高盐胁迫对凡纳滨对虾消化及免疫相关酶活力的影响

为探讨高盐对凡纳滨对虾(Litopenaeus vannamei)消化及免疫相关酶活力的影响,实验设置了30、40、50、60共4个盐度梯度。对虾体长(7.84±0.68)cm,养殖密度333尾/m~3,每个梯度设3个平行,实验周期30d。取血淋巴、肌肉、肝胰腺等组织,检测其超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、碱性磷酸酶(AKP)和酸性磷酸酶(ACP)及蛋白酶、脂肪酶、淀粉酶活力。结果表明,盐度显著影响凡纳滨对虾肝胰脏中胃蛋白酶、脂肪酶、淀粉酶的活力(P0.05);随着盐度增加,消化相关酶活力均不断下降,处理间差异显著(P0.05);盐度对凡纳滨对虾不同组织的免疫指标产生影响,表现为随着盐度升高,血淋巴中,AKP活力逐渐升高,ACP、CAT和SOD活力均表现为先升高后降低;肌肉中,AKP、ACP和SOD活力呈现先升高后降低的变化趋势;肝胰脏中,AKP活力呈现先降低后升高再降低的变化趋势,ACP活力高盐处理间差异不显著(P0.05),CAT活力先降低后升高,SOD活力盐度40后逐渐降低。实验结果初步说明,高盐显著影响凡纳滨对虾的消化及免疫相关酶活力,且盐度对不同组织中免疫酶活力影响存在一定的组织特异性,50以上的高盐胁迫对对虾消化和免疫相关酶活力的影响尤为显著。     

盐度、温度对卵形鲳鲹选育群体肝抗氧化酶活力的影响

通过盐度渐变和温度骤变的方法,分别研究了不同盐度(10、20、30、40)处理和不同温度(18.0℃、21.0℃、24.6℃、29.0℃、32.0℃)处理对卵形鲳鲹(Trachinotus ovatus)选育群体肝超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPX)活力的影响.在实验结束时,盐度10组酶活力与对照组无显著差异(P＞0.05),盐度20组SOD活力极显著低于对照组(P＜0.01);盐度40组120 h时,SOD和GPX活力极显著低于对照组(P＜0.01),CAT活力与对照组差异不显著(P＞0.05).18.0℃和21.0℃ SOD活力在1、3、6、12、24 h这5个取样时间点均高于对照组,CAT活力在实验结束时(24 h)极显著高于对照组(P ＜0.01);29.0℃SOD和CAT活力在实验结束时(24 h)显著高于对照组(P＜0.05);32.0℃ SOD和CAT活力在5个取样时间点均显著低于对照组(P＜0.05).结果表明,适当的盐度或温度变化可以改变卵形鲳鲹肝抗氧化酶活力,达到机体耐受极限时酶活力下降.     

盐度对脊尾白虾亲虾抱卵及其子代生长发育的影响

选用室内人工培育的性腺已发育到Ⅱ期的脊尾白虾亲虾,经逐级淡化后,盐度稳定在2、5、10、15、20、25、30,研究了盐度对雌虾抱卵、胚胎发育及子代生长发育的影响.结果表明： 在试验盐度范围内脊尾白虾性腺均可发育成熟,但在盐度2时无法完成抱卵,最适抱卵盐度为10~20;在抱卵盐度范围内,胚胎均可正常发育,盐度对胚胎发育速率影响显著,15、20、25盐度下的孵化时间显著低于其他盐度组;盐度对脊尾白虾幼体后的变态和存活无显著影响,但对仔虾的个体干质量影响显著,15和20盐度下仔虾的干质量显著高于其他盐度组;盐度对20日龄脊尾白虾的生长影响显著,其特定生长率随着盐度升高而逐渐增大,在盐度20时达到最大,之后开始降低;鳃Na⁺-K⁺ ATPase的mRNA相对表达量在高盐或低盐时均较高,表达量最低时的理论盐度为17.5,可能为脊尾白虾的等渗点.研究表明,脊尾白虾亲虾可在较广盐度范围内繁殖,20日龄脊尾白虾在其理论等渗点附近具有较快的生长速度.
     

