 10 min heat shock at 37°C. Under these conditions, over 40 spots of GUS transient activity were observed per immature embryo. Transient gene expression after electroporation was further demonstrated using an anthocyanin construct, which is specific for expression in plant cells.     

Expression of <Emphasis Type="Italic">cspH</Emphasis> upon nutrient up-shift in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

The gene cspH , which encodes one of the cold-shock proteins in Salmonella enterica serovar Typhimurium, has previously been reported to be induced during early exponential phase at 37°C. In the present study, the expression of cspH upon nutrient up-shift at 37°C was investigated and found to be affected by DNA gyrase and DNA-binding protein Fis. When cells at stationary phase were subcultured into a rich medium, the mRNA level of cspH increased dramatically prior to the first cell division. However, when the cells were treated with DNA gyrase inhibitors, cspH mRNA was not induced upon nutrient up-shift. The low level of DNA superhelical density at the cspH promoter in part affected the expression of cspH mRNA in vitro. In addition, a fis-deficient strain had a lower level of cspH mRNA than the wild-type upon nutrient up-shift. Finally, a cspH–lacZ construct, in which the putative binding region for Fis was deleted in the cspH promoter, expressed a low level of LacZ, in contrast to the native cspH–lacZ construct.This revised version was published online in July 2004 with corrections to Fig. 4.     

The proteomic response of sea squirts (genus Ciona) to acute heat stress: a global perspective on the thermal stability of proteins

Congeners belonging to the genus Ciona have disparate distributions limited by temperature. Ciona intestinalis is more widespread with a cosmopolitan distribution ranging from tropical to sub-arctic zones, while Ciona savignyi is limited to temperate-latitudes of the northern Pacific Ocean. To compare the heat stress response between congeners, we quantified changes in protein expression using proteomics. Animals were exposed to 22 °C, 25 °C, and 28 °C for 6 h, then recovered at a control temperature (13 °C) for 16 h (high heat stress experiment). In a second experiment we exposed animals to lower levels of heat stress at 18 °C, 20 °C, and 23 °C, with a 16 °C control. A quantitative analysis, using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry (with a 69% and 93% identification rate for Ciona intestinalis and Ciona savignyi, respectively), showed changes in a number of protein functional groups, including molecular chaperones, extracellular matrix proteins, calcium-binding proteins, cytoskeletal proteins and proteins involved in energy metabolism. Our results indicate that C. intestinalis maintains higher constitutive levels of molecular chaperones than C. savignyi, suggesting that it is prepared to respond faster to thermal stress. Systematic discrepancies between estimated versus predicted molecular masses of identified proteins differed between protein families and were more pronounced under high heat conditions, suggesting that thermal sensitivities are lower for cytoskeletal proteins and ATP-synthase than for any other protein group represented on 2D gels.     

Thermosensitization of tumor cells with inhibitors of chaperone activity and expression

Effects of inhibitors of the heat shock protein 90 (HSP90) chaperone activity and inhibitors of the heat shock protein (HSP) expression on sensitivity of HeLa tumor cells to hyperthermia were studied. It was found that nanomolar concentrations of inhibitors of the HSP90 activity (17AAG or radicicol) slowed down the chaperone-dependent reactivation of a thermolabile reporter (luciferase) in heat-stressed HeLa cells and slightly enhanced their death following the incubation for 60 min at 43°C. The inhibitors of HSP90 activity stimulated de novo induction of additional chaperones (HSP70 and HSP27) that significantly increased intracellular HSP levels. Treatment of the cells with 17AAG or radicicol along with an inhibitor of the HSP induction (e.g. quercetin or triptolide, or NZ28) completely prevented the increase in the intracellular chaperone levels resulting from the inhibition of HSP90 activity and subsequent heating. Combination of all three treatments (inhibition of the HSP90 activity + inhibition of the HSP induction + heating at 43°C for 60 min) resulted in more potent inhibition of the reporter reactivation and a sharp (2–3-fold) increase in cell death. Such enhancement of the cytotoxicity may be attributed to the “chaperone deficiency” when prior to heat stress both the functional activity of constitutive HSP90 and the expression of additional (inducible) chaperones are blocked in the cells.     

