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本研究旨在探讨一定理化条件下形成的蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞（SH-SY5Y）的毒性作用。采用高温（57±0.1）℃、pH 2.0甘氨酸溶液、振荡条件下孵育100 μmol·L-1的蛋清溶菌酶蛋白，使其聚集成为蛋清溶菌酶淀粉样纤维；透射电子显微镜观察蛋清溶菌酶淀粉样纤维的超微结构，ANS荧光法测定纤维的疏水性，圆二色谱法测定并计算纤维的二级结构含量；将此纤维作用于培养的SH-SY5Y神经细胞，MTT法检测细胞活力，核染色法检测细胞凋亡状况。透射电镜结果显示，蛋清溶菌酶在孵育4 d后形成了呈短杆状超微结构的淀粉样纤维；与天然蛋清溶菌酶蛋白相比，纤维的疏水性和二级结构β折叠含量均显著提高；且1~3 μmol·L-1纤维作用于SH-SY5Y细胞12、24和48 h均产生了显著的毒性作用（P<0.01），1、2和3 μmol·L-1纤维作用细胞12 h时的细胞活力分别为75.16%±15.51%、67.54%±12.13%和67.89%±10.26%，作用24 h的细胞活力分别为75.78±13.01%、58.41%±5.55%和61.90%±8.94%，作用48 h的细胞活力分别为71.59%±14.75%、55.65%±5.78%和46.45%±6.23%，细胞活力降低呈现明显的浓度依赖性和时间依赖性，且细胞核染色呈现典型的核凋亡变化。结果提示，蛋清溶菌酶在一定理化条件下可形成具有神经细胞毒性的淀粉样纤维，为解释朊蛋白或其他蛋白质形成淀粉样纤维的机制提供参考。 相似文献
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本研究旨在探讨高温酸性改造的蛋清溶菌酶对两种革兰阳性菌G+（金黄色葡萄球菌Staphylococcus aureus、枯草芽胞杆菌Bacillus subtilis）和两种革兰阴性菌G-（大肠杆菌Escherichia coli、鳗弧菌Vibrio anguillarum）的抑制作用。采用高温（57.0±0.1）℃、pH 2.0条件对蛋清溶菌酶进行改造，透射电镜观察改造蛋清溶菌酶的超微结构，8-苯胺基-1-萘磺酸（ANS）测定蛋清溶菌酶改造前后疏水性的变化，圆二色谱法与BeStSel软件分析改造后溶菌酶二级结构的改变，并采用牛津杯法测量改造后蛋清溶菌酶对供试菌的抑菌直径，生长曲线测定法测定改造后蛋清溶菌酶的最小抑菌浓度，Western blot检测4种供试菌菌液与菌体中溶菌酶含量变化。透射电镜结果显示，改造后蛋清溶菌酶为短杆状纤维结构；ANS和圆二色谱法结果显示，改造后蛋清溶菌酶疏水性增强，β折叠含量提高31.7%；牛津杯法显示，改造后蛋清溶菌酶对试验菌的抑菌强弱为鳗弧菌>枯草芽胞杆菌>金黄色葡萄球菌>大肠杆菌；生长曲线测定结果发现，鳗弧菌最小抑菌浓度为8 μmol·L-1，大肠杆菌、金黄色葡萄球菌和枯草芽胞杆菌的最小抑菌浓度为6 μmol·L-1；Western blot发现菌液中改造蛋清溶菌酶分子质量未发生改变，而菌体中溶菌酶含量和分子质量发生改变，革兰阳性菌和阴性菌均在10 ku处出现条带，且革兰阳性菌在22 ku处溶菌酶含量增加，但菌液上清均仅在14.3 ku处有条带。结果提示，改造蛋清溶菌酶对革兰阳性菌和阴性菌均具有较强的抑菌效果，为改造蛋清溶菌酶在畜牧业中的应用提供了基础数据。 相似文献
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为探明铜离子在朊蛋白构象转化中的作用机制，进一步研究构象病发生机制提供基础数据，利用DNA重组技术，绵羊朊病毒蛋白在大肠杆菌BL2l（DE3）中高效表达，获得绵羊PrP^C重组蛋白（OvPrP^C），与氯化铜在体外结合。对CuCl2处理后的蛋白进行圆二色谱（CD）和蛋白酶K（PK）消化试验进行转化调节后蛋白抗性分析。结果表明，OvPrP^C分子量为25172．80U，其二级结构主要由α-螺旋构成，含少量β-折叠。经过CuCl2体外调节后，OvPrP^C结构中α-螺旋减少，β-折叠增加。而蛋白酶K消化后，CuCl2作用后的蛋白产生了PK抗性。为进一步的神经细胞毒性试验提供了宝贵的材料，进而为探讨构象病发生机理提供有力的科学依据。 相似文献
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旨在探究GDF8基因（Growth differentiation factor 8）编码蛋白的结构和功能,对后期研究其生理机制提供理论依据。利用生物信息学技术对牛GDF8蛋白二级和三级结构进行了预测,分析了保守结构域、信号肽剪切位点、理化性质、亲疏水性、亚细胞定位和蛋白网络通路。结果显示,牛GDF8蛋白与人和小鼠具有相似的保守结构域,不稳定指数85.52,属于不稳定蛋白,其亲水性氨基酸明显多于疏水性氨基酸,为亲水性蛋白;牛GDF8蛋白在内质网分布的概率44.4%,在线粒体分布的概率33.3%,二级结构以无规则卷曲为主,折叠缠绕后形成三级结构;牛GDF8蛋白PPI网络图有11个节点,其KEGG通路主要与骨骼肌纤维发育、活化素受体信号通路、成肌细胞融合等相关。综上,推测GDF8蛋白存在于内质网和线粒体中,通过与亲水性蛋白结合调节机体生物学功能。 相似文献
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丝素蛋白是蚕丝的主要组成部分,具有较好的吸湿性、透气性、吸附性等。而现今人类生活环境中存在较多的有害金属离子,因此拓展丝素蛋白吸附金属离子的用途具有重要的意义。本文在前期研究已证实丝素蛋白具有较好的吸附重金属离子能力的基础上,进一步测定分析了不同状态下的丝素蛋白（溶液状、凝胶状、粉末状）对金属离子（以锌为例）的吸附作用,探讨了丝素蛋白二级结构的变化,阐明了丝素蛋白吸附金属离子的机理。结果显示：丝素蛋白吸附锌离子时,氢元素与氧元素参与了其反应,而氮元素也可能参与了反应,丝素蛋白肽链上的-OH、-CO-NH-以及-NH2与Zn2+通过配位键形成了配合物;且丝素蛋白β-折叠增加而无规卷曲减少,丝素蛋白与Zn2+形成的配合物具有更稳定配位结构;同时pH对该配位反应过程也存在一定的影响。 相似文献
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[目的]对非洲猪瘟病毒（African swine fever virus,ASFV）的pS273R蛋白进行生物信息学分析。[方法]采用生物信息学工具分析43株ASFV的pS273R基因序列同源性以及遗传进化关系,预测pS273R蛋白的理化性质、二级结构、磷酸化位点、糖基化修饰、亚细胞定位、三级结构。[结果 ]不同ASFV毒株的pS273R基因同源性较高,分离自中国的5个毒株的pS273R基因序列同源性为100%,与分离自其他国家的38个毒株的pS273R基因序列同源性在93.60%～100%;系统进化分析表明,中国毒株与欧洲分支的亲缘关系较近。pS273R蛋白由273个氨基酸组成,预测大小为31.58 kDa,是亲水性蛋白;二级结构以α-螺旋（Hh）、延伸链（Ee）、β-转角（Tt）、无规卷曲（Cc）4种形式为主,含28个磷酸化位点和2个潜在的N-糖基化位点,在宿主细胞的胞质中存在的可能性最高（P=47.8%）;三级结构是由二级结构α-螺旋、β-转角、β-折叠以及无规卷曲等经过旋转、折叠与卷曲等生物过程而形成。[结论]研究结果为p S273R蛋白结构和功能的深入解析以及ASFV抑制剂... 相似文献
