Comparative studies on temperature threshold for heat shock protein 70 induction in young and adult Murrah buffaloes

To know the temperature threshold for heat shock protein 70 (HSP70) induction in lymphocytes and to assess physiological changes, if any, in relation to HSP70 induction in young and adult Murrah buffaloes, this study was divided into two parts: I. In vivo study: where assay of HSP70 was performed in blood samples collected from acutely exposed young and adult Murrah buffaloes (n = 6) inside a climatic chamber at 40, 42 and 45 °C for 4 h and thermoneutral temperature (22 °C). Physiological parameters viz., rectal temperature, respiratory rate, pulse rate and skin temperature of different body parts were monitored to assess magnitude of stress in the animals owing to thermal exposure II. For in vitro study, equal numbers of lymphocyte cells were separated from blood collected from young and adult buffaloes and were subjected to four temperature treatments (38, 40, 42 and 45 °C) for 4 h. A significant increase (p < 0.05) in all the physiological parameters in both young and adult buffaloes was observed after exposure to 40, 42 and 45 °C for 4 h as compared to 38 °C. The average plasma HSP70 concentrations (ng/ml) were significantly higher (p < 0.05) at 40, 42 and 45 °C as compared to 38 °C in both young and adult and were higher in young than adult buffaloes at 38 and 45 °C. Heat shock protein 70 level in lymphocyte lysate showed highest concentration after 3-h exposure to all temperatures (40, 42 and 45 °C) in both young and adult buffaloes. The intensity of changes of all physiological parameters was more in young animals than in the adults indicating the greater susceptibility of younger animals to heat stress and was found to be changed at around 40 °C when animals were exposed to different temperatures, indicating the possibility that HSP70 production may be initiated at this temperature which is 2 or 3 °C higher than core body temperature.     

Purification, characterization and expression kinetics of heat shock protein 70 from Bubalus bubalis

The present work on Bubalus bubalis (buffalo) was designed to study heat shock protein 70 (HSP70) induction in lymphocytes, its purification and characterization. HSP70 induction and expression kinetics at different temperatures and time durations were also studied. HSP70 purification was carried out by immunoaffinity chromatography using adenosine di-phosphate (ADP-agarose column) and the characterization of the purified protein was done using western blotting by mouse monoclonal anti-HSP70. The molecular weight of HSP70 of buffalo lymphocytes was found to be approximately of 68 kDa and was less than that of bovine brain HSP70. The purified HSP70 was assessed using indirect inhibition enzyme-linked immunosorbent assay (ELISA). A good amount of HSP70 (1430 ηg HSP70/100 μl) was recovered after purification, out of the total 2040.40 ηg of HSP70/100 μl of cell supernatant. To assess the impact of temperature and time dependent variability in the induction and expression pattern of HSP70, buffalo lymphocytes were subjected to three different temperature treatments, viz.: (I) 38 °C for 48 h and further exposed the same cells at 45 °C for 3 h, (II) 42 °C for 3 h, and (III) 45 °C for 3 h, respectively. The respective cell viability was found to be 68%, 63%, and 51%. The HSP70 levels were 58.30 ± 4.37, 42.59 ± 9.04 and 21.95 ± 6.79 ng/million cells, respectively, at three temperature exposures. The results indicates that higher intensity and duration of temperature exposure cause higher HSP70 induction in buffalo lymphocytes to maintain cellular homeostasis with a threshold of thermal dose for maximum HSP70 expression. However immediate induction of HSP70 in the lymphocytes was dependent on magnitude of thermal exposure (stress level) and time of thermal exposure (stress duration). The present study on HSP70 and its induction will help likely to solve the problems related to the present scenario of thermo-adaptability in buffaloes.     

Synthesis of heat stress proteins in lymphocytes from livestock

Cultured bovine, equine, ovine and chicken lymphocytes responded to heat stress by the increased synthesis of a specific set of proteins known as heat stress proteins (HSP). Proteins with molecular weights of 70 and 90 kDa were synthesized in all species. Additional proteins were found in bovine, ovine and chicken lymphocytes. A time course of induction showed an increased synthesis of some of these proteins with only 30 min of heat stress and of several proteins with 60 min of heat stress. A specific monoclonal antibody was used to identify HSP70 as one of the stress proteins in bovine lymphocytes. Cells from these animals are capable of responding to heat stress by the production of heat stress proteins. These may be of physiological importance.     

