BACKGROUND & AIMS: :In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in alpha 2 beta 2 conformation (A. Pramanik et al., J. Bacteriol. 137:469-473, 1979). In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined. The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S. K. Spratt et al., J. Bacteriol. 158:535-542, 1984). Only 10 nucleotides separated the coding sequences for the two peptides, confirming the suggestion that these genes form an operon. The peptides encoded by fadB and fadA were 729 amino acids and 387 amino acids, respectively, in length. The larger and smaller peptides had predicted molecular masses of 79,678 and 40,876 Da, respectively. Recently, the sequence of the fadA gene was published in a separate report (Yang et al., J. Biol. Chem. 265:10424-10429, 1990). In this work, most of the DNA sequence for fadA was confirmed, and 10 errors were corrected. Three of these nucleotide changes resulted in five amino acid residue changes predicted in the carboxy terminus of the fadA-encoded peptide. By comparison to other peptide sequences, the alpha subunit encoded within fadB had 31% perfect identity with the rat peroxisomal enoyl-coenzyme A:hydratase-3-hydroxyacyl-coenzyme A dehydrogenase trifunctional enzyme over the entire length of the two peptides. In agreement with the work of Yang et al., the beta subunit encoded within fadA had 35 to 45% perfect identity with five thiolase genes from different eucaryotic sources over the entire length of the peptide.

背景与目标： 在大肠杆菌中，脂肪酸的 β-氧化所需的至少五种酶活性与由 α2β2构象中的两个亚基组成的多酶复合物相关 (a.Pramanik等人，J. Bacteriol. 137:469-473，1979)。在本工作中，已经确定了编码这两个亚基fadB和fadA的基因的DNA序列。转录的方向是从fadB到fadA，而不是从fadA到fadB，如前所述 (S. K. Spratt等人，J. Bacteriol. 158:535-542，1984)。只有10个核苷酸分离了两个肽的编码序列，证实了这些基因形成操纵子的暗示。fadB和fadA编码的肽长度分别为729个氨基酸和387个氨基酸。较大和较小的肽分别预测79,678和40,876 Da的分子量。最近，fadA基因的序列发表在单独的报告中 (Yang等人，J. Biol. Chem. 265:10424-10429，1990)。在这项工作中，fadA的大部分DNA序列得到了确认，并纠正了10个错误。这些核苷酸变化中的三个导致fadA编码肽的羧基末端预测的五个氨基酸残基变化。与其他肽序列相比，fadB内编码的 α 亚基与大鼠过氧化物酶体烯酰辅酶a具有31% 完美的同一性: 在两个肽的整个长度上hydratase-3-hydroxyacyl-coenzyme脱氢酶三官能酶。与Yang等人的工作一致，在fadA内编码的 β 亚基在肽的整个长度上与来自不同真核来源的五个硫解酶基因具有35至45% 完美的同一性。

BACKGROUND & AIMS: :Neurons within each layer of cerebral cortex express multiple members of the neurotrophin family and their corresponding receptors. This multiplicity could provide functional redundancy; alternatively, different neurotrophins may direct distinct aspects of cortical neuronal growth and differentiation. By neutralizing endogenous neurotrophins in organotypic slices of developing cortex with Trk receptor bodies (Trk-IgGs), we found that BDNF and NT-3 oppose one another in regulating the dendritic growth of pyramidal neurons. In layer 4, both endogenous and exogenous NT-3 inhibited the dendritic growth stimulated by BDNF. In contrast, in layer 6 both endogenous and exogenous BDNF inhibited dendritic growth stimulated by NT-3. These antagonistic actions of endogenous BDNF and NT-3 provide a mechanism by which dendritic growth and retraction can be dynamically regulated during cortical development, and suggest that the multiple neurotrophins expressed in developing cortex represent distinct components of an extracellular signaling system for regulating dendritic growth.

背景与目标： : 大脑皮层各层内的神经元表达神经营养蛋白家族的多个成员及其相应的受体。这种多样性可以提供功能冗余; 或者，不同的神经营养蛋白可能会指导皮质神经元生长和分化的不同方面。通过用Trk受体体 (Trk-igg) 中和发育中的皮层器官型切片中的内源性神经营养蛋白，我们发现BDNF和NT-3在调节锥体神经元的树突状生长方面相互对抗。在第4层中，内源性和外源性NT-3均抑制BDNF刺激的树突状生长。相反，在第6层中，内源性和外源性BDNF均抑制NT-3刺激的树突生长。内源性BDNF和NT-3的这些拮抗作用提供了一种机制，通过该机制可以在皮质发育过程中动态调节树突生长和收缩，并表明在发育中的皮质中表达的多种神经营养蛋白代表了调节树突生长的细胞外信号系统的不同成分。

