The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

Hsp70 chaperones assist protein folding by cycling between the ATP-bound T state with low affinity for substrates and the ADP-bound R state with high affinity for substrates. The transition from the T to R state is catalyzed by the synergistic action of the substrate and DnaJ cochaperones. The reverse transition from the R state to the T state is accelerated by the nucleotide exchange factor GrpE. These two processes, T-to-R and R-to-T conversion, are affected differently by temperature change. Here we modeled Hsp70-mediated protein folding under permanent and transient heat shock based on published experimental data. Our simulation results were in agreement with in vitro wild-type Escherichia coli chaperone experimental data at 25°C and reflected R-to-T ratio dynamics in response to temperature effects. Our simulation results suggested that the chaperone system evolved naturally to maintain the concentration of active protein as high as possible during heat shock, even at the cost of recovered activity after return to optimal growth conditions. They also revealed that the chaperone system evolved to suppress ATP consumption at non-optimal high growing temperatures.     

Heat- shock Induced Protein in Pea Leaves Which is Immunoreactive to Gro EL Antibody

A high molecular weight protein was found in pea ( Pisum sativum L. ) seedling by means of Westem-blotting, and it consisted of a ct (60.4 kD) and α β (65.5 kD) subunits. The protein had low ATPase activity. Its expression could be enhanced by 3 to 4 folds under heat-shock stress, but was not affected by exogenous ABA. The results of localization and 35S-Met labeling showed that it was a cytoplasmic protein and its synthesis was not inhibited by chloramphenicol.     

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.     

Chronic oral administration of pine bark extract (flavangenol) attenuates brain and liver mRNA expressions of HSPs in heat-exposed chicks

Exposure to a high ambient temperature (HT) can cause heat stress, which has a huge negative impact on physiological functions. Cellular heat-shock response is activated upon exposure to HT for cellular maintenance and adaptation. In addition, antioxidants are used to support physiological functions under HT in a variety of organisms. Flavangenol, an extract of pine bark, is one of the most potent antioxidants with its complex mixture of polyphenols. In the current study, chronic (a single daily oral administration for 14 days) or acute (a single oral administration) oral administration of flavangenol was performed on chicks. Then the chicks were exposed to an acute HT (40±1 °C for 3 h) to examine the effect of flavangenol on the mRNA expression of heat-shock protein (HSP) in the brain and liver. Rectal temperature, plasma aspartate aminotransferase (AAT), a marker of liver damage, and plasma corticosterone as well as metabolites were also determined. HSP-70 and -90 mRNA expression, rectal temperature, plasma AAT and corticosterone were increased by HT. Interestingly, the chronic, but not the acute, administration of flavangenol caused a declining in the diencephalic mRNA expression of HSP-70 and -90 and plasma AAT in HT-exposed chicks. Moreover, the hepatic mRNA expression of HSP-90 was also significantly decreased by chronic oral administration of flavangenol in HT chicks. These results indicate that chronic, but not acute, oral administration of flavangenol attenuates HSP mRNA expression in the central and peripheral tissues due to its possible role in improving cellular protective functions during heat stress. The flavangenol-dependent decline in plasma AAT further suggests that liver damage induced by heat stress was minimized by flavangenol.     

人脑胶质瘤中热休克蛋白70、Caspase-3 的表达及临床意义

目的:探讨热休克蛋白70、caspase-3在人脑胶质瘤中的表达和临床意义。方法:收集2008年7月～2010年12月汕头大学医学院第二附属医院神经外科手术获取的人脑胶质瘤组织标本35例,另选择20例脑外伤手术中切除的正常脑组织标本作为对照组。采用EnVision免疫组化方法检测脑胶质瘤组织和正常脑组织中热休克蛋白70、caspase-3的表达,并分析其与脑胶质瘤组织临床病理特征之间的关系。结果:HSP70在胶质瘤组和对照组中的阳性表达率分别为74.3%和25.0%,胶质瘤组的HSP70表达明显高于对照组(P<0.05)。caspage-3在胶质瘤组和对照组中的阳性表达率分别为34.3%和80.0%,胶质瘤组的caspage-3阳性表达率均明显低于对照组(P<0.01)。HSP70和caspase-3的阳性表达与胶质瘤的病理学分级、术后复发情况密切相关(P<0.05)。相关分析表明,胶质瘤组织中HSP70阳性表达率和caspase-3表达呈负相关(r=-0.568,P<0.05)。结论:胶质瘤组织中HSP70呈高表达,而caspase-3表达下调,两者均在胶质瘤浸润、复发等恶性演进过程中发挥重要作用;HSP70可能通过某些途径抑制caspase-3表达来抑制胶质瘤恶性细胞的凋亡发生。     

