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J F Alderete

Publications and source records attributed to J F Alderete.

102 records · Page 6Linked to original sources
PubMed1980

Surface characterization of virulent Treponema pallidum.

Characterization of the surface of Treponema pallidum was accomplished by [(125)I]lactoperoxidase-catalyzed iodination of intact organisms and sensitive radioimmunoprecipitation and gel electrophoresis technology. At least 11 outer membrane proteins with molecular weights ranging from 89,000 (89K) to 20K were identified, and all elicited high titers of antibody in experimentally infected rabbits. Proteins of 89.5K, 29.5K, and 25.5K previously implicated as ligands involved in attachment (J. B. Baseman and E. C. Hayes, J. Exp. Med. 151:573-586, 1980) were found to reside on the treponemal surface. Low levels of the 89.5K treponemal protein were released by high salt concentrations, whereas the remaining comigrating material was neither radioiodinated nor released with selective detergents. Other lower-molecular-weight (60K, 45K, and 30K) surface proteins were extracted with octyl glucoside detergent, suggesting their hydrophobic interaction with the external membrane. The molecular organization of surface proteins was studied by employing the cross-linker dithiobis(succinimidyl)-propionate, and data suggested the presence of a highly fluid envelope resulting in random collisions by the surface proteins. The biological function of the treponemal outer envelope proteins was evaluated using, as the indicator system, adherence of T. pallidum to monolayer cultures of eucaryotic cells. Trypsin treatment of motile, freshly harvested organisms decreased the extent of surface parasitism to normal rabbit testicular cells, reinforcing the idea of the proteinaceous nature and role of treponemal ligands for attachment. Other data supported functional and antigenic relatedness among the implicated ligands. Finally, brief periodate treatment of human epithelial (HEp-2) and normal rat testicular cells as well as casein-elicited rabbit peritoneal macrophages significantly reduced the extent of treponemal parasitism, suggesting a role of specific host membrane molecules as mediators of attachment.

Antibody Formation↗
PubMed1979

Surface-associated host proteins on virulent Treponema pallidum.

A surface coat of host serum proteins was detected on virulent Treponema pallidum by sodium dodecyl sulfate-gel electrophoresis. The loosely associated serum proteins could be removed by repeated washings in a protein-free medium. Washed T. pallidum retained the ability to readsorb numerous host proteins from rabbit serum as well as iodinated rabbit or human albumin. In addition, various avidly associated host serum proteins including albumin, alpha(2)-macroglobulin, transferrin, ceruloplasmin, immunoglobulin G, immunoglobulin M, and C3 were identified on the outer envelope of washed treponemes by an immunoadsorbent technique with protein A-bearing staphylococcus. Hyaluronidase treatment did not remove the avidly associated host proteins from the surface of washed treponemes, whereas trypsin treatment resulted in decreased levels of agglutination. Electrophoretic patterns of trypsin-treated treponemes showed that treponemal proteins as well as adsorbed host proteins were released concurrently by protease digestion. Reacquisition studies involving alpha(2)-macroglobulin and transferrin suggested the presence of noncompetitive binding sites for serum proteins on the treponemal outer envelope. Finally, differences among the T. pallidum preparations from individual rabbits with respect to incorporation of [(35)S]methionine, extent of agglutination with antisera, and length of time required for removal of avidly associated host proteins by trypsin treatment indicated biological variability among the treponemal populations.

Adsorption↗
PubMed1978

Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli.

Heat-stable enterotoxin (ST) produced by porcine strains of enterotoxigenic (ENT+) Escherichia coli has been purified to apparent homogeneity by sequential ultrafiltration, acetone fractionation, preparative gel electrophoresis, diethylaminoethyl Bio-Gel A ion-exchange chromatography, and Bio-Gel P-10 gel filtration. The enterotoxin, purified more than 1,500-fold, exhibited a molecular weight of 4,400, as determined by both sodium dodecyl sulfate-gel electrophoresis and gel filtration. A molecular weight of 5,100, representing 47 residues, was calculated from amino acid analysis data. The amino acid content was distinctive, with an unusually high proportion of cystines and few hydrophobic amino acids. A single amino-terminal residue, glycine, was observed. Purified ST was stable to heating (100 degrees C, 30 min) and did not lose biological activity after treatment with Pronase, trypsin, proteinase K, deoxyribonuclease, ribonuclease, and phospholipase C. Periodic acid oxidation and several organic solvents (acetone, phenol, chloroform, and methanol) had no effect on the biological activity of ST. Further, purified ST was stable to acid treatment at pH 1.0 but lost biological activity at pH values greater than 9.0. Neither lipopolysaccharide nor lipid contamination was evident in purified preparations. A characteristic absorption spectrum was observed during the course of the purification, which shifted from a maximum at 260 nm in crude preparations to 270 nm for the purified toxin. Antiserum obtained from rabbits immunized with ST or ST coupled to bovine serum albumin neutralized the action of the enterotoxin in suckling mice; however, passive hemagglutination and hemolysis titer assays suggested that ST is a poor antigen.

Amino Acids↗
PubMed1977

Nutrition and enterotoxin synthesis by enterotoxigenic strains of Escherichia coli: defined medium for production of heat-stable enterotoxin.

