Differentiation of the epidermis in turtle: an immunocytochemical, autoradiographic and electrophoretic analysis

Proteins involved in the process of cornification of turtle epidermis are not well known. The present immunocytochemical, electrophoretic and autoradiographic study reports on the localization patterns and molecular weights of keratins, which are cornification proteins, and of tritiated histidine in turtle epidermis. Alpha-keratins with a molecular weight of 40-62 kDa are present in the epidermis. Beta-keratin is mainly detectable in the stratum corneum of the carapace and plastron, but is rarely present or even absent in the corneous layer of limb, tail and neck epidermis. After electrophoresis and immunoblotting with an antibody against chicken scale beta-keratin, bands at 15-17, 22-24, and 36-38 kDa appeared. This antibody recognized weaker bands at 38-40 and 58-60 kDa in the soft epidermis. After reduction and carboxymethylation of proteins extracted from carapace and plastron, but not of proteins from the soft epidermis, protein bands at 15-17 and 35-37 kDa were found when using the anti-beta 1-keratin antibody. Loricrin-, filaggrin-, sciellin-, and transglutaminase-like immunostaining was detectable only in the transitional and lowermost corneous layers of the soft epidermis. Vesicular bodies in the transitional layer were immunolabeled by the anti-loricrin antibody, and weakly by the anti-filaggrin and anti-transglutaminase antibodies. In immunoblots, the anti-loricrin antibody reacted with a major band at 50-54 kDa in both carapace-plastron and soft epidermis. The anti-sciellin antibody detected major bands at 38-40 and 50 kDa in hard epidermis, and at 50 and 54-56 kDa in soft epidermis. Filaggrin-like immunostained bands were observed at 50-55 and 62-64 kDa. This immunostaining was probably due to a common epitope in filaggrin and some keratins. Histidine was evenly incorporated in the epidermis, and the ultrastructural study showed random labeling, often associated with keratin bundles of alpha and beta-keratinocytes. Histidine-labeled protein bands were not found in the carapace-plastron. In the soft epidermis, weakly labeled bands at 15-20, 25, and 45-60 kDa were found occasionally. The latter bands probably represented neo-synthesized keratins as was also indicated by the ultrastructural autoradiographic analysis. In conclusion, our study suggests that proteins with epitopes that they have in common with cornification proteins of mammalian epidermis are also present in the epidermis of turtle.     

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.     

The consensus workshops for the detection of autoantibodies to intracellular antigens in rheumatic diseases

In 1988 and in 1989 consensus workshops were organized in order to define the interlaboratory concordance in detecting autoantibody specificities in selected sera from patients with rheumatoid disorders and to determine the possible causes of discrepancies. In total 20 sera were tested for the presence of antibodies against nRNP, Sm, Ro (SS-A), La (SS-B), Scl-70, centromeric antigens, ribosomal RNP and Jo-1. The methods used for detection by the 28 European laboratories who participated included immunofluorescence, counter-immunoelectrophoresis, immunodiffusion, immunoblotting and ELISA.

The results showed that only a combination of two or more techniques was able to detect all specificities with an adequate efficiency. Recommendations to improve the efficiency of autoantibody detection and to standardize laboratory protocols are given.     

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots: Identification of glycophorin and band 3 variant forms

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with ¹²⁵I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.     

Synaptophysin: A reliable marker for medulloblastomas

Summary Synaptophysin is an acidic, integral membrane glycoprotein (M_r 38000) of presynaptic vesicles in various neurons and neuroendocrine cells, and in tumours derived from such cells. By indirect immunofluorescence microscopy of cryostat sections, using the monoclonal antibody SY 38 to synaptophysin, a consistent positive immunoreactivity was observed in all medulloblastomas (n= 6) and neuroblastomas (n=3) as well as a ganglioneuroma and a glioneuronal hamartoma. The presence of synaptophysin in medulloblastomas was confirmed biochemically by immunoblotting experiments. For purpose of comparison, the expression of intermediate-sized filament (IF) proteins was also examined. While neurofilament proteins were consistently expressed in the neuroblastomas (3/3), the ganglioneuroma and the glioneuronal hamartoma, IF distribution in medulloblastomas was variable. A neurofilament-positive type of tumour (1/6) could be distinguished from vimentin-expressing neoplasms (4/6) by immunocytochemistry. These data indicate that synaptophysin is a reliable marker for medulloblastomas as well as other differentiated and undifferentiated neuronal tumours and in this respect is superior to the more heterogeneous expression patterns of IF proteins in these tumours.     