嗜水气单胞菌感染对背角无齿蚌5种免疫相关酶活力的影响

本研究以背角无齿蚌为材料,利用嗜水气单胞菌为诱导刺激物,对背角无齿蚌进行注射感染,在注射后3、6、12 、24、48h分别取血淋巴、肝胰腺,测定其超氧化物歧化酶（SOD）、酚氧化酶 (PO)、酸性磷酸酶 (ACP)、碱性磷酸酶 (AKP) 及过氧化氢酶 (CAT) 的活力,并研究各项酶活力的变化规律。结果表明：除3h肝胰腺外,实验组的SOD活力均不同程度地高于对照组;血清实验组的PO活力开始显著高于对照组,然后降低,而肝胰腺实验组的PO活力持续高于对照组;另外,与对照组相比,实验组中血淋巴与肝胰腺的ACP、AKP和CAT活力在不同的时间段虽然有所增强,但两者之间无显著差异。因此,认为SOD、PO活性可以作为背角无齿蚌免疫抗病功能指标参数,而ACP、AKP及CAT活性能否作为该参数还有待于进一步研究。     

邻苯二甲酸二丁酯(DBP)胁迫对三疣梭子蟹亚急性毒性

研究不同浓度邻苯二甲酸二丁酯(DBP)对三疣梭子蟹相关酶活的影响及组织损伤。设置了三个处理浓度(0.03 mg/L、0.2 mg/L和0.4 mg/L)分别在第0、5、10、15、20 d进行取样。结果表明,随着DBP浓度的增大,三疣梭子蟹死亡率升高。在0.03 mg/L浓度组胁迫下,血清超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和碱性磷酸酶(AKP)活性呈现升高-下降交替波动,过氧化物酶(POD)活性在第10 d低于对照组(P<0.05),其余时间与对照组持平,酸性磷酸酶(ACP)活性在第5 d较对照组显著升高(P<0.05),此后一直保持对照组水平;在0.2 mg/L浓度组胁迫下,血清SOD活性呈现先升高后下降的趋势,POD活性变化不明显,CAT活性先下降后升高,此后恢复到对照组水平,ACP活性在第15 d出现最高值,其余时间与对照组持平,AKP活性呈现先升高后恢复的波动趋势;0.4 mg/L浓度组SOD、CAT和AKP活性呈现先升高后下降的趋势,POD活性和ACP活性在第20 d较对照组低(P<0.05),其余时间与对照组持平。组织切片观察可见,DBP胁迫损伤了三疣梭子蟹肝胰腺和鳃组织,DBP浓度越大,胁迫时间越长,损伤越明显。     

红毛五加多糖AHP-II对THP-1源巨噬细胞中酶活性的影响

本研究检测红毛五加多糖AHP-II对THP-1巨噬细胞内多种酶活性的影响。通过PMA诱导THP-1成为巨噬细胞,用不同浓度的AHP-II与巨噬细胞共孵育48 h后,测定超氧化物歧化酶(SOD)、琥珀酸脱氢酶(SDH)、酸性磷酸酶(ACP)、ATP酶的活性及溶菌酶(LZM)含量变化。结果显示AHP-II能显著增强细胞内的SOD、SDH、ACP和ATP酶的活性及LZM的含量。     

