核蛋白基本单元的合成—O—乙基—UMP—丙氨酸甲酯

在合成磷酰蛋白的模型化合物Ｎ－磷酰化氨基酸（１）的基础上,首次合成了有机磷生命化学的模型化合物－核蛋白基本单元Ｎ－磷酰化氨基酸（２）,并对上述物质进行了分离,鉴定,比较了两种模型的合成方法,这对在小分子水平上,深入研究磷酰基的参与作和蛋白与核酸之间的互相作用机理提供了较好的模型化合物。     

N－（膦羧甲基）α－甘氨酸的合成与应用

叙述了Ｎ－（膦羧甲基）α－甘氨酸的化学合成及其在除草剂方面的应用。该化合物有五条合成路线：曼尼斯反应、加成反应、取代反应、氧化还原反应和其他反应。其中曼尼斯反应较简便,可由甘氨酸、甲醛和亚磷酸酯一步合成标题化合物。Ｎ－（膦羧甲基）α－甘氨酸是一种高效广谱内吸传导型叶片喷药除草剂,它能有效地控制世界上危害最大的７８种恶性杂草中的７６种,还能“斩草除根”。除草机理为竞争性抑制５－烯醇丙酮莽草酸－３－磷酸合成酶（ＥＰＳＰａｓｅ）。     

2．5分辨率单斜Al－（L－丙氨酸）胰岛素晶体结构研究

空间群为Ｐ２１的Ａ１－（Ｌ－丙氨酸）胰岛素晶胞内,一个不对称单位含有一个六聚体,应用差值Ｆｏｕｒｉｅｒ技术,立体化学制最小二来技术和Ｘ—ＰＬＯＲ程序并辅以电子密度图的人工拟合,解析了分辨率ＡＩ—（Ｌ－丙氨酸）胰岛素（Ａｌ－Ｌ－ＡｌａⅠ）的晶体结构。最终Ｒ因子为２０．６％,与标准键长与键角的均方根偏差分别为和４．１９°,从电子密度图与模型的拟合来看,六聚体中每条Ａ链的Ａｌ位置替换的Ｌ—Ａｌａ清晰可见,每条Ｂ链Ｎ端Ｂ１—Ｂ８伏段都为α螺旋构象,形成了Ｂ１—Ｂ１９的连续α螺旋段。     

微生物发酵合成L－缬氨酸－~（15）N

采用含有稳定同位素￣（１５）Ｎ－硫铵为主要氮源的专用发酵培养液配方和相应的工艺条件,在国内首次用微生物直接发酵研制成Ｌ－缬氨酸－￣（１５）Ｎ高丰度精制产品。产品￣（１５）Ｎ丰度９７．６８％,反比原料￣（１５）Ｎ－硫铵丰度下降０．５３％,Ｌ－缬氨酸－￣（１５）Ｎ产酸率最高达４％以上（未校正）。每克￣（１５）Ｎ－硫铵可得到１克以上的Ｌ－缬氨酸－￣（１５）Ｎ（分析值）。提取精制收率平均为８０－９０％（单程）,最高达到９５％以上（二次提取）。实际每克￣（１５）Ｎ－硫铵可得０．６克左右的Ｌ－缬氨酸－￣（１５）Ｎ精制产品。     

基因工程菌发酵合成L－苏氨酸－N~（15）

采用含有稳定同位素１５Ｎ－硫酸铵为主要氮源的专用发酵培养基配方和提取精制条件,在国内、外首次采用基因工程菌ＡＡ７（ｐＴＨ２）（ＡＨＶｒＡＥＣｒ,Ｔｈｒ－Ｎ－,Ｈｏｍｒ,Ａｐｒ）直接发酵方法研制Ｌ－苏氨酸－Ｎ１５高丰度精制产品。每ｍｏｌ１５Ｎ－硫酸铵实际得到０．６３８ｍｏｌＬ－苏氨酸－Ｎ１５,产品Ｎ１５丰度达９９．０９％,仅比原料１５Ｎ－硫酸铵丰度下降０．４２％,提取精制得率高达９２．８３％。     

作物籽粒中NO-3-N和NO-2-N的积累与氮肥种类和数量的关系

１９９６～１９９７在芸豆（云南）、红小豆（河北）、大豆（黑龙江）和花生（山东）绿色食品产地,研究了施用有机肥和化肥对这些作物籽粒中ＮＯ＾－３、ＮＯ＾－２的累积特点。结果表明,芸豆和红小豆不论施用有机肥还是尿素,土壤中的速效氮与籽粒中ＮＯ＾－２的灰色关联最好;施用有机肥的最大关联度分别出现在土壤库内部（全氮与速效氮之间）或籽粒库内部（ＮＯ＾－３、ＮＯ＾－２之间）;而施用化肥的最大关联度分别出现在土壤     