卵巢癌基因1和癌超甲基化基因1蛋白在人卵巢上皮性肿瘤中的表达及临床意义

目的检测卵巢癌基因1(OVCA1)和癌超甲基化基因1(HIC1)蛋白在人卵巢上皮性肿瘤中的表达,并探讨其与卵巢上皮性肿瘤临床病理特征的联系。方法免疫印迹法检测20例正常卵巢组织、20例良性卵巢上皮性肿瘤组织、20例交界性卵巢上皮性肿瘤组织及39例卵巢上皮性癌组织中OVCA1和HIC1蛋白的表达。结果①卵巢癌和交界性卵巢肿瘤中OVCA1的蛋白表达量明显低于正常卵巢组织和良性卵巢肿瘤组织,差异有统计学意义(P<0.05)。②在卵巢癌中,低分化组中OVCA1的蛋白表达量低于高分化组,差异有统计学意义(P<0.05);临床Ⅲ+Ⅳ期中OVCA1的蛋白表达量低于临床Ⅰ+Ⅱ期,差异有统计学意义(P<0.05)。③与正常卵巢组织、良性卵巢肿瘤组织和交界性卵巢肿瘤组织相比,卵巢癌组织中HIC1的蛋白表达量显著降低,差异有统计学意义(P<0.05)。④HIC1蛋白表达量的变化与卵巢癌的病理类型、分级和临床分期无关。结论 OVCA1和HIC1蛋白表达水平的下调与卵巢上皮性癌的发生相关,OVCA1可能参与了卵巢上皮性癌的发展过程。     

用反向斑点杂交法检测结核杆菌对链霉素和乙胺丁醇的耐药性

目的建立结核分支杆菌对链霉素（SM）和乙胺丁醇（EMB）耐药基因突变的快速检测方法。方法根据结核分支杆菌标准株H37Rv序列,自行设计针对rpsL和embB基因常见耐药突变的系列寡核苷酸探针,制成膜芯片,并对64例结核分支杆菌临床株的基因突变情况进行检测。结果在34株SM耐药菌中,有25株被检出在rpsL基因分析部位发生突变,检出率为73．5％（25／34）,其中23株（67．6％）为第43位密码子AAG→AGG突变,2株（5．9％）为第88位密码子AAG→AGG突变;在32株EMB耐药菌中,有19株在embB基因分析部位发生突变,检出率为59．4％（19／32）,其中12株（37．5％）为ATG→GTG突变;6株（18．8％）为ATG→ATA突变;1株（3．1％）为ATG-＊CTG突变。膜芯片检出的突变与基因测序结果一致。结论用膜芯片检测结核分支杆菌对链霉素和乙胺丁醇的耐药性,具有较高的特异性和敏感性,可作为常规药敏方法的补充,用于指导开展早期、有效的临床化疗。     

自身免疫性肝病患者自身抗体免疫学特点分析

我们对自身免疫性肝病患者血清中出现循环的自身抗体进行分析,探讨自身抗体在鉴别自身免疫性肝病及病毒性肝炎中免疫学之间的关系。一、资料与方法1.研究对象:收集2000年10月至2005年6月来我院门诊及入院就诊的肝功能生化指标。ALT≥40 U/L,且反复异常,HBsAg阴性的3500例患者。参照国际自身免疫性肝炎组修订的评分标准和美国肝病学会原发性胆汁性肝硬化(PBC)指导建议,结合相关临床资料及部分患者肝组     

结核分枝杆菌H37Rv株菌体抗原及分泌抗原分析

目的 对比分析人型结核枝杆菌（结核菌）H37Rv株固体培养菌、液体培养菌及分泌蛋白中抗原成分的差异，寻找结核菌的主要免疫抗原。方法 应用SDS-聚丙烯酰胺凝胶电泳技术、免疫印迹技术及单克隆抗体技术分析人型结核菌H37Rv株固体培养、液体培养及其分泌蛋白中的抗原成分。结果 固体培养菌与液体培养菌具有基本一致的多肽抗原成分，其主要成分分子量为79000、64000、54000、50000、28000～38000、23000等，而分泌蛋白差异稍大，其主要成分分子量为66000、63000～65000、55000、42000、32000～34000、24000、16000等。单克隆抗体进一步证明66000、55000、16000为分泌蛋白。多克隆抗体免疫印迹分析表明，菌体主要免疫原有79000、54000、38000～50000、28000蛋白成分，而分泌蛋白主要免疫原为分子量66000、55000～64000、30000～34000、24000、16000成分。结论 人型结核菌H37Rv株菌体蛋白和分泌蛋白具有不同的主要免疫原，将有助于认识H37Rv株的总体蛋白组成及寻找与结核病相关的特异性抗原成分。     

RA33/36抗原的纯化及相应抗体的检测

目的纯化RA33/36抗原,观察其检测效率。方法从小鼠Ehrlich腹水癌细胞中提取RA33/36粗抗原,使用肝素-琼脂糖凝胶CL-6B对RA33/36粗抗原进行亲和层析,SDS-聚丙烯酰胺凝胶(SDS-PAGE)和免疫印迹法(IBT)检测富含RA33/36的组分,比较粗抗原和纯化抗原的检测效果。结果SDS-PAGE和IBT显示RA33/36抗原主要存在于缓冲液C的洗脱峰中,纯化抗原较粗抗原杂蛋白条带明显减少,且更加清晰,在检测RA33/36抗体时得到了相同的阳性和阴性结果。结论纯化的RA33/36抗原较粗抗原提高了抗体检测效率,可广泛应用于临床。