高温胁迫下绿豆象内参基因筛选及其Hsp70基因的表达特性

为明确Hsp70蛋白在绿豆象Callosobruchus chinensis高温耐受性中的作用,该研究筛选并克隆β-actin、α-tubulin、β-tubulin、EF1-α、GAPDH、Hsc70、AK和RPL40八个候选内参基因,采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术测定其在绿豆象雌雄成虫体内的表达量,通过geNorm、BestKeeperr和NormFinde软件对这8个候选内参基因的表达稳定性进行评价,选择最适宜的内参基因对不同高温胁迫下绿豆象体内Hsp70基因的表达特性进行测定。结果表明,8个候选基因引物均有良好的特异性和引物扩增效率,引物扩增效率介于91.6%～108.1%之间。高温胁迫下,β-tubulin基因较其他7个候选基因有较小的平均变异度、稳定值和标准差,为最适宜的内参基因。39、42和45℃高温处理1 h后绿豆象雌雄成虫体内Hsp70表达水平较对照显著上调,当处理时间延长至3 h后,Hsp70基因的相对表达量较对照也有不同程度上调,但差异不显著,表明绿豆象Hsp70基因可以响应高温胁迫,Hsp70蛋白在绿...     

烟草Hsc70-2的克隆及对马铃薯Y病毒侵染烟草的促进作用

【目的】马铃薯Y病毒(Potato virus Y,PVY)是危害烟草、马铃薯、番茄、辣椒等主要农作物的重要病毒,给农业生产带来巨大损失。论文旨在克隆Hsc70-2蛋白在本氏烟(Nicotiana benthamiana)中的基因序列并分析其生物学信息,研究NbHsc70-2蛋白对PVY侵染烟草的影响,为进一步解析PVY的侵染机制提供理论依据。【方法】以本氏烟为材料,克隆NbHsc70-2蛋白的基因编码序列(coding sequence,CDS),利用MEGA 6.0进行多序列比对并构建系统进化树;利用实时荧光定量PCR(qRT-PCR)分析NbHsc70-2在本氏烟各组织中的表达水平;采用在线软件BaCeILo和SignalP 4.0对NbHsc70-2进行生物信息学分析;构建NbHsc70-2-RFP融合蛋白确定该蛋白的亚细胞定位;研究PVY处理对本氏烟叶片中NbHsc70-2的影响;利用激光共聚焦观察PVY-GFP处理对NbHsc70-2-RFP定位的影响;构建NbHsc70-2 VIGS沉默体系及瞬时表达载体,采用qRT-PCR比较NbHsc70-2在沉默/过表达后对PVY表...     

Some aspects of murine experimental listeriosis

A set of experiments was carried out in order to approach the complex nature of L. monocytogenes infections from different aspects. Experiment 1 showed that Listeria are able to gain admission to body by numerous ways and both subcutaneous and oral entry can lead to fatal septicemia. It also gave slight support to the theory of direct neural transmission of Listeria to the brain and indicated the possibility that intestinal absorption after oral exposition at least partly occurs via lymphatic vessels. No inflammatory reaction could be caused to mice by ocular flushing with Listeria suspension.The second trial proved that there are vast differences in the animal pathogenecity of Listeria strains — even among those of the same serotype. In experiment 3A the abolishing effect of dextran sulfate on the early resistance of mice to Listeria was confirmed and it turned out that cortisone at a therapeutic dose level did not bring about that phenomenon. Levamisole granted no conspicuous enhancement of resistance in this acute challenge; however, the results of the immunity test (3B) suggested that levamisole may be beneficial during the induction phase. On the other hand, starvation appeared to impair long-term immunity. Likewise, in experiment 4 starved mice were quite susceptible to acute challenge with Listeria. Raised ambient temperature, on the contrary, prominently increased the survival rate of the animals.Owing to the fairly small number of animals these results should be regarded as preliminary starting points to further studies.     