BACKGROUND & AIMS: :Matrix metalloproteinases (MMPs) have been the subject of intense research because of their roles in tumor metastasis and in the rise and spread of degenerative diseases such as osteo- and rheumatoid arthritis. A preliminary class of 140 druglike, small-molecule matrix metalloproteinase-3 inhibitors, intended as starting scaffolds for optimization and synthesis, has been designed in silico using a series of highly predictive three-dimensional quantitative structure-activity relationship models, including comparative molecular field analysis and comparative molecular similarity indices analysis, with docking and scoring. Thalidomide was chosen as the skeleton on which to base the new lead series, as it moderately inhibits MMP-3, is antiangiogenic, and lends itself easily to structural modifications. Most of the new compounds demonstrate medium to high predicted biological activity and good bioavailability as estimated by the octanol-water partition coefficient ClogP. Compound 102 in particular exhibits extremely favorable predicted activity against MMP-3; is moderately bioavailable; satisfies Lipinski's Rule of Five; and shows promise for further optimization, synthesis, and experimental evaluation as a potential adjunct anticancer or antirheumatic therapeutic.

背景与目标： : 基质金属蛋白酶 (MMPs) 由于其在肿瘤转移以及退行性疾病 (如骨关节炎和类风湿性关节炎) 的兴起和传播中的作用而成为深入研究的主题。初步设计了140类药物小分子基质metalloproteinase-3抑制剂，作为优化和合成的起始支架，使用一系列高度预测性的三维定量构效关系模型，包括比较分子场分析和比较分子相似性指数分析，对接和得分。沙利度胺被选为新铅系列的骨架，因为它适度抑制MMP-3，具有抗血管生成作用，并且易于进行结构修饰。根据辛醇-水分配系数ClogP估计，大多数新化合物表现出中等至高的预测生物活性和良好的生物利用度。化合物102尤其表现出对MMP-3极其有利的预测活性; 具有适度的生物利用度; 满足Lipinski的五法则; 并显示出有望进一步优化，合成和实验评估作为潜在的辅助抗癌或抗风湿治疗剂。

BACKGROUND & AIMS: :Apicomplexan parasites are the cause of numerous important human diseases including malaria and AIDS-associated opportunistic infections. Drug treatment for these diseases is not satisfactory and is threatened by resistance. The discovery of the apicoplast, a chloroplast-like organelle, presents drug targets unique to these parasites. The apicoplast-localized fatty acid synthesis (FAS II) pathway, a metabolic process fundamentally divergent from the analogous FAS I pathway in humans, represents one such target. However, the specific biological roles of apicoplast FAS II remain elusive. Furthermore, the parasite genome encodes additional and potentially redundant pathways for the synthesis of fatty acids. We have constructed a conditional null mutant of acyl carrier protein, a central component of the FAS II pathway in Toxoplasma gondii. Loss of FAS II severely compromises parasite growth in culture. We show FAS II to be required for the activation of pyruvate dehydrogenase, an important source of the metabolic precursor acetyl-CoA. Interestingly, acyl carrier protein knockout also leads to defects in apicoplast biogenesis and a consequent loss of the organelle. Most importantly, in vivo knockdown of apicoplast FAS II in a mouse model results in cure from a lethal challenge infection. In conclusion, our study demonstrates a direct link between apicoplast FAS II functions and parasite survival and pathogenesis. Our genetic model also offers a platform to dissect the integration of the apicoplast into parasite metabolism, especially its postulated interaction with the mitochondrion.

背景与目标： : Apicomplexan寄生虫是许多重要人类疾病的病因，包括疟疾和与艾滋病相关的机会性感染。这些疾病的药物治疗并不令人满意，并受到耐药性的威胁。叶绿体样细胞器apicoplast的发现提出了这些寄生虫特有的药物靶标。apicoplast定位脂肪酸合成 (FAS II) 途径是一种与人类类似FAS I途径根本不同的代谢过程，它代表了一个这样的目标。然而，apicoplast FAS II的特定生物学作用仍然难以捉摸。此外，寄生虫基因组还编码用于合成脂肪酸的其他且潜在的冗余途径。我们已经构建了酰基载体蛋白的条件无效突变体，这是弓形虫中FAS II途径的核心组成部分。FAS II的丢失严重损害了寄生虫在培养中的生长。我们显示FAS II是激活丙酮酸脱氢酶所必需的，丙酮酸脱氢酶是代谢前体乙酰辅酶a的重要来源。有趣的是，酰基载体蛋白敲除还导致apicoplast生物发生缺陷，并导致细胞器丢失。最重要的是，在小鼠模型中体内敲除apicoplast FAS II可从致命的攻击感染中治愈。总之，我们的研究证明了apicoplast FAS II功能与寄生虫存活和发病机理之间的直接联系。我们的遗传模型还提供了一个平台，可以将apicoplast整合到寄生虫代谢中，尤其是其与线粒体的假定相互作用。