N－糖链在纤连蛋白分泌及纤连蛋白受体与配体结合中的作用

应用能阻断糖蛋白Ｎ－糖链合成的衣霉素（ＴＭ）,研究了Ｎ－糖链缺失对ＨＴ１０８０细胞分泌纤连蛋白（Ｆｎ）以及纤连蛋白受体（ＦｎＲ）与配体结合的影响。结果发现,１μｇ／ｍｌ的ＴＭ可抑制Ｎ－糖链的合成（此时,３Ｈ－甘露糖掺入下降６３％）,但细胞分泌Ｆｎ的量仅下降３３％,这主要是由于蛋白合成受ＴＭ抑制（２５％）而引起,因而,Ｎ－糖链缺失可能并不影响Ｆｎ的分泌。而在同样条件下,单个细胞结合１２５Ｉ－Ｆｎ的量显著下降,显示Ｎ－糖链的缺失可能导致了膜上ＦｎＲ总量或其与配体结合的亲和力的改变。ＴＭ处理组的ＦｎＲ的内吞率与对照组相比较无明显差异,提示受体分子中的Ｎ－糖链缺失不影响其内吞过程．     

叠氮水杨酰胺类化合物对呼吸链琥珀酸-细胞色素c还原酶的抑制作用

合成了３－叠氮基－Ｎ－正癸烷基水杨酰胺和５－叠氮基－Ｎ－正癸烷基水杨酰胺并检测了它们对呼吸链酶系从琥珀酸到细胞色素ｃ段电子传递活性的抑制作用．两种化合物对琥珀酸－泛醌还原酶的抑制能力基本相同,而５位叠氮基取代物对泛醌－细胞色素ｃ还原酶的抑制能力较３位叠氮基取代物为强．它们与泛醌反应抑制剂３－硝基－Ｎ－正癸烷基水杨酰胺相比较,其抑制性质基本相似,只是抑制能力较后者为弱     

L－NNA及NO供体对延髓腹外侧头端区神经元自发放电的影响

在麻醉大鼠观察了静注ＮＯ合成酶抑制剂Ｎ－硝基左旋精氨酸（Ｌ－ＮＮＡ）和ＮＯ供体──硝普钠（ＳＮＰ）和ＳＩＮ－Ｉ对血压、心率和延髓腹外侧头端区（ＲＶＬＭ）神经元自发放电活动的影响,旨在探讨Ｌ－ａｒｇ：ＮＯ通路对动脉血压调节的中枢作用部位。所得结果如下：（１）静注Ｌ－ＮＮＡ后,平均动脉压（ＭＡＰ）升高,心率（ＨＲ）加快,１１个ＲＶＬＭ神经元自发放电频率增加。这些变化发生于给药后５ｍｉｎ,持续时间达３０ｍｉｎ以上。（２）静注ＳＮＰ后,ＭＡＰ降低,ＨＲ加快,２３个ＲＶＬＭ神经元自发放电频率降低,且有剂量依赖性。ＳＮＰ作用发生快,持续时间短。为了排除脑缺血的影响,还特意向一侧颈动脉内注射相同剂量ＳＮＰ,结果引起ＭＡＰ轻度降低,而ＨＲ无明显改变,但ＲＶＬＭ神经元自发放电频率仍显著降低。（３）静注另一ＮＯ供体ＳＩＮ－Ｉ后,ＭＡＰ降低,１１个ＲＶＬＭ神经元自发放电频率降低．与ＳＮＰ的效应基本一致。以上结果提示,ＲＶＬＭ是Ｌ－ａｒｇ：ＮＯ通路实现动脉血压调节的一个中枢作用部位。     

单斜Al－（L－丙氨酸）胰岛素初步晶体学研究

用微量气相扩散方法在含酚的柠檬酸缓冲体系中,得到了可供Ｘ射线晶体学分析用的Ａｌ－（Ｌ－丙氨酸）胰岛素晶体,晶体衍射能力为２．５Ａ。经Ｘ射线衍射分析确定,该晶体属于单斜晶系,空间群为Ｐ２_１,晶胞参数：ａ＝６１．５Ａ,ｂ＝６２．２Ａ,ｃ＝４８．３Ａ,α＝γ＝９０．０°,β＝１１０．９°。晶胞中每个结晶学不对称单位含有一个Ａｌ－（Ｌ－丙氨酸）胰岛素六聚体。     

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.     

