WHERE ARE THE GATES IN GAP JUNCTION CHANNELS?

1. In the formation and function of gap junction channels two types of gates ought to be discriminated: the docking gate and the channel gates proper. The docking gate is involved in the transformation of a closed hemichannel to a patent gap junction channel. By definition the trigger mechanism for this gate and maybe even the gate itself is contained within the extracellular loops of the gap junction proteins, the connexins. The channel gates proper determine the open and closed states of the complete gap junction channels. 2. Probing the docking gate by mutagenesis of connexins and by synthetic peptides indicates that this gate is the consequence of complex interactions between a large fraction of the amino acids comprising the extracellular loops. Probably both inter- and intra-molecular interactions are involved, and disulfide exchange may be entailed in the stabilization of the open and closed states. 3. Of the various effectors on the channel gate(s) the voltage effects have obtained the most scrutiny to date. The response of gap junction channels and hemichannels is diverse, the various channels respond differently to transjunctional and membrane potential. No equivalent to the S4 segment representing the voltage sensor in other voltage dependent ion channels is present in the connexin sequences, instead mutations in various segments of connexins have been reported to affect the voltage dependence of gap junction channels. To understand the complexity of voltage effects on gap junction channels, non-connexin peptides may need to be considered as voltage sensors or as modifiers thereof.     

A newly recognized point mutation in the cytochrome b558 heavy chain gene replacing alanine57 by glutamic acid,in a patient with cytochrome b positive X-linked chronic granulomatous disease

Molecular genetic analysis was performed in a patient with cytochrome b positive X-linked chronic granulomatous disease. A previous Southern blot study, using a cytochrome b heavy chain cDNA as probe, revealed a Pst I restriction fragment pattern for the cytochrome b heavy chain gene (CYBB) different to that of normal individuals. Since restriction length polymorphism with Pst I has never been observed in control individuals and no abnormal restriction fragment patterns in the patient's CYBB was detected with seven other enzymes used, we focussed on the single Pst I site in the CYBB cDNA as being the only mutation site responsible for his disease. A fragment of the patient's cDNA which included the Pst I site was amplified by reverse polymerase chain reaction, and loss of the Pst I site in the fragment was confirmed by incubation with Pst I. Subsequent sequence analysis of the fragment revealed a point mutation in the Pst I site (cytosine to adenine), substituting glutamic acid for alanine at position 57.     

采用基因配型行同种异体肾移植术

目的 探讨基因配型技术在同种异体肾移植中的应用。方法采用顺序特异引物聚合酶链反应法(PCR-SSP)分别进行供、受体的基因分型，为3例配型适当的终末性肾病患者行同种异体肾移植术，并观察其术后肾功能恢复情况。结果所有接受肾移植的患者于术后2d肾功能恢复至正常水平，无急性和超急性排异发生。随访11-15个月，各项检查指标均正常。结论 基因配型的准确率较之传统的血清学配型显著增加，显著提高同种异体肾移植的成功率。     

Rapid and simple isolation of DNA from agarose gels

Summary We present a simple method for the isolation of DNA from agarose gels that is economic, fast, and independent of electrical equipment. DNA fragments of up to 6 kb can be easily extracted within 5 min using a disposable plastic syringe and filter paper. Total extraction of DNA fragments between 10 and 20 kb in size is achieved by concentrating the DNA flushed from the gel in a DNA-binding column.     

rhTPO在大肠杆菌中的表达、纯化及生物活性分析

目的：探讨人促血小板生成素(human thromlbopoietin，hTPO)在大肠杆菌中表达、分离纯化及生物学活性初步鉴定。方法：利用RT-PCR法从人胎肝细胞中扩增目的基因片段，将其定向插入pQE30表达质粒T5启动子下游的多克隆区，转化大肠杆菌M15，得到pQE30-TPO的工程菌，诱导目的蛋白表达、纯化。将表达产物给血小板减少模型小鼠尾静脉注射，观察注射后不同时间血小板量的改变。结果：经异丙基硫代-β-D-半乳糖苷(isopropylthio-β-D-galactoside IPTG)诱导培养，该工程菌可以产生单一特异性的高表达蛋白条带。将纯化后的表达蛋白注射小鼠，结果显示对实验性小鼠血小板减少症具有一定的治疗作用。结论：在大肠杆菌中成功地高效表达了重组hTPO，该产物具有促血小板生成的活性。     

