Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

Inhibitory Effects of Akacid®plus on Growth and Aflatoxin Production by Aspergillus parasiticus

The effects of Akacid plus, a novel guanidine-based polymer first introduced as a biocidal and disinfectant agent were studied on Aspergillus parasiticus growth and its aflatoxin (AF) productivity. The fungus was cultured on yeast extract-sucrose (YES) broth in presence of various twofold serial dilutions of 25% Akacid plus (1.5-96 microL/50 mL medium) and then incubated in shaking condition with 150 rev./min at 28 degrees C for 96 h. Based on obtained results, Akacid plus was found to significantly inhibit both growth and aflatoxin B1 (AFB1) synthesis in very low concentrations in a dose-dependent manner. Fungal growth inhibition was determined in the range of 9.6-99.6% in mycelia exposed to the total concentration range of 1.5-48 microL. A final concentration of 96 microL was necessary to completely inhibit the growth of fungus. Under similar conditions, AFB1 synthesis was found to be strongly inhibited by 8.1-98.0% in presence of 1.5-24 microL Akacid plus with a maximum of 100% by 48 microL concentration. With respect to the unique physico-chemical properties of Akacid plus, its marked inhibitory effects on A. parasiticus growth and its AFB1 synthesis shown for the first time in this study make it a promising candidate for application in prevention programmes of AF contamination of susceptible crops.     

The adduct formation between the thioguanine-polyamine ligands and DNA with the AP site under UVA irradiated and non-irradiated conditions

The AP sites are representative of DNA damage and known as an intermediate in the base excision repair (BER) pathway which is involved in the repair of damaged nucleobases by reactive oxygen species, UVA irradiation, and DNA alkylating agents. Therefore, it is expected that the inhibition or modulation of the AP site repair pathway may be a new type of anticancer drug. In this study, we investigated the effects of the thioguanine-polyamine ligands (^SG-ligands) on the affinity and the reactivity for the AP site under UVA irradiated and non-irradiated conditions. The ^SG-ligands have a photo-reactivity with the A-F-C sequence where F represents a tetrahydrofuran AP site analogue. Interestingly, the ^SG-ligands promoted the β-elimination of the AP site followed by the formation of a covalent bond with the β-eliminated fragment without UVA irradiation.     

Catechol-based inhibitors of bacterial urease

Targeted covalent inhibitors of urease were developed on the basis of the catechol structure. Forty amide and ester derivatives of 3,4-dihydroxyphenylacetic acid, caffeic acid, ferulic acid and gallic acid were obtained and screened against Sporosarcinia pasteurii urease. The most active compound, namely propargyl ester of 3,4-dihydroxyphenylacetic acid exhibited IC₅₀?=?518?nM and$k_{\mathit{inact}}/K_i$?=?1379?M^?1?s^?1. Inhibitory activity of this compound was better and toxicity lower than those obtained for the starting compound – catechol. The molecular modelling studies revealed a mode of binding consistent with structure-activity relationships.     

In-vitro evaluation of the antimicrobial activity of Bauhinia variegata,locally known as koiralo

Bauhinia variegata, commonly known as Koiralo is considered as medicinal plant in Nepal and India. The alcoholic extract of this plant was found to have antimicrobial activity against Bacillus subtilis (ATCC 6635) Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus (ATCC 29213) and Vibrio cholerae. The largest zone of inhibition (18 mm) was found to be exhibited against B. subtilis. For this organism the minimum bactericidal concentration (MBC) of the crude extract was 0.39 mg/ml. The extract was found to be more effective against gram-positive than gram-negative bacteria. The antimicrobial activity of the extract was found to be decreased during purification.     

Differences between cellular mechanisms of ATP-and noradrenaline-induced inhibition of visceral smooth muscles under conditions of selective and combined activation of M<Subscript>2</Subscript> or M<Subscript>3</Subscript> cholinoreceptors