Inhibition of Jack Bean Urease by N-(n-butyl)thiophosphorictriamide and N-(n-butyl)phosphorictriamide: Determination of the Inhibition Mechanism

N-(n-butyl)thiophosphorictriamide (NBPT) and its oxygen analogue N-(n-butyl)phosphorictriamide (NBPTO) were studied as inhibitors of jack bean urease. NBPTO was obtained by spontaneous conversion of NBPT into NBPTO. The conversion under laboratory conditions was slow and did not affect NBPT studies. The mechanisms of NBPT and NBPTO inhibition were determined by analysis of the reaction progress curves in the presence of different inhibitor concentrations. The obtained plots were time-dependent and characteristic of slow-binding inhibition. The effects of different concentration of NBPT and NBPTO on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships for a one-step enzyme-inhibitor interaction, qualified as mechanism A. The inhibition constants of urease by NBPT and NBPTO were found to be 0.15 μM and 2.1 nM, respectively. The inhibition constant for NBPT was also calculated by steady-state analysis and was found to be 0.13 μM. NBPTO was found to be a very strong inhibitor of urease in contrast to NBPT.     

Inhibitions of degradation of rat liver aldolase and lactic dehydrogenase by N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine or leupeptin in vivo

When injected into rats, leupeptin and E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine), potent thiol protease inhibitors of microbial origin, inhibited cathepsin B (EC 3.4.22.1) and cathepsin L (EC 3.4.22.-) in the lysosomal fraction of liver. Both compounds strongly inhibited cathepsin B, but E-64 had more effect than leupeptin on cathepsin L. Neither compound inhibited cathepsin D (EC 3.4.23. 5). E-64 reduced the apparent turnover rate of aldolase (EC 4. 1.2.13) markedly and the turnover rates of lactic dehydrogenase (EC 1.1.1.27) and total soluble protein slightly. Leupeptin had apparently less effect on degradation of those enzymes, but significant effect on degradation of aldolase. These results indicate that proteinases, which are sensitive to inhibition by E-64 or leupeptin, especially cathepsin L and cathepsin B may be important in degradation of aldolase.     

Speciation of N-(phosphonomethyl)glycine (glyphosate) in aqueous solutions containing major components of natural fluids

ABSTRACT

Potentiometric measurements in different media and at different temperatures were performed in order to determine protonation constants of glyphosate and formation constants for Ca²⁺ and Mg²⁺ complexes, together with their dependence on ionic strength and ?H⁰ values. Calorimetric measurements were also performed to determine ?H⁰ of protonation. Potentiometric measurements in mixed Ca²⁺ and Mg²⁺ solution allowed us to determine the formation of a mixed metal species. Thermodynamic parameters reported in this work can be used for studying the complete speciation of glp in electrolyte solutions containing the major ionic components of natural fluids.     

Synthesis of methylated chitosan containing aromatic moieties: Chemoselectivity and effect on molecular weight

N-Arylated chitosans were synthesized via Schiff bases formed by the reaction between the primary amino group of chitosan with aromatic aldehydes followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Treatment of chitosan containing N,N-dimethylaminobenzyl and N-pyridylmethyl substituents with iodomethane under basic conditions led to quaternized N-(4-N,N-dimethylaminobenzyl) chitosan and quaternized N-(4-pyridylmethyl) chitosan. Methylation occurred at either N,N-dimethylaminobenzyl and N-pyridylmethyl groups before the residual primary amino groups of chitosan GlcN units were substituted. The total degree of quaternization of each chitosan varied depending on the extent of N-substitution (ES) and the sodium hydroxide concentration used in methylation. Increasing ES increased the total degree of quaternization but reduced attack at the GlcN units. N,N-dimethylation and N-methylation at the primary amino group of chitosan decreased at higher ES’s. Higher total degrees of quaternization and degrees of O-methylation resulted when higher concentrations of sodium hydroxide were used. The molecular weight of chitosan before and after methylation was determined by gel permeation chromatography under mild acidic condition. The methylation of the N,N-dimethylaminobenzyl derivative with iodomethane was accompanied by numerous backbone cleavages and a concomitant reduction in the molecular weight of the methylated product was observed. The antibacterial activity of water-soluble methylated chitosan derivatives was determined using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria; minimum inhibitory concentrations (MIC) of these derivatives ranged from 32 to 128 μg/mL. The presence of the N,N-dimethylaminobenzyl and N-pyridylmethyl substituents on chitosan backbone after methylation did not enhance the antibacterial activity against S. aureus. However, N-(4-N,N-dimethylaminobenzyl) chitosan with degree of quaternization at the aromatic substituent and the primary amino group of chitosan of 17% and 16–30%, respectively, exhibited a slightly increased antibacterial activity against E. coli.     