镉对长江华溪蟹免疫相关酶活性的影响

采用浸浴法研究了镉(Cd2+)对长江华溪蟹Sinopotamon yangtsekiense免疫系统相关酶的影响.Cd2+处理组浓度分别为7.25 mg/L、14.5 mg/L、29mg/L、58 mg/L和116 mg/L,同时设对照组.分别在Cd2+处理24 h、48h、72 h和96 h取血清进行酚氧化酶(PO)、超氧化物歧化酶(SOD)、酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活力测定.结果显示,在相同处理时间,随着cd2+浓度增加,PO和SOD活力均呈现先升后降的趋势,且分别于24 h、7.25 mg/L和96 h、14.5 mg/L时达到最大值.当处理浓度相同、作用时间延长时,ACP活力先升后降,且在48 h、58 mg/L时升至最高,AKP活力则持续上升.表明Cd2+对长江华溪蟹免疫相关酶活力具有不同程度的影响,且其作用和效果具有浓度及时间效应.PO和SOD活力变化能够灵敏反映Cd2+对水生动物的胁迫程度,可作为指示镉污染的生物学指标.     

高温胁迫对厚壳贻贝摄食、代谢和相关酶活性的影响

为了探讨高温胁迫对厚壳贻贝生理代谢和免疫功能的影响,测定了不同温度胁迫水平(20→25℃; 20→30℃)下厚壳贻贝的滤水率、耗氧率、排氨率以及不同组织己糖激酶(HK)、丙酮酸激酶(PK)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)和超氧化物歧化酶(SOD)的活性。结果表明:25℃胁迫3 h后厚壳贻贝滤水率升高至0.56 L·g-1·h-1,但与对照组相比无显著变化,30℃处理后滤水率则显著降低;高温胁迫处理后,厚壳贻贝的耗氧率与排氨率均显著升高,25℃与30℃处理组间耗氧率无显著差异,但30℃处理组排氨率显著低于25℃;与对照组相比,外套膜和鳃HK活性无显著变化,但肝胰腺HK活性显著降低,鳃部HK活性显著高于外套膜和肝胰腺;外套膜PK活性显著升高,在30℃达到最大值,而25℃胁迫处理组鳃PK活性却显著降低;高温胁迫后外套膜ACP活性显著降低,25℃处理组肝胰腺ACP活性显著降低,但各处理组间鳃ACP活性无显著差异; 25℃下外套膜AKP活性显著升高,30℃时鳃和肝胰腺AKP活性显著低于对照组; 30℃胁迫下外套膜SOD活性显著升高,鳃和肝胰腺SOD活性无显著变化,肝胰腺SOD活性显著低于鳃和外套膜。实验表明,一定温度变化范围内,厚壳贻贝可通过调节自身代谢水平应对温度升高带来的胁迫,但短时间内大幅升温显著影响厚壳贻贝的摄食、代谢和免疫反应。厚壳贻贝代谢酶和免疫酶活性具有组织特异性。     

基于原核表达的副溶血性弧菌类毒素疫苗对海水鱼类免疫保护作用

目的:实现副溶血性弧菌溶血毒素基因tdh克隆与表达,并以表达产物TDH作免疫原,研究其对海水鱼免疫活性的影响.方法:PCR扩增tdh;构建重组质粒(pET-28-TDH);IPTG诱导表达;SDS-PAGE检测:以纯化的TDH融合蛋白脱毒为类毒素免疫原免疫健康真鲷,测定其血清中过氧化氢酶(CAT)、酸性磷酸酶(ACP)、超氧化物歧化酶(SOD)和碱性磷酸酶(AKP)活性变化;用副溶血性弧菌病原攻毒,检测免疫保护作用.结果:tdh基因成功克隆并表达;表达蛋白相对分子量为21 kDa,证实为TDH蛋白;类毒素免疫真鲷后,其血清中总超氧化物歧化酶(T- SOD)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)和过氧化氢酶(CAT)活力在注射后48h、24h、24h、2Ah达到最高,最高点分别高于对照组77％、328％、75％、381％;72 h后均恢复至对照组水平.结论:类毒素对真鲷免疫系统具有明显的刺激作用;类毒素免疫后对攻毒的真鲷保护率达50％.     

