Enantiodiscrimination of chiral alpha-aminophosphonic acids by mass spectrometry.

Diastereomeric clusters between first-group metal ions (M(+)) and chiral alpha-aminophosphonic acids (A and B) have been readily generated in the gas phase by electrospray ionization (ESI) and their fragmentation investigated by mass spectrometry. The complexes studied had the general formula [MA(S)B(2)](+) and [MA(R)B(2)](+), where M = H, Li, Na, or K, A(S) and A(R) are the two enantiomers of a given acid A, and B is a reference alpha-aminophosphonic acid of defined configuration. Collision-induced decomposition (CID) of [MA(S)B(2)](+) and [MA(R)B(2)](+) leads to fragmentation patterns characterized by [MAB](+)/[MB(2)] abundance ratios which depend on the configuration of ligand A. These different spectral features were correlated to the different stability of the diastereomeric [MA(S)B(2)](+) and [MA(R)B(2)](+) complexes in the gas phase. The results have been discussed in the light of MM2 Molecular Mechanics Force Field calculations.     

蜘蛛牵引丝蛋白cDNA的扩增、克隆与序列分析

蜘蛛是一种能在同一生物体内产生具有不同功能的多种丝的生物。蜘蛛丝的本质是蛋白质。牵引丝 (Ｄｒａｇｌｉｎｅｓｉｌｋ)是由蜘蛛的主壶腹腺 (Ｍａｊｏｒａｍｐｕｌｌａｔｅ ,ＭＡ)产生的 ,其较高的抗张强度 (4× 10 9Ｎ ｍ2 )与弹性 (35 % ) [1 ,2 ] ,使之成为一种有广阔应用前景的生物材料。目前 ,国外已有实验室通过构建ｃＤＮＡ文库的方法获得Ｎｅｐｈｉｌａｃｌａｖｉｐｅｓ中的两个牵引丝蛋白Ｓｐｉｄｒｏｉｎ1与Ｓｐｉｄｒｏｉｎ2的ｃＤＮＡ片段[3 ,4] ;但国内尚未见有相关报道。本文介绍的工作是利用简单便捷的ＰＣＲ技术对牵引丝…     

The binding of aluminum to mugineic acid and related compounds as studied by potentiometric titration

The phytosiderophores, mugineic acid (MA) and epi-hydroxymugineic acid (HMA), together with a related compound, nicotianamine (NA), were investigated for their ability to bind Al(III). Potentiometric titration analysis demonstrated that MA and HMA bind Al(III), in contrast to NA which does not under normal physiological conditions. With MA and HMA, in addition to the Al complex (AlL), the protonated (AlLH) and deprotonated (AlLH₋₁) complexes were identified from an analysis of titration curves, where L denotes the phytosiderophore form in which all the carboxylate functions are ionized. The equilibrium formation constants of the Al(III) phytosiderophore complexes are much smaller than those of the corresponding Fe(III) complexes. The higher selectivity of phytosiderophores for Fe(III) over Al(III) facilitates Fe(III) acquisition in alkaline conditions where free Al(III) levels are higher than free Fe(III) levels.     

Positive and negative modulation of virus infectivity and envelope glycoprotein incorporation into virions by amino acid substitutions at the N terminus of the simian immunodeficiency virus matrix protein

The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal alpha-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within alpha-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the beta-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.     

High-performance liquid chromatographic determination of 1,3-diethyl-2-thiobarbituric acid–malonaldehyde adduct in fish meat

The reaction conditions of 1,3-diethyl-2-thiobarbituric acid (DETBA)–malonaldehyde (MA) adduct formation were examined in order to analyze MA in fish tissue by high-performance liquid chromatography. A reaction mixture containing 4 mM butyl hydroxytoluene was heated at 100°C for 150 min and the DETBA–MA adduct formed was separated by a Inertsil ODS column for 20 min. The detection limit was 5 pmol.     

Behavioral,metabolic, and molecular correlates of lower insulin sensitivity in Mexican-Americans