N-(2-mercaptopropionyl)-glycine,a diffusible antioxidant,activates HIF-1 by inhibiting HIF prolyl hydroxylase-2: Implication in amelioration of rat colitis by the antioxidant

We investigated anti-colitic effects of N-(2-mercaptopropionyl)-glycine (NMPG), a diffusible antioxidant, in TNBS-induced rat colitis model and a potential molecular mechanism underlying the pharmacologic effect of the antioxidant. NMPG alleviated colonic injury and effectively lowered myeloperoxidase activity. Moreover, NMPG substantially attenuated expression of pro-inflammatory mediators in the inflamed colon. NMPG induced hypoxia-inducible factor-1α (HIF-1α) in human colon carcinoma cells, leading to elevated secretion of vascular endothelial growth factor (VEGF), a target gene product of HIF-1 involved in ulcer healing of gastrointestinal mucosa. NMPG induction of HIF-1α occurred by inhibiting HIF prolyl hydroxylase-2 (HPH-2), an enzyme that plays a major role in negatively regulating HIF-1α protein stability. In in vitro Von Hippel-Lindau protein binding assay, the inhibitory effect of NMPG on HPH-2 was attenuated by escalating dose of ascorbate but not 2-ketoglutarate, cofactors of the enzyme. Consistent with this, cell-permeable ascorbate significantly attenuated NMPG induction of HIF-1α in cells. Our data suggest that NMPG is an anti-colitic antioxidant that exerts its pharmacologic effects at least partly through activation of an ulcer healing pathway, HIF-1-VEGF.     

Synthesis and biological activities of peptidomimetic analogues of compound A71623, a potent and selective CCK-A receptor agonist

The involvement of cholecystokinin receptors in thephenomenon of satiety has been the impetus forsignificant research efforts, leading to the designand synthesis of CCK-A selective agonists for thepossible treatment of obesity. The Abbott laboratorieshave described a novel series of pseudotetrapeptidesrepresented by compound A71623, a highly potent andselective peripheral receptor agonist, but possessingvery poor bioavailability. Starting from thestructural requirements of this series of compounds,a peptidomimetic study was investigated, especiallyfocusing on the N-terminal part of A71623. Usingstandard coupling methods, introduction of unnaturalaromatic amino acids bearing a 2-carboxyethyl sidechain on their -amino group, along withbackbone length modulation, afforded selective analogues,presenting a highly modified peptidic backbone. Fromour two lead compounds, further optimization is underdevelopment, tending towards nonpeptidic structures.     

Effects of calmodulin antagonists on human ovarian cancer cell proliferation in vitro

Examined were effects of calmodulin antagonists (W-5 and W-7) on proliferation of two kinds of human cell lines, designated HR and KF, derived from serous cystadecarcinoma of the ovary. Although both W-5 and W-7 inhibited their cell proliferation in vitro, the degree of inhibition was more marked with W-5 rather than with W-7. HR cells had higher sensitivity to cisplatinum than KF cells, while KF cells had higher sensitivity to adriamycin. Combinations of calmodulin antagonists and anti-cancer drugs resulted in adjuvant effects with regard to the inhibition of their cell proliferation in vitro.     

Interaction of local anesthetics with calmodulin

A crude folic acid antagonist, previously designated as X-methyl folate was studied. Five components were found to inhibit the growth of Streptococcus faecalis. 9-Methyl folic acid was the major bioactive constituent by weight (5%), but because of its low specific activity, contributed only 1% of the biological activity of the crude reaction product. The most active compound accounted for 40% of the activity and constituted 0.04% of the crude product by weight. Spectroscopic data suggest this to be a novel folic acid analogue.     

Structural analysis of peptide fragments following the hydrolysis of bovine serum albumin by trypsin and chymotrypsin

Peptide bond hydrolysis of bovine serum albumin (BSA) by chymotrypsin and trypsin was investigated by employing time-resolved fluorescence spectroscopy. As a fluorescent cross-linking reagent, N-(1-pyrenyl) maleimide (PM) was attached to BSA, through all free amine groups of arginine, lysine, and/or single free thiol (Cys34). Time-resolved fluorescence spectroscopy was used to monitor fluorescence decays analyzed by exponential series method to obtain the changes in lifetime distributions. After the exposure of synthesized protein substrate PM-BSA to chymotrypsin and trypsin, it is observed that each protease produced a distinct change in the lifetime distribution profile, which was attributed to distinct chemical environments created by short peptide fragments in each hydrolysate. The persistence of excimer emission at longer lifetime regions for chymotrypsin, as opposed to trypsin, suggested the presence of small-scale hydrophobic clusters that might prevent some excimers from being completely quenched. It is most likely that the formation of these clusters is due to hydrophobic end groups of peptide fragments in chymotrypsin hydrolysate. A similar hydrophobic shield was not suggested for trypsin hydrolysis, as the end groups of peptide fragments would be either arginine or lysine. Overall, in case the target protein’s 3D structure is known, the structural analysis of possible excimer formation presented here can be used as a tool to explain the differences in activity between two proteases, i.e. the peak’s intensity and location in the profile. Furthermore, this structural evaluation might be helpful in obtaining the optimum experimental conditions in order to generate the highest amount of PM-BSA complexes.