Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods.

In vertebrate photoreceptors, light reduces cyclic GMP concentration and closes cGMP-activated channels to induce a hyperpolarizing response. As Ca2+ can permeate the channels and the Na(+)-Ca2+ exchanger continuously extrudes Ca2+, closure of the channel results in a reduction of the inter-rod Ca2+ concentration. This is believed to be one of the mechanisms of light-adaptation produced by activation of guanylate cyclase. Effects of Ca2+ on the cGMP phosphodiesterase (PDE) have been reported, but their physiological significance has remained unclear. We have perfused the inside-out preparation of a frog rod outer segment (I/O ROS, originally termed truncated ROS, and find that Ca2+ in a physiological range regulates the light-activation of PDE. Therefore, PDE regulation by Ca2+ must be involved in light-adaptation in rods. The effect is mediated by a newly found protein which binds to disk membranes at high Ca2+ concentrations and prolongs PDE activation.     

Calcium and light adaptation in retinal rods and cones

Retinal rods and cones respond to light with a membrane hyperpolarization. This hyperpolarization is mediated by an ionic conductance (the light-regulated conductance) that is kept open in darkness by cyclic GMP acting as a ligand, and which closes in the light as a result of an increase in cGMP hydrolysis triggered by illumination. Calcium ions appear to have a role in this phototransduction process: they provide negative feedback between the conductance, which is permeable to Ca2+ (refs 4, 5), and the concentration of cGMP, which is sensitive to Ca2+ (refs 6-8). This feedback down-regulates the sensitivity to light of a photoreceptor and probably contributes to the important phenomenon of light adaptation in vision. It is still not clear, however, how much of the light adaptation is actually attributable to this Ca2+ feedback. We have examined the responses of amphibian rods and cones to light with the Ca2+ feedback removed. Normally, the response of a cell to a step of light rises transiently to a peak, but rapidly relaxes to a lower level, indicative of light adaptation. When the feedback is removed, however, the relaxation of the response is completely absent; furthermore, the steady response levels at different light-step intensities are well predicted by a statistical superposition of invariant single-photon responses. We therefore conclude that the Ca2+ feedback underlies essentially all light adaptation in rods and cones.     

Cyclic GMP-sensitive conductance of retinal rods consists of aqueous pores

The surface membrane of retinal rod and cone outer segments contains a cation-selective conductance which is activated by 3',5'-cyclic guanosine monophosphate (cGMP). Reduction of this conductance by a light-induced decrease in the cytoplasmic concentration of cGMP appears to generate the electrical response to light, but little is known about the molecular nature of the conductance. The estimated unitary conductance is so small that ion transport might occur via either a carrier or a pore mechanism. Here we report recordings of cGMP-activated single-channel currents from excised rod outer segment patches bathed in solutions low in divalent cations. Two elementary conductances, of approximately 24 and 8 pS, were observed. These conductances are too large to be accounted for by carrier transport, indicating that the cGMP-activated conductance consists of aqueous pores. The dependence of the channel activation on the concentration of cGMP suggests that opening of the pore is triggered by cooperative binding of at least three cGMP molecules.     

Effect of ions on the light-sensitive current in retinal rods

The effect of ions on the light-sensitive current of retinal rods was studied by sucking the inner segment into a tightly fitting capillary with the outer segment projecting into a flowing solution. This new method showed that the light-sensitive pathway, in which Na+ is the normal carrier of current, has an ionic selectivity different from that of other known sodium channels. Externàl calcium has a striking effect on the current, which increased about 20-fold when all calcium was removed. Reducing the sodium concentration gradient greatly prolonged the response to a flash of light, as would be expected if internal calcium blocks sodium channels and if light releases calcium which is subsequently extruded by a sodium-calcium exchange mechanism.     

Encoding social signals in the mouse main olfactory bulb

Mammalian urine releases complex mixtures of volatile compounds that are used in reproduction, territoriality and conspecific recognition. To understand how such complex mixtures are represented in the main olfactory bulb, we analysed the electrophysiological responses of individual mitral cells to volatile compounds in mouse urine. In both males and females, urine volatile compounds evoke robust responses in a small subset of mitral cells. Fractionation of the volatile compounds using gas chromatography showed that out of the hundreds of compounds present, mitral cells are activated by single compounds. One cohort of mitral cells responded exclusively to male urine; these neurons were activated by (methylthio)methanethiol, a potent, previously unknown semiochemical present only in male urine. When added to urine, synthetic (methylthio)methanethiol significantly enhances urine attractiveness to female mice. We conclude that mitral cells represent natural odorant stimuli by acting as selective feature detectors, and that their activation is largely independent of the presence of other components in the olfactory stimulus.     

