Dynamics of Protein-Protein Interactions at the MscL Periplasmic-Lipid Interface

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.     

Cellular localization and developmental changes of the different isoforms of divalent metal transporter 1 (DMT1) in the inner ear of rats

Divalent metal transporter 1 (DMT1) is generally considered to be the major transmembrane protein responsible for the uptake of a variety of divalent cations. Four isoforms of DMT1 have been identified in mammalian cells encoded by a single gene that differ both in their N- and C-terminal sequences with two mRNA isoforms possessing an iron response element (IRE) motif downstream from the stop codon on the message. Two distinct promoter sites regulate production of the 1A or 1B isoforms (translation starts at exon 2) for both the +IRE or –IRE species of the transporter resulting in the generation of four distinct configurations of this protein. Prior studies from our laboratory using cochlear organotypic cultures isolated from postnatal day three rats (P3) have demonstrated that Mn causes significant and selective damage to sensory hair cells and auditory nerve fibers and spiral ganglion neurons in a time and concentration dependent manner. Since DMT1 plays a critical role in controlling the uptake of a variety of essential and toxic metals into the cochlea, we compared the distribution and developmental changes of the 1A, +IRE and ?IRE isoforms in rat inner ear. Results reveal that all three isoforms of DMT1 are selectively expressed in different cell populations within the cochlea and, additionally, demonstrate their cellular and subcellular distribution changes with development.     

Theoretical and Experimental Study of DNA Helix-Coil Transition in Acidic and Alkaline Medium

Abstract

The theoretical approach to the calculation of the influence of selective binding of small ligands on DNA helix-coil transition has been described in the previous paper (Lando D.Yu., J. Biomol. Struct. Dyrt., (1994)). In the present paper that method is used for the study of DNA protonation and deprotonation in acidic and alkaline medium by theoretical analysis of pH effect on DNA heat denaturation.

The mechanism of DNA protonation in acidic medium and pK values of nucleotides are well known. It gave us an opportunity to check the theory without any fitting of pK values. A good agreement between experimental and calculated functions T_m(pH) and ΔT(pH) (melting temperature and melting range width) obtained for acidic medium proved the validity of the theory. However, for alkaline medium there was not even qualitative agreement when the agreed-upon mechanism of deprotonation was considered. Looking into the cause of the discrepancy, we have studied the DNA melting for different mechanisms of deprotonation by calculation of T_m(pH) and ΔT(pH). As a result, it has been established that the discrepancy is due to deprotonation of bonded GC base pairs of helical DNA regions (pK= 11).

It was shown that the early known protonation and newly found deprotonation of helical DNA essentially stabilised double helix in alkaline and acidic medium.     

双歧杆菌对大肠癌细胞ccL187cAMP,cGMP影响的实验研究

双歧杆菌对维持机体正常微生态平衡具有重要作用。本文研究了长双歧杆菌对大肠癌细胞ｃｃＬ１８７内第二信使ｃＡＭＰ、ｃＧＭＰ浓度的影响,对长双歧杆菌的对数期培养物分别进行下列处理：活菌、死菌、代谢产物作用ｃｃＬ１８７细胞２小时后分别收集ｃｃＬ１８７细胞内的ｃＡＭＰ、ｃＧＭＰ浓度。实验表明长双歧杆菌的不同状态及代谢产物对ｃＡＭＰ、ｃＧＭＰ产生显著影响,且存在时间依赖关系,三种处理作用ｃｃＬ１８７细胞内的ｃ     

蝮蛇抗栓酶对微循环作用的实验与临床研究

对实验性失血性休克的４０只大鼠和１５只家兔静注不同剂量的抗栓酶,用激光多普勒微循环血流计测定肾表面固定部位的微循环血流量,发现用药后血流量显著增加且与尿量相一致;阻断６０只沙鼠颈动脉造成脑缺血模型,在颅顶开窗用微循环显微镜观察用药后软脑膜微循环的变化,发现微循环血流速度加快并显示一定的冲击力,且能减少白细胞数及贴壁滚动;用２０只家兔做成ＤＩＣ模型,给抗栓酶后观察肠系膜微循环,发现毛细血管开放数增多,微循环流态改善。观察３３例脑血栓形成期病人用抗栓酶治疗前后甲襞微循环的变化,发现治疗后微循环总积分值改善,流态改善更明显     