河流型水牛热休克蛋白70的原核表达及其对精液抗冻性的影响

试验旨在克隆河流型水牛热休克蛋白70（heat shock protein 70,HSP70）基因的CDS区序列,构建HSP70原核表达载体,诱导表达HSP70融合蛋白,进一步研究HSP70蛋白对水牛精液抗冻性的影响。以摩拉水牛精子基因组DNA为模板,采用PCR方法扩增HSP70基因CDS区序列,将PCR产物与pET30a质粒连接构建重组原核表达质粒,诱导表达HSP70融合蛋白,将获得的表达蛋白作为稀释液组分添加到水牛精液中制作冷冻精液并评定其活力。结果显示,试验成功扩增获得HSP70基因CDS区长为2 155 bp的片段;SDS-PAGE及Western blotting分析显示,在70 ku处出现一条明显条带,且表达产物可与相应的抗体发生反应;对添加有HSP70蛋白的水牛冷冻精液活力评定结果显示,终浓度为2、4、8 mg/mL的HSP70能提高冷冻精液活力,且8 mg/mL HSP70组冻精活力最高,但各组间差异均不显著（P>0.05）。综上所述,本试验成功表达了水牛HSP70蛋白,获得了高纯度、有活性的HSP70融合蛋白,外源添加HSP70蛋白可提高水牛冷冻精液的活力,为进一步研究水牛HSP70蛋白的抗冻性保护机理奠定了理论基础。     

热应激雏鸡免疫器官中热休克蛋白70的定位研究

通过检测热休克蛋白70（heat shock protein,HSP70）在免疫器官及细胞上的分布规律,研究热应激雏鸡免疫器官中热休克蛋白70的定位及表达。结果显示：HSP70在常温饲养雏鸡的胸腺、脾脏、法氏囊中均有明显的表达,在胸腺、脾脏的细胞中主要以胞浆表达为主,而法氏囊中则以胞核表达为主,脾脏中HSP70的表达强度最为稳定。热应激时,胸腺中HSP70的IOD值随日龄增长而逐渐降低,末期显著低于对照组（P〈0．05）;脾脏中HSP70的IOD值随日龄增长逐渐降低,末期稍低于对照组（P〉0．05）;法氏囊中HSP70的IOD值与饲养日龄间无明显规律,末期高于对照组（P〉0．05）。结果表明,热应激对雏鸡胸腺的损伤明显,胸腺细胞通过自身合成HSP70抵抗热应激的能力较差,而对脾脏和法氏囊影响较小。     

HSP70在急性热应激蛋鸭脾脏表达变化的研究

采用半定量RT-PCR与Western-blotting方法检测相同温度、不同时间(3、5、7h)热应激处理鸭脾脏组织中HSP70基因的表达水平,以研究热应激对HSP70基因表达的影响。结果表明:与对照组相比,热应激组蛋鸭脾脏HSP70 mRNA转录水平在热应激进行到3h时呈现下降趋势,但是差异不显著;而进行到5h时则出现显著(P0.05)的回升;热应激组蛋鸭脾脏HSP70蛋白表达水平在热应激进行到5h时出现显著下降(P0.05),表明急性热应激对蛋鸭脾脏组织HSP70基因表达具有重要影响。     

Prospects of HSP70 as a genetic marker for thermo-tolerance and immuno-modulation in animals under climate change scenario