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试验旨在对多杀性巴氏杆菌recN基因进行克隆和原核表达，并对其表达蛋白进行生物信息学分析。参照GenBank中recN基因序列（登录号：CP003313.1）设计1对引物，通过PCR扩增获得目的基因片段，构建pET-28a（+）-recN重组质粒并转化E.coliDH5α感受态细胞，提取质粒进行酶切鉴定，将鉴定正确的重组质粒转化E.coliBL21（DE3）感受态细胞，经IPTG诱导表达，对融合蛋白进行SDS-PAGE及Western blotting鉴定。结果表明，试验成功克隆了大小约为1 677 bp的recN基因序列，通过诱导表达的His-Tag融合蛋白大小约为66.94 ku，主要以包涵体形式存在。经生物信息学分析，recN蛋白的分子式为C2735H4428N786O855S16，消光系数为24 785，不稳定系数为43.99，属于不稳定蛋白；理论等电点（pI）为5.62，为酸性蛋白；总平均亲水性为-0.316，与试验表达的包涵体蛋白性质相同，即同为疏水性蛋白；recN蛋白在哺乳动物网织红细胞的半衰期为30 h，在酵母（体内）中的半衰期>20 h，在大肠杆菌（体内）中的半衰期>10 h；二级结构主要以α-螺旋（64.87%）及无规则卷曲（21.00%）为主；经疏水性分析，预测该蛋白有3个高疏水性区域和9个高亲水性区域。本试验结果为进一步探究多杀性巴氏杆菌recN基因的功能提供了参考依据。 相似文献
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本研究旨在对芦花鸡的干扰素诱导的跨膜蛋白-3（interferon-induced transmembrane protein 3，IFITM3）基因进行克隆及生物信息学分析。根据GenBank中鸡IFITM3基因序列（登录号：NM_001350061.1）设计特异性引物，利用RT-PCR技术对芦花鸡IFITM3基因进行克隆、测序和生物信息学分析。结果表明，芦花鸡IFITM3基因片段大小为414 bp，与GenBank中发表的鸡IFITM3基因序列（登录号：NM_001350061.1）同源性为99.9%；编码137个氨基酸。IFITM3基因理论分子质量为14.95 ku，理论等电点（pI）为6.89，不稳定系数为33.98，脂肪系数为103.14，平均疏水指数为0.300，存在3个明显的疏水区，无信号肽，存在2个跨膜区，分别位于第46-68、101-123位氨基酸处；二级结构预测显示，IFITN3蛋白主要以无规则卷曲为主，存在多个α-螺旋和β-折叠区域。本研究成功克隆了芦花鸡IFITM3基因，为进一步研究IFITM3蛋白功能及阐明其抗病毒分子机制奠定了理论基础。 相似文献
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Cavana P Capucchio MT Bovero A Ripanti D Catalano D Scaglione FE Miller J Blunden T Farca AM 《Veterinary pathology》2008,45(3):347-351
This report describes an uncommon case of nonamyloidotic fibrillary glomerulonephritis. A 5-year-old female European cat was presented with nephrotic syndrome. Serum biochemistry and urinalysis revealed a mild increase in cholesterol, low total protein, severe hypoalbuminemia, and high proteinuria with a high protein-to-creatinine ratio. An histologic examination revealed an interstitial nephritis and a diffuse glomerulonephritis, with multifocal thickening of the Bowman's capsule. Transmission electron microscopy showed widespread fibrillary deposits in the glomerular basement membrane and in the mesangium. These fibrils ranged between 18 and 26 nm in diameter and were Congo red negative, which allowed their differentiation from amyloid. Immunohistochemistry demonstrated expression for immunoglobulin M (IgM) and immunoglobulin G (IgG) within the mesangium. Renal deposits of Congo red-negative amyloid-like fibrils have been described in humans, horses, monkeys, and dogs. This is the first report of noncongophilic fibrillary glomerulopathy in a cat. 相似文献
