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Biomedical subjects

J F Miller

Publications and source records attributed to J F Miller.

At least 181 records · Page 10Linked to original sources
PubMed1989

Expression in transgenic mice of class I histocompatibility antigens controlled by the metallothionein promoter.

To study the effects of increased expression of major histocompatibility complex class I molecules on the development of self-tolerance, transgenic mice were produced that expressed the H-2Kb gene under the control of the metallothionein promoter. Administration of zinc enhanced transgene expression in liver, kidney and exocrine pancreas. No evidence suggestive of an autoimmune response was found in transgene-expressing tissues in mice otherwise allogeneic to H-2Kb. Despite this lack of responsiveness in vivo, T cells could be stimulated in vitro to lyse H-2Kb-bearing target cells. No infiltration was detected in transgenic mice after irradiation and reconstitution with bone marrow cells. When spleen cells were used for reconstitution, however, dense lymphocytic infiltration was seen, particularly in the portal tracts of the liver, and this was accompanied by piecemeal necrosis and apoptosis of periportal hepatocytes. This aggressive response progressively diminished with time, and by 12 weeks after reconstitution many of the portal tracts were free of infiltration while the others showed no accompanying necrosis. The picture at this stage was similar to that seen in chronic persistent hepatitis. These results suggest that, in addition to negative selection in the thymus, peripheral mechanisms not involving clonal deletion or permanent clonal anergy can prevent immune responses to self molecules.

Animals↗
PubMed1989

Purification and characterization of glia maturation factor beta: a growth regulator for neurons and glia.

A protein has been isolated from bovine brains by using a modification of the procedure used to purify glia maturation factor. The method consists of ammonium sulfate precipitation, chromatography with DEAE-Sephacel, Sephadex G-75, and hydroxylapatite columns, passage through a heparin-Sepharose column, and finally fractionation by reverse-phase HPLC with a C4 column. The isolated protein reacts strongly with the mouse monoclonal antibody G2-09 and has a molecular weight of approximately 17,000 and an isoelectric point of pH 4.9. The N terminus is blocked, but tryptic digestion releases 28 peptides, 8 of which have been sequenced. The total known residues add up to more than two-thirds of the entire 140-residue protein, estimated from amino acid composition, and show no sequence homology with any known protein. Reversible thermal renaturation greatly enhances its biological activity. The purified protein stimulates differentiation of normal neurons as well as glial cells. It inhibits the proliferation of the N-18 neuroblastoma line and the C6 glioma line while promoting their phenotypic expression. We designate this protein glia maturation factor beta.

Amino Acids↗
PubMed1989

Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins.

The bvg locus of Bordetella pertussis is required for coordinate regulation of several factors associated with virulence. The control system is modulated by various environmental signals, including low temperature, MgSO4, and nicotinic acid. The nucleotide sequence of the bvg region has been determined and three open reading frames, bvgA, bvgB, and bvgC, are present. Twelve-base-pair linker insertion mutations in any of these open reading frames result in a Bvg- phenotype. The predicted protein products of bvgA and bvgC share homology with a family of prokaryotic regulatory proteins that respond to environmental stimuli and are members of two-component sensory transduction systems. We propose a model in which BvgB and the N-terminal portion of BvgC are localized in the periplasm. Environmental signals are recognized, transduced to the cytoplasmic portion of BvgC, and then transmitted to BvgA, a positive regulator of transcription.

Amino Acid Sequence↗
PubMed1989

CD4 and CD8 molecules can physically associate with the same T-cell receptor.

The expression of the cell-surface glycoproteins CD4 and CD8 on functionally mature T cells is usually mutually exclusive and correlates with class II and class I major histocompatibility complex (MHC) restriction, respectively. CD4 and CD8 function by binding to class II and class I MHC molecules on the antigen-presenting cell (APC), thereby increasing the adhesion between the T cell and the APC. From antibody-blocking studies and from cocapping and comodulation experiments, CD4 and CD8 come into close physical contact with their appropriately restricted T-cell receptor (TCR) at the time of antigen recognition. By the use of affinity chromatography followed by two-dimensional diagonal gel electrophoresis, we have identified a Mr 43,000 disulfide-bonded heterodimer copurifying with CD4. This protein was identified as the TCR by its removal after preclearing with the anti-TCR antibody F23.1 and by its generation after protease digestion of the same peptides as the TCR from this clone. When CD4 and CD8 were similarly isolated from an unusual CD4+ CD8+ class II-restricted T-cell clone, the TCR was identified as associating with either accessory molecule in the absence of activation. Therefore, CD4 and CD8 do not distinguish between class I- and class II-restricted TCRs in their ability to form membrane complexes, indicating a need for both the TCR and its associated accessory molecule to recognize the same individual MHC molecule on the APC to optimize TCR triggering.

Antibodies, Monoclonal↗
PubMed1989

The bvgA gene of Bordetella pertussis encodes a transcriptional activator required for coordinate regulation of several virulence genes.