控制性打开二硫键-葡聚糖修饰对大豆分离蛋白表面活性性能及结构的影响

为提高大豆分离蛋白的表面活性,试验采用过氧乙酸控制性的打开蛋白质的二硫键,并在此基础上加入葡聚糖进行复合修饰。结果表明:随着二硫键断开程度的增加,接枝度明显提升,褐变度与其有相同的变化趋势。当二硫键断开率为46%时,表面活性最理想。其中乳化性和乳化稳定性提高107.56%和175%;起泡性和起泡稳定性提高83.63%和105%;荧光图谱表明随着二硫键断开率的增加,荧光强度出现先升高后降低的趋势,而糖基化复合产物荧光强度依次降低;SDS-PAGE进一步证实,在糖基化过程中7S亚基最先消失,二硫键断开后,可以加速11S酸性亚基与葡聚糖发生糖基化反应。     

Synergistic effect of insulin and selenium in combination on inhibiting myocardial apoptosis in rats with diabetic cardiomyopathy

AIM: To investigate the effect of insulin and selenium in combination on the apoptosis and the expression of Ku70, acetylated Ku70, Bax and cytochrome C in myocardial cells of the rats with diabetic cardiomyopathy(DCM), and to explore the mechanism of insulin and selenium in their synergistic anti-DCM effect. METHODS: SD rats (n=50) were randomly grouped into control, DCM, DCM with insulin treatment (DCM+In) group, DCM with selenium treatment (DCM+Se) group, and DCM with insulin and selenium combination treatment (DCM+In+Se) group. Mitochondrial membrane potential (MMP) was measured by flow cytometry. The cell apoptosis was observed by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The levels of Ku70, Bax and cytochrome C were examined by Western blot. The acetylation status of Ku70 was detected by co-immunoprecipitation.RESULTS: The rats in DCM group showed marked cell apoptosis compared with the control rats. The levels of Ku70 and acetylated Ku70 declined significantly compared with control group. Bax significantly translocated from cytoplasm into mitochondria and cytochrome C translocated from mitochondria into cytoplasm compared with control group. Compared with DCM+In group or DCM+Se group, insulin and selenium in combination significantly inhibited the apoptosis, down-regulated Ku70 and acetylated Ku70 levels, and prevented Bax and cytochrome C translocation.CONCLUSION: Insulin and selenium synergistically inhibits myocardial apoptosis by regulating Ku70 acetylation and inhibiting Bax translocation.     

99mTc-labeled dextran for mammary lymphoscintigraphy in dogs

Lymphoscintigraphy is the technique of choice for sentinel lymph node detection in women with early breast cancer, but there is limited information evaluating the value of this technique in animals. We investigated mammary lymphatic drainage in 25 young female mongrel dogs by intramammary injection of 18.5 MBq of 99mTc-dextran (70,000 Da). Lymph node anatomical referencing was obtained using an external marker, bone scintigraphy, or scintiscanning the body contour. Cranial and caudal thoracic mammary glands drained into the cranial sternal lymph node and axillary lymph center. The cranial thoracic mammary gland also drained into the superficial cervical lymph node in two of five animals. The cranial abdominal gland was drained by the axillary lymph center. The caudal abdominal mammary gland was drained by the superficial inguinal lymph node in all animals and simultaneously by medial iliac lymph nodes in four of five animals. In one dog, this mammary gland was also drained by the mediastinal and the superficial cervical lymph nodes. The inguinal mammary gland was drained by superficial inguinal lymph nodes and simultaneously via the medial iliac lymph node in one animal. Lymphatic communications between lymph nodes were identified in 11 of 25 (44%) animals. 99mTc-dextran mammary lymphoscintigraphy was easy and rapid to perform and may provide valuable information for further studies.     