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An eosinophilic substance (ES) is usually observed in the mouse nasal septum and increases in volume with aging. It has been described as amyloid in textbooks and one report. However, it has been described as "not amyloid" in other reports because there was a negative reaction to Congo red. In this study, the ES was investigated histopathologically and electron microscopically to determine whether it was amyloid or not. The ES was only observed at the interstitium of clear HE-stained nasal glands in the septum, in which 2 kinds of glands were present (dark and clear stained by HE). The volume of the ES was small in young mice and large in older ones. Neither nasal gland degeneration nor inflammation resulted, even if a large amount of the ES was observed. The ES reacted negatively to Congo red but was strongly positive to periodic acid-Schiff reaction with prior diastase treatment. In the electron microscope observation, the ES consisted of amorphous material and collagen, but no nonbranching fibrils. Similar amorphous material was also observed in the nasal gland epithelial cells and was connected to the material in the rough endoplasmic reticulum. The above-mentioned findings indicated that the ES was not amyloid and suggested the ES might consist of not only collagen but also complex carbohydrate, which was produced by the nasal gland epithelial cells. 相似文献
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Burrough ER Myers RK Hostetter SJ Fox LE Bayer BJ Felz CL Waller KR Whitley EM 《Veterinary pathology》2012,49(4):616-620
Two cases of feline thymoma with amyloid deposition were encountered between 1982 and 2010. Neoplastic cells were separated by abundant, pale eosinophilic, homogeneous material that was congophilic and birefringent. Ultrastructurally, the neoplastic cells were connected by desmosomes, and the extracellular deposits were composed of nonbranching, hollow-cored fibrils, 8-10 nm in diameter. In the case with sufficient archived tissue for additional sections, the amyloid remained congophilic following potassium permanganate incubation, and the neoplastic cells were immunoreactive for pancytokeratin. The histologic, histochemical, ultrastructural, and immunohistochemical features of both neoplasms are consistent with epithelial-predominant thymoma with the unusual feature of intratumoral amyloid deposition. The affinity of the amyloid for Congo red following potassium permanganate incubation is consistent with non-AA amyloid. The ultrastructural findings were consistent with amyloid production by the neoplastic epithelial cells. 相似文献
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Hartig W Leifsson PS Nielsen OL 《Journal of veterinary medicine. A, Physiology, pathology, clinical medicine》2005,52(9):447-453
Amyloidosis represents a heterogenous group of diseases that have in common the deposition of fibrils composed of proteins of beta-pleated sheet structure, a structure which can be specifically identified by histochemistry using the Congo red or similar stains. Amyloid consists primarily of the amyloid fibrils but also of the amyloid P component (AP). This component, which is identical with the serum counterpart (SAP), is found in all types of human amyloid, and immunohistochemical identification of AP has been proposed