BACKGROUND & AIMS: The effect of epinephrine on leucine and phenylalanine kinetics was measured by using the stable isotope amino acid tracers L-[1-(13)C]leucine and L-[phenyl-2H5]-phenylalanine in the postabsorptive state and during the intravenous administration of a standard amino acid solution with respect to the amino acid load. Infusion of epinephrine (plasma concentrationapproximately 3600 pmol/L) decreased leucine and phenylalanine and increased ketoisocaproate plasma concentrations and increased the metabolic clearance rate of leucine and phenylalanine. Epinephrine neither influenced leucine or phenylalanine flux nor leucine oxidation or leucine net balance. Hyperaminoacidemia from amino acid infusion reduced endogenous leucine release and stimulated leucine oxidation and nonoxidative disposal of leucine, resulting in a dose-dependent increase in leucine net balance. Epinephrine did not influence any changes in amino acid kinetics during parenteral amino acid administration. Therefore, we conclude that epinephrine had no catabolic effects on amino acid metabolism and no negative effect on the utilization of a parenterally offered amino acid solution in healthy humans.

背景与目标： 肾上腺素对亮氨酸和苯丙氨酸动力学的影响是通过使用稳定同位素氨基酸示踪剂L-[1-(13)C] 亮氨酸和L-[phenyl-2H5]-苯丙氨酸在后吸收状态和静脉内给药期间测量的。标准氨基酸溶液相对于氨基酸负荷输注肾上腺素 (血浆浓度约3600 pmol/L) 降低亮氨酸和苯丙氨酸，增加酮异己酸血浆浓度，增加亮氨酸和苯丙氨酸的代谢清除率。肾上腺素既不影响亮氨酸或苯丙氨酸通量，也不影响亮氨酸氧化或亮氨酸净平衡。氨基酸输注的高氨基酸血症降低了内源性亮氨酸释放并刺激亮氨酸氧化和亮氨酸的非氧化处理，导致亮氨酸净平衡的剂量依赖性增加。肾上腺素在肠胃外给药期间不影响氨基酸动力学的任何变化。因此，我们得出的结论是，肾上腺素对健康人的氨基酸代谢没有分解代谢作用，对肠胃外提供的氨基酸溶液的利用也没有负面影响。

BACKGROUND & AIMS: MyristoylCoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational covalent attachment of a rare cellular fatty acid, myristate, to the N-terminal Gly residue of a variety of eukaryotic proteins. The myristoyl moiety is often essential for expression of the biological functions for these proteins.

Attachment of C14:0 alone provides barely enough hydrophobicity to allow stable association with membranes. The partitioning of N-myrisotylproteins is therefore often modulated by "switches" that function through additional covalent or noncovalent modifications. Candida albicans, the principal cause of systemic fungal infection in immunocompromised humans, contains a single NMT gene that is essential for its viability. The functional properties of the acylCoA binding site of human and C. albicans NMT are very similar. However, there are distinct differences in their peptide binding sites. An ADP ribosylation factor (Arf) is included among the few cellular protein substrates of the fungal enzyme. Alanine scanning mutagenesis of an octapeptide derived from an N-terminal Arf sequence (GLYASKLS-NH2) disclosed that Gly1, Ser5, and Lys6 play predominant roles in binding. ALYASKLS-NH2 is an inhibitor competitive for peptide [Ki(app) = 15.3 +/- 6.4 microM] and noncompetitive for myristoylCoA. Remarkably, replacement of the N-terminal tetrapeptide with an 11-aminoundecanoyl group results in a competitive inhibitor (11-aminoundecanoyl-SKLS-NH2) that is approximately 40-fold more potent [Ki(app) = 0.40 +/- 0.03 microM] than the starting octapeptide. Removal of Leu-Ser from the C-terminus generates a competitive dipeptide inhibitor (11-aminoundecanoyl-SK-NH2) with a Ki(app) of 11.7 +/- 0.4 microM, equivalent to that of the starting octapeptide. A derivative dipeptide inhibitor containing a C-terminal N-cyclohexylethyl lysinamide moiety has the advantage of being more potent (IC50 = 0.11 +/- 0.03 microM) and resistant to digestion by cellular carboxypeptidases. Rigidifying the flexible aminoundecanoyl chain results in very potent general NMT inhibitors (IC50 = 40-50 nM). Substituting a 2-methylimidazole for the N-terminal amine and adding a benzylic alpha-methyl group with R stereochemistry to the rigidifying element produces even more potent inhibitors (IC50 = 20-50 nM) that are up to 500-fold selective for the fungal compared to human enzyme. A related less potent member of this series of compounds is fungistatic. Its growth inhibitory effects are associated with a reduction in cellular protein N-myristoylation, judged using cellular Arf as a reporter. These studies establish that NMT is a new antifungal target.

