A soluble SNARE drives rapid docking, bypassing ATP and Sec17/18p for vacuole fusion

Membrane fusion requires priming, the disassembly of cis-SNARE complexes by the ATP-driven chaperones Sec18/17p. Yeast vacuole priming releases Vam7p, a soluble SNARE. Vam7p reassociation during docking allows trans-SNARE pairing and fusion. We now report that recombinant Vam7p (rVam7p) enters into complex with other SNAREs in vitro and bypasses the need for Sec17p, Sec18p, and ATP. Thus, the sole essential function of vacuole priming in vitro is the release of Vam7p from cis-SNARE complexes. In 'bypass fusion', without ATP but with added rVam7p, there are sufficient unpaired vacuolar SNAREs Vam3p, Vti1p, and Nyv1p to interact with Vam7p and support fusion. However, active SNARE proteins are not sufficient for bypass fusion. rVam7p does not bypass requirements for Rho GTPases,Vps33p, Vps39p, Vps41p, calmodulin, specific lipids, or Vph1p, a subunit of the V-ATPase. With excess rVam7p, reduced levels of PI(3)P or functional Ypt7p suffice for bypass fusion. High concentrations of rVam7p allow the R-SNARE Ykt6p to substitute for Nyv1p for fusion; this functional redundancy among vacuole SNAREs may explain why nyv1delta strains lack the vacuole fragmentation seen with mutants in other fusion catalysts.     

The human megakaryocytic cell line UT‐7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor

We have demonstrated that a unique megakaryocytic cell line UT‐7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT‐7/TPO was found to express GPVI and its associate signal molecule of FcRγ (Fc receptor γ chain). When UT‐7/TPO was stimulated with the GPVI agonist convulxin, the [Ca²⁺]i (intracellular Ca²⁺) was elevated in a convulxin concentration‐dependent manner, and [Ca²⁺]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT‐7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)‐1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca²⁺]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin‐stimulated platelets failed to produce TX, it is suggested that UT‐7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT‐7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.     

西藏块菌属的分类研究

首次报道产于西藏的块菌属３种,包括２个新种和１个新记录种。新种是刘氏块菌ＴｕｂｅｒｌｉｕｉＡ．Ｓ．Ｘｕｓｐ．ｎｏｖ．和西藏块菌ＴｕｂｅｒｘｉｚａｎｇｅｎｓｅＡ．Ｓ．Ｘｕｓｐ．ｎｏｖ,新记录种是少孢块菌Ｔｕｂｅｒｏｌｉｇｏｓｐｅｒｍｕｍ（Ｔｕｌ．＆Ｃ．Ｔｕｌ．）Ｔｒａｐｐｅ．标本全部保存于西藏高原生态研究所标本室（ＨＸＺＥ）。     

玉米苗中DIMBOA与几种酚酸类物质抑菌活性比较

本文从室内培养的7日龄玉米幼苗中提取、分离、鉴定了抗性次生化合物丁布(2,4-d ihydroxy-7-m ethoxy-2H-1,4-benzoxazin-3(4H)-one,D IMBOA),并就该物质对玉米纹枯病病原菌立枯丝核菌(Rhizoctonia solani)的活性与三种酚酸类物质(阿魏酸、对羟基肉桂酸和咖啡酸)进行了离体比较研究。结果表明,丁布(D IMBOA),对立枯丝核菌有很强的生物活性,在浓度为50μg/mL时即可抑制立枯丝核菌菌丝的生长,抑制率为18.52%。阿魏酸、对羟基肉桂酸和咖啡酸,这三种酚酸在浓度250μg/mL时对立枯丝核菌菌丝的生长有抑制作用,抑制率分别为26.30%、8.50%和6.30%。不仅如此,丁布与对羟基肉桂酸之间、以及三种酚酸两两组合之间还存在一定的协同作用。在浓度相等的情况下,丁布与对羟基肉桂酸的等量混合液的抑菌率显著高于这两种物质单独存在时的抑菌率之和;同样,对羟基肉桂酸与阿魏酸的等量混合液的抑菌率比单一的对羟基肉桂酸溶液的抑菌率高18.89%,比单一的阿魏酸溶液的抑菌率高13.33%;对羟基肉桂酸与咖啡酸的等量混合液,抑菌率比两者单独试验时分别高9.63%和14.83%;阿魏酸与咖啡酸的混合液,抑菌率比两酸单独试验时分别高11.48%和22.23%。这一结果提示植物体内产生适当比例不同次生化合物的组合对植物抗病性的提高是至关重要的。     

Synergistic interaction between Gdf1 and Nodal during anterior axis development

Growth and Differentiation Factor 1 (GDF-1) has been implicated in left-right patterning of the mouse embryo but has no other known function. Here, we demonstrate a genetic interaction between Gdf1 and Nodal during anterior axis development. Gdf1-/-;Nodal+/- mutants displayed several abnormalities that were not present in either Gdf1-/- or Nodal+/- single mutants, including absence of notochord and prechordal plate, and malformation of the foregut; organizing centers implicated in the development of the anterior head and branchial arches, respectively. Consistent with these deficits, Gdf1-/-;Nodal+/- mutant embryos displayed a number of axial midline abnormalities, including holoprosencephaly, anterior head truncation, cleft lip, fused nasal cavity, and lack of jaws and tongue. The absence of these defects in single mutants indicated a synergistic interaction between Nodal and GDF-1 in the node, from which the axial mesendoderm that gives rise to the notochord, prechordal plate, and foregut endoderm originates, and where the two factors are co-expressed. This notion was supported by a severe downregulation of FoxA2 and goosecoid in the anterior primitive streak of double mutant embryos. Unlike that in the lateral plate mesoderm, Nodal expression in the node was independent of GDF-1, indicating that both factors act in parallel to control the development of mesendodermal precursors. Receptor reconstitution experiments indicated that GDF-1, like Nodal, can signal through the type I receptors ALK4 and ALK7. However, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for the effects of GDF-1 and Nodal during anterior axis development. These results indicate that GDF-1 and Nodal converge on ALK4 in the anterior primitive streak to control the formation of organizing centers that are necessary for normal forebrain and branchial arch development.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

Background

Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.

Methods

Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X₇ receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X₇ receptor.

Results

Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X₇ receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X₇ receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X₇ receptor channel plays a significant role in mediating the ATP release.

General Significance

We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.     

我国H7N2亚型禽流感病毒CK/HB/1/02株分离鉴定

从鸡组织中获得了一株分离物,能凝集鸡红细胞,经负染后电镜观察可见球形、外被囊膜的病毒颗粒,直径约90～100nm;经血凝抑制和神经氨酸酶抑制试验鉴定为H7N2亚型禽流感病毒(Avian in fluenza virus,AIV),命名为A/Chicken/Hebei/1/2002(H7N2)(简称CK/HB/1/02).将该病毒接种SPF鸡,测得静脉接种致病指数(IVPI)为0.00,剖检可见实验鸡多种组织器官有出血性变化,判为低致病力AIV;接种后7d从实验鸡泄殖腔棉拭中回收到病毒,并在血清中检测到H7亚型AIV抗体.经RT-PCR扩增了病毒HA1基因片段(约1.1kb),测定其核苷酸序列并与GenBank中的序列比较.结果表明,该病毒的HA1基因序列与AIV标准株A/Afri.Star./Eng Q/79(H7N1)的HA1基因同源性最高,为99.4%;与以色列和意大利H7N2AIV的同源性较高,为96.8%～98.2%;与美国H7N2病毒的同源性很低,约为81.0%;其HA裂解位点的氨基酸序列为KGR-GLF-,符合低致病力AIV的特征.     

Structure of a Peptidal Antibiotic P168 Produced by Paecilomyces lilacinus (Thom) Samson

( + )-α-Kainic acid (1) was synthesized by starting from a building block, N-Boc-3-acetoxyallylglycine ethyl ester (2). The key intermediate, a methyl 4-[(tert-butoxycarbonyl)prenylamino]-5-hydroxy-2-pentenoate derivative (9), was prepared from 2 in eight synthetic steps. After converting 10 into a methyl ester (11), intramolecular ene-carbocyclization of 11 gave a pyrrolidine derivative (12), which was converted to 1 in a moderate yield.