Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Effects of heat shock on ovary development and hsp83 expression in Tribolium castaneum (Coleoptera: Tenebrionidae)

Heat shock affects reproductive performance in insects including Tribolium castaneum. In this study, the effects of heat shock on ovary development and hsp83 expression in T. castaneum were investigated. Two lines of T. castaneum, H line and C line, from the same base population were established and maintained for five successive generations. In each generation, the newly hatched beetles (within 3 h after eclosion) in the H line were treated with a heat shock at 40°C for 1 h, and those in the C line were raised at normal temperature (30°C) as control treatment. Four traits related to ovary development were measured for the beetles of the fifth generation: days from eclosion to laying the first eggs (T_o), days from eclosion to laying the first hatchable eggs (T_h), ovariole size on the third day after eclosion, and pupal mass of their offspring. The results showed that the beetles of the H line had a significantly longer pre‐oviposition period (0.6 more days) and smaller ovariole size than those of the C line. No significant difference in pupal mass was observed. Applying heat shock to the offspring of the fifth generation of both lines led to significantly higher hsp83 expression in offspring of the C line than in offspring of the H line. Within each line, the hsp83 expression level in offspring with heat shock was significantly higher than that of offspring without heat shock, but the difference in the C line was much larger than that in the H line. We infer from these results that a tradeoff between heat resistance, registered as hsp83 expression, and ovarian development operates under heat stress in T. castaneum. 2009 Wiley Periodicals, Inc.     

Expression of gamma-tubulin during the development of nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Gamma-tubulin is a centrosomal protein found in microtubule organizing centres (MTOCs) in cells from many different organisms, and has several properties, which makes it a candidate for both the initiation of microtubule assembly and anchorage. Gamma-tubulin is encoded by a single gene tbg-1 in Caenorhabditis elegans. In this paper tbg-1 was studied to understand the essential role of gamma-tubulin in C. elegans. Essential role of tbg-1 expression was confirmed by the disruption of the gene expression by gamma-tubulin anti-sense RNA production in vivo under the heat shock promoter that caused lethality in the nematodes. Expression of tbg-1 deduced from Northern blot analysis during the development revealed differential expression in different developmental stages. Using tbg-1::lacZ fusion gene expression studies in the germ line transformed worms, it was further revealed that gamma-tubulin expression was observed through out the development from embryonic and post-embryonic stages.     

Real‐time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions

The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real‐Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL‐TcA‐CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV‐A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up‐regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real‐time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management.     

Facing warm temperatures during migration: cardiac mRNA responses of two adult Oncorhynchus nerka populations to warming and swimming challenges

The main findings of the current study were that exposing adult sockeye salmon Onchorhynchus nerka to a warm temperature that they regularly encounter during their river migration induced a heat shock response at an mRNA level, and this response was exacerbated with forced swimming. Similar to the heat shock response, increased immune defence‐related responses were also observed after warm temperature treatment and with a swimming challenge in two different populations (Chilko and Nechako), but with some important differences. Microarray analyses revealed that 347 genes were differentially expressed between the cold (12–13° C) and warm (18–19° C) treated fish, with stress response (GO:0006950) and response to fungus (GO:0009620) elevated with warm treatment, while expression for genes involved in oxidative phosphorylation (GO:0006119) and electron transport chain (GO:0022900) elevated for cold‐treated fish. Analysis of single genes with real‐time quantitative PCR revealed that temperature had the most significant effect on mRNA expression levels, with swimming and population having secondary influences. Warm temperature treatment for the Chilko population induced expression of heat shock protein (hsp) 90α, hsp90β and hsp30 as well as interferon‐inducible protein. The Nechako population, which is known to have a narrower thermal tolerance window than the Chilko population, showed even more pronounced stress responses to the warm treatment and there was significant interaction between population and temperature treatment for hsp90β expression. Moreover, significant interactions were noted between temperature treatment and swimming challenge for hsp90α and hsp30, and while swimming challenge alone increased expression of these hsps, the expression levels were significantly elevated in warm‐treated fish swum to exhaustion. In conclusion, it seems that adult O. nerka currently encounter conditions that induce several cellular defence mechanisms during their once‐in‐the‐lifetime migration. As river temperatures continue to increase, it remains to be seen whether or not these cellular defences provide sufficient protection for all O. nerka populations.     