应用电泳技术分析飞行员富含三酰甘油脂蛋白亚组分及其代谢特征

目的 分析富含三酰甘油脂蛋白(TRLs)分子组成及其代谢特征.方法应用脂蛋白电泳技术,对30例飞行员空腹和脂肪负荷餐后血浆做脂蛋白分析,以扫描图面积表示各亚组分构成,观察脂蛋白亚组分变化特点.结果 30例飞行员TRLs清除延迟发生率46.67%.TRLs分子脂蛋白亚组分由极低密度脂蛋白(VLDL)、中密度脂蛋白(IDL)、低密度脂蛋白(LDL)、乳糜微粒(CM)和脂蛋白a[LP(a)]组成.TRLs清除延迟典型扫描图特征为LDL左侧区面积增高,呈双峰或多峰.LDL、VLDL区面积增高,伴有高密度脂蛋白(HDL)面积降低.结论 高三酰甘油血症致动脉粥样硬化作用是在代谢水平异常所致的TRLs清除延迟,TRLs清除延迟是核心环节.     

肝脏移植后的免疫损伤与热休克蛋白的表达

目的 探讨肝脏移植后的免疫损伤与几种主要分子伴侣(热休克蛋白)表达状况之间的内在规律。方法 34份移植后肝脏穿刺标本和10份正常肝脏标本，分为A(无排斥反应)组、B(轻／中度急性排斥反应)组、C(重度急性排斥反应)组、D(慢性排斥反应／肝纤维化)组、E(对照)组。进行HSP60、HSP70、HSP90、HO—1等四种分子伴侣免疫组织化学分析和图像分析。结果 B和C组各指标间无统计学差异，A、D、E与B、C组间有统计学差异。HSP60在移植肝脏中表达降低，排斥反应发生时高；HSP70和HSP90在移植肝脏中升高。HO—1肝脏移植后升高。结论 不同种类的热休克蛋白依据自身表达的特点对移植后的免疫损伤作出反应，体现了细胞的自我保护机制。     

妇科疾病中人乳头瘤病毒感染的分子流行病学调查

目的:探讨各种妇科疾病与人乳头瘤病毒感染的关系,建立不同妇科疾病和不同年龄段妇女人乳头瘤病毒感染的流行病学资料库。方法:应用反向点杂交技术分别对疾病组1 512例病人标本和正常组500例标本进行HPV检测,所得数据用SPSS 13.0软件包进行统计分析。结果:正常组500例标本中,阳性标本101例,感染率为20.2%,疾病组1 512例标本中,阳性标本523例,感染率为34.6%,其中双重感染82例,占阳性标本的15.7%,多重感染34例,占阳性标本的6.5%,而高危感染340例,感染率为22.5%,占阳性标本的65.0%,其中单纯HPV16亚型感染155例,占所有阳性标本的29.6%;不同年龄段、不同性生活史、不同妇科疾病以及不同首次性生活年龄的HPV感染检出率都有显著性差异。结论:正常组和疾病组的HPV感染率有显著性差异(2χ=36.37,P<0.01)。在HPV感染的普查中可以有重点的选择高危人群,这样更加有针对性,从而大大降低普查成本。     

钙调素拮抗剂亲脂区与亲水区之间连接链距离的研究

目的：研究钙调素拮抗剂亲脂区与亲水区之间连接链最佳的距离。方法：采用Fibonacci寻找法设计并合成了不同长度侧链的1，2－二苯乙烯类钙调素拮抗剂。结果：它们的拮抗活性表明，随着连接亲水部分氨基与亲脂苦环部分的碳链的增长，其活性增强。分子图形学研究显示，随着碳链的增长，氨基与钙调素Glu84，87的羧基的距离缩短，静电相互作用增强，拮抗剂与钙调素的结合能增加，并在碳链个数7左右达到一个极值。结论：在1，2-二苯乙烯类钙调素拮抗剂中，连接亲脂区与亲水区的脂肪链的碳原子的最佳个数在7左右。     

人白细胞介素7基因克隆及其真核表达载体的构建

目的：构建人白细胞介素7（hIL-7）的真核表达载体。方法：从人脾脏组织的总RNA中，用RT－PCR方法扩增出编码成熟hIL-7的cDNA，将其克隆于pMD18-T质粒中，并进行序列测定。再将hIL-7cDNA以正义及反义插入真核、原核双重表达载体pBK-CMV质粒，转化至大肠杆菌DH5α。最后将表达的lacz-hIL-7重组蛋白行SDS－PAGE电泳，并作考马斯亮蓝染色分析，western blot鉴定。结果：hIL-7序列测定结果与预期一致。pBK-CMV-hIL-7（正义插入）的无隆表达重组蛋白，western blot证实此蛋白为IL－7。结论：成功构建了hIL-7的真核表达载体，为进一步研究hIL-7的抗肿瘤作用创造了条件。