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.     

叶用莴苣热激蛋白LsHsp70-3701基因的克隆及高温胁迫下的表达分析

为探讨热激蛋白Hsp70基因在高温胁迫下的响应机制及其分子机理,分析Hsp70基因与叶用莴苣耐热性的关系,本研究通过同源克隆及RACE技术,克隆了叶用莴苣热激蛋白Ls Hsp70-3701基因的c DNA全长序列,利用实时荧光定量PCR(qRT-PCR)分析该基因在叶用莴苣热敏品种P-S11和耐热品种G-S59中高温胁迫下的表达差异。该基因c DNA全长为2 191 bp,开放阅读框为1 950 bp,编码649个氨基酸,具有Hsp70家族特有的标签序列,与拟南芥、番茄、小麦等物种的Hsp70同源性达到90%以上。qRT-PCR分析显示,高温胁迫下该基因在2个品种中的表达均上调,在耐热品种中总体表达水平显著高于热敏品种,且在42℃高温下热敏品种P-S11中基因表达随着胁迫时间的增加受到抑制,而耐热品种G-S59中则能长时间保持较高的表达水平。结果表明,Ls Hsp70-3701基因可能与叶用莴苣耐热性相关。本研究为有效解决叶用莴苣高温抽薹问题提供了数据支持。     

Induced thermotolerance and expression of heat shock protein 70 in sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

ABSTRACT:   Thermal limits, induced thermotolerance and the expression of heat shock protein 70 (Hsp70) in an echinoderm Apostichopus japonicus were studied. The sublethal and lethal temperatures for the juveniles were 30 and 34°C, respectively; a previous sublethal heat shock exposure (30°C, 2 h) could increase the survival rates of the sea cucumbers when they were exposed to 34°C. This induced thermotolerance could last for at least 2 days. Levels of Hsp70 increased substantially after sublethal heat shock exposure and linearly decreased with time. This result indicated that a close relationship existed between the induction of thermotolerance and the levels of Hsp70 in A. japonicus.     

Mycobacterium salmoniphilum infection in farmed Atlantic salmon, Salmo salar L

Multiple greyish‐white visceral nodules containing abundant rapidly growing and acid‐fast bacteria, subsequently identified as Mycobacterium salmoniphilum, were detected in moribund and newly dead market‐sized fish during a period of increased mortality in an Atlantic salmon, Salmo salar, farm in western Norway. Isolates cultured from diseased fish were phenotypically consistent with Mycobacterium sp. previously isolated from Atlantic salmon [MT 1890 (= NCIMB13533), MT1892, MT1900 and MT1901] in the Shetland Isles, Scotland. Partial sequences of 16S rDNA, ribosomal RNA internal transcribed spacer (ITS1), 65‐kDa heat‐shock protein (Hsp65) and β subunit of RNA polymerase (rpoB) revealed 97‐99% similarity with M. salmoniphilum type strain ATCC 13758^T. The source of infection was not confirmed. Koch’s postulates were fulfilled following experimental challenge of Atlantic salmon with field isolate NVI6598 ( FJ616988 ). Mortality was recorded in experimentally infected fish; however, the infection remained subclinical in the majority of affected fish over the 131‐day challenge period.     

间接免疫荧光方法检测小鼠睾丸和附睾组织Hsp70的表达

采用间接免疫荧光方法对小鼠睾丸和附睾组织Hsp70的表达进行检测.结果表明,小鼠睾丸组织Hsp70呈极强的表达(+++),并且在各级生精细胞及支持细胞都呈极强表达;小鼠附睾头、附睾体、附睾尾的Hsp70呈现极强表达(+++),主要在附睾上皮细胞,而管腔内精子偶见阳性染色.说明间接免疫荧光方法能够特异性、原位地检测小鼠睾丸和附睾组织Hsp70的表达,而且操作简单,适用于研究Hsp70在精子发生和成熟过程的作用.     