A defined medium has been developed which supports synthesis of heat-stable enterotoxin (ST) by porcine and bovine strains of enterotoxigenic (ENT+) Escherichia coli in levels equivalent or better than a complex Casamino Acids-salts medium. The medium components did not support production of heat-labile enterotoxin (LT) but were similar for ST synthesis by ENT+ strains producting only ST and those which produced ST in addition to LT. The amino acids in Casamino Acids found to be necessary for growth and enterotoxin synthesis were proline, serine, aspartic acid, and alanine. Maximal growth and toxin levels were obtained after 8 h of incubation. Improved growth, but not an increase in synthesis of ST, was observed in the presence of Mg2+, Mn2+ and Fe3+ compared with Mg2+ alone. A chelator, tricine, was necessary for maximal cell densities,, probably to solubilize trace ions and make them more available to the bacteria. Increased growth was observed upon addition of glucose to both complex and defined media; however, glucose as well as gluconate and pyruvate appeared to cause repression of toxin synthesis. Addition of vitamins, oleic acid, or DL-lactic acid to the defined medium slightly increased levels of ST.

Amino Acids↗
PubMed1977

Repression of heat-stable enterotoxin synthesis in enterotoxigenic Escherichia coli.

Five different carbon sources were examined for their ability to control synthesis of heat-stable enterotoxin (ST) by enterotoxigenic (ENT+) Escherichia coli grown in either a defined medium containing four amino acids or a minimal salts medium. No ST activity was observed when D-glucose, D-gluconate, and L-arabinose were added separately to the defined medium, whereas glycerol and pyruvate decreased toxin levels. Similar results were obtained using a minimal salts medium, except with pyruvate, which did not support growth. Inhibition of ST synthesis by D-glucose was overcome by the addition of 3 X 10(-3) M cyclic adenosine 3',5'-monophosphate. Glucose repression of beta-galactosidase synthesis under conditions optimal for inhibition of ST synthesis was also reversed by exogenous cyclic adenosine 3',5'-monophosphate in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside. The data suggest that control mechanisms for the synthesis of plasmid gene products of bacterial pathogens are similar to those exerted on the host chromosome.

Cyclic AMP↗
PubMedDate not supplied

Trichomonas vaginalis: evaluating capsid proteins of dsRNA viruses and the dsRNA virus within patients attending a sexually transmitted disease clinic.

Some isolates of Trichomonas vaginalis, the number one, non-viral sexually transmitted disease agent, are infected with one or several distinct double stranded (ds)-RNA virus. Immune rabbit anti-capsid serum (IRS) reacted with the capsid protein of purified dsRNA virus of a subset of the virus-infected T. vaginalis isolates. A monoclonal antibody (mAb) that recognized the capsid protein reactive with the IRS was generated. Analysis of the virus capsid protein of virus-infected isolates by probing nitrocellulose blots with mAb revealed diversity among immunoreactivity and in the size of the reactive capsid protein. Despite difficulties in visualizing virus within parasites by cross-section electron microscopy, gold-conjugated mAb readily labeled the cytoplasm of virus-positive trichomonads. Finally and importantly, isolates infecting patients attending an STD clinic, 75% of which were virus-positive isolates, had capsid protein of the same size detected by mAb present in all dsRNA viruses.

Animals↗
PubMedDate not supplied

Trichomonas vaginalis has two fibronectin-like iron-regulated genes.

BACKGROUND: Trichomonas vaginalis, a protozoan parasite of the human urogenital tract, interacts with fibronectin (FN), a glycoprotein of the extracellular matrix. We, therefore, attempted to identify genes of this eukaryote encoding FN-binding proteins. METHODS: A cDNA clone, C1, representing an incomplete gene was obtained from an expression library based on its FN-binding ability and was characterized. The full-length 378-bp gene encoding a 14.8-kDa protein of 125 amino acids was obtained. RESULTS: The amino acid sequences revealed homology with the type III-14 repeat of the heparin-binding domain at the carboxyl terminal end of FN. This fibronectin-like protein gene, flp1, was single copy in all the T. vaginalis isolates examined. Levels of flp1 transcript were elevated in cells grown under low-iron conditions. Another low-iron-regulated gene, flp2, with 70 and 67.5% identity to flp1 at the nucleotide and amino acid levels, respectively, was recovered from the trichomonad genome. Both flp1 and flp2 had consensus Inr promoter-like elements immediately adjacent to the start codon. flp2 also contained an additional Inr element followed by an ATG 24-bp within the gene. CONCLUSIONS: Unlike flp2, the flp1 gene had AU-rich destabilizing elements in the 3'-untranslated region (UTR).

Amino Acid Sequence↗
PubMedDate not supplied

Alternating phenotypic expression of two classes of Trichomonas vaginalis surface markers.

The antigenic heterogeneity of Trichomonas vaginalis is due in part to the membrane disposition of immunogens (repertoire A) among some but not all isolates and among subpopulations of trichomonads of certain isolates. Heterogeneous T. vaginalis isolates undergo phenotypic variation for the A repertoire of immunogens. The presence of immunogens on (A+ phenotype) or the absence of them from (A- phenotype) trichomonal surfaces clearly influences the virulence traits of the pathogenic human trichomonads. A- parasites, for example, possess an enhanced ability to cause cytadherence-dependent killing of HeLa cells as compared with A+ parasites. A relation between phenotype of repertoire A and adherence was further substantiated by fractionating parasites of a parent T. vaginalis isolate, yielding adherent and nonadherent subpopulations. Trichomonad proteins were identified as putative adhesin candidates (repertoire B); adhesins were synthesized only by adherent (B+ parasites). Flow cytofluorometry of B+ and B- (nonadherent) subpopulations with antibody to proteins of the A repertoire demonstrated corresponding A- B+ and A+ B- phenotypes, respectively. Data support the hypothesis that trichomonads of some isolates of T. vaginalis undergo alternating expression of at least two classes of surface markers.

Animals↗