低盐度胁迫对银鲳幼鱼肝脏抗氧化酶、鳃和肾脏ATP酶活力的影响

通过逐级降低水体盐度的方法,将银鲳幼鱼分别在盐度25、20、15和10的条件下饲养120 h,检测不同盐度下、不同时间点银鲳幼鱼肝脏中抗氧化酶、鳃和肾脏ATP酶的活力.结果表明:随着盐度的降低和处理时间的延长,肝脏中超氧化物歧化酶(SOD)和谷胱甘肽-S-转移酶(GST)活力总体表现出先升后降的趋势(P<0.05);而过氧化氢酶(CAT)的活力除在盐度20的24 h和盐度15的48 h略有上升外,其他各时间点的酶活力均低于对照组(P<0.05);谷胱甘肽过氧化物酶(GPX)活力表现出逐步升高的趋势(P<0.05);谷胱甘肽还原酶(GR)活力在盐度15的24 h时出现上升,随后下降到较低水平(P<0.05).鳃和肾脏中Na+/K+-ATP酶和Ga2+/Mg2+-ATP酶活力总体均表现为先升后降的趋势(P<0.05),只是在两种器官中ATP酶上升的起始盐度和时间有所不同.适当降低水体盐度可以激活和增强银鲳幼鱼肝脏中的抗氧化酶、鳃和肾脏ATP酶活力,消除机体中过多的活性氧自由基和稳定细胞内外渗透压平衡.但不同酶被激活具有一定的组织器官特异性和时序性,而且当达到机体的耐受极限后,酶活力反被抑制.     

Immune responses and gene expression in hepatopancreas from Macrobrachium rosenbergii challenged by a novel pathogen spiroplasma MR-1008

Freshwater prawn Macrobrachium rosenbergii inoculated with 100 μl novel pathogen spiroplasma strain MR-1008 in logarithmic phase (10⁸ spiroplasmas ml^?1) were examined for alkaline phosphatase (AKP) activity, acid phosphatase (ACP) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, as well as expressions of 7 immune related genes in hepatopancreas after 1–28 d. Hematoxylin-eosin (HE) staining showed obvious pathological features in hepatopancreas connective and epithelial tissue. Enzyme activity analyze showed that hepatopancreas AKP and ACP activity increased markedly (P < 0.05) when inoculated with spiroplasma MR-1008 after 5 d and 10 d, respectively. SOD enzyme activity changed less obviously and slightly increased at 1 day post-inoculation, but CAT activity decreased significantly after 5 d inoculation. The expression levels of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), peroxinectin (PE), α2-macroglobulin (α2M), AKP, ACP, CAT, and copper/zinc SOD (Cu, Zn-SOD) genes in the hepatopancreas were examined by Real-Time PCR (qRT-PCR) and the results demonstrated that these immune related genes were induced by challenge with spiroplasma MR-1008. The results suggested that the prawn immune responses could be activated or inhibited by spiroplasma MR-1008, and that the hepatopancreas also plays key roles in innate immunity for defense against the pathogen.     

两种鼠蚤在新羽化和吸血后不同时间三种酶的组织化学研究

采用组织化学技术,研究了不等单蚤Monopsyllus anisus (Rothschild)和缓慢细蚤Leptopsylla segnis (Schonherr)新羽化和吸血后24 h、48 h和72 h碱性磷酸酶、酸性磷酸酶和三磷酸腺苷酶的分布和活性。显微摄影及定量图像分析结果显示：新羽化蚤碱性和酸性磷酸酶主要存在于中肠、神经细胞核、精子束头部、精巢附腺、射精管、输卵管和受精囊附腺中;三磷酸腺苷酶各组织中均有分布。吸血消化后,两种蚤中肠3种酶活性均有增强;除碱性磷酸酶在消化72 h 后酶活性有所下降外,其余不同消化时间酶活性增强程度上不存在显著差异。两种蚤卵母细胞发育成熟过程中3种酶活性亦见增强。     

