A new route to "bifunctional" chelating agents: conversion of amino acids to analogs of ethylenedinitrilotetraacetic acid.

Via the amides, α-amino acids can be converted to analogs of ethylenedinitrilotetraacetic acid, with retention of configuration at the asymmetric carbon. 1-(p-Carboxymethoxybenzyl)-ethylenedinitrilotetraacetic acid has been prepared from l-tyrosine and then coupled as the iron(III) chelate to 1,2-diaminoethane or to human serum albumin. The iron(III) chelate is effective in preventing EDTA carboxyl groups from reacting via carbodiimide coupling with amino groups. After the coupling reaction, iron is readily removed from the chelate upon reduction with ascorbate. These new procedures possess several attractive features for the use of “bifunctional” chelating agents in biophysical studies.     

A simple and sensitive assay procedure for ribonucleotide reductase system

A sensitive, rapid, and accurate assay for measuring ribonucleotide reductase activity, including the reduction of CDP, UDP, GDP, and ADP to their corresponding deoxyribonucleotides, is described. Using polyethyleneimine-cellulose thin-layer chromatography, I was able to separate the substrates, the reaction products, and the associated hydrolytic products from each other. This procedure allowed me to measure all of the products formed without dephosphorylation or acid hydrolysis. It has been adapted to study the ribonucleotide reductase system of bacteriophage T4-infected cells.     

Liquid chromatographic analysis of amino acids: Using dimethylamino-azobenzenesulfonyl chloride and hypersil ODS column to analyze the composition of apo B peptides

A reliable high-performance liquid chromatography (HPLC) method is described for the separation of dimethylamino-azobenzenesulfonyl-amino acid (DABS-AA). The separation is accomplished by reversed-phase chromatography on a Hypersil ODS column (4.6×150 mm) withe a Hewlett-Packard liquid chromatography system. In addition to the developed sample and solvent preparation procedure, this precolumn modification method using dimethylaminoazobenzene sulfonyl chloride (DABS-CL) for amino acid analysis is proved reliable and sensitive. Five apolipoprotein B-100 tryptic peptides, two of them containing cysteine, were demonstrated as examples for the general application of this method in amino acid analysis. It is a useful method for analysis of cysteine- and cysteine-containing peptide and, furthermore, for determination of sulfhydryl and disulfide linkages of proteins.     

Characterization and reactivity of broiler chicken sera to selected recombinant Campylobacter jejuni chemotactic proteins

Campylobacter jejuni, a Gram-negative rod bacterium, is the leading causative agent of human acute bacterial gastroenteritis worldwide. Consumption and handling of raw or undercooked poultry are regarded as a major source for human infection. Because bacterial chemotaxis guides microorganisms to colonization and invasion in the host cells, proteins involved in chemotactic processes can be novel targets for vaccine development. In this communication, we report amplification, cloning and expression of the C. jejuni chemotactic proteins in an Escherichia coli expression system. A total of 15 chemotactic protein genes were successfully expressed. These recombinant proteins were confirmed by nucleotide sequencing, SDS-PAGE analysis and immunoblot analysis of six-His and hemagglutinin tags. Twelve recombinant chemotactic proteins were further tested whether they were antigenic using sera from broiler chickens older than 4 weeks. The immunoblot results show that each chicken serum reacted to a variety of the recombinant proteins, but all sera reacted to the Cjj0473 gene product (annotated as a methyl-accepting chemotaxis protein), suggesting that anti-Campylobacter antibodies may be prevalent in the poultry population. These antibody screening results provide a rationale for further evaluation of the Cjj0473 protein as a potential vaccine for broilers to improve human food safety.     

Drosophila notal bristle as a novel assessment tool for pathogenic study of Tau toxicity and screening of therapeutic compounds

To elucidate the Tau gain-of-toxicity functional mechanism and to search for potential treatments, we overexpressed human Tau variants (hTau) in the dorsal mesothorax (notum) of Drosophila. Overexpression of Tau variants caused loss of notal bristles, and the phenotype was used for evaluating toxicity of ectopic Tau. The bristle loss phenotype was found to be highly associated with the toxicity of hyperphosphoryled Tau in flies. We have shown that the bristle loss phenotype can be rescued either by reducing Glycogen synthase kinase 3β (GSK3β)/Shaggy (Sgg) activity or overexpressing Bβ2 regulatory subunits of PP2A. Elevated expression of the Drosophila Bβ2 homolog, Twins (Tws), also alleviated neuritic dystrophy of the dorsal arborization (da) neuron caused by Tau aggregation. Additionally, lowering endogenous Tau dosage was beneficial as it ameliorated the bristle loss phenotype. Finally, the bristle loss phenotype was used to evaluate the efficacy of potential therapeutic compounds. The GSK3β inhibitor, alsterpaullone, was found to suppress toxicity of Tau in a concentration-dependent manner. The notum of Drosophila, thus, provides a new tool and insights into Tau-induced toxicity. It could also potentially assist in screening new drugs for possible therapeutic intervention.     

Effect of particle size on the rate of enzymatic hydrolysis of cellulose

The effect of particle size on enzymatic hydrolysis of cellulose has been investigated. The average size of microcrystalline cotton cellulose has been reduced to submicron scale by using a media mill. The milled products were further subjected to hydrolysis using cellulase. High cellulose concentration (7%) appeared to retard the size reduction and resulted in greater particles and smaller specific surface areas than those at low concentration (3%) with the same milling time. Initial rate method was employed to explore the rate of enzymatic hydrolysis of cellulose. The production rate of cellobiose was increased at least 5-folds due to the size reduction. The yield of glucose was also significantly increased depending upon the ratio of enzyme to substrate. A high glucose yield (60%) was obtained in 10-h hydrolysis when the average particle size was in submicron scale.