视神经损伤后视网膜细胞凋亡的研究

目的：探讨凋亡在视神经撞击和挤压的损伤导致视网膜视网膜神经节细胞死亡中的作用。方法：应用活细菌悬液荧光染色法和流式细胞仪ＰＩ染色法检测损伤后多膜神经元凋亡的形态和数据。结果：荧光染色法见视野内有散在细胞出现核染色质凝聚现象（不规则核块）,流式细胞仪ＤＮＡ直方图见正常对照组有直而高的Ｇ０／Ｇ１峰和低平的Ｇ２峰,在Ｇ０／Ｇ１之前有一细胞群体大约占细胞总数的１．６３％,损伤组在Ｇ０／Ｇ１之前有一低小的亚     

Effects on the photoresponse of calcium buffers and cyclic GMP incorporated into the cytoplasm of retinal rods

It is generally accepted that the light response in retinal rods involves a reduction of ionic permeability (predominantly to Na+) in the plasma membrane of the outer segment and that this is mediated by an internal messenger which diffuses between the disk and plasma membranes. There is controversy, however, over the identity of the diffusible substance; two alternative schemes have received widespread support (for review see refs 1,2). According to the 'calcium hypothesis', light stimulates the release into the cytoplasm of calcium, leading to the blockage of channels which are normally open in darkness, whereas based on the 'cyclic nucleotide hypothesis', cyclic GMP causes the opening of channels in the dark, but is hydrolysed by a light-activated phosphodiesterase. We report here effects of introducing calcium buffers and cyclic GMP into the rod cytoplasm by means of a patch pipette, which seem to be inconsistent with the calcium hypothesis.     

New signals from the invasive front

Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumours. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but they are the least well understood at the molecular level. Revealing their mechanisms is one of the main challenges for exploratory and applied cancer research. Recent experimental progress has identified a number of molecular pathways and cellular mechanisms that underlie the multistage process of metastasis formation: these include tumour invasion, tumour-cell dissemination through the bloodstream or the lymphatic system, colonization of distant organs and, finally, fatal outgrowth of metastases.     

扩展青霉碱性脂肪酶cDNA的克隆和表达

采用TRIzol试剂一步法所抽提的总RNA的D（260）／D（280）值为1.82,甲醛变性电泳呈现真核微生物所特有的28S rRNA和18S rRNA条带。根据扩展青霉所产碱性脂肪酶（Lip PE)N末端20个氨基酸残基序列和真核生物mRNA3‘端具有poly(A)等所提供的生物信息,采用RT－PCR技术和3‘－RACE法扩增了Lip PE成熟肽编码区和3‘非编码区的cDNA,直接将该PCR产物克隆至pUCm-T载体中。序列分析表明,该碱性脂肪酶含有258个氨基酸,其中保守的五肽序列为Gly-His-Ser-Leu-Gly。进一步采用Clot Tech公司的SMART^TM PCR cDNA文库构建试剂盒,扩增、克隆和测定了自转录起始点至编码区的cDNA片段,从而完成了Lip EP完整cDNA的分析测定。最后将编码完整脂肪酶蛋白的cDNA克隆至pGEX-5X-3表达载体中,在大肠杆菌BL21中进行IPTG诱导表达,SDS－PAGE检测结果表明,所表达的GST－Lip EP融合蛋白分子质量约为53ku;免疫印迹（Western Blotting)技术证明了所克隆的cDNA确为编码扩展青霉WMC20718脂肪酶的基因。     

频谱重叠信号分离的循环平稳算法

利用信号的循环平稳性研究了分离频谱重叠信号的算法,并以直接序列扩频系统为例进行了性能分析.该算法在接收端设置了频移滤波器,以便同时利用信号的时间相关性及频谱相关性来实现信号的分离.通过对循环平稳性和频谱相关性的分析,得出了频移滤波器的结构并采用自适应算法确定多个频移分量的最优权值.仿真结果表明,该算法可有效分离频谱重叠信号,在大干扰功率时,误码率与时域滤波器和简单直扩系统比较均低约2个数量级.     