Differential induction of branchial carbonic anhydrase and NA(+)/K(+) ATPase activity in the euryhaline crab,Carcinus maenas,in response to low salinity exposure

The time course of induction of activity of carbonic anhydrase (CA) and Na/K ATPase, two enzymes that are central to osmotic and ionic regulation in the eyryhaline green crab, Carcinus maenas, was measured in response to a transfer from 32 to 10 ppt salinity. CA activity was low in all gills in crabs acclimated to high salinity. Activity was induced in the posterior three gills (G6-G9) starting at 96 hr following transfer to low salinity, with activity peaking at seven post-transfer. Na/K ATPase activity in posterior gills was already high in crabs acclimated to 32 ppt salinity, and it did not increase as a result of transfer to 10 ppt. Acclimation of crabs to hypersaline (40 ppt) conditions resulted in uniformly low levels of Na/K ATPase activity, and transfer from 40 ppt to 10 ppt stimulated a four-fold induction of activity in the posterior gills that was evident by seven days of low salinity exposure. Low salinity stimulates the activity of both enzymes, but a different degree of salinity change appears to be necessary to cause the induction of each enzyme. The Na/K ATPase activity is already high at a salinity (32 ppt) at which the crab is still an osmotic and ionic conformer. CA activity, however, even when expressed in low levels, is still present in excess of what is needed to supply counterions at a rate adequate to match the rate of active ion transport. It is possible that two strategies exist for the regulation of these two enzymes that coincide with the crab's intertidal and estuarine lifestyle: short-term modulation of activity of highly expressed enzyme (Na/K ATPase) and long-term modulation of enzyme concentration by changes in gene expression (CA). For all ranges of low salinity exposure, crabs undergo hemodilution, cell swelling, and subsequent cell volume readjustment as evidenced by the increase in concentration of TNPS in the hemolymph. This response takes place before the induction of enzyme activity, and it could serve as the initial signal in the induction pathway.     

Effects of dietary live yeast Hanseniaspora opuntiae C21 on the immune and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

A feeding experiment was conducted to determine effects of Hanseniaspora opuntiae C21 on immune response and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Sea cucumbers were fed with either diets containing C21 at 10⁴, 10⁵ and 10⁶ CFU g^?1 feed or a control diet for 30–50 days, respectively. After feeding for 30 days and 45 days, five sea cucumbers from each tank were sampled for immunological analyses. Results indicated that C21 significantly improved the phagocytic activity in coelomocytes of sea cucumbers (P < 0.05). Moreover, C21 administration significantly enhanced lysozyme (LSZ), phenoloxidase activity (PO), total nitric oxide synthase (T-NOS), superoxide dismutase (SOD), alkaline phosphatase (AKP) and acid phosphatase (ACP) activities in coelomic fluid, and LSZ, T-NOS, AKP and ACP activities in coelomocytes lysate supernatant (CLS) of sea cucumbers (P < 0.05). After feeding for 45 days, 10 sea cucumbers from each dose group were challenged with V. splendidus NB13. Cumulative incidence and mortality of sea cucumbers fed with C21 were found to be lower than those of control group. After feeding for 50 days, sea cucumbers in 10⁴ CFU g^?1 C21 treatment and control tanks were subjected to acute salinity changes (from 30 to 20) for 24 h in the laboratory, and the immunological parameters were measured to evaluate the immune capacities of the A. japonicus. Phagocytic, LAZ and T-NOS activities of C21-treated group were higher than those of control group, indicating that salinity stress tolerance of sea cucumber was enhanced by C21. The present results showed that a diet supplemented with C21 could stimulate the immune system of juvenile A. japonicus thus enhancing their resistance against V. splendidus.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

The effects of gradual or abrupt changes in salinity on digestive enzymes activity of Caspian kutum,Rutilus kutum (Kamensky, 1901) larvae