Effect of temperature on vegetative growth among isolates of Metarhizium anisopliae and M. flavoviride

Effects of temperature on vegetative growth on a semi-synthetic medium of 22 isolates of Metarhizium anisopliae and 14 isolates of M. flavoviride were determined. The majority of isolates of both species grew between 11 and 32°C; several isolates grew at 8 and 37 °C. None of the isolates grew at 40 °C. Relative growth rate, calculated from the maximum growth rate for each isolate, was significantly affected by temperature and isolate, with significant isolate * temperature interactions. The maximum absolute growth rates among the isolates ranged from 2.5 mm to 5.9 mm/day. Optimal temperatures were generally between 25 and 32 °C with several isolates exhibiting optimal growth at temperatures as high as 32 °C. Overall, relative growth rates were greater in isolates of M. anisopliae than M. flavoviride at temperatures of 25 °C or lower; conversely mean relative growth rates were greater in M. flavoviride than M. anisopliae at temperatures higher than 25 °C. However, the two most cold tolerant isolates at 8 °C were M. flavoviride and the three most heat tolerant at 35 °C were M. anisopliae. Since temperature growth responses varied considerably between isolates, strain selection according to thermal tolerance may be warranted when choosing a strain for development as a microbial control agent.This revised version was published online in October 2005 with corrections to the Cover Date.     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

水稻胁迫相关基因OsPM1启动子的克隆与分析

依据NCBI数据库OsPM1的序列信息,采用PCR技术扩增获取OsPM1的2 100bp的启动子序列。利用PLACE预测启动子的顺式作用元件分析表明,启动子内含有大量与胁迫相关的顺式作用元件,主要有ABA响应相关元件、脱水响应元件、低温响应元件、热激响应元件和转录因子结合元件。构建OsPM1的启动子和GUS基因融合表达载体,转入拟南芥。组织化学染色分析结果显示,非生物胁迫处理前,幼苗中GUS基因表达水平很低;干旱、低温、高盐等胁迫处理后,GUS基因表达量显著升高。研究表明,OsPM1的启动子能够显著提高在干旱、高盐和低温处理后下游基因的表达水平。     

Survival kinetics of Salmonella enterica serotype senftenberg (S. senftenberg) after heat and acid stress

The survival kinetics of two clinical isolates of Salmonella senftenberg were studied after heat and acid stress. The strains survived better at 53 and 55 °C after heat shock of 30 min at 50 °C or overnight heat adaptation at 45 °C. An increase in the decimal reduction time, D, of heat-shocked [10.2 min (53 °C) and 9.37 min (55 °C)] and heat-adapted [8.12 min (53 °C) and 7.8 min (55 °C)] cells was observed compared with the non-stressed cells [6.87 min (53 °C), 6.56 min 55 °C)]. A significant difference was also observed in the survival of acid-adapted to acid non-adapted S. senftenberg bacteria.     

Polybrene-mediated transfection of cultured lepidopteran cells: Induction of aDrosophila heat shock promoter

Summary We have introduced hsp-cat plasmid DNA intoSpodoptera frugiperda (Lepidoptera: Noctuidae) cells by transfection with purified DNA (1 to 48 μg/ml) mixed with the polycation polybrene (100 μg/ml) in serum-free Grace's medium. The hsp-cat construct contains a gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT), whose expression is controlled by a promoter derived from aDrosophila heat shock protein (hsp) gene. Expression of CAT activity in transfectedSpodoptera cells was induced by a 2-h heat shock at 43°C. The temperature of the heat shock was based on conditions that maximized the expression of endogenous heat shock protein genes in these cells. CAT activity was maximal in cells that were exposed to the heat shock 2 d after transfection; by 4 d, activity was diminished, and little activity was detectable after 6 d. Transfection frequencies, which varied with DNA concentration and ranged as high as 6000 per million cells, were determined using a histochemical staining procedure. This work was supported by grant 88-37263-4020 from the United States Department of Agriculture, Washington, DC, and by the University of Minnesota Experiment Station. This is contribution 17,543 from the University of Minnesota Experiment Station, St. Paul, MN.     