回春生及其中药合剂对脾虚动物肠道双歧杆菌的调整作用

本实验用大黄建立脾虚动物模型,以其肠道双歧杆菌水平为主要指标,配以电镜检查,观察回春生菌种及其四君子汤合剂对脾虚动物肠道菌群的调整作用。结果表明,单用回春生即可明显提高肠道双歧杆菌水平,但总体效果不如具有益气健脾作用的四君子汤。用回春生的目的在于直接补充肠道所需的双歧杆菌;四君子汤则主要使肠绒毛的形态恢复正常而改善微群落的生境。利用回春生中药合剂可作用于上述两个环节,产生协同作用,使疗效明显提高。     

人参不同部位皂甙对衰老的人胚肺成纤维细胞影响的细胞化学研究

应用细胞化学的定性、定位,显微分光光度计定量分析的方法,测定人参不同部位皂甙对低代龄和高代龄人胚肺成纤维细胞内多糖类(PAS反应)、酸性非特异性酯酶(ANAE)、碱性磷酸酶(ALP)、酸性磷酸酶(ACP)、单胺氧化酶(MAO)含量的影响,实验结果表明:人参根、果、茎叶皂甙(SRG、SFG、SSLG)使低代龄细胞的多糖类、MAO含量下降或上升,对ANAE台量没有显著影响。但对高代龄细胞,SFG、SRG、SSLG则显著增加了多糖类、ANAE、ALP、ACP的相对含量,同时降低了MAO的含量。本文提示,人参不同部位皂甙均可以提高衰老细胞内多糖类,ALP、ACP、ANAE的相对含量,降低MAO的含量,而对年青细胞的影响则很不一致。     

复方回春生对鲤暴发性肝炎治疗效果的初步研究报告

本文报道了1990年抚顺市腰堡水库网箱养殖鲤越冬中爆发性肝炎的血液学研究结果:其GPT、血脂、血清球蛋白明显高于健康鲤;血糖、血浆总蛋白量、血清白蛋白量则明显低于健康鲤;A/G明显降低。本实验应用微生态制剂复方回春生(Bifidobiogen Complex)作为饵料添加剂投喂后,检测其各项指标的变化。结果表明:投喂组鲤的GPT、血脂、血清球蛋白明显下降,均低于对照组;血糖、血浆总蛋白量、血清白蛋白量则明显上升;A/G明显升高,均超过对照组。投喂组鱼体肥满度增加,增重快,尾均重超过对照组。     

鸡卵清蛋白基因启动子调控GFP基因在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞的表达

特异性扩增家鸡卵清蛋白基因上游调控序列 1340bp～ +16 5 5bp片段和第一内含子 +49bp～ +16 5 5bp片段 ,去除pG FP N2载体自身的CMV启动子 ,分别构建了P_2.9koval GFP和P_1.5koval GFP两种表达载体 ,经测序和酶切鉴定表达载体构建正确。采用脂质体转染法分别将这两种载体、pGFP N2 (阳性对照 )质粒及阴性对照转染鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞。用荧光倒置显微镜观测绿色荧光蛋白的表达。结果表明 :两种表达质粒在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞中都可以表达荧光蛋白。结果既显示卵清蛋白第一内含子对基因的表达起到一定的调控作用 ,也显示卵清蛋白启动子对输卵管上皮细胞和卵巢细胞不存在特异性 ,并且不存在种属差异性。     

小窝蛋白-1在细胞衰老及衰老相关疾病中的作用

胞膜小窝(caveolae)是细胞质膜内陷所形成的囊状结构.小窝蛋白(caveolin)是胞膜小窝区别于其它脂筏结构的特征性蛋白分子,维持胞膜小窝的结构和功能,包括3个家族成员小窝蛋白-1、小窝蛋白-2和小窝蛋白-3.其中,小窝蛋白-1是参与胆固醇平衡、分子运输和跨膜信号发放事件的主要结构成分,从而调节细胞的生长、发育和增殖．小窝蛋白-1在细胞衰老中起着重要调控作用,主要通过p53-p21及p16-Rb信号通路抑制细胞增殖、酪氨酸激酶的级联反应,调控粘连信号级联、胰岛素信号及雌激素信号系统等途径调控衰老进程．衰老过程中不同器官小窝蛋白-1变化趋势不尽一致．近年研究还发现,小窝蛋白-1与神经系统退行性疾病、糖尿病、动脉粥样硬化等衰老相关疾病密切相关,通过调节多条信号通路参与这些疾病的发生发展．本文结合最新研究进展,对小窝蛋白-1在细胞衰老进程的作用及参与衰老相关疾病进行综述．