Isolation and characterization of a naturally occurring inhibitor from mung bean (Vigna radiata) seedlings for serine hydroxymethyltransferase

A naturally occurring inhibitor of serine hydroxymethyltransferase (EC2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98-degrees-C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.

Part I. Synthesis of L-amino acid oxidase by a serine- or glycine-requiring strain of neurospora. Part II. Studies concerning multiple electrophoretic forms of tyrosinase in neurospora

Part I: Synthesis of L-Amino Acid Oxidase by a Serine- or Glycine-Requiring Strain of Neurospora

Wild-type cultures of Neurospora crassa growing on minimal medium contain low levels of L-amino acid oxidase, tyrosinase, and nicotinarnide adenine dinucleotide glycohydrase (NADase). The enzymes are derepressed by starvation and by a number of other conditions which are inhibitory to growth. L-amino acid oxidase is, in addition, induced by growth on amino acids. A mutant which produces large quantities of both L-amino acid oxidase and NADase when growing on minimal medium was investigated. Constitutive synthesis of L-amino acid oxidase was shown to be inherited as a single gene, called P110, which is separable from constitutive synthesis of NADase. P110 maps near the centromere on linkage group IV.

L-amino acid oxidase produced constitutively by P110 was partially purified and compared to partially purified L-amino acid oxidase produced by derepressed wild-type cultures. The enzymes are identical with respect to thermostability and molecular weight as judged by gel filtration.

The mutant P110 was shown to be an incompletely blocked auxotroph which requires serine or glycine. None of the enzymes involved in the synthesis of serine from 3-phosphoglyceric acid or glyceric acid was found to be deficient in the mutant, however. An investigation of the free intracellular amino acid pools of P110 indicated that the mutant is deficient in serine, glycine, and alanine, and accumulates threonine and homoserine.

The relationship between the amino acid requirement of P110 and its synthesis of L-amino acid oxidase is discussed.

Part II: Studies Concerning Multiple Electrophoretic Forms of Tyrosinase in Neurospora

Supernumerary bands shown by some crude tyrosinase preparations in paper electrophoresis were investigated. Genetic analysis indicated that the location of the extra bands is determined by the particular T allele present. The presence of supernumerary bands varies with the method used to derepress tyrosinase production, and with the duration of derepression. The extra bands are unstable and may convert to the major electrophoretic band, suggesting that they result from modification of a single protein. Attempts to isolate the supernumerary bands by continuous flow paper electrophoresis or density gradient zonal electrophoresis were unsuccessful.

Study of Proteinuria: Isolation of Proteins from the Nephrotic Syndrome

One of the most puzzling phenomena of abnormal renal physiology is the occurrence of the nephrotic syndrome. The syndrome has been defined by a collection of clinical and pathological symptoms, but there is no correlation between the clinical and pathological symptoms nor is the etiology of the syndrome known. Proteinuria is probably the most distinguishing feature in the nephrotic syndrome, and there are two possible explanations for its occurrence: (1) the excessive amounts of protein found in nephrotic urine could be due to an increased basement membrane permeability in the glomerulus of the kidney or (2) dysproteinemia. An attempt has been made to evaluate the theory of dysproteinemia in connection with the syndrome. The albumin fractions of nephrotic urine have been studied for their amino acid composition by separating them from the urine by paper electrophoresis, hydrolyzing them, and identifying the amino acids present by two-dimensional chromatography. There seem to be no variations in the qualitative makeup of nephrotic albumin from that of normal albumin, but the literature shows that there are some slight variations in the quantitative amino acid composition of nephrotic albumin compared with normal albumin. More extensive and highly developed experimentation along the lines of protein structure and composition must be done before it can conclusively be stated that dysproteinemia is of importance in the nephrotic syndrome.

Efeitos quimicos do recuo do cromo em alguns complexos hexacoordenados no estado solido

Os efeitos químicos do recuo do 51Cr formado pela reação (n,y) foram investigados nos compostos: [Cr(NH3) 5ONO] (NO3)2, [Cr(NH3) 5NO3] (NO3)2 e [Cr(NH3)5 NCS] (NO3)2, irradiados no estado sólido. Após dissolução dos cristais, os fragmentos resultantes da irradiação foram analisados por cromatografia com resina trocadora de íons e eletroforese em papel. A distribuição inicial das espécies é discutida em termos da formação de complexos polinucleares e da não ocorrência de conversão interna em compostos de cromo (III). Os efeitos de reversão térmica indicam uma considerável influência do oxigênio atmosférico e de defeitos cristalinos no comportamento químico das espécies.

Occurrence of Cello-Oligosaccharides in the Apoplast of Auxin-Treated Pea Stems

Treatment of pea (Pisum sativum L.) hypocotyl segments with indole-3-butyric acid, which promotes segment elongation, increased the solubilization of both xyloglucan and cello-oligosaccharides in the apoplast of auxin-treated pea stems. The cello-oligosaccharides were isolated from the apoplastic solution with a charcoal/Celite column and were identified as cellobiose, cellotriose, and cellotetraose after subsequent thin-layer chromatography and paper electrophoresis. Cello-oligosaccharides in the apoplastic fraction were monitored using cellobiose dehydrogenase. Both xyloglucan and cello-oligosaccharides appeared to be formed concurrently within 30 min after treatment with the auxin, and the cello-oligosaccharides increased with stem elongation even after 2 h. The total activity of cellulase did not increase for up to 4 h.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

Use of the FAR guide to present a pedagogical analogical model of gel electrophoresis in year 10 Science

This paper is a qualitative, practice based study describing the use of the Focus-Action-Reflection (FAR) Guide (Harrison and Treagust, 2000) to address the shortcomings of a pedagogical analogical model in Year 10 Science. The aim of this paper is to present my experience of the FAR Guide in relation to an analogical model that gave rise to perceived shortcomings by both teachers and students. This study found the FAR Guide to be a highly valuable tool, transforming the presentation of the analogical model, and enabling students to develop a deeper understanding of the nature of scientific knowledge.

Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

Recent advances in the study of biomolecular interactions by capillary electrophoresis

Capillary electrophoresis (CE) has been abundantly used in the study of molecular interactions owing to such advantages as short analysis time, low sample size requirement, high separation efficiency, and flexible applications. The focus of this paper is to 2 review recent studies and advances (mainly from 1998 to now) in biomolecular interactions using CE. Five CE modes: zone migration CE, affinity CE, frontal analysis (FA), Hummel-Dreyer (HD) and vacancy peak (VP) are cited and compared. Quantitative aspects of the thermodynamics and kinetics of biomolecular interaction are reviewed. Several biomolecular binding systems, including protein-protein (polypeptide), protein-DNA (RNA), protein(polypeptide)-carbohydrate, protein-small molecule, DNA-small molecule, small molecule-small molecule, have been well characterized by CE. CE is shown to be a powerful tool for the determination of the binding parameters of various bioaffinity interactions.

Determination of arsenic species by capillary zone electrophoresis with large-volume field-amplified stacking injection

Determination of arsenic species by large-volume field amplified stacking injection-capillary zone electrophoresis (LV-FASI-CZE) is reported in this paper. Whole column injection was employed. The optimum buffer pH for the separation of weak acids was discussed. It was found that the optimum buffer to analyze the stacked arsenate (As(V)), monomethylarsonate (MMA), and dimethylarsinate (DMA) was 25 mm phosphate at pH 6.5. However, the optimum buffer to analyze the concentrated arsenite (As(III)) was 20 mm phosphate - 10 mm borate at pH 9.28. The limits of detection of the method developed were 0.026 mg/L for As(III), 0.023 mg/L for As(V), 0.043 mg/L for MMA, and 0.018 mg/L for DMA. An enrichment factor of 34-100 for several arsenic species was obtained. In the end, this method was applied to determine the arsenic concentration in the environmental reference materials to show the usefulness of the method developed.

Capillary electrophoresis of urinary normal and modified nucleosides of cancer patients

This paper gives a capillary electrophoretic method for the separation of 15 urinary normal and modified nucleosides from cancer patients in less than 40 min. A 500 mmx50 mu m uncoated capillary column (437.5 mm to window) was used. The effects of the voltage and the sodium dodecyl sulfate (SDS) concentration in the buffer on the separation were studied. With reproducibilities of migration times better than 1.2% (R.S.D.) and determined concentrations better than 5-25%, depending on the concentrations of nucleosides in the urine, the analytical characteristics of the method were food. Using this developed method, the concentrations of 13 normal and modified nucleosides, extracted on a phenyl boronic acid affinity chromatography column, in 25 urines from patients of 14 kinds of cancer were determined. The levels (nmol/mol creatinine) of modified nucleosides in urines from cancer patients were increased as compared with those in normal urines. (C) 1998 Elsevier Science B.V.

Simultaneous determination of pethidine and methadone by capillary electrophoresis with electrochemiluminescence detection of tris(2,2 '-bipyridyl)ruthenium(II)

In this paper, we described a simple and rapid method, capillary electrophoresis with electrochemiluminescence (CE-ECL) detection using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)), to simultaneously detect pethidine and methadone. Analytes were injected to separation capillary of 67.5 cm length (25 mu m i.d., 360 mu m o.d.) by electrokinetic injection for 10 s at 10 kV.

Identification of herb Acanthopanax senticosus (Rupr. Et Maxim.) harms by capillary electrophoresis with electrochemical detection

In this paper, a rapid, high efficient, sensitive and inexpensive approach based on a combination of simple ultrasonic extract and capillary electrophoresis (CE) separation with electrochemical detection (ED), is described to identify herbs by comparing their CE-ED profiles (namely, CE-ED electropherograms). The proposed method takes advantage of ultrasmall sample volume, low consumption of organic solvent, simple sample pretreatment and easy cleanup procedure. It was applied to analyze the CE-ED profiles of stems of herb Acanthopanax senticosus (Rupr. Et Maxim.) Harms from different sources and different parts (roots, rhizomes, stems and leaves) of this herb. By comparing peak number, peak height and peak height ratio, we found that the CE-ED profiles showed big differences for the herbs from the different sources and the different parts of this herb. In addition, the distribution of bioactive compounds (isofraxidin, rutin and chlorogenic acid) in the different parts of this herb and their content variations affected by the source were studied with the CE-ED method. Based on their own unique CE-ED profiles, these herbs from the different sources and the different parts of this herb could be easily distinguished. Therefore, the proposed approach could be used as a rapid, high efficient and sensitive method for the identification of herbal medicines.