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Biomedical subjects

D B Young

Publications and source records attributed to D B Young.

At least 55 records · Page 3Linked to original sources
PubMed1999

Construction and murine immunogenicity of recombinant Bacille Calmette Guérin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin.

Three recombinant strains of Mycobacterium bovis Bacille Calmette Guerin (rBCG) were prepared in which the immunogenic B subunit of human Escherichia coli heat labile enterotoxin (LT-Bh) was expressed either as a cytoplasm protein, a cell wall associated lipoprotein or a secreted protein. Intraperitoneal immunisation of mice with these rBCG induced IgG and IgA antibodies to LT-Bh and shifted the serum IgG subclass response to subsequent challenge with purified LT-Bh from IgG1 to an IgG2a. Oral administration of recombinant BCG induced mucosal and serum IgA antibodies to LT-Bh which peaked four months after immunisation. Antibody responses were greater when LT-Bh was expressed as a secreted protein or lipoprotein rather than in the cytoplasm. Oral vaccination with recombinant BCG may be an effective approach, particularly to induce mucosal IgA and prime for a serum TH1 recall response.

Adjuvants, Immunologic↗
PubMed1999

CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex.

CBF1 is a member of the CSL family of DNA binding factors, which mediate either transcriptional repression or transcriptional activation. CSL proteins play a central role in Notch signaling and in Epstein-Barr virus-induced immortalization. Notch is a transmembrane protein involved in cell-fate decisions, and the cytoplasmic domain of Notch (NotchIC) targets CBF1. The Epstein-Barr virus-immortalizing protein EBNA2 activates both cellular and viral gene expression by targeting CBF1 and mimicking NotchIC. We have examined the mechanism of CBF1-mediated repression and show that CBF1 binds to a unique corepressor, CBF1 interacting corepressor (CIR). A CIR homolog is encoded by Caenorhabditis elegans, indicating that CIR is evolutionarily conserved. Two CBF1 mutants that were unable to bind CIR did not function as repressors, suggesting that targeting of CIR to CBF1 is an important component of repression. When expressed as a Gal4 fusion protein, CIR repressed reporter gene expression. CIR binds to histone deacetylase and to SAP30 and serves as a linker between CBF1 and the histone deacetylase complex.

Amino Acid Sequence↗
PubMed1999

Inverse relationship between potassium intake and coronary artery disease in the cholesterol-fed rabbit.

We tested the hypothesis that a low level of dietary potassium intake would exacerbate the severity of vascular lesion formation in rabbit coronary arteries during high cholesterol intake. Two groups of nine rabbits were studied for 6 weeks while eating a diet containing 2% cholesterol and 0.9% sodium. The normal potassium group consumed a diet containing 1.5% potassium and the low potassium group consumed a diet containing 0.4% potassium. After 6 weeks the animals were killed, the hearts were removed, and blood samples were withdrawn from the abdominal aorta immediately before removing the heart. The hearts were sectioned and slides were prepared and fixed with hematoxylin and eosin. The numbers of normal and abnormal vessels, and those with foam cells in the subintima, were counted in selected sections. Plasma potassium concentration in the normal and low potassium intake groups averaged 4.27 +/- 0.27 mmol/L and 3.90 +/- 0.11 mmol/L, respectively. No differences between the groups were observed in plasma cholesterol or body weight gain. The percentages of abnormal arteries in the groups were 4.20 +/- 0.35 in the normal intake group and 6.36 +/- 0.50 in the low intake group, 51% greater in the normal intake group (P < .001). These results support the hypothesis that low potassium intake exacerbates the severity of subintimal lesion development in the coronary arteries.

Animals↗
PubMed1999

Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene.

A genomic DNA region, designated RD1, that is present in virulent and clinical strains of Mycobacterium tuberculosis and M. bovis, has been shown to be deleted in bacillus Calmette Guérin (BCG). The DNA segments corresponding to three open reading frames (ORFs: ORF-10, ORF-14 and ORF-15) of the RD1 region, that are deleted in BCG strains, were amplified from M. tuberculosis genomic DNA by polymerase chain reaction (PCR), subcloned into pGEX-4T vector system and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass. The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots. When pooled human sera from 11 tuberculosis (TB) patients were used as the source of antibodies, only GST-ORF-14 fusion protein reacted in Western immunoblots. The protein corresponding to ORF-14 was then purified to near homogeneity and isolated free of its fusion partner (GST) by treating the purified GST-ORF-14 fusion protein with thrombin protease. In Western immunoblots, the purified ORF-14 protein reacted with antibodies in 26 of 57 human sera (46%) from TB patients while no reactivity was seen with 11 sera from M. bovis BCG-vaccinated healthy subjects. Interestingly, sera from nine of 15 (60%) long-term contacts of TB patients also had antibodies reactive to the ORF-14 protein. These results suggest that the ORF-14 protein in combination with other immunodominant proteins could be useful in the serodiagnosis of individuals infected with M. tuberculosis.

