饲料蛋白水平对低温应激下吉富罗非鱼血清生化指标和HSP70 mRNA表达的影响

在水温27℃条件下, 分别对吉富罗非鱼(27.642.79) g投喂3组不同蛋白水平的饲料, 养殖周期56d。饲养结束后, 进行14℃低温应激实验, 研究低温应激后0-24h内, 鱼体血清生化指标与肝脏HSP70　mRNA表达量的变化。结果表明, 短期投喂50%蛋白水平的饲料可以提高罗非鱼血清中葡萄糖、总蛋白、甘油三酯与胆固醇水平以及血清谷丙转氨酶与谷草转氨酶活力。在低温应激后, 50%蛋白组血清葡萄糖、总蛋白、甘油三酯与胆固醇水平呈下降趋势, 而血清皮质醇水平与谷草转氨酶活力表现为先上升后下降变化。25%和38%蛋白组血清葡萄糖水平与谷丙转氨酶活力在应激期间呈上升趋势, 而谷草转氨酶与碱性磷酸酶活力和总蛋白与胆固醇水平呈先上升后下降变化。各实验组溶菌酶活力与肝脏HSP70 mRNA的表达量呈先上升后下降的变化。由此可知, 短期投喂高蛋白饲料可以提高罗非鱼血液中蛋白、血糖与脂肪含量, 增强鱼体抗应激能力, 同时, 高蛋白水平的饲料也会引起肝脏产生分解压力以及增加饲料成本。在生产中, 需要根据实际需要合理确定饲料的蛋白含量。     

饲料脂肪水平对吉富罗非鱼生产性能、营养物质消化及血液生化指标的影响

为探讨吉富罗非鱼对脂肪的适宜需求量,将630尾[(2.63±0.16)g]吉富罗非鱼随机分成6个脂肪组的饲料组(1.73%、3.71%、5.69%、7.67%、9.64%和16.55%),每组设置3个重复,每个重复35尾,第1组为对照组,投喂基础日粮(含脂肪1.73%),另外5组为试验组,在基础日粮中分别添加2%、4%、6%、8%、15%的鱼油,饲养90d后测定生长、饵料系数、营养物质表观消化率及血液常规生化指标。结果显示,随着饲料脂肪水平提高,增重率和特定生长率呈现一个先上升后下降的趋势(P＜0.05),蛋白质效率极显著地提高(P＜0.01),饵料系数极显著地下降(P＜0.01)。增重率与饲料脂肪水平的二次多项式回归分析显示,吉富罗非鱼获得最高增长所需饲料的最佳脂肪水平为9.34%。饲料脂肪水平对粗蛋白表观消化率和饲料干物质表观消化率无显著影响(P>0.05),饲料脂肪水平增加显著提高了粗脂肪和磷的表观消化率(P＜0.05)。未添加鱼油的1.73%组血液中白蛋白和白球比均显著高于其他组(P＜0.05),随着饲料脂肪水平的提高,胆固醇的浓度及碱性磷酸酶的活性极显著地上升(P＜0.01)。饲料脂肪水平对血糖浓度有显著影响(P＜0.05),对甘油三酯浓度、谷丙和谷草转氨酶的活性无显著影响(P>0.05)。结果表明,饲料中的脂肪水平可以促进吉富罗非鱼对脂肪和磷的表观消化率,但是脂肪水平过高会对鱼体增重及血液生化参数产生负作用,从生产上来说吉富罗非鱼鱼种对脂肪的适宜需求量为7.67%-9.34%。     