CaM对小鼠胎儿成纤维细胞Hsp70表达的影响

[目的]证实小鼠胎儿成纤维细胞中钙调蛋白 (calmodulin,CaM) 基因参与热应激诱导型热休克蛋白70(Hsp70)的基因表达并明确其作用条件.[方法]取12.5 d孕鼠胚胎制备成纤维细胞(MEF),上述细胞随机分为对照组和热应激组:对照组在37℃条件下培养,热应激组包括I组(39℃、0.5 h),Ⅱ组(39℃,1 h),Ⅲ组(39℃,1.5 h),Ⅳ组(41℃、0.5 h),V组(41℃、1 h),Ⅵ组(41℃、1.5 h);每组3个重复.将选择的热应激组和对照组细胞分别加入不同浓度(25、50及100 mm=ol·L<'-1>)钙调蛋白拮抗剂W7,运用RT-PCR检测Hsp70、CaM mRNA表达量.[结果] 39℃和41℃热应激组Hsp70 mRNA表达量显著高于常温对照组(P<0.05):39℃应激1h的Hsp70 mRNA表达量极显著高于其它各组(P<0.01);39"C应激0.5 h和1 h CaM mRNA表达量极显著高于其它各组(P<0.01);41℃热应激组的CaM mRNA表达量与常温对照组比较差异不显著(P>0.05).在培养液中加100 mmol·L<'-1> W7、分别于37℃和39℃处理1 h后,其成纤维细胞的Hsp70 mRNA表达量极显著低于相应对照组 (P<0.01).[结论]中度热应激条件下,Hsp70和CaM基因表达量呈正相关,且39℃应激1 h可以显著诱导Hsp70和CaM基因表达;CaM通过某种途径参与了Hsp70基因表达.     

三叶斑潜蝇热激蛋白Hsp90基因的克隆及其在高温胁迫下的表达分析

采用RT-PCR及RACE技术克隆了三叶斑潜蝇Hsp90基因全长cDNA序列,并用实时定量RT-PCR的方法检测其在不同发育阶段受到高温胁迫后的表达水平。该基因的cDNA序列全长2 408 bp,开放阅读框为2 145 bp,编码714个氨基酸;5′非编码区为151 bp,3′非编码区为112 bp。该基因推导的氨基酸序列与其他昆虫同源序列比较有很高的相似性（80%~99%）。聚类分析结果显示三叶斑潜蝇与美洲斑潜蝇和南美斑潜蝇的亲缘关系最近。实时荧光定量PCR检测结果表明三叶斑潜蝇Hsp90基因的表达受到热胁迫的诱导,诱导3龄幼虫最大表达量的温度比诱导其他发育阶段的温度低,在43 ℃时预蛹和蛹的表达量在整个生命周期中最高,在检测的高温胁迫条件下,雄虫比雌虫的表达量更高。该结果为阐明三叶斑潜蝇胁迫耐受能力及其对其他潜蝇种群的取代机制奠定了分子基础。     

冷暴露仔猪PBMC中Hsp72与血浆TNF-α、IL-6的变化规律

为探讨急性冷应激对仔猪免疫系统的影响及机制,本试验将18头仔猪随机分成常温对照组、冷暴露组和冷暴露并糖皮质激素受体(GRs)阻断组.常温组在18℃～20℃下而冷暴露各组在4℃～8℃环境下分栏饲养.试验期持续12 h,在冷暴露开始后六个时间点对18头仔猪采血并分离血浆和外周血单个核细胞(PBMC),检测Hsp72和TNF-α、IL-6的水平.实验结果显示:冷暴露组IL-6水平在冷暴露6 h时比开始前有显著升高(P＜0.01),TNFα水平在冷暴露1 h 时显著下降(P＜0.01),3 h和6 h又显著升高(P＜0.01),且IL-6与TNF-α呈中等相关(r=0.4198);GRs阻断组IL-6水平在冷暴露6 h时显著升高,TNF-α水平在各时间点差异不显著;冷暴露各组Hsp72在冷暴露1 h,3 h,6 h,9 h,12 h时均显著升高(P＜0.01),且受体阻断组TNF-α与Hsp72呈中等强度负相关(r=0.6444).这些结果表明:急性冷暴露能使血浆IL-6水平升高和PBMC中Hsp72 的表达增强,而使TNF-α的水平降低,且阻断糖皮质激素对它们之间的相关性有一定影响.     

天津地区番茄褪绿病毒的分子检测与基因组部分序列分析

为了确定天津番茄产区是否发生了番茄褪绿病毒病,了解该病毒天津分离物的主要基因是否发生变异,对该地区发生的To CV进行了分子检测,并对该病毒的外壳蛋白(CP)和热激蛋白70类似物(Hsp70h)的核苷酸和氨基酸进行了序列分析。结果表明,天津分离物CP蛋白的核苷酸和氨基酸序列与已报道的具有代表性的不同国家的分离物相似性均在97.3%以上。Hsp70h蛋白的核酸和氨基酸序列与已报道的具有代表性的不同国家的分离物相似性均在97.2%以上。表明CP和Hsp70h蛋白是2个最保守的蛋白。这是首次在分子水平上证明番茄褪绿病毒在天津地区为害。