背景与目标： 肉豆蔻酰辅酶a : 蛋白质N-肉豆蔻酰转移酶 (NMT) 催化稀有细胞脂肪酸肉豆蔻酸酯与多种真核蛋白质的N末端Gly残基的共翻译共价连接。肉豆蔻酰部分对于表达这些蛋白质的生物学功能通常是必不可少的。
C14的附着 :0仅提供了足够的疏水性，无法与膜稳定结合。因此，N-肉豆蔻基蛋白的分配通常由 “开关” 调节，该开关通过其他共价或非共价修饰起作用。白色念珠菌是免疫功能低下的人体全身性真菌感染的主要原因，它包含一个对其生存能力至关重要的NMT基因。人与白色念珠菌NMT的酰基辅酶a结合位点的功能特性非常相似。然而，它们的肽结合位点存在明显差异。真菌酶的少数细胞蛋白底物中包括ADP核糖基化因子 (Arf)。衍生自N-末端Arf序列 (GLYASKLS-NH2) 的八肽的丙氨酸扫描诱变揭示Gly1、Ser5和Lys6在结合中起主要作用。ALYASKLS-NH2是一种对肽 [Ki(app) = 15.3 +/- 6.4 microM] 有竞争力的抑制剂，对肉豆蔻酰辅酶a无竞争力。值得注意的是，用11-氨基十一酰取代N-末端四肽导致竞争性抑制剂 (11-氨基十一酰-skls-nh2) 的效力比起始八肽高约40倍 [Ki(app) = 0.40 +/- 0.03微米]。从C末端去除Leu-Ser会产生竞争性二肽抑制剂 (11-氨基十一酰-sk-nh2)，Ki(app) 为11.7 +/- 0.4微米，相当于起始八肽。含有C-末端N-环己基乙基赖氨酰胺部分的衍生物二肽抑制剂具有更有效 (IC50 = 0.11 +/- 0.03微米) 和对细胞羧肽酶消化的抗性的优点。硬化柔性氨基十一酰链会产生非常有效的通用NMT抑制剂 (IC50 = 40-50nm)。用2-甲基咪唑代替N-末端胺并将具有R立体化学的苄基 α-甲基添加到硬化元件中，产生甚至更有效的抑制剂 (IC50 = 20-50nm)，其对真菌的选择性与人酶相比高达500倍。该系列化合物中一个相关的效力较低的成员是真菌抑制剂。使用细胞Arf作为报告基因，其生长抑制作用与细胞蛋白N-肉豆蔻酰化的减少有关。这些研究表明，NMT是一种新的抗真菌靶标。

BACKGROUND & AIMS: Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III1 module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III1. Proteins containing the fourth and fiftBiomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706U.S.A. Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conh as 70 kDa deletion mutants lacking I4 and I5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both competed only partially for binding of 125I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I4 and I5 and that interactions in addition to that of I4 and I5 with III1 are important for cell-surface-mediated fibronectin polymerization.

背景与目标： 纤连蛋白原纤维的组装发生在附着于底物的细胞的表面，并由分子的N末端70 kDa部分中的第一至第五种I型模块介导。纤连蛋白的第一个III型模块 (III1) 不存在于70 kDa部分中，包含纤连蛋白70 kDa N末端区域的构象依赖性结合位点，提示细胞表面纤连蛋白上的III1模块可以用作底物附着细胞上纤连蛋白N末端的结合位点。为了探索这种可能性，我们比较了含有前五个I型模块中一个或几个缺失的突变重组70 kDa蛋白与成纤维细胞和iii1结合的能力。含有第四和第五生物分子化学和医学的蛋白质，威斯康星大学麦迪逊分校，WI 53706u.S.A.纤连蛋白原纤维的组装发生在附着于底物的细胞的表面，并由分子的N末端70 kDa部分中的第一至第五种I型模块介导。纤连蛋白的第一个III型模块 (III1) 不存在于70 kda部分中，包含也与细胞表面结合的缺乏I4和I5的conh as 70 kda缺失突变体，并且缺乏I1-3和I4-5的缺失突变体都仅部分竞争125i标记的纤连蛋白或70 kda蛋白的结合。这些数据表明，纤连蛋白的N端部分通过I4和I5与III1结合，并且除了I4和I5与III1的相互作用外，还对细胞表面介导的纤连蛋白聚合很重要。

BACKGROUND & AIMS: The stability and activity of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus were studied as a function of pH and temperature. In this paper we focus on three points(1) the long-term stability of the protein to irreversible denaturation at high temperature; (2) the short-term stability of the protein to reversible temperature-driven unfolding; and (3) the dependence of its activity on temperature.

