Oxidative stress induced heat shock factor phosphorylation and HSF-dependent activation of yeast metallothionein gene transcription.

Metallothioneins (MTs) are a class of low-molecular-weight, cysteine- rich metal-binding proteins that function in metal detoxification and oxidative stress protection. We demonstrate that transcription of the Saccharomyces cerevisiae MT gene CUP1 is strongly activated by the superoxide anion generator menadione. This activation is exacerbated in a strain lacking the gene encoding Co, Zn superoxide dismutase (SOD1).

Steroid therapy of chronic hepatitis: characteristics associated with response in anti-hepatitis C virus-positive and -negative patients.

Objectives: The goals of this study were to examine responses to corticosteroid-containing therapy in non-B chronic hepatitis patients with different anti-hepatitis C virus (HCV), autoantibody, and biochemical test results and to determine what factors correlate with response.

Methods: Patients with a prior or current history of steroid therapy for putative autoimmune or chronic non-A, non-B hepatitis were assessed. Responses during the first 6 months of therapy were categorized as "complete" (normal aminotransferases for > or = 1 month), "partial" ( > 50% reduction), or "no response.

Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions.

Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves.

Toxic metal-responsive gene transcription.

Metals play a dual role in biological systems, serving as essential co-factors for a wide range of biochemical reactions yet these same metals may be extremely toxic to cells. To cope with the stress of increases in environmental metal concentrations, eukaryotic cells have developed sophisticated toxic metal sensing proteins which respond to elevations in metal concentrations. This signal is transmitted to stimulate the cellular transcriptional machinery to activate expression of metal detoxification and homeostasis genes.

Chlamydia trachomatis antibodies in serum and ejaculate of male patients without acute urethritis.

In a prospective study, the prevalence of specific antibodies against Chlamydia trachomatis in serum and seminal plasma evaluated by a genus specific immunofluorescence test in 101 men without acute urethritis was investigated. The results were compared to the clinical diagnosis, cell culture of urethral swabs, demonstration of DNA particles by Polymerase Chain Reaction (PCR) in the ejaculate and signs of genital inflammation by counting peroxidase-positive leukocytes and elastase level in semen. The objective was to evaluate the significance of chlamydial antibodies in genital infection.

Ex vivo purging of allogeneic marrow with L-Leucyl-L-leucine methyl ester. A phase I study.

L-Leucyl-L-leucine methyl ester (LLME) is a lysosomatropic compound that is converted by dipeptidyl peptidase I to metabolites that are membranolytic for cytotoxic T cells, NK cells, and LAK cells. Ex vivo treatment of murine marrow with LLME ameliorates acute graft-versus-host-disease (GVHD), which led to consideration of a clinical study. A phase I study design was initiated to evaluate the effects of ex vivo purging of allogeneic marrow on engraftment, since LLME also suppresses human progenitor cells.

Sequencing and linkage analysis of a Coxiella burnetii 2.1 kb NotI fragment.

Most of the Coxiella burnetii isolates (72 of 80) available in our institute share a 2.1 kb NotI fragment. Sequence analysis revealed two incomplete open reading frames (ORF) for putative genes hemA and bcR coding for glutamyl-tRNA-reductase and bicyclomycin resistance protein, respectively.

Quantification of Coxiella burnetii by polymerase chain reaction (PCR) and a colorimetric microtiter plate hybridization assay (CMHA).

A colorimetric microtiter plate hybridization assay (CMHA) for the quantitative determination of Coxiella burnetii DNA after amplification by externally controlled polymerase chain reaction (PCR) is described. The quantification assay is based on an enzyme linked immunosorbent assay (ELISA) format. Cloned DNA, representing a sequence complementary to an internal part of the diagnostic amplicon, was noncovalently attached to the wells of a microtiter plate.

Protection from T helper cell-mediated graft-versus-host disease by the presence of an MHC class I alloantigen is associated with perturbation of MHC class II-restricted responses by class I-derived peptides.

