A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.     

The cellular and molecular regulation of 1,25(OH)2D3 production

The synthesis of 1,25(OH)₂D₃ is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH)₂D₃ itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH)₂D₃ production.     

XX true hermaphroditism in southern African blacks: an enigma of primary sexual differentiation.

A high incidence of 46,XX true hermaphroditism exists among southern African blacks. The gonadal distribution and clinical presentation of 38 patients are described. The aim of our study on 11 families with histologically proven XX true hermaphroditism was to determine whether a common genetic or environmental etiology could be identified. Pedigree analysis excluded the presence of a simple inheritance pattern, and no constant environmental factors could be implicated. Hybridization studies with Y chromosome--specific probes (pDP132, pDP61, pDP105, pDP31, pDP97, and pY431-HinfA) excluded the presence of a large portion of Yp in these patients. It is possible that smaller portions of the Y chromosome or one or more X-linked or autosomal mutations, either interacting and/or with incomplete penetrance, are present.     

Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens

Genes encoding antigens of Eimeria acervulina were cloned from cDNA expression libraries prepared from the sporozoite and merozoite stages in order to examine humoral and cellular immune responses to this protozoan parasite. Two clones expressing surface antigens were characterized by DNA hybridization studies to identify homologous genomic DNA fragments. The proteins they encode were identified by 125I-labeling, immunoblotting, immunofluorescence, and T-cell activation experiments. One, designated cSZ-1, encodes a 130-kDa beta-galactosidase fusion protein which represents a portion of a p240/p160 immunodominant sporozoite surface antigen. Immunofluorescence studies using anti-cSZ-1 sera and live or 1% paraformaldehyde-fixed E. acervulina sporozoites have confirmed this surface locale. Purified cSZ-1 fusion protein, which is not recognized by sera from E. acervulina-infected chickens, induced the activation of immune T lymphocytes in vitro. Another cDNA clone, designated cMZ-8, gives rise to a 150-kDa fusion protein and encodes a portion of a p250 immunodominant merozoite surface antigen. This was established by immunoblotting of 125I-labeled merozoite proteins with anti-cMZ-8 sera and immunofluorescence staining of live and 1% paraformaldehyde-fixed E. acervulina merozoites. Purified cMZ-8 is recognized by sera from E. acervulina-infected chickens and induces a significant activation of immune T lymphocytes in vitro.     

Molecular cloning, expression, and analysis of the genes of the homoprotocatechuate catabolic pathway of Escherichia coli C.

The molecular cloning and fine-structure analysis of the homoprotocatechuate (hpc) catabolic pathway genes of Escherichia coli C are described. The genes were located in two operons, hpcBCDEF and hpcGH, that were very closely linked. A regulatory gene, hpcR, involved in the expression of both operons was also identified. Various subclones isolated in the study were useful in the production of chemical intermediates of the pathway. The availability of one such compound facilitated the discovery of a previously unrecognized isomerase involved in the catabolic sequence.     

Thermodynamic bookkeeping when nucleotides bind. Applications of the theory of linked functions

The thermodynamic theory of linked functions was used to determine the numbers of modifier ions involved when nucleotides dissociate. Nucleotide dissociation constants, obtained spectrophotometrically using Dowex-1 resin as a model system, were plotted on log/log paper with respect to the modifier concentrations. The slopes of the lines represent the net number of modifier molecules/ions involved in the dissociation. Varying numbers of nucleotides are bound to the resin because the resin capacity is determined by the total number of charges bound. The nucleotides bind to the resin at comparable diffusion-limited rates, irrespective of how tightly they bind. When ATP binds at pH 6.8, 4 chlorides, 4 formates, 2 succinates or 1.4 citrates are displaced, indicating that the fully charged (ATP4-) nucleotide binds. By comparing ATP, ADP and AMP it was possible to evaluate the contributions of the adenosine moiety and each phosphate to the binding. Between pH 2 and 3, where ATP has two negative charges, ATP binds largely as the trianion, displacing 2.7 chlorides and 0.7 protons. In the presence of 4 mM magnesium, 0.58 magnesiums facilitate the dissociation by chelating 58% of the liberated ATP. Calcium behaved similarly to magnesium but aluminum, at pH 6.8, promoted the binding of ATP as an (A1.ATP)3- complex with the concomitant liberation of three chloride ions. These experimental thermodynamic stoichiometries were found to be independent of the concentrations of the other modifiers present. Thermodynamic linkage stoichiometries can be evaluated from log K vs. log (modifier) plots when a direct determination of modifier binding is impossible.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of phenelzine and tranylcypromine on the release of prostaglandins from the rat mesenteric vascular bed

We investigated the effects of phenelzine and tranylcypromine on the release of prostacyclin, thromboxane A2, prostaglandin E2, and prostaglandin E1 from the isolated perfused rat mesenteric vascular bed. Perfusion of the preparation with phenelzine in concentrations of 15, 45, and 135 microM for 150 min led to attenuated release of all four prostaglandins measured. Inhibition generally occurred with the lowest dose used and was most prominent with the highest concentration. Tranylcypromine also decreased prostaglandin formation. However, low doses were not effective in the suppression of prostacyclin release. Both drugs had an inhibitory effect on production of prostaglandin E1, which is a metabolite of dihomo-gamma-linolenic acid, the precursor of arachidonic acid, but this was only shown to be significant with phenelzine. In this work we demonstrate that phenelzine and tranylcypromine have an inhibitory effect on the production of 2-series prostaglandins derived from arachidonic acid, and possibly a similar effect on prostaglandins of the 1-series derived from dihomo-gamma-linolenic acid.