不同植物脂肪源对吉富罗非鱼生长性能、肌肉脂肪酸和生理生化指标的影响

配制了6种等氮等脂的饲料饲喂(12.320.02) g的吉富罗非鱼8周, 探讨罗非鱼对不同脂肪源的利用效果,筛选出适合吉富罗非鱼的植物脂肪源。6种饲料中分别添加大豆油(SO)、棕榈油(PO)、棉籽油(CO)、菜籽油(RO)、磷脂(SL)和1:1:1:1:1大豆油-棕榈油-棉籽油-菜籽油-磷脂混合油(MIX)。结果显示: (1)菜籽油组特定生长率显著高于棕榈油组、棉籽油组、磷脂油组和混合油组(P0.05), 大豆油组显著高于棉籽油组和棕榈油组(P0.05)。菜籽油组饲料系数显著低于棉籽油组、磷脂油组和棕榈油组(P0.05), 与其他各组无显著性差异(P0.05); (2)肌肉脂肪酸明显受饲料脂肪源影响, 棕榈油组肌肉脂肪酸组成与饲料脂肪酸组成相关性最大(P0.05), 棉籽油组肌肉脂肪酸组成与饲料脂肪酸组成相关性最小(P0.05); (3)棕榈油组天冬氨酸转氨酶活性显著高于菜籽油组(P0.05)。棉籽油组高密度脂蛋白胆固醇含量和低密度脂蛋白胆固醇含量显著高于菜籽油组(P0.05), 大豆油组肝脏脂蛋白酯酶活性显著高于棉籽油组和菜籽油组(P0.05)。研究结果表明: 菜籽油、大豆油可以作为吉富罗非鱼饲料中良好的脂肪源, 棉籽油不利于吉富罗非鱼生长。     

急性冷应激对牦牛乳腺上皮细胞 HSP70 mRNA 表达的影响

本文主要研究了急性冷应激对牦牛乳腺上皮细胞热休克蛋白７０ （Heat stress protein,ＨＳＰ７０） 表达量的影响。采用实时荧光定量ＰＣＲ 技术,以急性冷刺激１０℃ 为典型研究环境,分析了ＨＳＰ７０ ｍＲＮＡ 表达的变化规律。结果显示,乳腺上皮细胞在１０℃分别冷处理２ ｈ、４ ｈ、６ ｈ 和８ ｈ,其ＨＳＰ７０ ｍＲＮＡ 的表达量变化均不显著（Ｐ ＞０. ０５）;分别在１０℃冷处理２ ｈ、４ ｈ、６ ｈ 和８ ｈ,再复温培养４ ｈ,ＨＳＰ７０ ｍＲＮＡ 的表达量均极显著增加（Ｐ ＜０. ０１）,于６ ｈ 达到峰值;在１０℃先冷处理４ ｈ,然后分别复温２ ｈ、４ ｈ、６ ｈ 和８ ｈ,ＨＳＰ７０ ｍＲＮＡ 的表达量亦均显著增加（Ｐ ＜０. ０１）,并于４ ｈ 达到峰值。结论：急性冷应激诱导牦牛乳腺上皮细胞ＨＳＰ７０ 表达量的增加不是发生在冷处理过程中,而是发生在复温过程中,并且在一定范围内随冷处理时间的增长表达量增高。     

低温胁迫对苜蓿幼苗存活及生理生化指标的影响

用秋眠级为2级至9级的11个苜蓿品种,每品种被随机分为对照组和5个处理组,研究低温胁迫和苜蓿品种对幼苗存活和叶片生理生化指标的影响。结果表明:低温胁迫和苜蓿品种对幼苗存活率、叶片生理生化指标的影响均极显著,而且二者对它们的互作效应也极显著。子叶期幼苗在-7.5℃下处理12 h,存活率仅为3.26%。幼苗在-5℃下处理12 h,存活率为54.73%,但叶片游离脯氨酸含量、过氧化物酶活性、质膜相对透性和根系活力极显著提高;每天把幼苗置于-5℃下2 h至3 h锻炼7天,幼苗存活率可提高到95%以上,叶片游离脯氨酸、可溶性糖、可溶性蛋白质含量,超氧化物歧化酶活性、根系活力和质膜相对透性均极显著提高。结果表明:4级至9级品种的存活率极显著低于2级至3级的品种;6级至9级品种叶片游离脯氨酸含量、过氧化物酶活性、质膜相对透性极著显低于2级至3级的品种。     