Lethal graft-vs-host disease (GVHD) develops after transfer of CTL-depleted spleen and bone marrow cells from C57BL/6 (B6) mice to irradiated MHC class II disparate (B6xbm12)F1 recipients. Onset of lethal GVHD is significantly delayed in (bm1xbm12)F1 recipients of the same donor inoculum despite the additional MHC class I disparity (H-2Kbm1). To investigate the basis of this protective effect, hybridomas were generated from T cells activated in vivo during GVHD in both strain combinations.

CD27-CD27 ligand/CD70 interactions enhance alloantigen-induced proliferation and cytolytic activity in CD8+ T lymphocytes.

To study the role of CD27-CD27 ligand (CD27L)/CD70 interactions in the generation of murine allospecific T cell responses, SF9 cells or cell membranes expressing recombinant human CD70 were added to in vitro MLC containing C57BL/6 (H-2b) responder cells and class I and II MHC disparate H-2b/d stimulator cells. Alloantigen-specific CTL generation, CD8+ T cell proliferation, and levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase activity were enhanced in the presence of human CD27L/CD70 expressed on SF9 cell membranes. Enhancement of CD8+ T cell responses occurred in the absence of any discernible effects on CD4+ T cell proliferation or IL-2 responses.

Cellular blebbing in superficial colonic epithelial cells occurring with murine graft-versus-host disease.

Subnuclear blebbing of the superficial colonic epithelium, a rarely described light and electron microscopic change in graft-versus-host disease (GVHD), was studied in a murine model of GVHD. Severity of changes induced by transfer of various donor T cell subsets to irradiated, allogeneic recipients, and association with more severe alterations such as erosions and ulceration were evaluated. By light microscopy the basal region of the superficial enterocytes was greatly expanded by eosinophilic to amphophilic, flocculent, sometimes vacuolated material.

Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1.

Elevations in gene dosage of the transcriptional regulatory protein yAP-1 in Saccharomyces cerevisiae can elicit pronounced phenotypic increases in tolerance of a variety of drugs including the toxic heavy metal cadmium. While a large elevation in cadmium tolerance occurs in response to overproduction of yAP-1, the target genes under yAP-1 control have not yet been identified that are responsible for this increase. We show here that the YCF1 gene, encoding a likely integral membrane protein, is required for yAP-1 to exert its normal effects on cadmium tolerance.

Heat shock transcription factor activates yeast metallothionein gene expression in response to heat and glucose starvation via distinct signalling pathways.

Metallothioneins constitute a class of low-molecular-weight, cysteine-rich metal-binding stress proteins which are biosynthetically regulated at the level of gene transcription in response to metals, hormones, cytokines, and other physiological and environmental stresses. In this report, we demonstrate that the Saccharomyces cerevisiae metallothionein gene, designated CUP1, is transcriptionally activated in response to heat shock and glucose starvation through the action of heat shock transcription factor (HSF) and a heat shock element located within the CUP1 promoter upstream regulatory region. CUP1 gene activation in response to both stresses occurs rapidly; however, heat shock activates CUP1 gene expression transiently, whereas glucose starvation activates CUP1 gene expression in a sustained manner for at least 2.

Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.

Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome XVI, caused a loss of copper resistance in strains which possessed CUP1 and ACE1, as well as in the cup1 ace1 deletion strain.

Reactivity of hybridomas derived from T cells activated in vivo during graft-versus-host disease.

To examine the specificity of T helper cells activated during murine graft-vs-host disease (GVHD), T cell hybridomas from GVHD spleens and livers were generated and analyzed. CTL-depleted C57BL/6 (B6) donor cells were injected into irradiated (B6 x bm12)F1 or (bm1 x bm12)F1 recipient mice. Five or fourteen days later, cells from livers and spleens were fused directly with the TCR-deficient (alpha beta)- BW5147 thymoma line.

