Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-c

This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO 2 substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures. © 2011 Delivopoulos, Murray.

菜花烙铁头蛇毒C���型凝集素基因的克隆与序列分析

从菜花烙铁头蛇(Trimeresurus jerdonii)的毒腺中提取mRNA,采用RT－PCR技术进行体外扩增，将扩增产物克隆到PMD18－T载体中，最后筛选出一个编码凝集素的基因，命名为TJL。由TJL基因序列推导的氨基酸序列中包含分别由23和135个氨基酸残基组成的信号肽和成熟肽。氨基酸序列比较分析表明，TJL含有半乳糖结合位点和钙离子结合位点，与蝰科蛇互凝集素TSL、PAL、APL和RSL的同源性较高(87.4%～90.4%)，与眼镜蛇科蛇互凝集素BML的同源性较低(61.5%)。

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15,536 Da) and beta (14,873 Da). The mature amino acid sequences of the a (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers Of (alphabeta)(2) and (alphabeta)(4) with molecular weights of 63,680 and 128,518 Da, respectively. The (alphabeta)(2) complex is stabilized by an interchain disulfide bridge between the two alphabeta-heterodimers, whereas the stabilization of the (alphabeta)(4) complex seems to involve non-covalent interactions between the (alphabeta)(2) complexes. (C) 2002 Elsevier Science Ltd. All rights reserved.

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR). (C) 2002 Elsevier Science Inc. All rights reserved.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) lb is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLCgamma2. Mucetininduced phosphorylation of the Fcgamma chain of platelet was greatly promoted by inhibition of alpha(llb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream Of alpha(llb)beta(3) activation are involved in dephosphorylation of Fcgamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(llb)beta(3). Inhibition Of alpha(llb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation Of alpha(llb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxaneA(2) are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Purification and properties of Aspartate aminotransferase (E.C. 2.6.1.1) from skeletal muscle of Cirrhina mrigala

Aspartate aminotransferase (E.C. 2.6.1.1.) from the skeletal muscle of fresh water fish Cirrhina mrigala has been purified 40 fold by ammonium sulphate fractionation, adsorption on alumina Csub(8) gel and chromatography using DEAE-cellulose column and the properties of the purified enzyme studied. The pH optimum of the enzyme is 7.8. The Km value of aspartic acid and 2-oxoglutaric acid are found to be 2.8 x 10sub(-3) M and 1.0 x 10sub(-4) M respectively. The activity of enzyme is inhibited by p-chloromercurybenzoate, hydroxylamine hydrochloride and sodium cyanide. The inhibition by pchloromercurybenzoate is reversed by reduced glutathione, B-mercaptoethanol and cysteine. Dicarboxylic acids such as maleic acid, malic acid and succinic acid inhibit the enzyme activity. The enzyme is not activated by any of the metal ions tested and heavy metal ions such as mercury and silver strongly inhibit the enzyme activity.

B itte r an d Sw e e t /Um am i Ta s te Re c ep to rs w ith D iffe ren tly Evo lu tio n a ry Pa thw ays

通过生物信息学和系统发育学分析,研究了苦味受体和甜味/鲜味受体的进化途径。结果显示,苦味受体 和甜味/鲜味受体在进化上具有远相关,并且具有不同的进化途径,提示这可能是导致这些受体具有不同功能,传 导不同味觉的原因。

On the reaction of marine fishes in interrupted A.C. of 50 Hz

The reactions of marine fishes in interrupted A. C. off uniform electrical field and constant current density have been studied in order to determine the optimum effective periods of shock: for narcosis and fixation. Higher effective periods were required with gradual decrease in potential difference between head and tail, which in turn depended on the length of fish and its position in electrical field. The body voltage of fish varied directly with the length and inversely with the rise of angle between their body axis and field lines. The fish subjected to higher effective period took longer time for recovery from narcosis. The effect of impulse current was manifested by increased rate of gill movements in Platax tefora, Haetodon ollaris, Crysophus burda, Siganus vermiculatus and Scolopsis leucotaenia, and lowered rate of movements in others when subjected to different effective periods inspective of length.

Reaction of fishes to the underwater A.C. field

A comparative study on the effect of A.C. field on Puntius ticto, Heteropneustis fossilis and Tilapta mossambica was carried out using a slowly rising field intensity. Well defined reactions appeared in the species of fish with slight specific variations, depending on their orientation in the electrical field, on reaching the field intensity to specific value. These reactions can be distinguished as first reaction, when the fish perceive the surrounding field, jerky swimming when parallel to the current lines (longitudinal oscillotaxis), the static position finally adopted by the fish sooner or latter depending on the potential gradient (transverse oscillotaxis), and a state of muscular rigidity (tetanus). After switching off the current, a hypnotic condition prevailed in the treated fishes before returning to their normal swimming condition. The orientation of fish body in the field had an important bearing on the behaviour reactions and current thresholds necessary for those reactions. Initial reaction, jerky swimming between electrodes and hypnosis after stoppage of current appeared in fishes earlier when the fish body was in parallel to the current lines, whereas fishes responded to transverse oscillotaxis quickly when perpendicular to current lines.

Response of fishes to the underwater D.C. field

Response to external electric field (D. C.) of three different varieties of fish namely Puntius ticto, Heteropneustis fossilis and Tilapia mossambica having different anatomical and behavioural characteristics were studied. Clearly distinguished reactions occurred one after another m all the varieties of fish with the increase in field intensity with minor specific variations. These reactions can be broadly classified into initial start (first reaction), forced swimming (galvanotaxis), slackening of body muscle (galvanonarcosis) and state of muscular rigidity (tetanus). The orientation of the organism (projection of nervous element) to the surrounding field has been found to have important bearing on the behaviour reactions. Clearly differentiated anodic taxis and true narcosis set in when fish body axis was parallel to the lines of current conduction. But with greater angle between the body axis and the current lines, fish did not show well marked reactions. Fish body, when perpendicular to current lines responded for anodic curvature and off balance swimming followed by tetanus.

Polymorphisms in the C-type lectin genes cluster in chromosome 19 and predisposition to severe acute respiratory syndrome coronavirus (SARS-CoV) infection

Background: Polymorphisms of CLEC4M have been associated with predisposition for infection by the severe acute respiratory syndrome coronavirus (SARS-CoV). DC-SIGNR, a C-type lectin encoded by CLEC4M, is a receptor for the virus. A variable number tandem

Relation between weight and linear measurement of shell in C. madrasensis (Preston)

Relation of weight to height, length and breadth in the Indian backwater oyster Crassostrea madrasensis (Preston) is reported. The relative importance of the variables on weight was found to be height, length and breadth in their order of preference. The multiple regression V = -0.4017 + 0.46743 X + 0.8278 Y + 0.1130 Z can be used to estimate the meat weight (logarithm) for given dimensions of length, height and breadth (all in logarithms). An exponential relation between weight and height is also observed.

HIV-1 C���型密码子优化gp120基因重组腺病毒载体的构建及表达

目的 构建含有HIV-1 C���型gp120基因重组腺病毒载体,并在293细胞中表达gp120蛋白.方法 PCR扩增,获得HIV-1 C���型gp120片段,定向克隆入腺病毒转移载体pTrack-CMV,线性化后转化至含有腺病毒骨架载体pAd-easy-1的大肠埃希菌BJ5183,获得重组子prAd-gp120,Pac���酶切纯化后转染293细胞,包装成复制缺陷型重组腺病毒vAd-gp120.结果 经PCR、酶切及DNA测序,插入片段大小、方向正确,获得了具有感染力的vAd-gp120重组腺病毒;通过Western 印迹检测,重组腺病毒在293细胞中表达出分子量为120 kD的蛋白.结论 成功构建了含有HIV-1 C���型gp120基因重组腺病毒载体,并获得该基因的表达.