Heat stress induced by long periods of high ambient temperature decreases animal productivity, leading to heavy economic losses. This devastating situation for livestock production is even becoming worse under the present climate change scenario. Strategies focused to breed animals with better thermo-tolerance and climatic resilience are keenly sought these days to mitigate impacts of heat stress especially in high input livestock production systems. The 70-kDa heat shock proteins (HSP70) are a protein family known for its potential role in thermo-tolerance and widely considered as cellular thermometers. HSP70 function as molecular chaperons and have major roles in cellular thermotolerance, apoptosis, immune-modulation and heat stress. Expression of HSP70 is controlled by various factors such as, intracellular pH, cyclic adenosine monophosphate (cyclic AMP), protein kinase C and intracellular free calcium, etc. Over expression of HSP70 has been observed under oxidative stress leading to scavenging of mitochondrial reactive oxygen species and protection of pulmonary endothelial barrier against bacterial toxins. Polymorphisms in flanking and promoter regions in HSP70 gene have shown association with heat tolerance, weaning weight, milk production, fertility and disease susceptibility in livestock. This review provides insight into pivotal roles of HSP70 which make it an ideal candidate genetic marker for selection of animals with better climate resilience, immune response and superior performance.     

夏季奶牛血液中热休克蛋白的表达量

选择40头体质健康的中国荷斯坦奶牛,根据胎次和泌乳天数,按照随机区组试验设计分为Ⅰ组（30kg/d）、Ⅱ组（30～35kg/d）、Ⅲ组（35～40kg/d）和Ⅳ组（40kg/d）。热应激前、热应激前期、热应激中期、热应激后期和热应激后分别于尾静脉采血,用ELISA试剂盒测定热休克蛋白（HSP）27,70,90的表达量。结果显示,Ⅳ组HSP27表达量最高,Ⅱ组表达量最低,Ⅳ组、Ⅲ组和Ⅰ组均显著高于Ⅱ组（P〈0.05）。HSP70表达量各组间没有明显差异,但随产奶量呈线性增加（P〈0.05）。HSP90的表达量,Ⅳ组和Ⅲ组明显高于Ⅱ组（P〈0.05）。HSP27的表达量热应激后差异较大;HSP70的表达量各组整个过程差异较大;HSP90的表达量在热应激前、热应激前期和热应激后差异较大。总之,在热应激过程,高产奶牛血清中热应激蛋白的表达量较高,HSP70表达量随产奶量呈线性增加,而不同热应激蛋白的变化规律差异较大。     

断奶应激对奶水牛犊牛血液生理生化指标和免疫功能的影响

为研究断奶应激对奶水牛犊牛的影响,选用5头健康犊牛,分别于断奶前1 d、断奶后1、14和21 d进行采血,测定犊牛的血常规、血清生化指标和免疫指标。结果显示,奶水牛犊牛的白细胞数、淋巴细胞数、红细胞数、单核细胞数、红细胞压积、天冬氨酸转氨酶活性、血糖含量、皮质醇含量、免疫球蛋白G含量在断奶前后无显著变化（P > 0.05）;中性粒细胞数在断奶后21 d显著低于断奶前1 d（P < 0.05）;血清乳酸脱氢酶、丙氨酸转氨酶活性在断奶后显著高于断奶前（P < 0.05）;血清尿素氮含量在断奶后21 d显著高于断奶前1 d（P < 0.05）;血红蛋白含量在断奶21 d显著低于断奶前1 d（P < 0.05）。结果表明断奶应激不仅影响了奶水牛犊牛的生理机能,而且降低了其免疫力和抵抗力。     

运输应激对山羊血清和空肠中HSP27、HSP70和HSP90含量的影响

为探讨运输应激对山羊血清和空肠中HSP27、HSP70和HSP90 3种热休克蛋白含量的影响,试验选取12只赣西山羊,随机分为对照组、运输应激2 h组和运输应激6 h组,应用双抗体夹心ELISA法测定山羊血清和空肠中3种热休克蛋白浓度。结果表明:与对照组相比,运输2 h山羊血清中HSP70和HSP90极显著升高(P<0.01),空肠中HSP70和HSP90显著升高(P<0.05),血清和空肠中HSP27有升高趋势(P>0.05);与对照组相比,运输6 h后血清中HSP70和HSP90分别极显著和显著升高(P<0.05和P<0.01),血清中HSP27含量以及空肠中3种蛋白含量均无显著变化(P>0.05);与运输2 h相比,运输6 h后3种蛋白含量都极显著降低(P<0.01)。结果显示,山羊对运输应激在细胞水平的适应性调节与血清中HSP70和HSP90蛋白水平升高有密切联系,运输2 h山羊通过上调HSP70和HSP90对应激导致的空肠损伤发挥细胞保护作用。     

