Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.     

Enhancement of albumin expression in bone tissues with healing rat fractures

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.     

Fracture healing as a post-natal developmental process: molecular,spatial, and temporal aspects of its regulation

Fracture healing is a specialized post-natal repair process that recapitulates aspects of embryological skeletal development. While many of the molecular mechanisms that control cellular differentiation and growth during embryogenesis recur during fracture healing, these processes take place in a post-natal environment that is unique and distinct from those which exist during embryogenesis. This Prospect Article will highlight a number of central biological processes that are believed to be crucial in the embryonic differentiation and growth of skeletal tissues and review the functional role of these processes during fracture healing. Specific aspects of fracture healing that will be considered in relation to embryological development are: (1) the anatomic structure of the fracture callus as it evolves during healing; (2) the origins of stem cells and morphogenetic signals that facilitate the repair process; (3) the role of the biomechanical environment in controlling cellular differentiation during repair; (4) the role of three key groups of soluble factors, pro-inflammatory cytokines, the TGF-beta superfamily, and angiogenic factors, during repair; and (5) the relationship of the genetic components that control bone mass and remodeling to the mechanisms that control skeletal tissue repair in response to fracture.     

Are gap junction membrane plaques implicated in intercellular vesicle transfer?

Gap junction channels are concentrated in specialised plaques of plasma membrane where cells are in close apposition. In this communication evidence is provided showing that these specialised regions of membrane also provide a site for vesicular transfer between cells. Vesicle distribution in eye lenses was found to generally reflect the reported distribution of gap junction membrane plaques. In certain areas of the lens gap junction membrane plaques and vesicles could be seen to form combined, complex structures. Ultrastructure of the vesicle and gap junction membrane plaque complexes was consistent with the vesicles moving through membrane plaques from one lens fibre cell to the next. To investigate whether transport of substances was consistent with intercellular vesicle transfer, transport of various markers was investigated. Time course experiments showing the rate of uptake of various markers into the lens did not show dramatic differences for molecules smaller or larger then gap junction pores formed by connexons. While considered as a primary intercellular transport mechanism in the lens, connexon pores were not the sole agent mediating the observed transport. Other reported mechanisms of intercellular transport in the lens can only account for the movement of relatively small molecules. Vesicular transport may therefore be a major form of transport into the outer lens layers for larger molecules. Implicit in these observations is a new hypothesis for intercellular vesicle movement via gap junction membrane plaques. Intercellular vesicle movement could possibly provide a path for large molecules associated with intact vesicles to be transported into the eye lens tissue.     

Tight Junctions of the Blood–Brain Barrier

1. The blood–brain barrier is essential for the maintainance and regulation of the neural microenvironment. The blood–brain barrier endothelial cells comprise an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. The latter is realized by the tight junctions between the endothelial cells of the brain microvasculature, which are subject of this review. Morphologically, blood–brain barrier-tight junctions are more similar to epithelial tight junctions than to endothelial tight junctions in peripheral blood vessels.2. Although blood–brain barrier-tight junctions share many characteristics with epithelial tight junctions, there are also essential differences. However, in contrast to tight junctions in epithelial systems, structural and functional characteristics of tight junctions in endothelial cells are highly sensitive to ambient factors.3. Many ubiquitous molecular constituents of tight junctions have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin, and 7H6. Signaling pathways involved in tight junction regulation comprise, among others, G-proteins, serine, threonine, and tyrosine kinases, extra- and intracellular calcium levels, cAMP levels, proteases, and TNF. Common to most of these pathways is the modulation of cytoskeletal elements which may define blood–brain barrier characteristics. Additionally, cross-talk between components of the tight junction– and the cadherin–catenin system suggests a close functional interdependence of the two cell–cell contact systems.4. Recent studies were able to elucidate crucial aspects of the molecular basis of tight junction regulation. An integration of new results into previous morphological work is the central intention of this review.     

地塞米松复合罗哌卡因臂丛神经阻滞对儿童肱骨髁上骨折患儿术后镇痛的影响

摘要 目的：探讨地塞米松复合罗哌卡因臂丛神经阻滞（BPB）对儿童肱骨髁上骨折患儿术后镇痛效果的影响。方法：择期行肱骨髁上骨折手术的患儿140例，随机分组为对照组70例与试验组70例。麻醉后两组均于超声引导下实施BPB，其中对照组予以0.25%罗哌卡因药液，试验组予以0.25%罗哌卡因、0.1 mg/kg地塞米松所组成的混合药液。记录两组患儿痛觉阻滞时间；于患儿苏醒后10 min、术后2 h、术后6 h、术后12 h及术后24 h，采用FLACC评分对患儿疼痛程度进行评估；记录两组患儿术后24 h内镇痛药物使用情况；记录两组患儿术后首次下床活动时间和术后住院时间；记录两组术后24 h内不良反应发生情况。结果：与对照组相比，试验组痛觉阻滞时间显著延长（P<0.05）。与对照组相比，试验组术后2~24 h的疼痛评分均显著降低（P<0.05）。试验组术后24 h布洛芬混悬液使用次数显著少于对照组（P<0.05），曲马多使用率显著低于对照组（P<0.05）。与对照组相比，试验组下床活动时间提前（P<0.05），术后住院时间缩短（P<0.05）。两组不良反应发生率无统计学差异（P>0.05）。结论：地塞米松复合罗哌卡因行BPB能够为肱骨髁上骨折患儿提供良好术后镇痛效果，利于患儿术后恢复。     