Normoxic and anoxic heat output of the freshwater bivalves Pisidium and Sphaerium

In fasting Pisidium amnicum and Sphaerium corneum, regular periods of behavioural and metabolic quiescence were shown to occur in the normoxic, constant environment of the flow-through chamber of a heat-flow microcalorimeter. The metabolic rate was suppressed to 7.5% of normal at 10° C and to 8.5–9.7% at 20° C for periods exceeding the period of active metabolism by a factor of 3.5 at 10° C and 8.3 at 20° C. The rate of heat output during normoxic quiescence was equal to that during environmental anoxia, suggesting spontaneous achievement of body anoxia by complete shell closure. The mass-specific integrated heat output during closure periods was independent of size. Parallel observations on clam behaviour suggested that metabolic quiescence coincided with shell closure, and bursts of heat flow with active ventilation. Shell closure was accompanied by pronounced bradycardia, down to 20% of the active rate. In a constant environment, the rhythmic quiescence is regulated by shell closure which is probably triggered by lack of food. Regular quiescence of fasting bivalves may conserve energy reserves considerably, the amount depending on the possible excretion rate of the end products, and the post-quiescence recovery costs, which were not measured. Heat output during the active period was close to the average metabolic rate found earlier for Sphaeriidae. However, all the values determined so far are likely to be underestimates of the natural metabolism because the effects of digestion and growth are not included.     

Controlled Expression of Recombinant Proteins in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using β-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 °C. In contrast, a short non-damaging heat-treatment at 38 °C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 °C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.     

The growth of Epidermophyton floccosum and E. stockdaleae at different temperatures

The ability of 17 strains of genus Epidermophyton (15 strains belonging to Epidermophyton floccosum, one to E. floccosum var. nigricans and one to E. stockdaleae) to grow at different temperatures (4 °C, 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C) was stated.The strains were inoculated on Sabouraud Dextrose Agar and regularly controled over a period of 14 days when the plates were incubated at 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C, and over a period of 70 days when the temperature was 4 °C. The optimal growth of E. floccosum was observed at 28 °C and 31 °C, and no signs of growth were recorded neither at 4 °C nor at 40 °C. The optimal development of E. stockdaleae was observed at 25 °C and 28 °C. This species grew from 4 °C to 31 °C.     

The Role of Antioxidant Systems in the Cold Stress Response of Escherichia coli

The response of aerobically grown Escherichia coli cells to the cold shock induced by the rapid lowering of growth temperature from 37 to 20°C was found to be basically the same as the oxidative stress response. The enhanced sensitivity of cells deficient in two superoxide dismutases, Mn-SOD and Fe-SOD, and the increased expression of the Mn-SOD gene, sodA, in response to cold stress were interpreted as both oxidative and cold stresses are due to a rise in the intracellular level of superoxide anion. The long-term cultivation of E. coli at 20°C was also accompanied by the typical oxidative stress response reactions—an enhanced expression of the Mn-SOD and catalase HPI genes and a decrease in the intracellular level of reduced glutathione (GSH) and in the GSH/GSSG ratio.     

Effect of High Temperature on Protein Expression in Strawberry Plants

Strawberry plants (Fragaria×ananassa Duch.) cvs. Nyoho and Toyonoka were exposed to temperatures of 20, 33, and 42 °C for 4 h, and protein patterns in leaves and flowers was analyzed by 2-dimensional polyacrylamide gel electrophoresis and immunoblotting. In leaves and flowers of both cultivars, the content of most proteins decreased, but a few new proteins appeared in response to heat stress. These heat shock proteins (Hsps) were detected in the range of 19 – 29 kDa in leaves, and 16 – 26 kDa in flowers. The intensity of a 43 kDa protein spot increased in response to heat stress in Nyoho flowers, but not in Toyonoka flowers. The peaHsp17.7 antibody recognized one band at approximately 26 kDa in leaves, and two bands at approximately 16 and 17 kDa in flowers of both cultivars. These results show that the effects of heat stress on Hsp synthesis in strawberry plants differ between plant organs and between cultivars.