We determined whether lower insulin sensitivity persists in young, nonobese, nondiabetic Mexican-American [MA; n = 13, 27.0 +/- 2.0 yr, body mass index (BMI) 23.0 +/- 0.7] compared with non-Hispanic white (NHW; n = 13, 24.8 +/- 1.5 yr, BMI 22.8 +/- 0.6) males and females after accounting for cardiorespiratory fitness (maximal O(2) uptake), abdominal fat distribution (computed tomography scans), dietary intake (4-day records), and skeletal muscle insulin-signaling protein abundance from muscle biopsies (Western blot analysis). MA were significantly less insulin sensitive compared with their NHW counterparts when estimated by homeostatic model assessment of insulin resistance (MA: 1.53 +/- 0.22 vs. NHW: 0.87 +/- 0.16, P < 0.05) and the revised quantitative insulin sensitivity check index (MA: 0.45 +/- 0.08 vs. NHW: 0.58 +/- 0.19, P = 0.05). However, skeletal muscle protein abundance of insulin receptor-beta (IRbeta), phosphatidylinositol 3-kinase p85 subunit, Akt1, Akt2, and GLUT4 were not significantly different. Differences in indexes of insulin sensitivity lost significance after percent dietary intake of palmitic acid, palmitoleic acid, and skeletal muscle protein abundance of IRbeta were accounted for. We conclude that differences in insulin sensitivity between nonobese, nondiabetic MA and NHW persist after effects of chronic and acute exercise and total and abdominal fat distribution are accounted for. These differences may be mediated, in part, by dietary fat intake.     

Degradation of iprodione by a soil Arthrobacter-like strain.

A bacterial strain able to transform iprodione was isolated from a fast iprodione-degrading soil by enrichment procedures. Transformation was detected through 3,5-dichloroaniline production as measured by a rapid colorimetric method. The strain, MA6, was tentatively identified as an Arthrobacter sp. When it was incubated with MA6 in a minimum mineral medium (pH 6.5), iprodione (8.8 mumol/liter) was transformed into two major metabolites that were identified by high-performance liquid chromatography analysis: 3,5-dichlorophenylcarboximide (metabolite 1) and (3,5-dichlorophenylurea) acetic acid (metabolite 2), which was produced after ring cleavage of the former product. These products were synthesized in the laboratory and compared with metabolites 1 and 2 which were formed during iprodione degradation. Small quantities of 3,5-dichloroaniline also appeared in the bacterial culture but did not substantially increase between the first and second days of incubation. In contrast, in the sterile control medium, iprodione was spontaneously transformed into hydantoic acid and an iprodione isomer. Chemical and biological transformations of iprodione seem to occur through two different pathways. One biological degradation pathway is proposed.     

Hair analysis and self-report of methamphetamine use by methamphetamine dependent individuals

The questions of whether the dose of drug that is consumed corresponds to drug concentration levels in hair and how results of hair analyses can be interpreted are still debated. The aim of this study was to investigate (1) whether there is a correlation between doses of Methamphetamine (MA) use and MA concentration levels in hair and (2) whether results of hair analyses can be used to estimate dose, frequency, and patterns of MA use. In this study, segmental hair analysis was performed through consecutive 1cm as well as 1-4 cm (=3 cm) segmental hair lengths. MA dependent individuals (n=9) provided information on doses (0.25-4 g/day) of MA use as well as the frequency of MA use. The concentrations of MA and its metabolite amphetamine (AP) in hair were determined using gas chromatography/mass spectrometry (GC/MS). One-way analysis of variance (ANOVA) test was performed to evaluate whether MA and AP concentrations in consecutive 1cm length segmental hair were consistent with the history of MA use. The cumulative doses of MA use calculated from the daily dose and the frequency during 1-4 months were well correlated to the concentrations of MA and AP in 1-4 cm segmental hair length (correlation coefficient, r=0.87 for MA and r=0.77 for AP). The results from this study show the patterns and histories of MA use from MA dependent individuals and could assist in the interpretation of hair results in forensic toxicology as well as in rehabilitation and treatment programs.     

HIV-1 Matrix Protein Binding to RNA

The matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein plays multiple roles in the viral replication cycle. One essential role is to target PrGag proteins to their lipid raft-associated phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] assembly sites at the plasma membranes of infected cells. In addition to this role, several reports have implicated nucleic acid binding properties to retroviral MAs. Evidence indicates that RNA binding enhances the binding specificity of MA to PI(4,5)P₂-containing membranes and supports a hypothesis in which RNA binding to MA acts as a chaperone that protects MA from associating with inappropriate cellular membranes prior to PrGag delivery to plasma membrane assembly sites. To gain a better understanding of HIV-1 MA-RNA interactions, we have analyzed the interaction of HIV MA with RNA ligands that were selected previously for their high affinities to MA. Binding interactions were characterized via bead binding, fluorescence anisotropy, gel shift, and analytical ultracentrifugation methods. Moreover, MA residues that are involved in RNA binding were identified from NMR chemical shift data. Our results indicate that the MA RNA and PI(4,5)P₂ binding sites overlap and suggest models for Gag-membrane and Gag-RNA interactions and for the HIV assembly pathway.     