全髋关节置换术与双极人工股骨头置换术治疗老年股骨颈骨折的临床疗效

目的：探讨全髋关节置换术（THA）与双极人工股骨头置换术（BHA）治疗老年股骨颈骨折的临床疗效。方法：选择2013 年7 月-2015 年3 月我院收治的老年股骨颈骨折患者90 例,根据手术方法不同将患者分为全髋关节置换组（THA 组）和双极人工股 骨头置换组（BHA 组）,每组45 例。观察并比较两组患者的手术时间、术中出血量、住院时间、术后并发症的发生率及手术效果。结 果：两组患者的手术时间、术中出血量及住院时间比较,差异无统计学意义（P>0.05）;THA 组并发症的发生率明显低于BHA 组, 差异具有统计学意义（P<0.05）;术后1 年,两组手术优良率比较,差异无统计学意义（P>0.05）;术后两年及三年,THA 组手术优良 率明显高于BHA 组,差异具有统计学意义（P<0.05）。结论：THA和BHA 治疗老年股骨颈骨折均具有良好的临床疗效,但THA具 有更好的远期疗效,而且术后并发症的发生率较低。     

人工半肩关节置换治疗肱骨近端粉粹骨折的疗效及影响因素分析

目的:探讨人工半肩关节置换治疗肱骨近端粉粹骨折的疗效及其影响因素。方法:选择2010年1月-2013年12月间我院收治的肱骨近端粉粹骨折患者80例,随机分为研究组和对照组,每组40例,研究组应用人工半肩关节置换治疗,对照组应用切开复位内固定治疗。对比两组的术中情况、术后疼痛强度以及不良情况发生情况,术后应用Constant-Murlay评分评价两组肩关节功能。应用多因素Logistic回归分析分析疗效影响因素。结果:研究组手术时间、术中出血量均显著低于对照组(P<0.05);研究组Constant-Murlay中疼痛、ADL、ROM和总分评分显著优于对照组(P<0.05);手术前,两组患者VAS评分无统计学差异(P>0.05),手术后两组患者VAS评分显著降低,术后5 d、15 d、30 d和60 d,研究组VAS评分显著低于对照组(P<0.05);对照组患者肌力不足、异位骨化等不良反应发生率显著高于对照组(P<0.05)。以Constant-Murlay总分为因变量,以年龄、性别、是否合并内科疾病、受伤原因、骨折分型、受伤时间为自变量,经Logistic分析显示,年龄、合并内科疾病、骨折分型、受伤时间是肱骨近端粉碎性骨折疗效的影响因素(P<0.05)。结论:人工半肩关节置换治疗肱骨近端粉粹骨折安全有效,具有很高的临床价值,值得临床推广,同时在手术中应对影响疗效的因素予以注意,提高治疗效果。     

生长激素-海藻酸钠-壳聚糖微胶囊促进骨折愈合的组织学研究

目的:观察生长激素-海藻酸钠-壳聚糖微胶囊促进兔挠骨骨折愈合的作用。方法:实验将新西兰兔80只,在制备新西兰兔右桡骨中段3mm骨缺损模型的基础上,随机分成四组:口服生长激素-海藻酸钠-壳聚糖微胶囊组、皮下注射生长激素组、口服空微胶囊组和生理盐水对照组。实验组口服生长激素-海藻酸钠-壳聚糖微胶囊和皮下注射生长激素,对照组口服空微胶囊。并于术后9、17、30、42d定期HE染色和地衣红染色观察各组的骨折愈合情况。结果:本实验HE染色结果表明,由于在骨缺损部位成纤维细胞产生的大量胶原纤维为基质,形成透明软骨及成骨细胞,骨小梁生长的基础,连接骨痂形成和骨髓腔贯通。而观察到生长激素微胶囊组各期提前生长及改建提前的形态。地衣红染色图像结果分析及直方图的分析表明:生长激素微胶囊组胶原纤维产生促进骨小梁提前形成,进而骨折处骨性骨痂的提前愈合和髓腔的提前贯通。结论:生长激素-海藻酸钠-壳聚糖微胶囊口服能促进骨折修复愈合。