Effects of mitochondrial ATP-sensitive potassium channel opener and hypoxic preconditioning on the hyperpolarization-activated current of sinoatrial node cells in neonatal rats

AIM: To compare the effects of hypoxic preconditioning (HP) and mitochondrial ATP-sensitive potassium (K_ATP) channel opener pretreatment on the hyperpolarization-activated current (I_f) in sinoatrial node cells.METHODS: Sinoatrial node cells were randomized to five groups: ① Control;②Hypoxia/reoxygenation (H/R);③ HP;④ Diazoxide (mitochondrial K_ATP channel opener)+H/R;⑤ 5-HD (mitochondrial K_ATP channel blocker)+HP.At the end of the experiment, I_f was recorded by whole-cell patch clamp technology.RESULTS: ①H/R significantly enhanced the current densities of I_f, shifted the current activation curve to more positive value by changing the half-activation voltage from (-98.9±2.4)mV to (-85.2±2.5) mV (P＜0.01).② Both diazoxide pretreatment and HP remarkably reduced the augmented current density caused by H/R and shifted the current activation curve to more negative value by changing the half-activation voltage to (-90.7±5.0) mV (P＜0.01) and (-92.2±1.9) mV (P＜0.01).③ 5-HD pretreatment blocked the effects of HP and reversed the half-activation voltage to (-86.3±2.7) mV (P＜0.01).CONCLUSION: The study demonstrates that both mitochondrial K_ATP channel opener pretreatment and HP make current density and kinetics of I_f to approach normal level and to maintain electrophysiological stability of sinoatrial node cells during H/R.     

FAD-dependent sulfhydryl oxidase activity of augmenter of liver regeneration protein and its activity motif

AIM:To establish a sensitive and effective method to determine whether augmenter of liver regeneration protein (ALRp) has the activity of sulfhydryl oxidase.METHODS:The cysteine at the position of 65 in CXXC motif contained in ALRp was changed into alanine through site-directed mutation.There were four groups in the experiment:① ALR group;② ALR-FAD group;③Mutated ALR-FAD group;④ Control group.Using GSH as substrate,at different time points,the concentration of thiol in each group was measured.RESULTS:The concentration of thiol groups in the ALR-FAD group was decreased during the time of the experiment.But the thiol in the other groups didn’t show any significant decrease.CONCLUSION:A sensitive and effective method to determine the sulfhydryl oxidase activity of ALRp was established.ALRp is a FAD-dependent sulfhydryl oxidase,and the conserved CXXC motif is indispensable to ALRp.     

Role of neutrophil elastase and myeloperoxidase in asthma of rats

AIM:To observe the changes of polymorphonuclear leukocytes (PMN), neutrophil elastase (NE) and myeloperoxidase (MPO) expression in rat asthma. METHODS:Eighteen rats were randomly divided into two groups on average, including asthma group and control group. The rat model of asthma was established by the ovalbumin (OVA) challenge methods. Blood PMN were isolated andpurified. The protein expression of MPO were detected by immunohistochemistry and chromatometry techniques. NE was detected by ELISA. RESULTS:(1) Immunohistochemistry showed that the levels of MPO expression around bronchus and in purified PMN in asthma group were significantly increased compared with those in control group (P<0.01). Activities of MPO were significantly increased in bronchoalveolar lavage fluid (BALF) and lung tissue in asthma group compared with those in control group (P<0.05, P<0.01). (2) Levels of NE were significantly increased in BALF and PMN in asthma group compared with control group (P<0.01). (3) Number of PMN were significantly higher in BALF, bronchus and lung tissue in asthma group than those in control group (P<0.01). CONCLUSION:Levels of PMN counting and expression of MPO and NEare significantly increased in experimental asthma rats. PMN may be involved in inflammation in asthma via increasing the levels of NE and MPO.     

香榧茎段离体培养再生植株的研究

对香榧茎段不定芽诱导研究的结果表明,不定芽诱导的最佳培养条件为:培养基为B5+KT0.1mg/L+IBA0.5mg/L+0.08%活性炭+2%葡萄糖,培养温度为20℃,光强不高于400lx,该条件下的不定芽诱导率可达55%。将不定芽培养在1/2B5+IBA0.1mg/L+0.08%活性炭+2%葡萄糖的生根培养基上,生根率最高达到40%。通过器官发生途径国内外首次建立了香榧离体再生体系和快繁体系,并获得了完整的再生植株,为今后开展香榧的遗传转化和品种改良奠定了基础。     

野生松乳菇的驯化试验初探

经过2年松乳菇共生型食用菌的驯化探讨,通过野生松乳菇采集、组织分离培养,借鉴于其它食用菌的配方,找出适合松乳菇生长的人工培养基。设计4个原种培养基配方,以配方1最佳。设计二级、三级种培养基配方7个,以配方7最佳。在室内外人工模拟栽培观察均有菌丝体长出。初步摸索出驯化栽培松乳菇的方法。     

日本矮紫薇花芽形态分化的研究

日本矮紫薇花芽由中上部侧枝和主枝顶芽发育而成,花芽分化从4月末开始至5月末结束,历时30d,包括花序分化和小花分化两个过程,分为形态分化前、开始分化期、花序原基分化期、花蕾分化期、小花花萼分化期、花瓣分化期、雄蕊分化期、雌蕊分化期8个时期,花序和小花分化的顺序分别是离心和向心的.花芽分化与春梢生长有一定的相关性.     

