Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70 genomic locus

Toxoplasma gondii is an important human and veterinary pathogen. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite and many of the other stressors associated with heat shock protein induction. Heat shock or stress induced activation of a set of heat shock protein genes, is characteristic of almost all eukaryotic and prokaryotic cells. Studies in other organisms indicate that heat shock proteins are developmentally regulated. We have established that increases in the expression of bag1/hsp30 and hsp70 are associated with bradyzoite development. The T. gondii hsp70 gene locus was cloned and sequenced. The regulatory regions of this gene were analysed by deletion analysis using beta-galactosidase expression vectors transiently transfected into RH strain T. gondii. Expression was measured at pH 7.1 and 8.1 (i.e. pH shock) and compared to the expression obtained with similar constructs using BAG1 and SAG1 promoters. A pH-regulated region of the Tg-hsp70 gene locus was identified which has some similarities to heat shock elements described in other eukaryotic systems. Green fluorescent protein expression vectors driven by the Tg-hsp70 regulatory region were constructed and stably transfected into T. gondii. Expression of green fluorescent protein in these parasites was induced by pH shock in those lines carrying the Tg-hsp70 regulatory constructs. Gel shift analysis was carried out using oligomers corresponding to the pH-regulated region and a putative DNA binding protein was identified. These data support the identification of a pH responsive cis-regulatory element in the T. gondii hsp70 gene locus. A model of the interaction of hsp70 and small heat shock proteins (e.g. BAG1) in development is presented.     

Bacterial heat shock protein 60 may increase epithelial cell migration through activation of MAP kinases and inhibition of alpha6beta4 integrin expression

Exogenous heat shock proteins may modify cell behavior of infected epithelium. The effect of heat shock protein 60 (hsp60) of Actinobacillus actinomycetemcomitans and Escherichia coli, and human recombinant hsp60 on migration of HaCaT skin keratinocytes was studied using the Boyden chamber assay. Hsp60 from different species increased cell migration by two- to fivefold and this effect was inhibited by ERK inhibitor PD 98059, p38 inhibitor SB 203580, and a function-blocking epidermal growth factor receptor (EGFR) antibody. Hsp60 reduced the expression of alpha6-integrin mRNA and its protein levels on the cell surface but had no effect on the expression of beta4, beta1, alpha1, alpha5 or alphav integrin subunits. Hsp60 also significantly inhibited cell adhesion to laminin-5, a ligand of alpha6beta4 integrin. These results suggest that exogenous hsp60 released from bacteria or inflammatory cells may promote epithelial cell migration through activation of EGFR and MAP kinases, and inhibition of alpha6beta4 integrin expression.     

70-kDa-heat shock protein presents an adjustable lectinic activity towards O-linked N-acetylglucosamine

Numerous works demonstrated that the dynamic O-GlcNAc glycosylation could protect against the proteasomal degradation by modifying the target proteins and the proteasome itself. Considering that Hsp70 is a crucial component in the quality control of protein conformation in the proteasomal pathway, we investigated the possibility that Hsp70 physically interacts with O-GlcNAc proteins through a lectinic activity. First, we demonstrate that in HepG2 cells, Hsp70 can specifically bind to O-GlcNAc residues but also is itself modified by O-GlcNAc. Second, when cells were deprived of glucose (nutrient stress), Hsp70 lectinic activity markedly increased whereas its glycosylation dramatically decreased. On the other hand, a 42 degrees C thermic stress did not affect any of these features. Lastly, the nature of O-GlcNAc modified proteins co-immunoprecipitating with Hsp70 was similar for cells submitted to the thermic and to nutrient stress. These results strongly suggest that O-GlcNAc influences protein stability through specific interaction with 70-kDa-heat shock protein members.     

Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis

Small heat shock proteins (sHsps) usually exist as oligomers that undergo dynamic oligomeric dissociation/re-association, with the dissociated oligomers as active forms to bind substrate proteins under heat shock conditions. In this study, however, we found that Hsp16.3, one sHsp from Mycobacterium tuberculosis, is able to sensitively modulate its chaperone-like activity in a range of physiological temperatures (from 25 to 37.5 degrees C) while its native oligomeric size is still maintained. Further analysis demonstrated that Hsp16.3 exposes higher hydrophobic surfaces upon temperatures increasing and that a large soluble complex between Hsp16.3 and substrate is formed only in the condition of heating temperature up to 35 and 37.5 degrees C. Structural analysis by fluorescence anisotropy showed that Hsp16.3 nonameric structure becomes more dynamic and variable at elevated temperatures. Moreover, subunit exchange between Hsp16.3 oligomers was found to occur faster upon temperatures increasing as revealed by fluorescence energy resonance transfer. These observations indicate that Hsp16.3 is able to modulate its chaperone activity by adjusting the dynamics of oligomeric dissociation/re-association process while maintaining its static oligomeric size unchangeable. A kinetic model is therefore proposed to explain the mechanism of sHsps-binding substrate proteins through oligomeric dissociation. The present study also implied that Hsp16.3 is at least capable of binding non-native proteins in vivo while expressing in the host organism that survives at 37 degrees C.     