as an adjunct to the universal, type-independent diagnosis of human amyloidosis. In the present study of animal amyloidosis, we compared the amyloid-specific Congo red stain with an immunohistochemical protocol using an anti-human SAP antibody for the identification of amyloid in formalin fixed tissue samples. The species and types of amyloidoses investigated were: (i) seven cows, one yak (Bos grunniens), and one sheep affected with amyloidosis of presumed AA type, (ii) one dog with a pancreatic endocrine tumour producing amyloid of presumed AIAPP type, (iii) two cats with presumed AIAPP-amyloidosis of the islets of Langerhans, one cat with presumed AA-amyloidosis, and one cat with an amyloid-producing odontogenic tumour. Intense immunostaining co-localized with amyloid, identified by its congophilia and green birefringence, using a protocol without any antigen retrieval in each of the seven cows, the yak and the sheep. The method seemed more sensitive in the ruminants than the Congo red stain, but was unable to detect amyloid in the dog and the cats regardless of the application of various antigen retrieval protocols. However, specific identification of amyloid still rests on the Congo red method or similar histochemical techniques. 相似文献
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E Gruys 《Veterinary pathology》1975,12(2):94-110
Electron micrsocopic studies of bovine kidneys with amyloidosis showed macrophage-like cells in the interstitial amyloid that were either disseminated or in small groups. They had rather uniform protracted vacuoles filled with parallel fibrils. Similar vacuoles were also found in some reticular interstitial cells. In some samples electron-dense vacuoles and vacuoles containing fibrils were seen. Protracted lysosomal enzyme activity was seen in macrophage-like cells and reticular interstitial cells; these areas of activity showed green birefringence with Congo red. The vacuoles were thought to be lysosomes, and the parallel amyloid fibrils could be a consequence of intracellular formation or of phagocytosis. 相似文献
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Mutsuga M Chambers JK Uchida K Tei M Makibuchi T Mizorogi T Takashima A Nakayama H 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2012,74(1):51-57
The binding of curcumin to senile plaques (SPs) and cerebral amyloid angiopathy (CAA) was examined in the aged brain of various animal species and a human patient with Alzheimer's disease (AD), together with its binding to neurofibrillary tangles (NFTs). Brain sections were immunostained with anti-amyloid β protein 1-42 (Aβ42) and anti-amyloid β protein 1-40 (Aβ40) antibodies. These sections were also stained with alkaline Congo red, periodic acid-methenamine silver (PAM), and curcumin (0.009% curcumin solution) with or without formic acid pretreatment. The sections from the AD brain were also immunostained for anti-paired helical filament-tau (PHF-tau), and were stained with Gallyas silver for NFTs. Some SPs in the AD, monkey, dog, bear, and