Effect of Weaning Stress on Blood Physiology and Biochemistry Indexes and Immune Function in Dairy Buffalo Calves

To study the effect of weaning stress on dairy buffalo calves,5 healthy calves were chosen and weaned,and the blood samples were collected at 1 day before weaning,and 1,14 and 21 days after weaning to analysis blood physiology and biochemistry indexes and immune function.The results showed that the count of white blood cell,lymphocyte,erythrocyte,monocytes and hematocrit,aspartate aminotransferase activity,blood glucose,cortisol and immunoglobulin G contents of dairy buffalo calves were no significant difference between before and after weaning treatments (P > 0.05).The count of neutrophils at 21 d after weaning were significantly lower than that of 1 d before weaning of dairy buffalo calves (P < 0.05).However,lactate dehydrogenase and alanine aminotransferase activity in weaned treatments were significantly higher than those of animals in the treatment before weaning (P < 0.05).The serum urea nitrogen of dairy buffalo calves at 21 d after weaning was significantly higher than that of 1 d before weaning (P < 0.05) and hemoglobin content at 21 d after weaning was significantly lower than that at 1 day before weaning (P < 0.05).It indicated that the physiological and immunity function of dairy buffalo calves were challenged under weaning stress.     

热应激绍兴鸭不同组织HSP70基因mRNA表达研究

研究蛋鸭热应激条件下不同组织中热休克蛋白70(HSP70)基因mRNA表达规律,为揭示蛋鸭热应激反应机理提供参考。60只绍兴蛋鸭25℃饲养20d,其中30只进行40℃热应激处理1h,采取荧光定量PCR方法分别测定不同组织中HSP70mRNA的表达水平。试验组与对照组相比,肝脏、脾脏和胰腺组织HSP70mRNA表达差异不显著,其他6种组织HSP70基因表达量均有不同程度的增加,试验组心脏、腿肌、垂体、下丘脑、胸肌、肾脏中HSP70基因表达量分别高于对照组3.6倍、2.8倍、2.7倍、2.4倍、2.2倍和1.57倍。     

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7-12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8-10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.     

Biochemical changes in heat exposed buffalo heifers supplemented with yeast

Serum electrolytes, minerals, blood biochemical and plasma enzymes were studied in heat exposed buffalo heifers supplemented with yeast powder in feed to alleviate the adverse effects of heat stress. Eighteen healthy Murrah buffalo heifers (270-280) kg were divided into three groups. Animals of group I were kept in shed and served as control, while group II and group III were exposed in a psychrometric chamber at 40°C for 4?hrs daily for 16?days continuously. The animals in group III were also supplemented with yeast powder at 10?g per animal per day. The serum sodium, potassium and chloride levels decreased significantly (P?相似文献   

HSP90 gene expression induced by aspirin is associated with damage remission in a chicken myocardial cell culture exposed to heat stress

1. To understand the potential protection of heat shock protein 90 (HSP90) induced by aspirin against heat stress damage in chicken myocardial cells, enzyme activities related to stress damage, cytopathological changes, the expression and distribution of HSP90, and HSP90 mRNA levels in the myocardial cells exposed to heat stress (42°C) for different durations with or without aspirin administration (1 mg/ml, 2 h prior) in vitro were investigated.

2. Significant increase of enzyme levels in the supernatant of heat-stressed myocardial cells and cellular lesions characterised by acute degeneration, karyopyknosis and karyorrhexis were observed, compared to non-treated cells. However, the lesions of cells treated with aspirin were milder, characterised by earlier recovery of enzyme levels to the control levels and no obvious heat stress-related cellular necrosis.

3. Stronger positive signals in the cytoplasm and longer retention of HSP90 signal in nuclei were observed in aspirin-treated myocardial cells than those of only heat-stressed cells. HSP90 level in the aspirin-treated myocardial cells was 11.1-fold higher than that in non-treated cells, and remained at a high level at the early stage of heat stress, whereas it was just 4.1-fold higher in only heat-stressed cells and returned rapidly to a low level.