Analysis of maslinic acid and gallic acid compounds as xanthine oxidase inhibitors in isoprenaline administered myocardial necrotic rats

ObjectiveThis research designed to analyze the in vivo and in silico ameliorative action of maslinic acid (MA) and gallic acid (GA) on reactive oxygen species generating enzyme xanthine oxidase (XO) in isoprenaline or isoproterenol (ISO) induced myocardial infarcted rats.MethodsAlbino Wistar rats were categorized into four groups with eight rats in each group. A dose of 15 mg/kg of MA and GA were pretreated to each MA and GA groups for seven days. A dose of 85 mg/kg of ISO administered to the ISO group along with MA and GA groups except normal group on two consecutive days of pretreatment. All animals sacrificed and the heart tissues were collected for the analysis of XO. The in silico molecular docking analysis of the compounds MA and GA with XO was analyzed by using Gold 3.0.1 software.ResultsXO enzyme levels were significantly increased in the heart homogenate of ISO administered rats when compared to normal rats. Pretreatment of MA and GA to ISO treated rats significantly brought XO enzyme to the near normal levels which indicate the protective action of MA and GA against myocardial necrosis. The in vivo results were further supported by the in silico molecular docking study which revealed the inhibition of XO enzyme by the formation of enzyme and ligand complex with the compounds MA and GA.ConclusionMA and GA compounds manifested the ameliorative effect against ISO administrated myocardial necrosis by inhibiting the free radical generating enzyme XO which is evidenced by both in vivo and in silico studies.     

The possible formation of nano-clusters of succinic acid and maleic acid in tetrahydrofuran due to incomplete solvation

In continuation of our work on the conformational analysis of succinic acid (SA) and maleic acid (MA) in different solvents, we present here the experimental dielectric and IR and also the ab initio Hartree–Fock calculations of the two dicarboxylic acids in tetrahydrofuran (THF). The dielectric measurements are carried out at microwave X-band frequency of 9.7 GHz and the calculations are performed at STO-3G and 6-31G(d) basis sets. The dielectric data and the dipole moment determined experimentally are compared with the dipole moment determined from the conformal analysis. It is seen that the dielectric properties of SA/MA in THF are much different from that of SA/MA in 1-4, dioxane (1-4D) that we had reported previously. The IR spectra of SA–THF system is also reported here. The present study indicates the possible formation of nano-clusters of SA/MA in THF due to incomplete solvation by THF.     

Effects of malic acid on rumen fermentation, urinary excretion of purine derivatives and feed digestibility in steers

The objective of this study was to evaluate the effects of malic acid (MA) supplementation on rumen fermentation, urinary excretion of purine derivatives (PDs) and whole gastro-intestinal tract feed digestibility in steers. Eight ruminally cannulated Simmental steers (465 ± 13 kg) were used in a replicated 4 × 4 Latin square design. The treatments were: control (without MA), LMA (MA-low), MMA (MA-medium) and HMA (MA-high) with 0.0, 7.8, 15.6 and 23.4 g MA per kg dry matter (DM), respectively. Diets consisted of corn stover and concentrate (60/40, DM basis). DM intake was approximately 9 kg per day, which was 90% of ad libitum intake including 5.4 kg corn stover and 3.6 kg concentrate. Ruminal pH (range of 6.91 to 6.56), ratio of acetate to propionate (range of 3.88 to 3.25), ammonia N (range of 9.03 to 6.42 mg/100 ml) and lactate (range of 91.25 to 76.31 mg/100 ml) decreased linearly as MA supplementation increased, whereas total volatile fatty acid (VFA) concentration (range of 55.68 to 61.49 mM) linearly (P < 0.05) increased with increase in MA supplementation. In situ ruminal neutral detergent fiber (aNDF) degradation of corn stover was improved but the crude protein (CP) degradability of concentrate mix was decreased with increasing the dose of MA. Urinary excretion of PDs was quadratically (P < 0.01) changed with altering MA supplementation (67.88, 72.74, 75.81 and 73.78 mmol/day for control, LMA, MMA and HMA, respectively). Similarly, digestibilities of DM, organic matter (OM), NDF and acid detergent fiber (ADF) in the total tract were also quadratically increased with increasing MA, and no differences in terms of CP and ether extract digestibility were observed. The results indicate that MA supplementation has the potential to improve rumen fermentation and feed digestion in beef cattle. The MA stimulates the digestive microorganisms or enzymes in a quadratic response. In the experimental conditions of this trial, the optimum MA dose was 15.6 g MA per kg DM.     

Solubility enhancement of mefenamic acid by inclusion complex with β-cyclodextrin: in silico modelling,formulation, characterisation,and in vitro studies

The aim of this study was to prepare and characterise inclusion complexes of a low water-soluble drug, mefenamic acid (MA), with β-cyclodextrin (β-CD). First, the phase solubility diagram of MA in β-CD was drawn from 0 to 21 × 10⁻³ M of β-CD concentration. A job’s plot experiment was used to determine the stoichiometry of the MA:β-CD complex (2:1). The stability of this complex was confirmed by molecular modelling simulation. Three methods, namely solvent co-evaporation (CE), kneading (KN), and physical mixture (PM), were used to prepare the (2:1) MA:β-CD complexes. All complexes were fully characterised. The drug dissolution tests were established in simulated liquid gastric and the MA water solubility at pH 1.2 from complexes was significantly improved. The mechanism of MA released from the β-CD complexes was illustrated through a mathematical treatment. Finally, two in vitro experiments confirmed the interest to use a (2:1) MA:β-CD complex.     