Detection of Coxiella burnetii in cow's milk using the polymerase chain reaction (PCR).

A PCR approach (transposon PCR) with primers based on repetitive transposon-like sequences, which--depending on the isolate--were found at a minimum frequency of 19 on the C. burnetii genome, was established for the highly sensitive and specific detection of C. burnetii.

[Detection of Chlamydia psittaci in vaginal discharge of cows: a necessary enlargement of bacteriologic diagnosis for the etiologic clarification of fertility disorders in the female cow].

Vaginal discharge from 119 dairy cows from 59 herds was examined bacteriologically, including application of the IDEIA Chlamydia test, to detect genus-specific chlamydial LPS-antigen. A putrid quality of specimens was closely correlated with isolation of Actinomyces pyogenes (p < 0.001).

A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein.

Members of the ATP binding cassette (ABC) protein superfamily transport a variety of substances across biological membranes, including drugs, ions, and peptides. The yeast cadmium factor (YCF1) gene from Saccharomyces cerevisiae is required for cadmium resistance and encodes a 1,515 amino acid protein with extensive homology to both the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR). S.

Comparison of dark-field microscopy, culture, and polymerase chain reaction (PCR) for detection of Borrelia burgdorferi in field-collected Ixodes ricinus ticks.

In a study based on 100 field-collected female Ixodes (I.) ricinus ticks from the surroundings of Giessen, dark-field microscopy (DFM), culture, and PCR were compared as procedures for detecting Lyme borreliosis spirochetes in ticks. By DFM, 16 ticks were found to be infected with spirochetes.

Is plasmid based differentiation of Coxiella burnetii in 'acute' and 'chronic' isolates still valid?

Ten human Coxiella burnetii isolates from french patients with acute hepatitis or chronic endocarditis were characterized according to their polymorphism in DNA restriction patterns and differentiated by plasmid-specific PCR. The aim of this investigation was to clarify if the present classification of so called 'acute' and 'chronic' Coxiella burnetii isolates--based on plasmid profile of a so far limited number of partly ancient isolates--could be confirmed with lately isolated organisms of this agent. The data obtained in this investigation indicate that this classification based on C.

Analysis of the entire nucleotide sequence of the cryptic plasmid QpH1 from Coxiella burnetti.

The complete plasmid QpH1 from Coxiella burnetti, isolate 'Nine Mile', phase I, was cloned as NotI fragment with a size of 37329 bp. The entire plasmid was sequenced by the chain termination method after EcoRI subcloning. 37 open reading frames coding for polypeptides larger than 100 amino acid residues were determined.

The 16S/23S ribosomal spacer region of Coxiella burnetti.

The 16S/23S spacer region of Coxiella burnetti isolate Nine Nile, phase 1, was sequenced. Sequence analysis revealed two tRNA coding regions for tRNA(Ile) and tRNA(Ala). DNA sequence alignment demonstrated significant homology with tRNA species from Pseudomonas aeruginosa and Rhodobacter sphaeroides, respectively.

Valvular endocarditis occurs as a part of a disseminated Coxiella burnetii infection in immunocompromised BALB/cJ (H-2d) mice infected with the nine mile isolate of C. burnetii.

BALB/cJ (H-2d) mice were injected intraperitoneally (ip) with cyclophosphamide 2 days after ip inoculation with Coxiella burnetii Nine Mile, phase I. Ten days after infection, disseminated microabscesses, granulomas, and microthrombi were observed in most organs, including spleen and liver, and in bone marrow. In addition, endocarditis of the atrioventricular and semilunar valves, characterized by macrophages and neutrophils, was present.

Monoclonal antibody based competitive ELISA for the detection of specific antibodies against Coxiella burnetii in sera from different animal species.

A competitive ELISA system for the detection of antibodies against Coxiella (C.) burnetii in cattle, sheep, goats, horses and humans is described. The ELISA is based on a biotinylated monoclonal antibody with specificity for C.