4. Overexpression of HSP90 mRNA in aspirin-treated cells was observed throughout the experiment, whereas HSP90 mRNA decreased significantly only in heat-stressed cells.

5. The early higher HSP90 expression induced by aspirin during heat stress was accompanied by decreased heat stress damage, suggesting that aspirin might play an important role in preventing myocardial cells from heat stress damage in vitro.

     

小鼠卵巢HSP70诱导表达的免疫荧光组织化学研究

试验采用免疫荧光组织化学法,检验了经历不同温度热应激后,在不同恢复期内小鼠卵巢组织HSP70的诱导表达,确立了HSP70诱导表达模式。结果表明：随着热应激温度的升高,卵巢组织HSP70的表达量增加,表达持续期延长,特别是经历了预应激小鼠的HSP70表达更显著。这说明在一定的热应激条件下。小鼠卵巢组织可发生HSP70的诱导表达,其表达量和表达持续期与热应激强度相关,而且热应激可显著促进HSP70的表达。     

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes

Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation.     

中药复方对慢性热应激蛋鸡生产性能和心脏组织HSP70表达量的影响

探讨中药复方对慢性热应激下蛋鸡生产性能和心脏组织HSP70表达量的影响。选取250只60日龄蛋鸡,随机分为空白对照组、维生素C组、复方Ⅰ、复方Ⅱ、复方Ⅲ组。每天记录各组的耗料量、蛋重、产蛋量、淘死数以及软、破蛋数量,采用ELISA法测定蛋鸡心脏组织HSP70表达量。结果显示,中药复方能够改善热应激状态下蛋鸡的生产性能,其中复方Ⅱ的效果最佳;中药复方组蛋鸡心脏组织HSP70表达量均低于空白对照组,其中方Ⅱ组下降的幅度最大,表明各中药复方均能提高热应激蛋鸡的生产性能,其作用机制可能与调控HSP70表达量有关。     

应激宁对HSP70在大鼠应激性溃疡中表达的影响

为了探讨热休克蛋白70(HSP70)在应激性溃疡中表达的变化及应激宁对其变化的影响,选用Wistar大鼠水浸-束缚应激(WRS)4 h的方法,建立应激性溃疡模型.用免疫组织化学方法检测胃黏膜组织HSP70的表达.结果表明,HSP70的表达主要分布在胃腺区,对照组大鼠胃黏膜组织中有散在的表达,应激组中HSP70阳性细胞数目较应激宁组增多,两者具有明显的差异(P＜0.05),而且,应激组和应激宁组中HSP70阳性细胞数目均比对照组明显增多(P＜0.01).表明应激宁对WRS后胃黏膜组织HSP70的表达具有调节作用,这也表明应激宁具有抗应激的作用.     

Prokaryotic expression of chicken interferon‐γ fusion protein and its effect on expression of poultry heat shock protein 70 under heat stress

Interferons have attracted considerable attention due to their vital roles in the host immune response and low induction of antibiotic resistance. In this study, total RNA was extracted from spleen cells of chicken embryos inoculated with Newcastle disease vaccine, and the full‐length chicken interferon‐γ (ChIFN‐γ ) gene was amplified by RT‐PCR. The full complementary DNA sequence of the ChIFN‐γ gene was 495 bp long and was cloned into the prokaryotic expression vector pProEX?HT_b. The plasmid was transformed into Escherichia coli DH5α and the expression of ChIFN‐γ was induced by isopropyl β‐D‐1‐thiogalactopyranoside. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis and Western blot results showed the expressed fusion protein had a molecular weight of approximately 18 kDa and was recognized by an anti‐His mAb. Moreover, ChIFN‐γ was found to demonstrate anti‐viral activity in vitro . To test the in vivo function of ChIFN‐γ in broilers under heat stress, a total of 100 broilers were randomly assigned to either a control group or a treated group, in which they were hypodermically injected with recombinant ChIFN‐γ. Results demonstrated ChIFN‐γ affects the messenger RNA expression levels of heat shock protein 70 (HSP70) in the heart and lung tissues, and decreases the concentration of HSP70 in serum. Therefore, we conclude recombinant ChIFN‐γ can reduce heat stress to some extent in vivo .