Multiple oligomeric structures of a bacterial small heat shock protein

Small heat shock proteins are ubiquitous molecular chaperones that form the first line of defence against the detrimental effects of cellular stress. Under conditions of stress they undergo drastic conformational rearrangements in order to bind to misfolded substrate proteins and prevent cellular protein aggregation. Owing to the dynamic nature of small heat shock protein oligomers, elucidating the structural basis of chaperone action and oligomerization still remains a challenge. In order to understand the organization of sHSP oligomers, we have determined crystal structures of a small heat shock protein from Salmonella typhimurium in a dimeric form and two higher oligomeric forms: an 18-mer and a 24-mer. Though the core dimer structure is conserved in all the forms, structural heterogeneity arises due to variation in the terminal regions.

Predicting shock response in uncertain structures using the Hybrid method

Using the Hybrid method (FE + SEA) it is possible to estimate the frequency response of an uncertain structure. The current work develops the Hybrid method to allow for time domain analysis of the shock response of a structure. Problems to be overcome when taking Hybrid method results into the time domain are a) the Hybrid method frequency response has no phase information, and b) the Hybrid method frequency response is smoothed in frequency and shows no modal peaks. In this paper the first problem has been overcome, using minimum phase reconstruction. Explanation of minimum phase reconstruction and its limitations are described, and application to shock problems described. © 2009 IOP Publishing Ltd.

The nonlinear heat release response of stratified lean-premixed flames to acoustic velocity oscillations

This paper analyzes the forced response of swirl-stabilized lean-premixed flames to high-amplitude acoustic forcing in a laboratory-scale stratified burner operated with CH4 and air at atmospheric pressure. The double-swirler, double-channel annular burner was specially designed to generate high-amplitude acoustic velocity oscillations and a radial equivalence ratio gradient at the inlet of the combustion chamber. Temporal oscillations of equivalence ratio along the axial direction are dissipated over a long distance, and therefore the effects of time-varying fuel/air ratio on the response are not considered in the present investigation. Simultaneous measurements of inlet velocity and heat release rate oscillations were made using a constant temperature anemometer and photomultiplier tubes with narrow-band OH*/CH* interference filters. Time-averaged and phase-synchronized CH* chemiluminescence intensities were measured using an intensified CCD camera. The measurements show that flame stabilization mechanisms vary depending on equivalence ratio gradients for a constant global equivalence ratio (φg=0.60). Under uniformly premixed conditions, an enveloped M-shaped flame is observed. In contrast, under stratified conditions, a dihedral V-flame and a toroidal detached flame develop in the outer stream and inner stream fuel enrichment cases, respectively. The modification of the stabilization mechanism has a significant impact on the nonlinear response of stratified flames to high-amplitude acoustic forcing (u'/U∼0.45 and f=60, 160Hz). Outer stream enrichment tends to improve the flame's stiffness with respect to incident acoustic/vortical disturbances, whereas inner stream stratification tends to enhance the nonlinear flame dynamics, as manifested by the complex interaction between the swirl flame and large-scale coherent vortices with different length scales and shedding points. It was found that the behavior of the measured flame describing functions (FDF), which depend on radial fuel stratification, are well correlated with previous measurements of the intensity of self-excited combustion instabilities in the stratified swirl burner. The results presented in this paper provide insight into the impact of nonuniform reactant stoichiometry on combustion instabilities, its effect on flame location and the interaction with unsteady flow structures. © 2011 The Combustion Institute.

The first study on the effects of microcystin-RR on gene expression profiles of antioxidant enzymes and heat shock protein-70 in Synechocystis sp PCC6803

Microcystins are heptapeptide toxins produced by cyanobacteria. Microcystin-RR(MC-RR) is a common variant among the 80 variants identified so far. There have been many investigations documenting the toxic effects of microcystins on animals and higher plants, but little is known on the toxic effects of microcystins on algae, especially at molecular level. We studied the effects of MC-RR on gene expression profile of a few antioxidant enzymes and heat shock protein-70 (Hsp70) in Synechocystis sp. PCC6803. After two days post-exposure, a high dose toxin (5 mg/l, about 4.8 x 10(-3) mM) significantly increased expression levels of the genes gpx1, sodB, katG, acnB, gamma-TMTand dnaK2, while a relatively low dose toxin (1 mg/l, about 9.63 x 10(-4) mM) induced a moderate and slow increase of gene expression. Our results indicate that MC-RR could induce the oxidative stress in Synechocystis sp. PCC6803 and the increase in gene expression of antioxidant enzymes and Hsp70 might protect the organism from the oxidative damage. in addition, cell aggregation was observed during the early period of exposure, which might be a specific oxidative stress reaction to MC-RR. (C) 2008 Elsevier Ltd. All rights reserved.