The bvg region of the respiratory pathogen Bordetella pertussis coordinately regulates the expression of several unlinked virulence determinants in response to environmental signals. The DNA sequence of the bvg region contains three genes (bvgA, bvgB, and bvgC). Transcription of a single-copy fusion consisting of the upstream region of a bvg-activated B. pertussis gene (fhaB) attached to the promoterless lac operon in Escherichia coli requires the entire bvgABC region in trans. Activation of the fhaB::lacZYA fusion is sensitive to the same environmental stimuli in E. coli that modulate the expression of bvg-activated genes in B. pertussis. Our data show that overexpression of the bvgA gene from a strong heterologous promoter results in transcriptional activation of the fhaB::lacZYA fusion even in the absence of the bvgB and bvgC products. Activation of fhaB transcription by bvgA overexpression in E. coli is no longer repressed by environmental conditions. The bvgA product has been identified by maxicell analysis as a 23-kilodalton protein. A B. pertussis mutant containing an in-frame deletion in bvgA was constructed. This mutant was nonhemolytic and no longer produced filamentous hemagglutinin and pertussis toxin. The mutation in this strain was complemented by returning the bvgA gene in trans. Transcriptional chloramphenicol acetyltransferase fusions to the fhaB and ptx promoter regions were returned to both the B. pertussis bvgA deletion mutant and its parental wild-type strain. Analysis of these strains indicated that the deletion mutant was defective in transcription of both ptx and fhaB. We conclude from these data that bvgA, bvgB, and bvgC comprise an operon encoding the components essential for coordinate regulation and sensory transduction. The BvgA protein is a transcriptional regulatory factor. The bvgB and bvgC products may be important in regulating the activity of BvgA in response to the changing environmental stimuli that B. pertussis encounters during the diseases whooping cough.

Bordetella pertussis↗
PubMed1989

Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli.

The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors. The virulence control system also responds to environmental signals. We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA. Single-copy lacZYA fusions to the B. pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg. In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E. coli as measured by ptxA-lacZ expression. Environmental signals that modulate expression of virulence genes in B. pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ. MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively. Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.

Bordetella pertussis↗
PubMed1989

MHC molecules and beta-cell destruction. Immune and nonimmune mechanisms.

Hyperexpression of major histocompatibility complex (MHC) molecules by islet cells is a prominent, early feature of islet pathology in insulin-dependent diabetes mellitus and concomitant with beta-cell failure after exposure of islets to specific cytokines or viruses. The transgenic expression of a class I MHC gene (H-2Kb) in the beta-cells of either syngeneic or allogeneic mice leads to beta-cell failure by a nonimmune mechanism. Several class II MHC transgenes, with one exception, have the same effect, but the expression of other transgenes that have products that are membrane proteins is not necessarily detrimental. Class I MHC molecules have been shown to interact directly with other membrane proteins. The inappropriate expression of MHC molecules could therefore interfere with key cellular functions. We postulate that the hyperexpression of MHC molecules in the beta-cell, e.g. in response to viruses, is a primary, nonimmune mechanism of beta-cell failure that precedes a secondary autoimmune response.

Animals↗
PubMed1989

Expression of class I histocompatibility antigens in defined tissues: effects on T cell function.

We have generated transgenic mice by introducing into fertilized eggs the major histocompatibility complex class I gene, H-2Kb, linked to various promoters that target gene expression to particular tissues. In one system the gene was expressed in the medullary epithelial cells of the thymus; these were unable to impose tolerance on differentiating T lymphocytes. These findings, together with those of other workers who showed that epithelial thymus grafts can impose tolerance, provide clear evidence of functional heterogeneity among thymus epithelial cells. In other transgenic models the gene was expressed in nonlymphoid tissues, such as the pancreatic islet beta cells or the hepatocytes, exocrine pancreas and kidney tubules. In all these cases the tissues were not subjected to autoimmune attack and the animals were specifically tolerant of H-2Kb-bearing cells in vivo, although some could generate cytotoxic T lymphocytes in vitro. Hence, although the intrathymic environment may be the dominant site for negative selection, it is clear that ancillary mechanisms exist in the periphery to ensure that tolerance is achieved to antigens not synthesized in the thymus.

Animals↗
PubMed1989

Transformation of Actinobacillus pleuropneumoniae and analysis of R factors by electroporation.

An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiple antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (tau = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism.

Actinobacillus↗
PubMed1988

High efficiency transformation of E. coli by high voltage electroporation.

E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.

DNA, Bacterial↗
PubMed1988

Diabetes in transgenic mice resulting from over-expression of class I histocompatibility molecules in pancreatic beta cells.

A class I histocompatibility gene, H-2Kb, linked to the rat insulin promoter, is overexpressed in the pancreatic beta cells of transgenic mice. The mice, whether syngeneic or allogeneic to the transgene, develop insulin dependent diabetes without detectable T cell infiltration, suggesting a direct, non-immune role for the transgenic class I molecules in the disease process.

Animals↗
PubMed1988

Endogenous immunoreactive glia maturation factor-like molecule in cultured rat Schwann cells.

Using the monoclonal antibody G2-09 raised against bovine glia maturation factor (GMF), we demonstrated that cultured rat Schwann cells and Schwannoma cells, but not their conditioned media, possessed endogenous GMF-like immunoreactivity. The presence of immunoreactive GMF correlated well with GMF bioactivity. The GMF-like factor in Schwann cells was characterized by immunodotting, immunofluorescence, immunoadsorption and immunoblotting. Immunofluorescence confirmed the intracellular location of GMF. Immunoadsorption completely eliminated the GMF-like bioactivity from the cell extracts. Immunoblotting identified a protein band with a molecular weight of 14,000. Thus, the evidence strongly supports the argument that the GMF-like factor in rat Schwann cells is identical with GMF from the bovine brain. The GMF-like molecule in Schwannoma cells showed properties similar to those in Schwann cells, but for unknown reasons was not detectable by immunofluorescence. The presence of GMF in cultured rat Schwann cells suggests that the factor may play a role in the peripheral nervous system.

Antibodies, Monoclonal↗
PubMed1988

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells.

A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates. Fluorochrome-antibody conjugates bound to cell-surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA. STA is a unique determinant detected in activated human T cells by the monoclonal antibody K-1-21, which also recognizes a cross-reactive conformation-dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxic/suppressor T cells and Leu 3a+ helper/inducer T cells but is not induced in the Leu 15+ population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells.

Animals↗