Results can be summarized as follows(a) the same first-order kinetic constant (0.020+/-0.003 min-1) was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 degrees C; (b) short-term stability experiments revealed different behaviour in two different pH ranges (6.5-8.0, 8.5-9.5), suggesting that the melting temperature is higher at alkaline than at neutral pH; (c) the dependence of activity on temperature was investigated at pH 7.0 and 9.0, and a discontinuity was observed in the Arrhenius plot of kcat values at pH 9.0. We also investigated the stability in the presence of guanidinium chloride at 20 degrees C either at pH 7.0 or at pH 9.0, and we present data that indicate that the unfolding mechanism closely approaches a two-state model at pH 7.0 and a more complex mechanism at pH 9.0. Satisfactory fitting of the equilibrium unfolding transition obtained by fluorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 degrees C the folded conformation is more stable than the unfolded conformation by (14. 7+/-1.2) kJ/mol at pH 7.0 and by (25.5+/-1.8) kJ/mol at pH 9.0.

背景与目标： 研究了硫酸亚砜中吲哚-3-甘油磷酸合酶的稳定性和活性随pH和温度的变化。在本文中，我们重点关注三点 (1) 蛋白质在高温下不可逆变性的长期稳定性; (2) 蛋白质在可逆温度驱动的去折叠的短期稳定性; (3) 其活性对温度的依赖性。
结果可以总结如下 (a) 在不同的ph值 (6.5，8.0和9.5) 来自80 ℃ 下的长期稳定性实验; (b) 短期稳定性实验揭示了在两个不同pH范围 (6.5-8.0，8.5-9.5) 下的不同行为，表明在碱性下的熔融温度高于在中性pH下; (c) 在pH 7.0和9.0下研究了活性对温度的依赖性，在pH 9.0下的kcat值的Arrhenius图中观察到不连续性。我们还研究了氯化胍在20 ℃ 下在pH 7.0或pH 9.0下的稳定性，并且我们提供的数据表明，展开机制在pH 7.0下接近两态模型，在pH 9.0下接近更复杂的机制。在pH 9.0下通过荧光测量获得的平衡展开转变的令人满意的拟合需要一个模型，该模型除了天然和展开形式之外还涉及稳定的中间体。在20 ℃ 下，折叠构象比未折叠构象在pH 7.0下稳定 (14. 7 +/-1.2) kJ/mol，在pH 9.0下稳定 (25.5 +/-1.8) kJ/mol。

BACKGROUND & AIMS: The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.

背景与目标： 流感血凝素 (HA) 的膜锚定亚基的氨基末端片段在膜融合中起着至关重要的作用，因此被称为融合肽。我们通过荧光，圆二色性和傅立叶变换研究了与野生型和几种融合和非融合突变体相对应的合成肽的二级结构，取向和对双层结构的影响。红外光谱。所有肽均包含 α-螺旋和 β 链构象的片段。在野生型融合肽中，所有残基的40% 为 α-二级结构，30% 为 β-二级结构。相比之下，非融合肽表现出较大的 β/α 二级结构比。基于各自吸收带的红外二色性，分别测量了野生型融合肽的 β 链的螺旋和酰胺羰基的有序参数。对于野生型肽的两个片段，发现了0.1-0.7范围内的顺序参数，这表明它们最有可能与膜法线成倾斜角度对齐。非融合肽而不是融合肽在1735厘米 (-1) 处诱导红外吸收带分裂，这被分配给脂质酯羰基键的拉伸振动。这种分裂报告了脂质酯羰基与融合肽的水和/或供氢基团之间形成的氢键的变化，与肽的 β/α 比相关，表明不成对的 β 链可能取代水分子并与脂质酯羰基氢键。在融合肽的极端N末端由单个氨基酸置换诱导的深刻结构变化进一步表明，当融合肽与脂质双层结合时，三级或四级结构相互作用可能很重要。