amyloid precursor protein transgenic mouse (APP Tg-mouse) brains contained congophilic materials, and were intensely positive for curcumin. In addition, curcumin labeled some diffuse SPs negative for Congo red in the AD, monkey, bear, and APP Tg-mouse brains. In all animals, CAA was intensely positive for both Congo red and curcumin. The specific curcumin staining activity was lost by formic acid pretreatment. In the AD brain, NFTs positive for PHF-tau and Gallyas silver were moderately stained with curcumin. These findings indicate that curcumin specifically binds to the aggregated Aβ molecules in various animals, and further to phosphorylated tau protein, probably according to its conformational nature. 相似文献
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K Uchida Y Miyauchi H Nakayama N Goto 《Nippon juigaku zasshi. The Japanese journal of veterinary science》1990,52(3):605-611
Amyloid angiopathy with cerebral hemorrhage and senile plaques was found in the brain of aged dogs. In all 9 cases examined, 13 to 19 years old, 6 males and 3 females, amyloid deposits were observed mostly in the wall of cerebral arterioles and capillaries showed hyaline degeneration. The accumulation of amyloid fibrils measuring about 10 nm in diameter was seen in the cerebral vessel wall by electron microscopy. The cerebral hemorrhage was observed in 6 of 9 dogs and 2 of them showed massive hemorrhage. The hemorrhagic foci were sometimes closely contact with the vessels involved in amyloid angiopathy. In addition, senile plaques being classified into 2 types were found in the cerebral cortex of 3 dogs. The first type was characterized by the accumulation of degenerative neurites and often contained granular argyrophilic material. The second type had a well-defined amyloid core with neuritic halo. Amyloid deposits were also found in or around intestinal vessel walls of 3 dogs. The amyloid deposited in the cerebral vessels, senile plaques and intestinal vessels showed characteristic green birefringence under the polarized light even after potassium permanganate treatment. 相似文献
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Tei M Uchida K Mutsuga M Chambers JK Nakayama H 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2012,74(4):481-483
Curcumin is a constituent phenol compound of turmeric, and has been used as a dietary spice and Indian medicine. Curcumin has been reported to inhibit the formation of amyloid β fibrils and aggregation. In this study, the binding activity of curcumin to various types of canine amyloid was examined. Tissue samples used were lesions of AA, AL, amyloid of canine amyloid-producing odontogenic tumor (Aapot), and senile cardiovascular amyloid (ScA). Curcumin stained all types of amyloid. The binding of curcumin to AA, ScA, and AL was lost by the KMnO(4) treatment, but Aapot maintained the binding. These findings indicate that curcumin binds several types of amyloid, while the binding sites of amyloid molecules might be different from that of Congo red. 相似文献