Protective effect of HSP70 on injuried myocardial cells induced by H2O2

AIM: To investigate the role of heat-shock protein 70(HSP₇₀) in the protection of myocardial cells against ischemic injury.METHODS: Myocardial cells were cultured in vitro. HSP₇₀ was induced by hyperthermia. H₂O₂-injured myocardial cells were divided into different groups. Flow cytometry, DNA Ladder and biochemistry methods were employed to observe the myocardial cells of different groups.RESULTS: Immunohistochemistry showed hyperthermia induced the up-regulation of HSP₇₀ in myocardial cells. Apoptotic rate, activity analysis of cytochrome C and succinic dehydrogenase in H₂O₂-injuried and HSP₇₀-protected groups were obviously different. Electron micrograph shomed hyperthermia alliviated myocardial cell injury induced by H₂O₂. CONCLUSION: HSP₇₀ delays apoptosis and protects against H₂O₂-induced myocardial cell injury.     

文心兰HSP70基因的克隆及表达分析

为研究文心兰热激蛋白70基因（命名为OnHSP70）的分子特性及表达特点,以文心兰‘柠檬绿’（Oncidium hybridum ‘Honey Angel’）为材料,采用RT-PCR技术克隆OnHSP70,利用生物信息学方法分析其分子特性,通过qRT-PCR技术分析其在不同组织及不同非生物胁迫处理下的表达特点。生物信息学分析表明：该基因开放阅读框为1944 bp,编码647个氨基酸,翻译的蛋白为稳定蛋白,属于HSP70超家族,其蛋白二级结构由41.11%的α-螺旋、17.77%的延伸链、7.73%的β-转角和33.38%的无规卷曲组成,具有2个高度保守的功能域。聚类分析表明：该蛋白与铁皮石斛和深圳拟兰的HSP70亲缘关系较近。qRT-PCR分析表明：文心兰HSP70在4种不同组织器官中具有表达差异性,在花中的表达量最高;高温处理后基因的表达量明显上升;40 ℃高温胁迫4 h时表达量达峰值;茉莉酸甲酯及水杨酸处理时表达量呈现下调响应。本研究为后期该基因功能研究及提高文心兰对高温的适应性提供理论基础。     

鸡Hsc70基因5′侧翼区SNP与耐热性状的相关分析

为研究Hsc70基因作为鸡抗病育种中一种分子标记的可能性,本试验利用克隆测序技术对清远麻鸡、广东温氏矮脚黄鸡、灵山鸡和隐性白羽洛克鸡4个品种共20个个体Hsc70基因的5′侧翼区进行多态性筛查,发现10处变异,包括8个SNPs和2个插入/缺失突变。通过分析,选取C.-521A〉C位点,利用PCR-RFLP方法分析了此位点在灵山鸡和隐性白羽洛克鸡群体中的基因型分布,所得分型结果与测定的耐热性状（T3、皮质酮、CD3＋、CD4＋和CD8＋）进行关联分析,结果表明：常温状态下（15℃）,在隐性白羽洛克鸡群体中,位点C.-521A〉C与CD3＋、CD4＋和CD8＋显著相关（P〈0.05）,其中AA基因型个体CD3＋值显著低于AC基因型个体,而CD4＋与CD8＋值显著高于AC基因型个体;热应激状态下（35℃）,在灵山鸡群体中,位点C.-521A〉C与CD8＋极显著相关（P〈0.01）,CC基因型个体CD8＋值极显著高于AA和AC基因型。     