扩展青霉碱性脂肪酶的克隆、表达及表达产物的活性分析

以扩展青霉为出发菌株,用Biospin RNA Simply P试剂盒提取其总RNA,反转录-聚合酶链式反应(RT-PCR)扩增出扩展青霉碱性脂肪酶结构基因.构建真核重组表达质粒pEL-pIC9,电转化His4缺陷型巴斯德毕赤酵母(Pichia pastoris)GS115,利用MD-MM平板及PCR方法筛选和鉴定出重组子,进行甲醇诱导表达.重组子发酵液经SDS-PAGE分析显示获得了分子量约28ku的1条特异条带,用橄榄油检验板法和NaOH滴定法测其活性,酶活可达225U/mL.结果表明,扩展青霉碱性脂肪酶基因在巴斯德毕赤酵母中能高效表达为有脂肪酶活性的高活力酶.     

马铃薯天冬氨酸蛋白酶抑制剂基因克隆和结构分析

以马铃薯基因组ＤＮＡ为模板并基于天冬氨酸蛋白酶抑制剂基因的ｃＤＮＡ序列所设计的两个引物，通过ＰＣＲ扩增得到６６６ｂｐ的天冬氨酸蛋白酶抑制剂基因编码区，并将此片段克隆到ｐＵＣ１８的ＳｍａＩ位点．序列分析结果表明该基因除去末端一终止密码子外其可译框架编码２２１个氨基酸残基，前导肽包括３２个氨基酸残基，故该抑制剂的成熟蛋白由１８９个氨基酸组成，第６７位的精氨酸为抑制胰蛋白酶的活性中心．与ｃＤＮＡ相比核苷酸的同源性为９４．３％，氨基酸的同源性为９０％．基因ＤＮＡ无内含子．     

多传感器阵列信号的分布式恒虚警检测

对于均匀Gauss背景中的Swerlling II型起伏目标的多传感器分布式恒虚警检测,为了充分利用阵列雷达接收信号间的相位关系,采用了相干检波信号处理方式.针对局部检测统计量信息量大,通信带宽小的特点,结合实际检测中信号强度未知等困难,提出了广义似然比恒虚警(GLR-CFAR)局部处理算法.仿真结果表明,该检测方式性能接近最优性能,传感器积累对信噪比改善明显,达到3～7 dB, 提高小信号目标的检测概率,且具有实现简单的优点.因此,此方法适合用于多雷达检测场合.     

地面装甲目标声信号的混沌特征提取

针对地面装甲目标辐射的噪声信号的非线性特性,为使智能地雷能够有效地识别目标,利用非线性动力学理论中的混沌原理对目标声信号进行特征提取。通过野外场地实验,采集到2种装甲目标在不同运行速度下的40组样本信号,采用改进C-C法求得信号时间序列的相空间重构参数——时延和嵌入维,再利用Wolf法得到了2种目标声信号的混沌特征量——最大Lyapunov指数。结果显示：同一目标声信号的最大Lyapunov指数相近,且与运动状态相关性不大;不同目标间声信号的最大Lyapunov指数相差较大,辨识度较高。结论证明,最大Lyapunov指数可以作为地面装甲目标识别的有效特征参量。     

甘蓝型油菜(Brassica napus)中一个未知功能蛋白基因的cDNA克隆

随着现代分子生物学的发展,许多物种,特别是拟南芥等模式生物的整个基因组序列以及其中一部分基因的功能已经比较清楚,但仍然存在大量功能未知的基因有待进一步研究,从已知片段出发,利用cDNA末端快速扩增（rapid amplification of cDNA ends,RACE）技术钓取基因全长,并结合生物信息学技术预测蛋白质结构,是研究未知基因功能的一种重要手段．     

数据文件的加密观

文章就数据库文件的加密，从现实应用入手，提出了对文件和文件中的数据进行加密的三种加密方式，同时以具体的程序应用代码分别指出了不同加密方式的特点及所适应的环境，希望为开发应用程序的同仁提供一定的参考。