A siRNA system based on HSP70 promoter results in controllable and powerful gene silencing by heat‐induction

RNAi is a powerful tool for gene‐specific knockdown and gene therapy. However, the imprecise expression of siRNA limits the extensive application of RNAi in gene therapy. Here we report the development of a novel controllable siRNA expression vector pMHSP70psil that is initiated by HSP70 promoter. We determined the efficiency of the controllable siRNA system by targeting the gama‐synuclein (SNCG) gene in breast cancer cells MCF‐7. The results show that the controllable siRNA system can be induced to initiate siRNA expression by heat‐induction. The silencing effect of SNCG occurs at a relatively low level (10.1%) at 37°C, while it is significantly increased to 69.4% after heat induction at 43°C. The results also show that the controllable siRNA system inhibits proliferation of cancer cells by heat‐shock. Therefore, this RNAi strategy holds the promise of the high efficiency in gene knockdown at targeted times and locations, avoiding systemic side effects. It provides, for the first time, an approach to control siRNA expression by heat‐shock. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1289–1297, 2013     

Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions

Aims: The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV‐C) treatments after exposure to different sublethal stresses that may be encountered in food‐processing environments. Methods and Results: Cronobacter sakazakii stationary growth‐phase cells (30°C, 24 h) were exposed to acid (pH 4·5, 1 h), alkaline (pH 9·0, 1 h), osmotic (5% NaCl, 1 h), oxidative (0·5 mmol l^?1 H₂O₂, 1 h), heat (47·5°C, 1 h) and cold (4°C, 4 h) stress conditions and subjected to the subsequent challenges: heat (60°C), PEF (25 kV cm^?1, 35°C), MS (117 μm, 200 kPa, 35°C) and UV‐C light (88·55 mW cm^?2, 25°C) treatments. The inactivation kinetics of C. sakazakii by the different technologies did not change after exposure to any of the stresses. The combinations of sublethal stress and lethal treatment that were protective were: heat shock–heat, heat shock–PEF and acid pH–PEF. Conversely, the alkaline shock sensitized the cells to heat and UV‐C treatments, the osmotic shock to heat treatments and the oxidative shock to UV‐C treatments. The maximum adaptive response was observed when heat‐shocked cells were subjected to a heat treatment, increasing the time to inactivate 99·9% of the population by 1·6 times. Conclusions: Cronobacter sakazakii resistance to thermal and nonthermal preservation technologies can increase or decrease as a consequence of previous exposure to stressing conditions. Significance and Impact of the Study: The results help in understanding the physiology of the resistance of this emerging pathogen to traditional and novel preservation technologies.     

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

A heat‐inducible CRE/LOXP gene induction system in medaka

We established three lines of transgenic medaka, a heat‐shock element (HSE) monitor line (hse‐GFP line), heat‐inducible driver lines (hse‐cre lines), and effector lines (gapdh‐loxP[DsRed]‐GFP lines). We employed these to comprehensively analyze gene induction at different time points in various tissues. These analyses demonstrate a good response of synthetic HSEs by heat treatment during embryogenesis and the mosaic gene induction by cre/loxP‐mediated recombination, thus providing practical information regarding the feasibility of a heat‐inducible cre/loxP‐mediated system in medaka. We also activated recombination by local heat‐treatment using a metal probe and an infrared laser. Our results collectively indicate that these lines allow us to perform lineage tracing and mosaic analysis and provide the platform to investigate gene functions at later developmental stage and adult. genesis 51:59–67, 2013. © 2012 Wiley Periodicals, Inc.     

Micronucleus‐Specific Bacterium Holospora elegans Irreversibly Enhances Stress Gene Expression of the Host Paramecium caudatum

ABSTRACT. The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear‐specific symbiont Holospora obtusa survived better than symbiont‐free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 °C, a usual growth temperature. We report herein that paramecia bearing the micronuclear‐specific symbiont Holospora elegans also acquire the heat‐shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock‐resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 °C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat‐shock resistance.     

Microwave improved Escherichia coli transformation

Aims: The calcium chloride chemical transformation of Escherichia coli is still the most widley used cloning method in small laboratories. Therefore, any practicable improvement in its transformation efficiency seems to be of general interest. Methods and Results: We found that giving calcium chloride competent cells a 1 min microwave pulse at the lowest power setting (180 W), instead of the classic 1–2 min 42°C heat‐shock step, increases the transformation efficiency around threefold (3.3 ± 0.5). Moreover, when both treatments were given in a 2‐min 42°C ? 5 min on ice ? 1 min microwave pluse sequence, an additional improvement of 1.6 was obtained, resulting in an overall increase in efficiency of approximately 5.3‐fold compared to classical heat shock. Conclusions: This transformation method significantly improves the classical heat shock treatment. Significance and Impact of the Study: This method might be useful to those laboratories that cannot afford an electroporation apparatus.