两种温度下大黄素对团头鲂生长、血液指标及肝脏 HSP70 mRNA表达的影响

实验采用2×2双因子设计, 研究两种温度下在饲料中添加大黄素对团头鲂(Megalobrama amblycephala)生长、血液生理反应及肝脏HSP70 mRNA表达的影响。选取300尾健康的团头鲂[初均重(7.29±0.01) g], 两种温度下(25℃、30℃)分别投喂不添加和添加25 mg/kg的大黄素配合饲料, 养殖时间为77d。结果显示: 在长期30℃高温下, 鱼体增重率(WGR)和特定生长率(SGR)、红细胞积压(HCT)、乳酸脱氢酶(LDH)显著升高, 饵料系数(FCR)和脏体比(VSI)、谷草转氨酶(AST)、总胆汁酸(TBA)显著下降(P0.05); 添加25 mg/kg大黄素, SGR和肝体比(HSI)、红细胞积压(HCT)、乳酸脱氢酶(LDH)显著升高, 饵料系数(FCR)和摄食率(FR)显著下降(P0.05)。温度和大黄素对谷丙转氨酶(ALT)和血糖(GLU)无显著影响(P0.05), 对尿素(UREA)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、补体3 (C3)、补体4 (C4)和肝脏HSP70 mRNA的相对含量存在显著交互作用(P0.05)。与常温对照组相比, 高温对照组UREA、TG、TC显著下降; 在高温下, 添加大黄素组UREA、TG、TC有升高趋势(P0.05), C3、C4和肝脏HSP70 mRNA的相对含量显著升高(P0.05)。以上结果表明, 在饲料中添加25 mg/kg大黄素有助于缓解长期高温对团头鲂血液生理的影响, 提高补体水平和肝脏HSP70 mRNA的相对含量, 促进鱼体的生长。     

褐马鸡生理常数及某些血液生化指标的研究

为了全面地了解褐马鸡的生理特点,本研究对褐马鸡的主要生理常数及血液生化指标的正常值进行了分析测定。结果表明:血沉值和血清磷、血糖、电泳白蛋白及α-球蛋白的含量在性别间存在显著地差异(P<0.05—0.01),其它各项指标性别间差异不显著(P>0.05)。     

急性热应激对西伯利亚鲟HSP70 mRNA表达、血清皮质醇和非特异性免疫的影响

为研究西伯利亚鲟(Acipenser baerii)对急性热应激的抗逆机理, 将体质量为(155.4719.50) g的鱼从17.5 ℃迅速转至27.5 ℃水中, 在1h和3h取样测定HSP70 mRNA表达变化、血清皮质醇和非特异性免疫指标。结果显示: 急性热应激时鳃、脾和脑的HSP70 mRNA表达量升高, 具有组织特异性, 热应激1h时鳃的表达量升高最快(P0.05), 3h时保持1h时的表达水平; 脾和脑热应激1h时表达量变化不显著, 在1h至3h时升高较快, 并且脑组织的表达量升高最快(P0.05)。热应激1h时血清皮质醇(Cortisol)含量迅速升高(P0.05), 之后快速回落。脾脏巨噬细胞呼吸暴发在热应激1h时显著升高(P0.05), 3h时降低。血清补体C3在1h时略有升高, 3h时显著性降低(P0.05)。血清溶菌酶活性(LZM activity)先升高后降低差异不显著。血清超氧化物歧化酶(SOD)活力随热应激时间延长逐渐降低, 3h时显著降低(P0.05)。血清丙二醛(MDA)含量随热应激时间延长逐渐降低, 差异不显著。以上结果表明: 1h的短暂急性应激增强了西伯利亚鲟的非特异性免疫, 3h的应激使免疫力和抗氧化能力显著下降; 在热应激过程中, HSP70表达升高, 其中鳃组织最快, 起到应激保护作用, 提高了机体热耐受力。     

美洲鲥应激后皮质醇激素和血液生化指标的变化

比较了美洲鲥(Alosa sapidissima)1龄幼鱼因环境改变和运输引起的血清皮质醇激素浓度和血液生化指标变化。结果表明,美洲鲥转入较小容器中后血清皮质醇含量迅速增加到对照组[(2.05±1.48)ng/ml,n=10]的10倍左右。运输2 h后美洲鲥血清皮质醇激素含量[(41.97±17.92)ng/ml,n=9]比运输前显著上升20倍左右。2 h运输后总蛋白、白蛋白、谷丙转氨酶、碱性磷酸酶、Ca2 比对照组极显著增加(P<0.01),白球比和乙酸胆碱酯酶、血糖、K 显著增加(P<0.05),谷草转氨酶和乳酸脱氢酶无显著变化(P>0.05),Cl-显著低于对照组(P<0.01)。血液指标明显变化表明,美洲鲥运输应激后,心脏和肝脏等组织一定程度受损,可能导致了较高的死亡率。     