Antibodies, Bacterial↗
PubMed1999

Assessment of immunity to mycobacterial infection with luciferase reporter constructs.

Protective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain of Mycobacterium tuberculosis and demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.

Animals↗
PubMed1999

Intron retention may regulate expression of Epstein-Barr virus nuclear antigen 3 family genes.

The nuclear antigen 3 family genes (EBNA-3, EBNA-4, and EBNA-6) of Epstein-Barr virus (EBV) are important for EBV-induced immortalization and survival of B lymphocytes. However, little is known about how the expression of these genes is regulated. Each of the EBNA-3, EBNA-4, and EBNA-6 genes consists of two exons separated by a small intron. Reverse transcriptase PCR assays revealed that the vast majority of the EBNA-3, EBNA-4, and EBNA-6 mRNA, expressed in transfected and EBV-infected B cells, retained intron sequences. Northern blot and S1 protection assays confirmed that most of the EBNA-3 mRNA contained intron. Examination of deletion mutants of EBNA-3 indicated that the EBNA-3 protein was not necessary for intron retention and that there was no splicing silencing element encoded in the EBNA-3 mRNA. Cell fractionation and RNA gradient analysis revealed that the unspliced EBNA 3 family mRNAs were transported into the cytoplasm and associated with the polysomes. However, Western blot analysis of FLAG-epitope tagged EBNA-3 gave no indication of the presence of splice variant protein forms of EBNA-3. In contrast, transiently transfected cells expressing EBNA-3 revealed a sixfold increase in EBNA-3 protein expression from the genomic EBNA-3 gene compared to EBNA-3 cDNA. These data show that the intronic sequences can influence EBNA-3 protein expression and suggest that intron retention may provide a means for the fine-tuning of expression of the individual EBNA 3 family genes.

Alternative Splicing↗
PubMed1999

IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

Amino Acid Sequence↗
PubMed1999

Vascular protective effects of potassium.

Potential mechanisms responsible for the cardiovascular protection associated with high levels of dietary potassium were investigated in in vitro and in vivo studies. Elevation of extracellular potassium concentration within the physiological range inhibited free radical formation from macrophages and endothelial cells, inhibited proliferation and thymidine incorporation of vascular smooth muscle cells, and reduced platelet sensitivity to thrombin and other agonists. The effects of potassium on several well-characterized animal models of vascular pathology were also studied. Acute elevation of plasma potassium concentration inhibited thrombus formation in the coronary artery of the dog and the carotid artery of the rabbit. In cholesterol-fed rabbits, 6 weeks of diet containing low potassium content increased by approximately 50% (compared with control rabbits) the number of arteries in the myocardium showing signs of arteriosclerotic lesion formation. And in rats and pigs, 4 weeks of eating high potassium-content diet reduced the severity compared with control animals of neointimal proliferation associated with balloon angioplasty. We propose that high levels of dietary potassium provide cardiovascular protection by inhibiting the function of the cells that are responsible for vascular lesion formation.

Animals↗
PubMed1998

Molecular cloning and functional analysis of a novel tetracycline resistance determinant, tet(V), from Mycobacterium smegmatis.

The nucleotide sequence and mechanism of action of a tetracycline resistance gene from Mycobacterium smegmatis were determined. Analysis of a 2.2-kb sequence fragment showed the presence of one open reading frame, designated tet(V), encoding a 419-amino-acid protein (molecular weight, 44,610) with at least 10 transmembrane domains. A database search showed that the gene is homologous to membrane-associated antibiotic efflux pump proteins but not to any known tetracycline efflux pumps. The steady-state accumulation level of tetracycline by M. smegmatis harboring a plasmid carrying the tet(V) gene was about fourfold lower than that of the parental strain. Furthermore, the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone blocked tetracycline efflux in deenergized cells. These results suggest that the tet(V) gene codes for a drug antiporter which uses the proton motive force for the active efflux of tetracycline. By primer-specific amplification the gene appears to be restricted to M. smegmatis and M. fortuitum.

Amino Acid Sequence↗
PubMed1998

An epitope delivery system for use with recombinant mycobacteria.

We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium vaccae. The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens. We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed in M. vaccae. The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice. This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis.

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.

Amino Acid Sequence↗
PubMed1997

Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix.

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes.

Animals↗
PubMed1997

IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Cell Cycle Proteins↗
PubMed1997

p38-2, a novel mitogen-activated protein kinase with distinct properties.

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

Activating Transcription Factor 2↗