沙门菌属外膜蛋白的抗原同源性分析

目的 分析沙门菌属外膜蛋白（outer membrane protein，OMP）的抗原同源性。方法 十二烷基肌氨酸钠法提取甲型副伤寒沙门菌、伤寒沙门菌、鼠伤寒沙门菌、大肠埃希菌、福氏志贺菌、肺炎克雷伯菌、阴沟肠杆菌和粘质沙雷菌的OMP。用甲型副伤寒沙门菌的OMP与完全佐剂制成乳油状，多次多部位免疫日本大耳兔，获得抗OMP抗体血清。35％饱和硫酸铵沉淀并纯化IgG，用HRP标记。SDS-PAGE法分离检测上述8种细菌的OMP。Western blot测定兔抗甲型副伤寒沙门菌OMP抗体与上述其他细菌OMP抗原的反应特异性。斑点印迹．酶联免疫吸附试验（Dot blot-ELISA）测定兔抗甲型副伤寒沙门菌OMP抗体与肠炎沙门菌、费氏枸橼酸杆菌、变形杆菌、铜绿假单胞菌、蜂房哈夫尼亚菌、棒状杆菌、嗜麦芽窄食假单胞菌、金黄色葡萄球菌、摩根摩根菌、屎链球菌、肺炎链球菌、新型隐球菌、热带念珠菌、光滑念珠菌和白色念珠菌提取物的反应性。结果 沙门菌属成员的OMP可与兔抗甲型副伤寒沙门菌OMP抗体血清发生特异性反应；其他革兰阴性菌OMP与之无反应；革兰阳性菌和真菌亦不发生反应。结论 沙门菌属OMP具有高度的抗原同源性，有别于其他细菌。     

新鲜羊膜致I型超敏反应的变应原性研究

研究新鲜人羊膜的变应原性及其致敏后发生I型超敏反应的可能性。建立豚鼠全身主动过敏实验模型。分新鲜羊膜组、新鲜蛋清组(阳性对照)和PBS液组(阴性对照),每组10只豚鼠。观察豚鼠在致敏期和激发后的反应,采用化学荧光法检测外周血组胺含量,血液流变分析系统检测4项血液流变学指标(全血高切变率黏度、全血低切变率黏度、血浆黏度、红细胞聚集指数)。致敏期间各组豚鼠的体重变化无明显差别(P>0.05);激发后羊膜组豚鼠与阴性组表现一致,无异常反应;羊膜组外周血组胺含量及4项血液流变学指标均与阴性对照无明显差别(P>0.05),与阳性对照有显著性差异(P<0.01)。经规范化无菌处理后的新鲜羊膜,一般不具有变应原性,不会引起I型超敏反应。     

ctB和ure I双基因融合表达及其免疫特性分析

目的构建霍乱毒素B亚单位(CtB)和幽门螺杆菌尿素膜通道蛋白(UreI)融合的原核表达质粒pET32a( )ctB/ureI,并初步研究融合蛋白CtB/UreI的表达特性和免疫特性。方法PCR从pUC18ctB中克隆ctB基因,定向在pET32a( )/ureI的ureI基因5′端插入ctB基因,构建ctB和ureI双基因原核表达质粒pET32a( )ctB/ureI,转该质粒于E.coliBL-21(DE3),经酶切和序列分析鉴定工程菌。IPTG诱导表达,HP-His亲和层析纯化,SDS-PAGE和Gel-ProAnalizer4分析,重组蛋白免疫BALB/c小鼠。用Westernblot和ELISA分析重组蛋白的免疫特性。结果工程菌含完整的ctB和ureI基因,与相对应基因的序列同源性分别为100%。在22℃,1mmol/LIPTG诱导4h后,重组蛋白的表达占菌体总蛋白12%,亲和层析纯化后蛋白纯度为94.3%。Westernblot表明重组蛋白分别能与相应的抗体反应,该蛋白免疫小鼠后能产生相应的IgG抗体。结论成功构建了能表达CtB/UreI蛋白的大肠杆菌表达菌株。对融合蛋白表达和纯化后,初步证明了该重组蛋白有CtB和UreI的双特异反应原性和免疫原性,为研究新型幽门螺杆菌疫苗奠定了坚实的基础。     