BACKGROUND & AIMS: :Our recent study demonstrated that defects in p110delta result in B-cell immunodeficiency that is very similar to that observed in BTK-deficient mice. We revealed that the p110delta fit the B-cell signal transduction complex and played a non-redundant role in the development and function of B cells. In humans, most children with primary B-cell immunodeficiency have mutations in the BTK, whereas a few have defects in the components of the B-cell signal transduction complex. But little is known about the genetic variation of p110delta in children with defects in B-cell immunodeficiency of unknown aetiology. Sixteen patients from 15 unrelated families and 112 normal controls underwent sequence analysis to identify genetic variations of the p110delta. Allele frequency in each group was also analysed and compared. We identified five single base-pair polymorphic nucleotide exchanges in both patient and control groups with similar allele frequencies, which did not contribute to the immunodeficiency. Three of them are novel (m.953A>G, m.1200C>T and m.1561A>G), and the m.953A>G and m.1561A>G nucleotide exchanges are non-synonymous (N253S and T456A, respectively). The novel m.1561A>G was in complete linkage disequilibrium with the known m.873A>G in our study of Taiwanese group. In addition, one novel single base-pair missense mutation, m.3256G>A (E1021K), was identified in one boy with typical clinical features of primary B-cell immunodeficiency and could not be found in either his family or the normal control population. By atomic structural analysis of the amino acid as well as the alignment comparison between species, it resulted in the replacement of the negative-charged amino acid E with the positive-charged amino acid K at codon 1021, located in the highly conservative and important catalytic functional domain. Our findings could shed light on further understanding the polymorphisms of p110delta in B-cell immunodeficiency and different populations. Moreover, the 3256G>A missense mutation raised the attention and warranted further extensive analysis to elucidate the role of p110delta in human immunodeficiency.

背景与目标： : 我们最近的研究表明，p110delta的缺陷导致b细胞免疫缺陷，这与BTK缺陷小鼠中观察到的非常相似。我们揭示了p110delta符合b细胞信号转导复合物，并且在b细胞的发育和功能中起着非冗余的作用。在人类中，大多数患有原发性b细胞免疫缺陷的儿童在BTK中存在突变，而少数儿童在b细胞信号转导复合物的成分中存在缺陷。但是，对于病因不明的b细胞免疫缺陷儿童中p110delta的遗传变异知之甚少。来自15个无关家庭和112个正常对照的16名患者进行了序列分析，以鉴定p110delta的遗传变异。还对各组的等位基因频率进行了分析和比较。我们在患者组和对照组中鉴定出五个具有相似等位基因频率的单碱基对多态性核苷酸交换，这对免疫缺陷没有贡献。其中三个是新颖的 (m.953A>G，m.1200C>T和m.1561A>G)，而m.953A>G和m.1561A>G核苷酸交换是非同义的 (分别为N253S和T456A)。在我们对台湾组的研究中，新颖的m.1561A>G与已知的m.873A>G完全处于连锁不平衡状态。此外，在一名具有原发性b细胞免疫缺陷典型临床特征的男孩中鉴定出一个新的单碱基对错义突变m.3256G>A (E1021K)，在其家人或正常对照人群中均找不到。通过氨基酸的原子结构分析以及物种之间的比对比较，它导致负电荷氨基酸E被密码子1021位的正电荷氨基酸K取代，位于高度保守和重要的催化功能域。我们的发现可能有助于进一步了解b细胞免疫缺陷和不同人群中p110delta的多态性。此外，3256G>A错义突变引起了人们的注意，并需要进一步广泛的分析来阐明p110delta在人类免疫缺陷中的作用。

BACKGROUND & AIMS: :Epithelial cells play an important role in orchestrating mucosal immune responses. In allergic-type inflammation, epithelial cells control the recruitment of eosinophils into the mucosa. Th2-type cytokine-driven release of eosinophil-active chemokines from epithelial cells directs eosinophil migration into the mucosal epithelium. CCR3, the main eosinophil chemokine receptor, regulates this process; however, the respective contribution of individual CCR3 ligands in eosinophil transepithelial migration is less well understood. Using an in vitro transepithelial chemotaxis system, we found that eotaxin-3 produced by IL-4-stimulated airway epithelial cells and CCR3 on eosinophils exclusively mediate eosinophil transepithelial migration. Eotaxin-3 protein levels were also increased in the nasal mucosal epithelium recovered from allergic patients as compared to non-allergic patients. Surprisingly, eotaxin-3 in IL-4-stimulated airway epithelial cells was predominantly cell surface bound, and the cell surface form was critical for eosinophil transepithelial migration. Eotaxin-3 cell surface association was partially glycosaminoglycan (GAG) dependent, but was completely protein dependent, suggesting that eotaxin-3 associates with both GAG and cell surface proteins. We thus provide evidence that cell surface-associated eotaxin-3 is the critical IL-4-dependent chemotactic signal mediating eosinophil transepithelial migration in the setting of allergic inflammation.