Coordinate and non-coordinate expression of the stress 70 family and other molecular chaperones at high and low temperature in spinach and tomato

Stress 70 molecular chaperones are found in all the major subcellular compartments of plant cells, and they are encoded by a multigene family. Twelve members of this family have been identified in spinach. The expression of the stress 70 molecular chaperones in response to heat shock is well-known and it appears that low temperature exposure can also stimulate their expression. However, it has been difficult to determine which member(s) of the family are specifically responsive to low temperature. This study was initiated to determine the levels of expression of the stress 70 family members and other selected chaperones in response to high and low temperature exposure. During heat shock of spinach, of the 10 stress 70 family members that were examined, all 10 showed increased RNA levels after one hour, and all showed down-regulation at longer durations of high temperature exposure. However, the response to low temperature was quite variable and complex. Some members were induced, some were transiently up-regulated, while others showed sustained up-regulation at a low non-freezing temperature. In comparison, the entirety of the molecular chaperone expression response of cold-sensitive tomato at the same low non-freezing temperature was even more dramatic with 11 of 15 molecular chaperones tested exhibiting elevated expression. The increased chaperone expression is consistent with the hypothesis that the biogenesis or stability of some proteins is compromised at low non-freezing temperatures. In contrast, mild freezing sufficient to cause injury of spinach did not materially activate chaperone expression.     

Expression of stress proteins of HSP70 family in response to cold in Myrmica ants from various geographic populations

Expression of HSP70 is induced by stress factors, including sublethal chilling. However, the role of HSP70 for overcoming the consequences of cold stress is not clear. If it is positive, the level of HSP70 expression might be higher in populations from cold climates. Using the immunoblotting technique we investigated dynamics of HSP70 expression in response to cold stress in two Myrmica species (M. rubra and M. ruginodis) from three localities of different latitudes (50, 60 and 67°N). The results showed that in the more thermophilic species Myrmica rubra, expression of HSP70 after cold stress was higher. Within both species, HSP70 expression did not show a direct relationship with latitude. The southernmost population of M. rubra and northernmost population of M. ruginodis displayed the fastest and the most intense response. However, two other populations of M. rubra were similar in timing and intensity of the response, while in M. ruginodis the intermediate population showed the slowest and weakest response. The data suggest that expression of HSP70 may play essential role for adaptation to cold only in the northernmost population of M. ruginodis     

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.     

大黄素、维生素C及其配伍对团头鲂抗拥挤胁迫的影响

选取1200尾健康的团头鲂(Megalobrama amblycephala Yih),体重为(133.44±2.11)g,随机分成4组,其中1组为对照组,投喂基础日粮(含50.3 mg/kg维生素C,以L-抗坏血酸-2-多聚磷酸酯为Vc源),另外3组为试验组,投喂饲料是在基础日粮中分别添加60 mg/kg大黄素、700 mg/kg Vc、60 mg/kg大黄素+700 mg/kg Vc。饲养60d后,从各池中取25尾规格基本一致的鱼,进行连续48h的拥挤胁迫(100 g/L)实验,分别于0h、12h、24h、48h取样分析团头鲂血液和肝脏的生化指标以及肝脏两种HSP70s mRNA的表达水平,并统计各组鱼的累积死亡率。结果表明,在拥挤胁迫前,与对照组相比,大黄素、Vc组显著提高了团头鲂血清总蛋白(TP)、溶菌酶(LSZ)和碱性磷酸酶(AKP)的水平,肝脏超氧化物歧化酶(SOD)的活性和诱导型HSP70 mRNA的表达水平,降低了血清皮质醇(COR)、甘油三酯(TG)以及肝脏丙二醛(MDA)的含量(P<0.05),且大黄素组还显著提高了肝脏过氧化氢酶(CAT)的活性(P<0.05);配伍组虽然血清TP、LSZ以及肝脏HSP70 mRNA的水平显著升高,肝脏MDA的含量也显著降低(P<0.05),但均未表现出协同增效作用。在拥挤胁迫后,与对照组相比,大黄素、Vc组不同程度地提高了团头鲂血清TP和AKP的水平,肝脏SOD和CAT的活性以及HSC70和HSP70 mRNAs的表达水平,降低了血清COR、葡萄糖(GLU)、谷丙转氨酶(GPT)、谷草转氨酶(GOT)、TG以及肝脏MDA的水平,而LSZ活性表现为先下降后升高;在配伍组中,这些指标虽然有类似以上的变化趋势,但大多差异不显著(P>0.05),且同样未表现出协同增效作用。统计表明,大黄素和Vc组鱼的累积死亡率在拥挤胁迫24h、48h均显著低于对照组(P<0.05),而配伍组与对照组的差异均不显著(P>0.05)。由此可见,在基础日粮中添加大黄素60 mg/kg或Vc 700 mg/kg,可提高团头鲂的非特异性免疫力、抗氧化能力以及两种HSP70s mRNA的表达水平,增强鱼体的抗应激能力。二者配伍则效果不佳,其相互作用的机理还有待于进一步研究。     