鼻内镜手术后筛窦黏膜转归的病理组织形态学观察及临床意义

目的：了解鼻内镜手术后修复各阶段筛窦黏膜的组织学转归情况，探讨术后筛窦黏膜转归过程的组织形态学规律，为完善治疗、提高手术治愈率提供科学的依据。方法：采用Messerklinger术式治疗22例成年慢性鼻窦炎（CS）及慢性鼻窦炎鼻息肉（CSNP）患者，分别在术后恢复的3个阶段进行形态学观察和光镜、电镜检查。结果：（1）术腔清洁阶段术腔分泌物潴留，光镜下上皮缺失、黏膜缺损，水肿，炎性细胞浸润，黏膜下出血；（2）黏膜转归竞争阶段可有囊泡、小息肉和肉芽生长，光镜下上皮细胞增生，部分杯状细胞增生，黏膜下腺体增生或减少；（3）上皮化完成阶段见术腔清洁，镜下大部分黏膜基本恢复正常，部分固有层为致密结缔组织取代，可见不典型腺体。结论：鼻内镜术后筛窦黏膜可以再生，鼻内镜术中、术后筛窦黏膜的正确保护和处理对黏膜再生修复有重要作用。     

五日热巴通体重组外膜蛋白HbpA的研制和抗原性分析

为克隆并表达五日热巴通体外膜蛋白HbpA的基因,并对其抗原性进行初步分析,采用PCR方法从五日热巴通体基因组DNA扩增hbpA基因,将目的基因片段插入原核表达质粒pET32a（＋）,构建重组质粒pET32a（＋）-hbpA;将构建的重组质粒转化大肠杆菌BL21（DE3）并诱导目的基因表达,以SDS-PAGE电泳以及免疫印迹实验分析表达的目的蛋白。SDS-PAGE电泳分析发现pET32a（＋）-hbpA转化菌高效表达一48kDa重组蛋白;免疫印迹分析发现该蛋白与其免疫血清发生强烈反应;间接免疫荧光分析发现该重组蛋白免疫血清能特异识别五日热巴通体。实验结果表明五日热巴通体外膜蛋白HbpA的基因已在大肠杆菌中高效表达。     

Allocentric and egocentric reference frames in the processing of three-dimensional haptic space

In an earlier experiment we showed that selective attention plays a critical role in rabbit eye blink conditioning (Steele-Russell et al. in Exp Brain Res 173:587–602, 2006). The present experiments are concerned to examine the extent to which visual recognition processes are a separate component from the motor learning that is also involved in conditioning. This was achieved by midline section of the optic chiasma which disconnected the direct retinal projections via the brainstem to the cerebellar oculomotor control system. By comparing both normal and chiasma-sectioned rabbits it was possible to determine the dependence or independence of conditioning on the motor expression of the eye blink response during training. Both normal and chiasma-sectioned animals were tested using a multiple test battery to determine the effect of this redirection of the visual input pathways on conditioning. All animals were first tested for any impairment in visual capability following section of the optic chiasma. Despite the loss of 90% of retinal ganglion cell fibres, no visual impairment for either intensity or pattern vision was seen in the chiasma animals. Also no difference was seen in nictitating membrane (NM) conditioning to an auditory signal between normal and chiasma animals. Testing for motor learning to a visual signal, the chiasma rabbits showed a complete lack of any NM conditioning. However the sensory tests of visual conditioning showed that chiasma-sectioned animals had completely normal sensory recognition learning. These results show that NM Pavlovian conditioning involves anatomically separate and independent sensory recognition and motor output components of the learning. This research was supported by S&W research grants ID# 1810 to ISR and ID# 7985 to JAC.     

丙型肝炎病毒p7膜蛋白的原子力显微镜研究

目的：以丙型肝炎病毒p7蛋白为模型，探讨原子力显微镜在膜蛋白方面的研究。方法：在云母表面准备p7蛋白样品以及将p7合并到磷脂双分子层中，进行扣击模式原子力显微镜的观察。结果：可清晰观察p7蛋白分子离子隧道结构。p7合并到磷脂双分子层中后，始终存在于DOPC液相中，并出现多个蛋白逐渐结合的现象。结论：原子力显微镜能够用于膜蛋白的基本结构特征的观察，同时可了解磷脂双分子层对膜蛋白所产生的影响，为后期细胞表面膜蛋白的原子力显微镜研究打下基础。     

Role of the hemin-binding protein 35 (HBP35) of Porphyromonas gingivalis in coaggregation

Hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis is one of the outer membrane proteins and has been reported to be a non-fimbrial coaggregation factor. In this study, a P. gingivalis HBP35-deficient mutant (MD774) was constructed from wild-type strain FDC381 by insertion mutagenesis in order to provide a better understanding of this protein's role in coaggregation. The intact cells and vesicles in FDC381 were found to have strong aggregation activities with Gram-positive bacteria. But neither the vesicles nor the intact cells showed aggregation activity in MD774. In addition, MD774 reduced autoaggregation activity. Immunoblot analysis of MD774 showed the presence of a non-maturated 45-kDa fimbrillin protein. Electron microscopy showed that the MD774 had no long fimbriae on the cell surface. Arg- and Lys-gingipain activity in MD774 was significantly decreased, compared with FDC381. Real-time RT-PCR demonstrated a significant reduction in the expression of gingipain-associated genes rgpA, rgpB, and kgp. In conclusion, we suggest that the reduction in coaggregation was caused by the combined reduction of a variety of molecules, including HBP35, gingipains, and fimbriae. Our results suggest that the HBP35 protein directly influences not only coaggregation as an adhesion molecule but also indirectly influences the expression of other coaggregation factors.     

Rapsyn carboxyl terminal domains mediate muscle specific kinase-induced phosphorylation of the muscle acetylcholine receptor

At the developing vertebrate neuromuscular junction, postsynaptic localization of the acetylcholine receptor (AChR) is regulated by agrin signaling via the muscle specific kinase (MuSK) and requires an intracellular scaffolding protein called rapsyn. In addition to its structural role, rapsyn is also necessary for agrin-induced tyrosine phosphorylation of the AChR, which regulates some aspects of receptor localization. Here, we have investigated the molecular mechanism by which rapsyn mediates AChR phosphorylation at the rodent neuromuscular junction. In a heterologous COS cell system, we show that MuSK and rapsyn induced phosphorylation of beta subunit tyrosine 390 (Y390) and delta subunit Y393, as in muscle cells. Mutation of beta Y390 or delta Y393 did not inhibit MuSK/rapsyn-induced phosphorylation of the other subunit in COS cells, and mutation of beta Y390 did not inhibit agrin-induced phosphorylation of the delta subunit in Sol8 muscle cells; thus, their phosphorylation occurs independently, downstream of MuSK activation. In COS cells, we further show that MuSK-induced phosphorylation of the beta subunit was mediated by rapsyn, as MuSK plus rapsyn increased beta Y390 phosphorylation more than rapsyn alone and MuSK alone had no effect. Intriguingly, MuSK also induced tyrosine phosphorylation of rapsyn itself. We then used deletion mutants to map the rapsyn domains responsible for activation of cytoplasmic tyrosine kinases that phosphorylate the AChR subunits. We found that rapsyn C-terminal domains (amino acids 212-412) are both necessary and sufficient for activation of tyrosine kinases and induction of cellular tyrosine phosphorylation. Moreover, deletion of the rapsyn RING domain (365-412) abolished MuSK-induced tyrosine phosphorylation of the AChR beta subunit. Together, these findings suggest that rapsyn facilitates AChR phosphorylation by activating or localizing tyrosine kinases via its C-terminal domains.     

Nano-opto-mechanical characterization of neuron membrane mechanics under cellular growth and differentiation

We designed and fabricated silicon probe with nanophotonic force sensor to directly stimulate neurons (PC12) and measured its effect on neurite initiation and elongation. A single-layer pitch-variable diffractive nanogratings was fabricated on silicon nitride probe using e-beam lithography, reactive ion etching and wet-etching techniques. The nanogratings consist of flexure folding beams suspended between two parallel cantilevers of known stiffness. The probe displacement, therefore the force, can be measured through grating transmission spectrum. We measured the mechanical membrane characteristics of PC12 cells using the force sensors with displacement range of 10 mum and force sensitivity 8 muN/mum. Young's moduli of 425 +/- 30 Pa are measured with membrane deflection of 1% for PC12 cells cultured on polydimethylsiloxane (PDMS) substrate coated with collagen or laminin in Ham's F-12K medium. In a series of measurements, we have also observed stimulation of directed neurite contraction up to 6 mum on extended probing for a time period of 30 min. This method is applicable to measure central neurons mechanics under subtle tensions for studies on development and morphogenesis. The close synergy between the nano-photonic measurements and neurological verification can improve our understanding of the effect of external conditions on the mechanical properties of cells during growth and differentiation.