Induction of Tetraploid Gynogenesis in the European Sea Bass (Dicentrarchus labrax L.)

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7–95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.     

八肽胆囊收缩素对家兔内毒素休克时肺动脉张力的影响

为探讨八肽胆囊收缩素(CCK-8)缓解内毒素休克(ES)时肺动脉血压(PAP)增高的机制,观察了CCK-8对脂多糖(LPS)引起家兔ES时PAP变化以及离体肺动脉环(PARs)张力改变的影响。实验用新西兰大耳白雄性家兔40只,分为颈静脉注入LPS(8mg／kg i．v.)复制的家兔ES模型、LPS注入前15min给CCK-8(15μg/kg,i.v.)、LPS注入前15min给CCK受体拮抗剂丙谷胺(Pro 1mg／kg,i.v.)、单独注入CCK-8(15μg/kg,i.v.)和注射生理盐水(对照)共5组。用生理记录仪监测平均动脉压(MAP)和PAP的变化;5h后制备PARs,应用血管张力测定技术,检测各组PARs张力。结果为：(1)ES时MAP降低、PAP升高,CCK-8可完全翻转ES时PAP的增高,而Pro加剧ES时PAP的增高;(2)LPS组的PARs对苯肾上腺素(PE)的收缩反应增强,对ACh内皮依赖性舒张反应降低,而CCK-8可逆转LP5的上述作用。上述结果提示CCK—8可缓解ES时的PAP升高,这可能与其调节肺动脉张力改变有关。     

Expression of small heat shock proteins HspB2, HspB8, Hsp20 and cvHsp in different tissues of the perinatal developing pig

Recently, we have described the developmental expression of the small heat shock proteins (sHsps) Hsp27/HspB1 and alphaB-crystallin/HspB5 in different tissues of pigs from almost full-term foetuses to three years old adults (P. Tallot, J. F. Grongnet, J. C. David, Biol. Neonate, 83, 281-288, 2003). The data described in this report extends this study to four other members of the sHsp family (Hsp20/HspB6, cvHsp/HspB7, MKBP/HspB2 and HspB8). We studied expression of these proteins in porcine lens, brain, heart, liver, kidney, lung, skeletal muscle, stomach, and colon, and found a ubiquitous expression of Hsp20 and HspB8 as earlier reported for Hsp27 and alphaB-crystallin. In contrast, cvHsp and HspB2 expression is essentially restricted to heart and muscle. During development, the sHsps tend to (temporarily) increase in stomach, liver, lung, kidney, hippocampus, and striatum, while expression in heart is more or less constant, and a large variation is found in sHsp expression patterns in skeletal muscle. In cerebellum and cortex a temporary decrease of Hsp20 and HspB8 is observed directly after birth. The major impact of this study is that each tissue seems to have a unique profile of sHsp expression, which varies during development and may reflect the need of a particular tissue to maintain at all stages an optimal chaperoning machinery to protect against physiological stress.     

Influence of Hypotonic Shock on Glutamate and GABA Uptake in Rat Brain Synaptosomes

Hyponatremia leads to hyperexcitability of neurons, seizures, and coma. It is well established that uptake of neurotransmitters is a sodium-dependent process. Therefore, we suggest that inhibition of neurotransmitter uptake can lead to the clinical manifestations of hyponatremia. Decreasing of sodium concentration down to 92 mM in incubation medium, which corresponds to lowering the osmolarity down to 230 mOsm/l, leads to a 45% decrease in glutamate uptake and a 46% decrease in gamma-aminobutyric acid (GABA) uptake. However, this effect was mediated by the nonspecific lowering of osmolarity rather than by decreasing sodium concentration. Hypotonic shock was able to reduce glutamate uptake in the presence of protein kinase inhibitors staurosporine and genistein, the phosphatase inhibitor okadaic acid, the phosphatidylinositol 3-kinase inhibitor wortmannin, and cytoskeleton modulators colchicine and cytochalasin B. Therefore, we suggest that intracellular signaling is not mediating the effect of osmolarity reduction on neurotransmitter uptake.