目录

为了表达牛环形泰勒虫（Theileria annulata）截短HSP70基因编码蛋白并研究该蛋白的结构与功能特性,本研究扩增牛环形泰勒虫HSP70目的基因并构建重组质粒pMD18-T-HSP70,选取其他种的同源HSP70蛋白序列构建系统进化树;利用生物信息学方法分析HSP70基因编码蛋白的氨基酸组成、基本理化性质、亲疏水性、跨膜区结构、信号肽、可能的磷酸化位点、亚细胞定位及蛋白的二级结构和三级结构;对重组蛋白HSP70进行蛋白互作网络分析;构建原核表达载体pET28a-HSP70,筛选诱导表达条件,镍柱纯化重组蛋白及检测反应原性。结果显示,牛环形泰勒虫HSP70蛋白序列与小泰勒虫的序列同源性较高,蛋白分子质量为42 ku,理论等电点（pI）为5.61,属于酸性亲水性蛋白,无跨膜区及信号肽;蛋白功能预测结果显示,HSP70包含32个可能的磷酸化位点,亚细胞定位分析显示该蛋白主要分布于细胞质。蛋白质二级结构中α-螺旋、β-转角、无规则卷曲、延伸链分别占39.18%、8.51%、30.41%和21.91%。蛋白互作网络构建结果显示,与HSP70相互作用的蛋白主要为HSP90家族成员,另外还有伴侣蛋白GrpE同系物,预示着HSP70可能在细胞内与HSP90形成复合体发挥作用。本试验成功构建原核表达载体,获得了大小约为48 ku的融合蛋白,以0.6 mmol/L IPTG于37 ℃诱导5 h,蛋白表达较好;点状印迹及Western blotting结果表明,表达产物可被自然感染的牛环形泰勒虫阳性血清识别,具有良好的反应原性。本试验结果为进一步探讨牛环形泰勒虫HSP70功能机制提供了理论依据。     

Extracellular heat shock protein 70 levels in tumour-bearing dogs and cats treated with radiation therapy and hyperthermia

Hyperthermia is a form of a cancer treatment which is frequently applied in combination with radiotherapy (RT) to improve therapy responses and radiosensitivity. The mode of action of hyperthermia is multifactorial; the one hand by altering the amount of the blood circulation in the treated tissue, on the other hand by modulating molecular pathways involved in cell survival processes and immunogenic interactions. One of the most dominant proteins induced by hyperthermia is the major stress-inducible heat shock protein 70 (Hsp70). Hsp70 can be found in the blood either as a free-protein (free HSP70) derived from necrotic cells, or lipid-bound (liposomal Hsp70) when it is actively released in extracellular vesicles (EVs) by living cells. The aim of the study was to evaluate the levels of free and liposomal Hsp70 before and after treatment with RT alone or hyperthermia combined with radiotherapy (HTRT) in dogs and cats to evaluate therapy responses. Peripheral blood was collected from feline and canine patients before and at 2, 4, 6 and 24 h after treatment with RT or HTRT. Hsp70 enzyme-linked immunosorbent assays (ELISAs) were performed to determine the free and liposomal Hsp70 concentrations in the serum. The levels were analysed after the first fraction of radiation to study immediate effects and after all applied fractions to study cumulative effects. The levels of free and liposomal Hsp70 levels in the circulation were not affected by the first singular treatment and cumulative effects of RT in cats however, after finalizing all treatment cycles with HTRT free and liposomal Hsp70 levels significantly increased. In dogs, HTRT, but not treatment with RT alone, significantly affected liposomal Hsp70 levels during the first fraction. Free Hsp70 levels were significantly increased after RT, but not HTRT, during the first fraction in dogs. In dogs, on the other hand, RT alone resulted in a significant increase in liposomal Hsp70, but HTRT did not significantly affect the liposomal Hsp70 when cumulative effects were analysed. Free Hsp70 was significantly induced in dogs after both, RT and HTRT when cumulative effects were analysed. RT and HTRT treatments differentially affect the levels of free and liposomal Hsp70 in dogs and cats. Both forms of Hsp70 could potentially be further investigated as potential liquid biopsy markers to study responses to RT and HTRT treatment in companion animals.     

