Effect of Gossypol on Trypomastigotes and Amastigotes of Trypanosoma cruzi

Bloodstream Trypanosoma cruzi trypomastigotes isolated from infected mice undergo reduction of motility and structural damages after 5 to 45 min exposure to gossypol at concentrations ranging from 5 to 50 μM. When 1% serum albumin is added to the incubation medium, no alterations of parasites are observed, even with 100 μM gossypol. Intracellular T. cruzi amastigotes in infected Vero cell cultures exposed to 5 μM gossypol for 2 h do not show changes. Incubation with 5 μM gossypol for 48 h produces complete disruption of host cells; however, the amastigotes they contain show only mineor alterations. The observations indicate that, in protein-rich media, gossypol is complexed into associations which have no activity on the different forms of the T. cruzi biological cycle.     

放线菌结瘤植物根与根瘤中有机氮化物的组分及其上运形式

杨梅、沙棘和赤杨三种放线菌结瘤植物根瘤、根部有机氮化物的组分中,都含有占总有机氮化物50％以上的尿囊酸,说明在它们的根瘤中合成了大量的酰脲;同时,三种植物结瘤植株的茎木质部提取物中也含有大量的尿囊酸,表明根瘤将其合成的酰脲向植物地上部位运送。三种植物的根瘤还将其合成的特定的氨基酸及酰胺向地上部位转运,其中杨梅根瘤将固定的氮素以Asn和Gln的形式输出,而根部则以Arg的形式向上转运;沙棘根瘤以Ash,Gln及Ser,赤杨根瘤以Cit的形式合成并转运固定的氮素;后两种植物的无根瘤植株,以NH_4~+为氮源时,在转运的氨基酸组分中Arg的比例明显提高。     

Genetic variation of recent Alu insertions in human populations

The Alu family of intersperesed repeats is comprised of ovr 500,000 members which may be divided into discrete subfamilies based upon mutations held in common between members. Distinct subfamilies of Alu sequences have amplified within the human genome in recent evolutionary history. Several individual Alu family members have amplified so recently in human evolution that they are variable as to presence and absence at specific loci within different human populations. Here, we report on the distribution of six polymorphic Alu insetions in a survey of 563 individuals from 14 human population groups across several continents. Our results indicate that these polymorphic Alu insertions probably have an African origin and that there is a much smaller amount of genetic variation between European populations than that found between other populations groups. Present address: Department of Pathology, Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1901 Perdido St., New Orleans, LA 70112 Correspondence to: M.A. Batzer     

Interactions between the regulatory regions of twoAdh alleles

A region (NS1) that acts like an enhancer is located approximately 300 bp upstream of the larval cap site in theAdh gene ofD. melanogaster. When this sequence is deleted (NS1), the gene fails to express ADH protein. Gene expression can be restored by placing a secondAdh gene with an intact enhancer elsewhere on the same plasmid. In these circumstances, both genes are expressed equally regardless of their orientation on the plasmid. In this report we further characterize the interactions that occur when a single enhancer activates expression from a proximal and distant promoter. We have made the following observations: (1) While the two genes are expressed equivalently, their expression relative to a plasmid carrying two intact genes is reduced by a factor of 2 to 6 depending on the orientation of the two genes. (2) The single enhancer drives expression of both genes on any given plasmid molecule. (3) The enhancer does not interact with theAdh gene from which the NS7 region (which spans the larval TATA box) is removed. (4) Expression of the NS1 gene can be restored by an intact gene when both are inserted together into theDrosophila genome via P element-mediated transformation. (5) Increasing the separation between the two genes on a plasmid by up to 15 kbp does not prevent the restoration of expression of the NS1 gene. We propose a model that explains how a single enhancer can stimulate equal expression from two genes.     

Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants

We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The -glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Act1) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration.     

可育的抗除草剂溴苯腈转基因小麦

报道了采用微粒轰击（Ｍｉｃｒｏｐｒｏｊｅｃｔｉｌｅ ｂｏｍ ｂａｒｄｍ ｅｎｔ） 幼胚将除草剂抗性基因导入小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ Ｌ．）的转化研究。实验共使用了１３ 个小麦品种, 从开花后１４～１８ ｄ 的籽粒中剥取幼胚, 植物表达质粒含有ＣａＭＶ ３５Ｓ启动子控制的除草剂溴苯腈抗性基因ｂｘｎ 以及筛选标记基因ＮＴＰⅡ。采用高压放电基因枪,用质粒ＤＮＡ 包被的钨粒轰击预培养３ ｄ 的幼胚。在含有卡那霉素类似物ｇｅｎｅｔｉｃｉｎ Ｇ４１８ｓｕｌｐｈａｔｅ 的ＭＳ培养基上, 经过多步骤筛选和分化, 从８００ 多个幼胚中获得了１６ 株转化苗。除草剂抗性鉴定和Ｓｏｕｔｈｅｒｎ 杂交分析证明, 其中４ 株为转基因植物,具有溴苯腈抗性, 并且自交可育。转化工作从分离幼胚到转化苗鉴定完毕, 最短时间为６ 个月, 因此, 该方法是一项快速有效的基因导入技术     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

甘蓝类无蜡粉亮叶性状遗传规律及其利用的研究

我们于1987年从普通结球甘蓝“迎春”品种自交二代群体中,发现了无蜡粉亮叶甘蓝突变株, 经过多年对其遗传规律进行的研究,认为这一无蜡粉亮叶性状是由一对隐性纯合基因控制。利用这一性状可培育结球甘蓝及其它甘蓝类具有这同一性状的新类型、新品种,提高其品质,更可作一代杂种利用的标记性状,充分发挥一代杂种的优势。     

Studies on activation and inhibition of cathepsin B from buffalo liver

Cathepsin B (EC 3.4.22.1) was purified from buffalo liver. The enzyme activity against-benzoyl-dl-arginine-naphthylamme (BANA) was substantially reduced by heat (above 37C) and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-1-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu²⁺ (20–200M) and Ca²⁺ (30–250 mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5-dithiobis(2-nitro-benzoic acid) (DTNB), andp-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.