Down-regulation of heme oxygenase-1 by SVCV infection

Heme oxygenase (HO-1) is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. In mammalian systems, viral infection or antigen expression can down-regulate the expression of HO-1. In turn, the induction of HO-1 or overexpression of HO-1 results in potent and direct antiviral activity that targets the replication of several mammalian viruses. In this study, the HO-1 gene of Cyprinus carpio was cloned, and the expression profile of HO-1 was investigated during spring viremia of carp virus (SVCV) infection. The results demonstrate that the expression of HO-1 was down-regulated during SVCV infection in the EPC cells and in common carp. These results indicated that SVCV infection could induce host oxidative stress, which may contribute to tissue injury in affect fish.     

Synthesis of characteristic proteins in nutrient-depleted cell suspension cultures of parsley

Cell suspension cultures of parsley (Petroselinum crispum) exhibited an altered pattern of protein synthesis after transfer from complete growth medium to water or medium containing no macronutrients. Similar changes occurred when cultures were grown in the original medium until the nutrients were depleted. The effect was reversible upon transfer to fresh medium and was not observed during regular subculturing of the cells. While total protein synthesis decreased sharply after nutrient depletion, the synthesis of a few characteristic proteins (starvation-related proteins, STPs) increased strongly. The protein labeled at highest rates with [³⁵S]methionine in vivo (STP 62) had an apparent molecular weight of about 62000 and a pI of about 6.3. Although its increased rate of synthesis was therefore easily detected by labeling in vivo, translation of mRNA in vitro did not give comparable results. Thus, regulatory control may be exerted mainly at the level of translation. Synthesis of STP ceased rapidly when heat shock (37° C) was applied under conditions of nutrient depletion, whereas heat-shock proteins were strongly induced.Abbreviations HSP heat-shock protein - STP starvation-related protein     

Pulpal responses to cavity preparation in aged rat molars

The dentin-pulp complex is capable of repair after tooth injuries including dental procedures. However, few data are available concerning aged changes in pulpal reactions to such injuries. The present study aimed to clarify the capability of defense in aged pulp by investigating the responses of odontoblasts and cells positive for class II major histocompatibility complex (MHC) to cavity preparation in aged rat molars (300–360 days) and by comparing the results with those in young adult rats (100 days). In untreated control teeth, immunoreactivity for intense heat-shock protein (HSP)-25 and nestin was found in odontoblasts, whereas class-II-MHC-positive cells were densely distributed in the periphery of the pulp. Cavity preparation caused two types of pulpal reactions based on the different extent of damage in the aged rats. In the case of severe damage, destruction of the odontoblast layer was conspicuous at the affected site. By 12 h after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border but subsequently disappeared together with HSP-25-immunopositive cells, and finally newly differentiated odontoblast-like cells took the place of the degenerated odontoblasts and acquired immunoreactivity for HSP-25 and nestin by postoperative day 3. In the case of mild damage, no remarkable changes occurred in odontoblasts after operation, and some survived through the experimental stages. These findings indicate that aged pulp tissue still possesses a defense capacity, and that a variety of reactions can occur depending on the difference in the status of dentinal tubules and/or odontoblast processes in individuals.This work was supported in part by a grant from MEXT to promote 2001-multidisciplinary research project (in 2001–2005), KAKENHI (B) to H.O. (no. 16390523), and Daiwa Securities Health Foundation, Japan.