Mutations in acid beta-galactosidase cause GM1-gangliosidosis in American patients.

We describe four new mutations in the beta-galactosidase gene. These are the first mutations causing infantile and juvenile GM1-gangliosidosis to be described in American patients. Cell lines from two patients with juvenile and from six patients with infantile GM1-gangliosidosis were analyzed. Northern blot analysis showed the acid beta-galactosidase message to be of normal size and quantity in two juvenile and four infantile cases and of normal size but reduced quantity in two infantile cases. The mutations are distinct from the Japanese mutations. All are point mutations leading to amino acid substitutions: Lys577-->Arg, Arg590-->His, and Glu632-->Gly. The fourth mutation, Arg208-->Cys, accounts for 10 of 16 possible alleles. Two infantile cases from Puerto Rico of Spanish ancestry are homozygous for this mutation, suggesting that this allele may have come to South America and North America via Puerto Rico. That these mutations cause clinical disease was confirmed by marked reduction in catalytic activity of the mutant proteins in the Cos-1 cell expression system.     

湖羊瘤胃微生物GH9家族葡聚糖酶基因IDSGLUC9-25的表达与功能表征

【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30-50℃下活性较高,在pH 4.0-8.0范围内能够保持较高的稳定性,经pH 4.0-8.0缓冲液处理1 h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73) U/mg;利用薄层色谱法(thin layer chromatography, TLC)和高效液相色谱法(high performance liquid chromatography, HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25 (EC 3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。     

-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of -cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

Preparation of the pure diastereomeric forms of S- (5′-deoxy-5′-adenosyl)-1-ammonio-4-methylsulfonio-2- cyclopentene and their evaluation as irreversible inhibitors of S-adenosylmethionine decarboxylase from Escherichia coli

The conformationally restricted S-adenosylmethionine analogue AdoMac (S-(5′-deoxy-5′-adenosyl)-1-ammonio-4-methylsulfonio-2-cyclopentene has been shown to act as an enzyme activated, irreversible inhibitor of theEscherichia coli form of the enzyme S-adenosylmethionine decarboxylase. Inactivation of the enzyme is presumably initiated by formation of an imine linkage between the inhibitor and the terminal pyruvate of the enzyme, followed by base-catalyzed elimination of methylthioadenosine and generation of a latent electrophile. Removal of the driving force for the elimination of methylthioadenosine resulted in a reversibly binding inhibitor. Thus, the thioether analogue corresponding to AdoMac, and the corresponding dihydro derivative (H₂-AdoMac), reversibly inhibit the enzyme. AdoMac was resolved into its four pure diastereomeric forms, and each diastereomer was evaluated as an irreversible inhibitor of the enzyme. The K_I values for the individual diastereomers range between 3.83 and 39.6 μM, with the cis-1S,4R diastereomer being the most potent inhibitor. However, the k_inact values for the four diastereomers are not significantly different, suggesting that the binding of each diastereomer to the enzyme is configuration-dependent, while the subsequent inactivation likely proceeds through a single intermediate which is formed from each of the four diastereomers. Since each pure diastereomer represents a distinct conformational mimic exhibiting restricted sidechain rotation, the data suggests that these and related analogues may be useful as conformational probes for the catalytic site of AdoMet-DC.     

盐磷胁迫对木麻黄和台湾相思种子萌发及幼苗生长的影响

恶劣环境下,人工海防林因面临养分胁迫而经营困难。为探讨盐、磷胁迫对主要海防林树种木麻黄和台湾相思种子萌发及生长的影响,该研究分别用不同浓度的NaCl(盐)和KH₂PO₄(磷)溶液处理种子和浇灌幼苗,测定种子萌发和幼苗生长指标。结果表明：(1)高盐胁迫显著抑制种子萌发,对幼苗生长有一定影响,但两种植物影响程度不同;台湾相思种子萌发耐盐性高于木麻黄,前者相对盐害率最大值为23.03%,后者为89.15%;随着盐浓度增加,木麻黄和台湾相思种子的发芽率、发芽势、发芽指数和活力指数均降低,对应最大值分别为38.70%、34.67%、18.70、0.055和76.67%、62.22%、48.46、6.11。(2)两种植物的株高和根长随盐浓度增加而降低,木麻黄和台湾相思株高分别为12.29～6.01 mm和48.27～17.33 mm,根长分别为8.57～1.45 mm和33.41～5.88 mm;台湾相思根、茎、叶生物量及根冠比均随盐浓度的增加逐渐减小,木麻黄各处理差异较小。(3)台湾相思的种子和幼苗较木麻黄更耐低磷环境,二者最适磷浓度存在差异;木麻黄种...     

哺乳动物透明带糖蛋白及其生物活性

透明带在哺乳动物的受精过程中发挥着重要作用,透明带化学成分及其生物活性的研究对于了解精卵识别、顶体反应的诱导等过程的分子机理具有重要意义．近10年来的研究表明透明带主要由糖蛋白（ZPGPs）组成,是在细胞质内合成后转移至透明带的.大多数动物的ZPGPs为4～5种,不同种类动物的ZPGPs的氨基酸序列存在高度同源性.在受精过程中,ZPGPs具有精子受体和诱导顶体反应的双重作用,ZPGPs含有N－连接和O－连接寡聚糖,这些寡糖链是ZPGPs行使生物活性不可缺少的部分．     

Antitumor agents-CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis

(+)-Thiocolchicine (2b) was prepared from (±)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (−)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (−)-camphanylated diastereomer, had potent activity in all assays comparable to that of (−)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.     

不同光学异构体组成的氯氰菊酯对敏感及抗性家蝇的神经电生理学研究

采用敏感家蝇(Musca domestica vicina L.)及由5种不同光学异构体组成的氯氰菊酯选育的抗性家蝇中胸足离体标本,观测五种药剂对足感觉神经纤维冲动发放的影响。各种异构体组成的氯氰菊酯均可引起感觉神经纤维发放的增加,然后逐渐降低,直至完全阻断。以阻断时间和加药剂量为参数,求出神经敏感度。结果表明：五种药剂作用于敏感家蝇的神经敏感度与室内生物测定的LD₅₀值(μg/头)无相关性,而与供试药剂中反α体与顺α体的比例有关,反α体所占比例越多的药物,神经敏感度越高。抗性家蝇的神经敏感度与敏感 家蝇相比大幅度下降,可以认为神经敏感度降低是家蝇对氯氰菊酯产生抗性的主要机制。抗性家蝇中,氯氰菊酯品系(RC₁)的抗生水平最高,但它的神经敏感度较其它抗性品系也高,由此推测,RC₁,的抗性机制与其它晶系有所不同。