Cloning and differential expression of three heat shock protein genes associated with thermal stress from the wolf spider Pardosa pseudoannulata (Araneae: Lycosidae)

Pardosa pseudoannulata is the main predatory natural enemy of crop pests in a paddy ecosystem. When P. pseudoannulata is exposed to unfavorable temperature conditions, the response of heat shock proteins could resist the damage, and is therefore, conducive to the organism’s rapid adaptation to the surrounding stress environment. In this study, we explored the roles of hsp70 and hsp90 genes in response to heat stress, using the rapid amplification of cDNA ends technique and cloned full-length cDNAs of Pphsp70, Pphsp83, and Pphsp90. The mRNA expression levels of the three genes under different temperature stresses (25, 28, 31, 34, 37, 40, and 43 °C) and with different duration stresses (4, 8, 12, 16, and 20 h) were analyzed by quantitative real-time polymerase chain reaction. The full-length cDNA of Pphsp70, Pphsp83, and Pphsp90 was 2331 base pair (bp), 2466 bp, and 2663 bp, respectively. Phylogenetic analysis of amino acid sequences of Pphsp70, Pphsp83, and Pphsp90 showed that the sequences had high homology with that of other spiders. The mRNA expression of all three genes was extremely significantly up-regulated at 43 °C. Moreover at 43 °C, the expression of all three genes in both female and male spiders at the duration of 4 h was the highest compared to that of other stress duration groups. Therefore, it can be inferred that the three genes of P. pseudoannulata play a crucial protective role in resistance in a high-temperature environment.     

Tissue-specific expression of heat shock proteins of the mouse in the absence of stress

The steady-state levels of four members of the heat shock proteins families (HSP84, HSC73, HSP71, and HSP25) were examined by immunoblot analysis of several different tissues of young and adult mice in the absence of stress. These hsps were detected in all tissues but their level was variable. The levels of HSC73 and HSP84 varied only slightly between different tissues in either young or adult mice, with the exception of skin where these hsps were found in reduced amounts. In contrast, the stress-inducible member of the HSP70 family, HSP71, was found to be expressed in all tissues but in amounts which differed by as much as two orders of magnitude between tissues. In general, the levels of both HSP71 and HSP25 were found to be tissue dependent, with higher levels found in tissues such as stomach, intestine, colon and bladder, tissues which are exposed to toxic environmental or metabolic products, and which may concentrate these substances by water resorption and/or be exposed to them for longer periods. The levels of HSP71 and HSP25 were generally positively correlated both in young and adult mice although this correlation was not found in certain tissues such as kidney, testes, and bone. Tissues of young mice contained lower amounts of HSP25 and HSP71 than were found in the same tissues from adults. We conclude that hsps are expressed in all tissues of the mouse in the absence of stress and that some organs, particularly those exposed to potentially toxic metabolites, show a higher level of expression of HSP71 and HSP25. © 1993Wiley-Liss, Inc.     

Roles of YB-1 under arsenite-induced stress: Translational activation of HSP70 mRNA and control of the number of stress granules

Background

When cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress.

Method

Using arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly.

Results

Arsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression.

Conclusion

In arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly.

General significance

Under stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.