Feeding rates affect heat shock protein levels in liver of larval white sturgeon (Acipenser transmontanus)

A two-week trial was conducted to study the effect of feeding rates on heat shock protein levels in larval white sturgeon. The larvae (30 day post hatch, 230 mg initial body weight) were fed a commercial feed (12.6% moisture, 49.5% crude protein. 20.7% Crude fat, and 8.6% ash) at 5, 15. or 25% body weight per clay (BW d(-1)). Liver heat shock proteins (Hsp) were measured before and after the larvae were subjected to a heat shock from 18 to 26 degrees C at 1 degrees C/15 min and maintained at 26 degrees C for 4 h thereafter. Before heat shock, larvae fed 5% BW d(-1) had significantly (P<0.05) lower final body weight, RNA/DNA ratio, whole body lipid and protein content, and Hsp60 and Hsp70 levels but higher protein efficiency ratio, and whole body moisture content than larvae fed the two higher feeding rates. Heat shock significantly induced Hsp60 and Hsp70 levels in the liver of all fish but they were lower in larvae fed the 5% than those fed 15 and 25% BW d(-1). Hsp70 level increased much more than Hsp60 after the heat shock Suggesting that Hsp70 is a more sensitive biomarker under our experimental conditions. (c) 2008 Elsevier B.V. All rights reserved.

Early Embryonic Exposure to Polychlorinated Biphenyls Disrupts Heat-Shock Protein 70 Cognate Expression in Zebrafish

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that have documented neurological effects in children exposed in utero. To better define neuronally linked molecular targets during early development, zebrafish embryos were exposed to Aroclor 1254, a mixture of PCB congeners that are common environmental contaminants. Microarray analysis of the zebrafish genome revealed consistent significant changes in 38 genes. Of these genes, 55% (21) are neuronally related. One gene that showed a consistent 50% reduction in expression in PCB-treated embryos was heat-shock protein 70 cognate (Hsc70). The reduction in Hsc70 expression was confirmed by real-time polymerase chain reaction (PCR), revealing a consistent 30% reduction in expression in PCB-treated embryos. Early embryonic exposure to PCBs also induced structural changes in the ventro-rostral cluster as detected by immunocytochemistry. In addition, there was a significant reduction in dorso-rostral neurite outgrowth emanating from the RoL1 cell cluster following PCB exposure. The serotonergic neurons in the developing diencephalon showed a 34% reduction in fluorescence when labeled with a serotonin antibody following PCB exposure, corresponding to a reduction in serotonin concentration in the neurons. The total size of the labeled neurons was not significantly different between treated and control embryos, indicating that the development of the neurons was not affected, only the production of serotonin within the neurons. The structural and biochemical changes in the developing central nervous system following early embryonic exposure to Aroclor 1254 may lead to alterations in the function of the affected regions.

Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis

The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5'- and 3'-untranslated region(5'- and 3'-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1-547 bp, but they did not exist in the region of 548-2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.

Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis

Chromosome manipulation for commercially valuable marine animals plays an important role in aquaculture. The special reproductive characteristics of shrimp make it difficult to control fertilization and synchronize egg development, so research on chromosome manipulation in shrimp has proceeded very slowly. In the present study, triploid shrimp Fenneropenaeus chinensis were induced by heat shocks and the optimal-inducing condition was screened at different spawning temperatures. Level of triploid induction for each treatment was evaluated by flow cytometry at nauplius stage. The highest level of triploid induction reached to more than 90%. Starting time for each treatment was very crucial for triploid induction in shrimp. One optimal treatment condition for triploid induction was heat shock (29-32 degreesC), starting at 18-20 min for duration of 10 min. These conditions varied depending on the temperature at spawning. Triploid level at embryo stage and nauplius stage was not different, suggesting the same hatching rate between diploids and triploids. Heat shock is a very effective way to induce triploids in this species, and can be easily used on large scale without any harmful effect on the environment as compared with chemical treatment. (C) 2003 Elsevier Science B.V. All rights reserved.

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamysfarreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd2+, Pb2+ and Cu2+ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment. (c) 2007 Elsevier Inc. All rights reserved.

Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity and regulation of a subset of cytosolic proteins. In the present study, the cDNA of Argopecten irradians HSP90 (designated AiHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of AiHSP90 was of 2669 bp, including an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids with predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. BLAST analysis revealed that AiHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in AiHSP90, which indicated that AiHSP90 should be a cytosolic member of the HSP90 family. Fluorescent real-time quantitative PCR was employed to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, the relative expression level of AiHSP90 transcript was up-regulated and reached maximal. level at 9 h after injection, and then dropped progressively to the original level at about 48 h post challenge. The results indicated that AiHSP90 was potentially involved in the immune responses against bacteria challenge in scallop A. irradian. (c) 2007 Elsevier Ltd. All rights reserved.