Our studies of the role of phospholipase C in inhibitory synaptic action upon visceral smooth muscles demonstrated that, under conditions of carbachol (CCh)-induced pre-activation of cholinoreceptors, ATP-or noradrenaline (NA)-evoked relaxation of these muscles is mediated by the phospholipase C-independent pathway, while the phospholipase C-dependent pathway does not manifest itself as a mechanism that determines the inhibitory effect of the above transmitters. Under conditions of pre-activation of muscarinic cholinoreceptors, ATP-and NA-induced relaxation is continued due to activation of inositol trisphosphate (InsP₃)-sensitive receptors despite the fact that the pathway of inhibition is phospholipase C-independent. This is confirmed by complete depression of the inhibitory effects of ATP and NA against the background of CCh-induced contraction after pre-incubation of the studied preparations together with 100 μM 2-APB, a blocker of InsP₃ receptors. Selective blockings of either M₂ or M₃ cholinoreceptors are accompanied by a complete loss of the ability of the above blocker of InsP₃ receptors (2-APB) to suppress ATP-and NA-induced contraction of smooth muscles in the state of CCh-induced contraction. It can be hypothesized that, under conditions of selective pre-activation of M₂ or M₃ cholinoreceptors, the mechanisms of intracellular signalling mediating the inhibition events are modified. The InsP₃-dependent pathway that determines both adrenergic and purinergic inhibition of smooth muscles is switched off, and the inhibitory action of neurotransmitters is realized under such conditions through the InsP₃-independent pathway. Therefore, in our study we first found differences between cellular mechanisms underlying ATP-and NA-induced inhibition of smooth muscles under conditions of selective activation of either M₂ or M₃ cholinoreceptors and the mechanisms underlying the relaxing action of inhibitory neurotransmitters under conditions of combined synergistic activation of cholinoreceptors of both the above-mentioned subtypes. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 22–31, January–February, 2007.     

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α₁-antitrypsin (α₁AT) deficiency cases. It leads to increased polymerization of the serpin α₁AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α₁AT and analyzed their fluorescence properties. α₁AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α₁AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α₁AT compared to M α₁AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B.     

Sesquiterpenoids and lignans from the roots of Valeriana officinalis L

Two new guaiane-type sesquiterpenoids, valerol A (1) and kessyl 3-acetate (2), together with nine known compounds, valeracetate (3), anismol A (4), orientalol C (5), spatulenol (6), 4α,10α-epoxyaromadendrane (7), (+)-8-hydroxypinoresinol (8), pinorespiol (9), pinoresinol 4-O-β-D-glucopyranoside (10), and 8-hydroxypinoresinol 4'-O-β-D-glucopyranoside (11) were isolated from the roots of Valeriana officinalis. The structures and relative configurations of 1 and 2 were elucidated on the basis of spectroscopic methods (1D- and 2D-NMR, MS, UV, and IR). These compounds were evaluated for inhibitory activity on acetylcholinesterase (AChE) and enhancing activity on nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells.     

小峰熊蜂头部乙酰胆碱酯酶测定条件的优化及其对六种常用杀虫剂的敏感性

小峰熊蜂Bombus hypocrita是我国优势熊蜂种群之一, 因其易于饲养、 群势较强且授粉性能优良而成为我国设施农业常用优良授粉蜂种, 但常受到以乙酰胆碱酯酶(AChE)为靶标酶的有机磷和氨基甲酸酯类杀虫剂的危害。 为合理规避这两类杀虫剂对熊蜂的危害, 同时也为完善熊蜂授粉配套技术和保护野生熊蜂资源提供理论基础, 本研究利用正交试验对小峰熊蜂头部乙酰胆碱酯酶活性的测定条件进行了优化, 并明确了6种常用有机磷和氨基甲酸酯类杀虫剂对乙酰胆碱酯酶活性的影响。结果表明： 各测定因素对小峰熊蜂乙酰胆碱酯酶活性测定影响的大小顺序依次为： 酶浓度＞pH＞温度＞底物浓度＞反应时间; 小峰熊蜂头部乙酰胆碱酯酶活性的最适反应条件为： 酶浓度0.25 g 蛋白质/L, 底物浓度0.8 mmol/L, pH值7.5, 温度40℃, 反应时间5 min。毒死蜱、 三唑磷、 丙溴磷、 异丙威、 仲丁威和残杀威6种杀虫剂对小峰熊蜂头部乙酰胆碱酯酶离体抑制作用均呈现明显的剂量-效应关系, 其抑制中浓度IC₅₀分别为0.39, 1.79, 0.42, 0.04, 0.43和0.63 mmol/L。这6种杀虫剂对小峰熊蜂AChE抑制作用的强弱依次为： 异丙威＞毒死蜱＞三唑磷＞仲丁威＞残杀威＞丙溴磷, 即小峰熊蜂对异丙威最敏感, 而对丙溴磷的敏感性最弱。     

酵母醇脱氢酶ADHI的纯化及动力学研究

本文报道了啤酒酵母醇脱氢酶组成型同工酶ＡＤＨＩ的快速高效的纯化方法。通过活性蓝色染料柱亲和层析的方法将该酶纯化至电泳均一,收率达４７％,对该酶的产物抑制及端点抑制动力学研究结果支持Ｗｒａｔｔｅｎ,Ｃｌｅｌａｎｄ提出的序列有序机制。