Comparative study of stress response,growth and development of uteri in post‐weaning gilts challenged with zearalenone and estradiol benzoate

The objective of this study was to evaluate the effects of zearalenone (ZEA) and estradiol benzoate (EB) on stress injury and uterine development in post‐weaning gilts. Thirty healthy post‐weaning female gilts (Duroc × Landrace × Large White) aged 28–32 days were randomly allocated to three treatments as follows: (a) basal diet (Control), (b) basal diet plus 1.0 mg/kg purified ZEA (ZEA) and (c) basal diet plus 0.75 ml (1.5 mg) EB per pig at 3‐days intervals by intramuscular injection (EB). The serum estradiol (E₂), the final and the increased vulvar area, uterine index, thickness of the myometrium and endometrium, and protein expression of heat shock protein 70 (HSP70) in ZEA group were higher than those in the control group (p < .05), but lower than those in the EB group (p < .05). The serum luteinizing hormone in ZEA group was lower than that of the control group (p < .05), but higher than that in the EB group (p < .05). Higher serum follicle‐stimulating hormone and progesterone were observed in the ZEA and control groups than those in the EB group (p < .05). The serum glutathione peroxidase activity in the ZEA group was lower than that in the control and EB groups (p < .001), and the malondialdehyde in the ZEA group was higher than that in the control and EB groups (p < .001). Moreover, the relative mRNA and protein expression of growth hormone receptor (GHR) and relative mRNA expression of HSP70 in the ZEA and EB groups were higher than those in the control group (p < .05). In conclusion, both ZEA (1.0 mg/kg) and EB (1.5 mg at 3 days intervals by intramuscular injection) stimulated vulvar swelling and uterine hypertrophy by disordering serum hormones and up‐regulating GHR expression, and induced stress by different mechanisms in this study. Furthermore, the observed up‐regulating HSP70 expression challenged by ZEA or EB may be part of the mechanism to resist stress injury.     

牛结核杆菌MPB70-ESAT-6融合蛋白的原核表达及抗原反应性分析

为增强单个蛋白的抗原性,利用(Gly4Ser)3柔性连接肽将牛结核杆菌MPB70和ESAT-6融合。采用重叠延伸PCR技术将牛结核杆菌mpb70与esat-6基因连接,获得融合基因mpb70-esat-6,连接至T-Vector pMD19中,获得克隆质粒pMD-70-esat-6。经Bam HⅠ、EcoRⅠ酶切、纯化,并与p ET28a(+)载体连接,构建了pET-70-esat-6重组表达质粒。SDS-PAGE发现,在27.2 ku处表达了融合蛋白MPB70-ESAT-6,Western blotting证实,MPB70-ESAT-6与牛结核阳性血清反应性良好。MPB70-ESAT-6融合蛋白的研究为牛结核病诊断抗原及相关疫苗研究奠定了基础。     

Reduced fasting periods increase intestinal permeability in chickens

Fasting of up to 24 hr has been shown to increase intestinal permeability (IP) in chickens. The aim of this study was to determine whether fasting duration of 4.5 and 9 hr increased IP and whether l ‐glutamine (a non‐essential amino acid) supplementation before fasting provided some protection of barrier function as shown in other species. Ross 308 male broilers (n = 96) were fed either a control diet or the same diet supplemented with 1% glutamine from d0 to d38 post‐hatch. On d37, the birds were assigned to single‐bird metabolism cages and were fasted for either 0, 4.5, 9 or 19.5 hr. This study design was 2 × 4 factorial with two levels of glutamine and four levels of fasting. Birds in the 0‐hr fasting group had free access to feed. All birds had ad libitum access to water. To measure IP on day 38, following their respective fasting periods, birds were administered two separate oral gavages of fluorescein isothiocyanate dextran (FITC‐d) followed by lactulose, mannitol and rhamnose (LMR) sugars, 60 min apart. Whole blood was collected from the jugular vein 90 min post‐LMR sugar gavage. FITC‐d and L/M/R ratios were measured by spectrophotometry and high‐performance ionic chromatography respectively. Lipopolysaccharide (LPS) endotoxins in plasma of the birds fed the control diet were also measured using chicken‐specific LPS antibody ELISA. Serum FITC‐d and plasma L/M and L/R ratios for 4.5, 9 and 19.5 hr were significantly (p < .05) higher compared to the non‐fasting group. However, IP was not different in the glutamine‐supplemented group (p > .05) compared to the control group. LPS concentrations measured by the ELISA were below the detectable range. We conclude that fasting periods of 4.5 and 9 hr increased IP compared to non‐fasted birds and dietary glutamine supplementation did not ameliorate changes in IP.