Feeding Response to Mercaptoacetate in Osborne-Mendel and S5B/PL Rats

SINGER, LORI K, DAVID A YORK, GEORGE A BRAY. Feeding response to mercaptoacetate in Osborne-Mendel and S5B/PL Rats. The purpose of this experiment was to determine if Osborne-Mendel (OM) rats, which are susceptible to dietary-induced obesity, and S5B/PL (S5B) rats, which are resistant to dietary-induced obesity, differ in their feeding responses to mercaptoacetate (MA), which blocks fatty acid oxidation, or 2-deoxy-D-glucose (2DG), which blocks glucose utilization. 2DG (100 mg/kg or 200 mg/ kg) increased food intake in both strains of rats on a high-fat diet (56% energy from fat). Mercaptoacetate (600 umol/kg) increased food intake in OM but not S5B rats on a high-fat diet. When maintained on a low-fat diet (10% energy from fat), MA (400 umol/kg or 600 umol/kg) stimulated food intake in OM rats, whereas S5B rats increased food intake only after the highest dose of MA (600 umol/kg). MA stimulated carbohydrate and protein intake in OM rats maintained on a macro-nutrient selection diet, whereas S5B rats maintained on this diet did not significantly increase intake of any mac-ronutrient after MA. These results demonstrate that OM and S5B rats have a similar food intake response to 2DG but a dissimilar response to MA. The variable response to MA in these strains may be due to a difference in peripheral or central signaling systems related to fatty acid oxidation or a difference in metabolic environments between the strains, which in turn affects the feeding response to MA. These studies suggest that a difference in control of fatty acid oxidation may account for the difference in susceptibility to obesity when eating a high-fat diet.     

Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats

Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05–5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca²⁺ retention capacity, and caused swelling in Ca²⁺-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients.     

Amino acid sequence analysis of the proteolytic cleavage products of the bovine immunodeficiency virus Gag precursor polypeptide.

Bovine immunodeficiency virus Gag proteins were purified from virions, and their amino acid sequences and molecular masses were determined. The matrix, capsid, and nucleocapsid (MA, CA, and NC, respectively) and three smaller proteins (p2L, p3, and p2) were found to have molecular masses of 14.6, 24.6, and 7.3 and 2.5, 2.7, and 1.9 kDa, respectively. The order of these six proteins in the Gag precursor, Pr53gag, is NH2-MA-p2L-CA-p3-NC-p2-COOH. In contrast to other retroviral MA proteins, the bovine immunodeficiency virus MA retains its N-terminal methionine and is not modified by fatty acids. In addition, the bovine immunodeficiency virus NC migrates as a 13-kDa protein in denaturing gel electrophoresis; however, its molecular mass was determined to be 7.3 kDa.     

Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism.

The interaction of the human immunodeficiency virus (HIV) Gag protein with the plasma membrane of a cell is a critical event in the assembly of HIV particles. The matrix protein region (MA) of HIV type 1 (HIV-1) Pr55Gag has previously been demonstrated to confer membrane-binding properties on the precursor polyprotein. Both the myristic acid moiety and additional determinants within MA are essential for plasma membrane binding and subsequent particle formation. In this study, we demonstrated the myristylation-dependent membrane interaction of MA in an in vivo membrane-binding assay. When expressed within mammalian cells, MA was found both in association with cellular membranes and in a membrane-free form. In contrast, the intact precursor Pr55Gag molecule analyzed in an identical manner was found almost exclusively bound to membranes. Both membrane-bound and membrane-free forms of MA were myristylated and phosphorylated. Differential membrane binding was not due to the formation of multimers, as dimeric and trimeric forms of MA were also found in both membrane-bound and membrane-free fractions. To define the requirements for membrane binding of MA, we analyzed the membrane binding of a series of MA deletion mutants. Surprisingly, deletions within alpha-helical regions forming the globular head of MA led to a dramatic increase in overall membrane binding. The stability of the MA-membrane interaction was not affected by these deletions, and no deletion eliminated membrane binding of the molecule. These results establish that myristic acid is a primary determinant of the stability of the Gag protein-membrane interaction and provide support for the hypothesis that a significant proportion of HIV-1 MA molecules may adopt a conformation in which myristic acid is hidden and unavailable for membrane interaction.