背景与目标： : 上皮细胞在协调粘膜免疫反应中起重要作用。在过敏型炎症中，上皮细胞控制嗜酸性粒细胞向粘膜的募集。Th2-type细胞因子驱动的嗜酸性粒细胞活性趋化因子从上皮细胞释放引导嗜酸性粒细胞迁移到粘膜上皮中。主要的嗜酸性粒细胞趋化因子受体CCR3调节这一过程; 然而，单个CCR3配体在嗜酸性粒细胞跨上皮迁移中的各自作用尚不清楚。使用体外经上皮趋化系统，我们发现IL-4-stimulated气道上皮细胞和嗜酸性粒细胞上的CCR3产生的eotaxin-3仅介导嗜酸性粒细胞经上皮迁移。与非过敏患者相比，从过敏患者恢复的鼻粘膜上皮中Eotaxin-3蛋白水平也增加。令人惊讶的是，IL-4-stimulated气道上皮细胞中的eotaxin-3主要是细胞表面结合的，细胞表面形式对于嗜酸性粒细胞经上皮迁移至关重要。Eotaxin-3细胞表面缔合部分依赖于糖胺聚糖 (GAG)，但完全依赖于蛋白质，这表明eotaxin-3与GAG和细胞表面蛋白均相关。因此，我们提供的证据表明，在过敏性炎症环境中，细胞表面相关eotaxin-3是介导嗜酸性粒细胞跨上皮迁移的关键IL-4-dependent趋化信号。

BACKGROUND & AIMS: Sixty-one strains of Mycobacterium tuberculosis complex and 47 strains of nontuberculous mycobacteria were analyzed for fatty acids and enzyme profiles. Cellular fatty acids were extracted from bacteria, methylated and analyzed by gas liquid chromatography operated either manually (Perkin-Elmer) or by the automatic Microbial Identification System. The major cellular fatty acids in all mycobacterial species were C161. Tuberculostearic acid was found in all species with the exception of Mycobacterium gordonae. The fatty acids with a carbon-length longer than 20 could be detected only by conventional gas chromatography. Strains of M.

tuberculosis had a high ratio of C260. For determination of branched-chain fatty acids, the MIS provided more definitive results. The data indicated that the fatty acid profiles could provide rapid species identification. The results of the enzyme profile analysis using API-ZYM strips showed 39 different patterns from 59 strains of M. tuberculosis, and 41 different patterns from 46 nontuberculous mycobacteria strains, suggesting that enzyme profiles can also be used for strain characterization within the same species.

背景与目标： 分析了61株结核分枝杆菌复合物和47株非结核分枝杆菌的脂肪酸和酶谱。从细菌中提取细胞脂肪酸，甲基化，并通过手动 (Perkin-Elmer) 或自动微生物识别系统操作的气相色谱法进行分析。所有分枝杆菌物种中的主要细胞脂肪酸为c161。除gordonae分枝杆菌外，所有物种中均发现了结核硬脂酸。碳长度大于20的脂肪酸只能通过常规气相色谱法检测。结核分枝杆菌菌株的c260比例很高。对于支链脂肪酸的测定，MIS提供了更明确的结果。数据表明，脂肪酸谱可以提供快速的物种鉴定。使用API-ZYM条带进行的酶谱分析结果显示，来自59株结核分枝杆菌的39种不同模式，以及来自46株非结核分枝杆菌的41种不同模式，这表明酶谱也可用于同一物种内的菌株表征。

BACKGROUND & AIMS: Low intensity ultrasound signals, similar to that employed in clinical therapy, are found to mediate differential gene transfer and expression of the Green Fluorescence Protein (GFP) reporter in two human prostate cancer cell lines, LnCap and PC-3. Cell suspensions in the presence or in the absence of GFP (44.5nM) were treated at 37 degrees C under a standing wave condition. Cells were exposed to either continuous wave, 932.7kHz ultrasound, or to several independent bursts, each burst comprising a 20% duty cycle (932.7kHz) sine wave. The burst "repetition" frequency was varied from 10Hz to 10kHz in several different experiments and each treatment received a net identical ultrasound energy exposure. Transient GFP expression levels in viable cells were monitored by flow cytometry. The findings revealed a strong ultrasound tone-burst frequency dependence on the transfection efficiencies. Interestingly, the ultrasound signal parameters which are routinely employed in clinical therapy did not yield any statistically significant enhancement in transfection efficiency relative to their sham counterparts.