中国杧果科学研究70年

新中国成立70年来,我国杧果科学研究在人才培养与团队建设、硬件平台、基础理论创新与技术研发等方面均取得了重要进展,有力支撑了我国杧果产业的创建和可持续发展。本文在概述世界和我国杧果地位、分布、规模的基础上,从科学研究发展历程、种质资源与遗传育种、土壤与肥水管理、耕作模式与花果管理、病虫害防控、采后贮运保鲜、产业经济等方面全面梳理了我国杧果研究取得的成就,分析了存在的不足并提出了未来的重点发展方向。     

Chronic otitis externa with heat shock protein 70-positive intranuclear inclusion bodies in the ceruminous gland epithelium of a Chihuahua dog

A Chihuahua dog showed persistent itching in the right ear canal. Anti-inflammatory medicines and prednisolone were ineffective and total ear canal ablation was performed. Histological diagnosis was chronic otitis externa. Eosinophilic intranuclear inclusion bodies (Cowdry type A and full-type) were occasionally observed in the ceruminous gland epithelium. The inclusion bodies were negative for nucleic acid and ultrastructurally composed of fibrous structures (approximately 10 nm in width). Viral infection was initially suspected, but polymelase chain reaction tests did not detect the expected viral genes. Immunohistochemistry revealed that the inclusion bodies were positive for heat shock protein 70 (HSP70), suggesting that these bodies could be protein aggregates including HSP70. The etiology of this lesion has not been elucidated, but chronic inflammation may influence the cytoplasm-to-nuclear transportation of HSP70. To the best of our knowledge, this is the first report of canine chronic otitis externa with HSP70-positive intranuclear inclusion bodies.     

Effect of thermal stress on Hsp70 gene expression and female reproductive performance of giant freshwater prawn,Macrobrachium rosenbergii

Using Hsp70 as a biomarker, thermal stress impinges on reproductive organs, ovary and hepatopancreas were being analyzed by determining the expression of Hsp70 mRNA inside the organs after the adult inter‐molt females were subjected to thermal treatment at 35, 30 and 28°C (Control). Results showed the expression of Hsp70 mRNA under thermal treatment of 35°C after 2 hr recovery in ovary were upregulated at 2, 4, 6, 12, 24 hr and 30 days compared to control whereas in hepatopancreas under similar treatment, the expression of Hsp70 mRNA were significantly higher than control at 6, 24 hr and 30 days. Frequency of reproductive molt at 35°C showed the ovary of females were failed to develop and only entered common molt along three consecutive molt cycles. For 30°C thermal treatment, the expression of Hsp70 mRNA was significantly higher than control after 2 hr recovery but returned to normal afterwards until 30 days’ thermal treatment. Maternal heat shock for 2 hr at 35°C were found to give significantly lower frequency of reproductive molt and longer duration of ovarian development and incubation period whereas maternal heat shock for 2 hr at 30°C gave lower frequency of reproductive molt, slower development of embryo and lower hatching success compared to untreated control. This study suggests that short and long‐term thermal stress at 30 and 35°C were found to affect the induction of Hsp70 mRNA in reproductive organs of Macrobrachium rosenbergii and also influence their reproductive performance.     

The effects of starving and feeding on Dover sole (Solea solea,Soleidae, Linnaeus, 1758) stress response and early larval development

In the view of an urgent necessity to improve the quantity and the quality of farmed fish species, there is a strong need to improve our basic knowledge on the effects of first feeding during the developmental stages of fish larvae. High mortality, mainly due to food deprivation or inappropriate food quality, has been observed in many larval fish species, but knowledge about the morphological, biochemical and molecular processes related to this topic is still poorly understood. The understanding of the early larval ontogeny as well as the larval nutritional requirements and the molecular and cellular mechanisms elicited by fish larvae during food deprivation and starvation are thus of primary importance. At this regard, this study investigates, in Dover sole larvae, the effects of starvation and starving/re‐feeding procedures at a morphological, histological, biochemical and molecular level. The results evidenced that starved larvae progressively decrease in growth, lipid content, affected their gastrointestinal tract and muscle development and increased cortisol and heat shock protein 70 levels. On the contrary, starved and re‐fed larvae showed, after the restoration of a favourable feeding condition, a compensatory growth. In conclusion, this is the first study analysing through a multidisciplinary approach the effects of food deprivation on the development of an important economic species, the Dover sole.