马白细胞介素18成熟蛋白(mEIL-18)基因在大肠埃希氏菌中的高效表达与纯化

用RT—PCR从经ConA刺激的马外周血单个核细胞（PBMC）中扩增出马白细胞介素18（Equine interleukin-18，EIL-18）前体蛋白（precursor EIL-18，pEIL-18）基因的cDNA，然后克隆至载体pCR2．1-TOPO中，鉴定并命名为pCR2．1-pEIL-18。自pCR2．1-pEIL-18中扩增马白细胞介素18成熟蛋白（mature EIL-18，mEIL-18）基因，并将其亚克隆至原核表达载体pET-28a（＋）中。将筛选出的阳性克隆进行测序、诱导表达并纯化其表达产物。结果表明，mEIL-18基因全长474bp，含1个开放阅读框，编码157个氨基酸的成熟蛋白；表达产物以可溶性和包涵体两种形式存在，经SDS-PAGE和Western—blot分析，重组蛋白相对分子量约为20ku，且具有免疫生物学活性。mEIL-18基因在大肠杆菌中高效表达并在非变性和变性条件下采用Ni^＋亲和层析纯化方法获得了高纯度的重组mEIL-18，为探究马白细胞介素18的生物学活性及其应用奠定了基础。     

表达猪繁殖与呼吸综合征病毒CH-1a株核衣壳蛋白重组鸡痘病毒的构建

从含有猪繁殖与呼吸综合征病毒（PRRSV）核衣壳蛋白（N）的质粒扩增出N基因，构建禽痘病毒转移载体。该载体含有禽痘病毒早晚期启动子LP2EP2控制之下的PRRSVN基因、P11启动下的报告基因lacZ以及用于同源重组的禽痘病毒基因组的片段。在转移载体转染亲本病毒S—FPV-017感染的鸡胚成纤维细胞（CEF）之后，采用蓝色表型筛选的方法，筛选到表达N基因的重组病毒，并对其进行了6轮蚀斑纯化。PCR方法鉴定证明重组病毒的基因组中含有完整PRRSVN基因，间接免疫荧光试验证明了PRRSVN蛋白在重组病毒感染的CEF细胞中获得表达，本研究为猪繁殖与呼吸综合征非复制型疫苗的研制打下了基础。     

高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流行病学分析

为了弄清2006年在我国南方一些猪场暴发的一种以高热、高发病率和高死亡率为特征的传染病的病因,我们从12个省市48个发病猪场采集临床样品进行了猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)的分离鉴定和分子流行病学分析。结果显示从48个猪场样品中均检测出了PRRSV,通过完整的ORF5和部分Nsp2基因序列比较分析发现来自12个省市不同猪场的PRRSV高度同源,而且在Nsp2基因上都有两个相同位点的缺失;从分离的15株PRRSV中选择湖南分离株HuN4人工感染5头60日龄的健康猪,结果5头猪分别在7 d～21 d内全部死亡,感染猪的临床症状和现地所见十分相似。本次试验结果表明新出现的以Nsp2基因部分缺失为特征的PRRSV对猪是高致病性的,可能是2006年大部分猪场暴发"高热综合症"的主要病因。     

A群猪轮状病毒JS株vp7基因序列分析

根据已发表的猪轮状病毒OSU毒株vp7基因核苷酸序列ORF两端保守区序列,设计一对特异引物,以猪轮状病毒JS毒株反转录cDNA为模板,通过PCR方法扩增出长约1000 bp目的片段。将其进行T-A克隆、序列测定和分析。结果表明,vp7基因全长1062 bp,含有一个981 bp的开放阅读框,编码326个氨基酸。与已知的15个毒株vp7全长基因的核苷酸及推导的氨基酸序列比较,同源性分别为74.5%～78.5%和75.2%～83.1%,核苷酸系统发育进化树结果表明,JS毒株与轮状病毒G9型参考毒株ICB2185、O-1亲缘关系较近,分为一个群,表明JS毒株血清型为G9型。目前,我国尚未见猪及其它动物轮状病毒G9型流行株的报道。