背景与目标： 发现与临床治疗中使用的低强度超声信号相似，可介导两种人前列腺癌细胞系LnCap和PC-3中的差异基因转移和绿色荧光蛋白 (GFP) 报告基因的表达。在驻波条件下，在37 ℃ 下处理存在或不存在GFP (44.5nm) 的细胞悬浮液。将细胞暴露于连续波、932.7khz超声波或几个独立的突发，每个突发包括20% 占空比 (932.7khz) 正弦波。在几个不同的实验中，突发 “重复” 频率从10Hz到10kHz不等，并且每种处理均获得净相同的超声能量暴露。通过流式细胞术监测活细胞中的瞬时GFP表达水平。研究结果表明，超声音调爆发频率对转染效率有很强的依赖性。有趣的是，与假手术相比，临床治疗中常规使用的超声信号参数在转染效率上没有任何统计学上的显着提高。

BACKGROUND & AIMS: Epoxyeicosatrienoic acids (EETs) are endothelium-derived arachidonic acid metabolites of cytochrome P450. They dilate coronary arteries, open K+ channels, and hyperpolarize vascular smooth muscles. However, the mechanisms of these smooth muscle actions remain unknown. This study examined the effects of EETs on the large-conductance Ca(2+)-activated K+ channel (KCa) in smooth muscle cells of small bovine coronary arteries. In cell-attached patch-clamp experiments, 11,12-EET produced a 0.5- to 10-fold increase in the activity of the KCa channels when added in concentrations of 1, 10, and 100 nmol/L. In the inside-out excised membrane patch mode, 11,12-EET was without effect on the activity of the KCa channel unless GTP (0.5 mmol/L) or GTP and ATP (1 mmol/L) were added to the bath solution. In the presence of GTP and ATP, the increase in the KCa channel activity with 11,12-EET in inside-out patches was comparable to that in cell-attached patches. This effect of 11,12-EET in inside-out patches was blocked by the addition of GDP-beta-S (100 mumol/L). In outside-out patches, 11,12-EET also increased the KCa channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-Gs alpha antibody (100 nmol/L) in the pipette solution completely blocked the activation of the KCa channels induced by 11,12-EET. An anti-G beta gamma or anti-Gi alpha antibody was without effect. We conclude that 11,12-EET activates the KCa channels by a Gs alpha-mediated mechanism. This mechanism contributes to the effects of EETs as endothelium-derived hyperpolarizing factors to hyperpolarize and relax arterial smooth muscle.

背景与目标： 环氧二十碳三烯酸 (EETs) 是细胞色素p450的内皮衍生的花生四烯酸代谢产物。它们扩张冠状动脉，打开K通道，并使血管平滑肌超极化。然而，这些平滑肌作用的机制仍然未知。这项研究检查了EETs对小冠状动脉平滑肌细胞中大电导Ca(2) 激活的K通道 (KCa) 的影响。在细胞附着的膜片钳实验中，当以1、10和100 nmol/L的浓度添加时，11,12-eet使KCa通道的活性增加了0.5至10倍。在由内向外切除的膜贴片模式中，11,12-eet对KCa通道的活性没有影响，除非将GTP (0.5 mmol/L) 或GTP和ATP (1 mmol/L) 添加到浴液中。在存在GTP和ATP的情况下，由内而外的贴片中11,12-eet的KCa通道活性增加与细胞附着的贴片相当。由内而外的贴片中11,12-eet的这种作用被添加GDP-β-S (100 mumol/L) 所阻断。在向外贴剂中，当将GTP和ATP添加到移液器溶液中时，11,12-eet也会增加KCa通道活性。在移液器溶液中添加特异性抗Gs α 抗体 (100 nmol/L) 完全阻断了由11,12-eet诱导的KCa通道的激活。抗G β γ 或抗Gi α 抗体无效。我们得出的结论是，11,12-eet通过Gs α 介导的机制激活了KCa通道。该机制有助于EETs作为内皮衍生的超极化因子，使动脉平滑肌超极化和松弛。

BACKGROUND & AIMS: OBJECTIVE:The scholarly dissemination of innovative medical education practices helps broaden the reach of this type of work, allowing scholarship to have an impact beyond a single institution. There is little guidance in the literature for those seeking to publish program evaluation studies and innovation papers. This study aims to derive a set of evidence-based features of high-quality reports on innovations in emergency medicine (EM) education. METHODS:We conducted a scoping review and thematic analysis to determine quality markers for medical education innovation reports, with a focus on EM. A search of MEDLINE, EMBASE, ERIC, and Google Scholar was augmented by a hand search of relevant publication guidelines, guidelines for authors, and website submission portals from medical education and EM journals. Study investigators reviewed the selected articles, and a thematic analysis was conducted. RESULTS:Our search strategy identified 14 relevant articles from which 34 quality markers were extracted. These markers were grouped into seven important themes: goals and need for innovation, preparation, innovation development, innovation implementation, evaluation of innovation, evidence of reflective practice, and reporting and dissemination. In addition, multiple outlets for the publication of EM education innovations were identified and compiled. CONCLUSION:The publication and dissemination of innovations are critical for the EM education community and the training of health professionals. We anticipate that our list of innovation report quality markers will be used by EM education innovators to support the dissemination of novel educational practices.

背景与目标：