This study investigates the effects of gradual or abrupt changes in rearing salinity on food transit time and digestive enzymes activity of Caspian kutum (Rutilus kutum) larvae. The larvae (532 ± 0.05 mg) were supplied and randomly allocated into 12 tanks at a density of 45 fish per tank. Experimental treatments were fresh water (salinity 0) [FW] as control, exposure to salinity 5 [T₁], and gradual transfer to salinity 10 in two steps of first to 5 h, then and after 12 h to a salinity of 10 [T₂], and abrupt change (direct transfer to a salinity of 10 [T₃]). Results showed at 8 h after start of feeding that the larvae intestine was filled with food pellets except in treatment T₁. Enzyme activity responded to salinity change as follows: the highest trypsin, amylase, and chymotrypsin activities were observed in T₁; however, these were not significantly different to treatment T₃ (P > 0.05). Trypsin activity peaks in the FW and T₂ groups occurred 8 h after feeding, and in T₃ and T₁ groups 5 h after feeding. Peak chymotrypsin and alkaline phosphatase activity was observed 5 and 8 h after feeding in all experiments, respectively. The highest α amylase activity in FW and T₂ groups occurred 5 h after feeding, while in T₃ and T₁ these peaks were observed 8 h after feeding. These results indicate that salinity had some noticeable effects on the activities of digestive enzymes after feeding.     

胎儿附睾的组织化学观察

本研究用光镜观察了在６例不同胎龄的胎儿及２例新生儿的附睾各段内ＡＫＰ、ＡＣＰ和ＳＤＨ在胎儿期附睾起始段的活性比其它各段都强,并且以３３周时为最强;至新生儿期,ＡＣＰ和ＳＤＨ在胎儿期附睾起始段的活性比其它各段都强并以３３周时最强,至新生儿期,ＡＣＰ和ＳＤＨ的组织化学活性及ＰＡＳ反应,并进行了半定量评估。结果显示：ＡＫＰ只在新生儿时期有微弱的活性;ＡＣＰ和ＳＤＨ的酶活性都比３３周时弱。ＰＡＳ阳生反应首先出现在６月龄的附睾各段,随后逐渐增强,至３３周时,附睾体、尾段的反应最为强烈,此后至新生儿期则轻微地减弱。根据文献对上述不同胎龄胎儿及新生儿附睾内酶活性及ＰＡＳ反应的变化进行了讨论。     

Separation and purification of the tonoplast ATPase and pyrophosphatase from plants with constitutive and inducible Crassulacean acid metabolism

Tonoplast vesicles were isolated from Kalanchoe daigremontiana Hamet et Pierrer de la Bâthie and Mesembryanthemum crystallinum L., exhibiting constitutive and inducible crassulacean acid metabolism (CAM), respectively. Membrane-bound proteins were detergent-solubilized with 2% of Triton X-100. During CAM induction in M. crystallinum, ATPase activity increases four-fold, whereas pyrophosphatase activity decreases somewhat. With all plants, ATPase and pyrophosphatase could be separated by size-exclusion chromatography (SEC, Sephacryl S 400), and the ATPase was further purified by diethylaminoethyl-ion-exchange chromatography. Sodium-dodecyl-sulfate electrophoresis of the SEC fractions from K. daigremontiana containing maximum ATPase activity separates several protein bands, indicating subunits of 72, 56, 48, 42, 28, and 16 kDa. Purified ATPase from M. crystallinum in the C₃ and CAM states shows a somewhat different protein pattern. With M. crystallinum, an increase in ATP-hydrolysis and changes in the subunit composition of the native enzyme indicate that the change from the C₃ to the CAM state is accompanied by de-novo synthesis and by structural changes of the tonoplast ATPase.Abbreviations CAM Crassulacean acid metabolism - DTT dithiothreitol - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - PPiase pyrophosphatase - SEC size exclusion chromatography - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol