Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.     

Epoxide hydrolase-catalyzed resolution of ethyl 3-phenylglycidate using whole cells of Pseudomonas sp.

A Pseudomonas sp. was isolated with enantioselective epoxide hydrolase activity to ethyl 3-phenylglycidate. Cells grown on sucrose and suspended in 10% (v/v) dimethyl formamide as co-solvent produced (2R,3S) ethyl 3-phenylglycidate with 95% ee and 26% yield in 12 h from 0.2% (w/v) of the racemate.     

Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity

Dual increases in nitric oxide ((*)NO) and superoxide anion (O(2)(*-)) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of (*)NO generation to offset the increase in O(2)(*-) that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase (*)NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of (*)NO and O(2)(*-). Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 microM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated (*)NO production and increased L(omega)-nitroargininemethylester (L-NAME)-inhibitable O(2)(*-) generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O(2)(*-) generation, confirming that during proliferation eNOS generates (*)NO. Our findings demonstrate that hsp90 plays an important role in maintaining (*)NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit (*)NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.     

A two-hit model for development of multiple endocrine neoplasia type 2B by RET mutations

Multiple endocrine neoplasia (MEN) type 2B mutations have been reported at methionine 918 or alanine 883 in the tyrosine kinase domain of the RET proto-oncogene. Recently, a new combination of two germline missense mutations at valine 804 and tyrosine 806 was identified in a patient with MEN 2B-like clinical phenotypes including medullary thyroid carcinoma, mucosal neuroma, and marfanoid habitus. In this case, valine 804 and tyrosine 806 were replaced with methionine and cysteine, respectively. In the present study, biological activities of RET with these new mutations were compared with those with known MEN 2A or MEN 2B mutations. The transforming activity of RET with the V804M/Y806C mutation was about 8- to 13-fold higher than that of RET with a single V804M or Y806C mutation. Like RET with the M918T or A883F MEN 2B mutation, the transforming activity of RET with the V804M/Y806C mutation was not affected by substitution of phenylalanine for tyrosine 905 that abolished the activity of RET with the MEN 2A mutation. On the other hand, substitution of phenylalanine for tyrosines 864 and 952 drastically diminished the activity of RET with the V804M/Y806C, M918T or A883F mutation, suggesting that these three mutant proteins have similar biological properties.     

Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (~60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.     

中国二叉蚜属研究及三新种记述(同翅目：蚜科)

本文记述中国二叉蚜属Schizaphis Borner 6种,其中包括3新种：贴梗海棠二叉蚜Schizaphis(Schizaphis)chaenometicola Zhang,sp.nov.,香草二叉蚜Schizaphis(Schizaphis)hierochlophaga Zhanget Chen,sp.nov.和长管二叉蚜Schizaphis(Schizaphis)longituberclata Zhang et Qiao,sp.nov.,并附亚属、种检索表。模式标本存放中国科学院动物研究所标本馆。     

中国五节根蚜亚科二新属五新种及一新亚种记述(同翅目:瘿绵蚜科)(英文)

作者系统整理了中国五节根蚜亚科Fordinae标本,发现2新属：离倍蚜属DimelaphisZhang,gen．nov．和拟孕蚜属SicifordaZhng,gen．nov．;5新种：榆钱毛根蚜ChaetogeolcaulmidrupaZhang,sp．nov,钝镰离倍蚜D．obtusifalcataZhang,sp．nov,金黄五节根蚜FordaauralentaZhngetQiao,sp．nov,毛多五节很蚜F.multicomaZhng,sp．nov．及六节拟孕蚜S．sexiarticulataZhang,sp．nov．二新亚种：麦拟根蚜沾化亚种ParacletuscimicformiszhanhuanusZhng,ssp.nov。模式标本保存在中国科学院动物研究所。     

棉铃虫抗药性基因频率的测定(英文)

本文介绍了用单个棉铃虫测定杀虫药剂抗性基因频率的灵敏方法。该技术是基于比较三个相等量单个棉铃虫的乙酰胆碱酯酶的活性,每个棉铃虫要用微量滴定板同时做3个试验（也可用Enppen－dorf管）（No．1－3）：1）不加抑制剂（No．1）;2）不加酶源（No．2）;3）加一定浓度的残杀威以抑制编码敏感AceS等位基因的乙酰胆碱酯酶,但不能抑制编码抗性AceR等位基因的乙酰胆碱酯酶。如果No．1和No．3的强度一样强,说明残杀威对乙酰胆碱酯酶的活性没有影响,那么基因型应是纯合抗性（AceRR,No3＝No．1）。No．2和地．3的强度一样弱时,说明残杀威完全抑制了乙酰碱酯酶的活性,那么基因型应是纯合敏感（AceSS,No．3＝No．2）。No．3的强度介于No．1（强）和No．2（弱）之间显示出残杀威部分地抑制了乙酰胆碱酯酶的活性;基因型是杂合型（AceSR,No．2＜No.3＜No．1）。作者用该方法测定了1995,1996两年从河北省邯郸和固安县棉田采集的棉铃虫,在邯郸棉铃虫抗药性高发地区,其抗性基因频率AceRR分别是68．3％和65．3％;但在棉铃虫抗药性低发区的固安县其抗性基因频率分别是23．9％和29．4％。     

黑曲霉3.795glaA基因及其5'调控区的克隆和序列分析

黑曲霉Ｔ２１是由黑曲霉３．７９５经诱变育种获得的糖化酶高产菌株,为阐明其高产的分子机制,由黑曲霉３．７９５克隆了糖化酶结构基因及其５′旁侧序列,并与黑曲霉Ｔ２１的相应序列进行了比较．由黑曲霉３．７９５菌丝体分离染色体ＤＮＡ,Ｓｏｕｔｈｅｒｎ杂交分析表明,糖化酶结构基因位于～２．５ｋｂ的ＥｃｏＲⅠ－ＥｃｏＲⅤ染色体ＤＮＡ片段上,在此ＥｃｏＲⅠ位点上游约１．０ｋｂ处有一ＳａｌⅠ位点．为构建糖化酶结构基因及其５′旁侧序列的基因组文库,该染色体ＤＮＡ分别用ＥｃｏＲⅠ＋ＥｃｏＲⅤ和ＥｃｏＲ＋ＳａｌⅠ消化,琼脂糖凝胶电泳分离并回收长度在１．０ｋｂ左右和２．５ｋｂ左右的ＤＮＡ片段,分别与ｐＵＣ１９载体连接后转化入Ｅ．ｃｏｌｉＤＨ５．用原位杂交方法筛选到了携带糖化酶基因编码区及其１５０５ｂｐ５′旁侧序列的阳性克隆．对克隆片段的ＤＮＡ序列进行了测定并与黑曲霉Ｔ２１的相应序列进行了比较,结果表明,在糖化酶基因编码区及其１５０ｂｐ３′非编码区内,未发现碱基差异,但在－３４０～－１５０５的５′上游区内发生了９个位置的碱基变化,包括缺失、插入和替换．这些结果表明,黑曲霉Ｔ２１与３．７９５的糖化酶产量的差异与其结构基因无关,但可能与其