Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.     

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and ³¹P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. ³¹P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.     

Decidual NK cells alter in vitro first trimester extravillous cytotrophoblast migration: a role for IFN-gamma

Abnormal placentation results in either inadequate (consequences: recurrent miscarriage, intrauterine growth restriction, and preeclampsia) or overzealous (consequences: placenta accreta, increta, and percreta) placentation. NK cells dominate in first trimester decidua and probably control extravillous cytotrophoblast (EVT) invasion. We examined this interaction in a novel way, using NK cells and villous explants from concordant first trimester pregnancies cocultured using a new collagen (two-dimensional) model of placentation. Decidual NK (dNK) cells exerted contact-independent inhibition of normal cytotrophoblast migration, associated with changes in the cytotrophoblast expression of metalloproteases-2 and -9, and plasminogen activator inhibitor-1. dNK cells did not affect EVT proliferation and apoptosis, and cell column formation. dNK cell effects were partially reversed by neutralizing Abs against IFN-gamma. We provide ex vivo human evidence of a direct role for dNK in modulating EVT differentiation as they form columns and then migrate from anchoring villi.     

渐危植物珊瑚菜种子活力和萌发率测定

种子传播与萌发关系到植物定居、繁育和分布等重要问题,种子萌发的成功率决定于种子本身的质量和习性,以及地表环境的空间异质性.     

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

Increased release of serotonin from rat ileum due to dexfenfluramine

Plasma levels of serotonin are elevated in primary pulmonary hypertension even after bilateral lung transplantation, suggesting a possible etiologic role. Serotonin is released primarily from the small intestine. Anorectic agents, such as dexfenfluramine, which can cause pulmonary hypertension, are known to inhibit potassium channels in vascular smooth muscle cells. We examined the hypothesis that dexfenfluramine may stimulate release of serotonin from the ileum by inhibition of K+ channels. In an isolated loop of rat ileum perfused with a physiological salt solution, the administration of dexfenfluramine, its major metabolite D-norfenfluramine, the potassium channel blocker 4-aminopyridine (5 mM), and caffeine (30 mM) increased serotonin levels in the venous effluent. Potassium chloride (60 mM) tended to increase serotonin levels. In genetically susceptible individuals, dexfenfluramine may induce pulmonary hypertension by increasing cytosolic calcium in enterochromaffin cells of the small intestine, thus releasing serotonin and causing vasoconstriction. This work indicates that dexfenfluramine and its major metabolite d-norfenfluramine can increase serotonin release from the small intestine.     

杉木与固氮和非固氮树种混交对林地土壤质量和土壤水化学的影响

第 1代人工杉木林皆伐后 ,3种不同的经营模式 ,即连载杉木纯林、杉木与固 N阔叶树混交林和杉木与非固 N阔叶树混交林 ,对林地土壤质量和土壤水化学的影响进行了比较。结果表明 ,在杉树与阔叶树混交经营模式下 ,土壤养分含量增加 ,物理性状改善 ,土壤生物活性提高 ,微生物商 (Cmic:Corg)上升 ,代谢商 (q CO2 )稍有下降 ,但杉木与固 N树种的混交对土壤质量的改善效果比杉木与非固 N树种混交好 ;相反 ,杉木连载只能导致林地土壤质量的逐渐恶化 ;土壤溶液中 ,主要来自于大气中的一些离子浓度 ,如 SO2 - 4,Cl- ,Na 和 Mg2 ,在杉木纯林中显著高于混交林 ,而主要受系统内影响较大的一些离子 ,如 K 和NH 4,NO- 3,在经营模式间变异较小 ;H 和 Al3 浓度也是杉木纯林比混交林高。另外 ,研究结果还表明 ,总有机 C、CEC和微生物 C与其它土壤理化性质与生物学性质之间存在着较好的相关性 ,所以可以将总有机 C、CEC和微生物 C作为红黄壤地区亚热带人工林土壤质量的指示指标     

羚牛肉、脂、蹄腺分泌物的生化分析

人类对于大型偶蹄类动物的利用已有较多先例,北美麝牛(Ovibous muschtus)近年在加拿大、美国等已经驯养,并以其毛绒作为一种特殊的毛丝工业原料。美洲野牛(Bison Americasus)已与家牛杂交生产肉用牛。我国青藏高原的牦牛(Bos grinus)与黄牛杂交而成的犏牛,已成为藏民的主要肉乳兼用牛。羚牛(Budorcas taxicolor)系稀有动物,为亚洲特产,分布于中国、印度、缅甸及不丹。中国的羚牛分布于川、陕、甘、滇、藏5省区,为分布区最广、数量最多、资源量丰富的国家(Wu,1985)。     

Variation in alpha-L-fucosidase properties among 28 inbred mouse strains: Six strains have high enzyme activity and heat-stabile enzyme with a variant pH-activity curve; twenty-two strains have low activity and heat-labile enzyme

Alpha-L-fucosidase in tissues of 28 inbred mouse strains varied with respect to three properties: high or low heat stability, a pH-activity curve with high or low relative activity at pH 2.8, and high or low activity. Alpha-L-fucosidase from six strains (A/J, BDP/J, LP/J, P/J, SEA/GNJ, and 129/J) had high heat stability, high pH 2.8 relative activity, and high activity, whereas the other 22 strains all had low heat stability, low pH 2.8 relative activity, and low activity. The heat-stability difference was seen in all organs tested (brain, liver, kidney, spleen, heart, skeletal muscle, lung, and testis) for two heat-stabile strains (P/J and 129/J) and four heat-labile strains (C57 BL/6J, C3H/HeJ, DBA/2J, and BALB/cJ) studied in detail. The findings suggested that two structural variants of alpha-L-fucosidase, probably genetically determined, exist in these 28 inbred mouse strains, although the presence of linkage disequilibrium between alleles of tightly linked structural and regulatory genes could not be excluded.This work was supported by grants from the National Institutes of Health (NS-15281 and NS-11766), the Muscular Dystrophy Association (H. Houston Merritt Clinical Center for Muscular Dystrophy and Related Diseases), the March of Dimes Birth Defects Foundation, and a generous gift from the Alexander Rapaport Foundation.     

YY1抑制效应的破坏可促进人乳头瘤病毒16型癌基因的转录

人乳头瘤病毒１６型（ＨＰＶ１６）癌基因的表达受病毒早期启动子Ｐ９７的控制。位于ＬＣＲ上ＹＹ１蛋白结合位点的破坏可明显提高Ｐ９７的活性。为了观测ＹＹ１位点破坏在全基因组范围内对病毒ｅ６／ｅ７基因转录的影响,将构建的带有ＬＣＲ特异性突变的重组ＨＰＶ１６全基因组ＤＮＡ和ＨＰＶ１６野毒株ＤＮＡ转染至培养细胞,同时组建ＨＰＶ１６Ｅ６反向序列ＲＮＡ体外转录质粒。ＲＮａｓｅ保护试验证实,突变ＨＰＶ１６ＤＮＡ在短