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Biomedical subjects

T M Wilson

Publications and source records attributed to T M Wilson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Down-regulation of apurinic/apyrimidinic endonuclease expression is associated with the induction of apoptosis in differentiating myeloid leukemia cells.

The human DNA repair enzyme apurinic/apyrimidinic endonuclease (APE/ref-1) is a multifunctional protein in the DNA base excision repair (BER) pathway that is responsible for repair of apurinic/apyrimidinic (AP) sites in DNA. DNA repair and programmed cell death both function using different mechanisms to protect the organism from the consequences of extensive cellular damage; however, little is known about the relationship of the DNA BER repair pathway to apoptosis. We have determined the relationship of a BER DNA repair enzyme, APE, to apoptosis using the myeloid leukemia cell line HL-60, which can be induced to differentiate down the granulocytic or monocytic/ macrophage pathway. Treatment of HL-60 cells with retinoic acid/DMSO (granulocytic) or phorbol 12-myristate 13-acetate (monocytic) results in apoptosis and in down-regulation of APE expression at both the RNA and protein levels. Moreover, double-labeling experiments using APE immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay for apoptosis demonstrate that individual cells undergoing apoptosis lose expression of APE regardless of their state of differentiation. Blocking apoptosis by overexpression of the bcl-2 proto-oncogene in HL-60 cells or by a bcr-abl-related mechanism in K562 cells and subsequent differentiation results in morphological differentiation but no loss of APE expression. These studies establish that down-regulation of APE expression is associated with programmed cell death in cells of the myeloid lineage.

Apoptosis↗
PubMed1997

Metabolism and disposition of the antifolate LY231514 in mice and dogs.

The metabolism and disposition of LY231514 was studied in mice and dogs. LY231514 is a novel pyrrotopyrimidine-based multi-target antifolate (MTA) showing broad in vivo antitumor activity in mouse models and is currently in phase II human clinical trials. Doses (iv) of the compound showed high plasma levels, resulting in AUC values of 30-33 micrograms-hr/ml for mice and dogs after 20 and 7.5 mg/kg doses, respectively. The compound was eliminated rapidly. Half-life values for mice and dogs were about 7 and 2 hr, respectively. In vitro plasma binding measured 56% in mice, 46% in dogs, and 81% in humans. Fecal elimination was the major excretion pathway in mice after single iv doses of [14C]LY231514. Urine constituted the major route of excretion in dogs. Parent LY231514 accounted for the majority of urinary radiocarbon in mice (90%) and dogs (68%). Minor metabolites were found in urine, but the amounts were too small to isolate or identify. Based on an earlier observation that LY231514 photodegraded to produce reaction products having similar retention times as these minor urinary isolates, a photo-oxidation system was developed which in fact produced these metabolites. Subsequently, these photolytically-produced materials were used as standards to identify two novel in vivo metabolites formed by oxidation of the pyrrolo-pyrimidine ring system of LY231514. The oxidative transformations are similar to those observed for tryptophan and other indoles in that the pyrrole ring is oxidized to give an amide; further oxidation cleaves this ring, one ring carbon is lost, and a ketone is formed.

Animals↗
PubMed1996

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.

Adoptive Transfer↗
PubMed1996

Differential expression of the apurinic / apyrimidinic endonuclease (APE/ref-1) multifunctional DNA base excision repair gene during fetal development and in adult rat brain and testis.

The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease is responsible for the repair of AP sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA binding capability of Jun-Jun homodimers and Fos-Jun heterodimers by altering their redox state and to be involved in calcium mediated transcriptional repression of the parathyroid hormone gene. Previous studies examining the tissue specific distribution of the AP endonuclease (APE) transcript and protein by Northern analysis and enzymatic assays, respectively, have shown that this gene is expressed in all tissues at relatively similar levels. In the current study, adult and fetal rat tissue sections were examined for the expression of the APE transcript in specific subpopulations of cells and during development by in situ hybridization. In the adult brain, the APE transcript showed a widespread, but heterogeneous pattern of expression. Predominant levels of transcript were detected in the suprachiasmatic nuclei, the supraoptic and paraventricular nuclei, the hippocampus and the cerebellum. During fetal development, transcript was detected in all somatic sites examined with very high levels in the thymus, liver and developing brain. Examination of the adult testis indicated that the expression of the transcript varies with the stage of spermatogenesis with the highest levels being present over round spermatids. These results provide evidence that the APE gene is not homogeneously expressed, but rather is found in subpopulations of cells in the brain and testes and during development.

Animals↗
PubMed1996

Single-strand conformation polymorphism analysis of RT-PCR products of UK isolates of barley yellow mosaic virus.

Single-strand conformation polymorphism (SSCP) analysis of the bipartite genomes of several UK isolates of barley yellow mosaic virus (Ba YMV) was done using fragments of cDNA amplified by RT-PCR. Isolates differed in their SSCP patterns in several regions, but in no case was the pattern able to distinguish between common and resistance-breaking strains. In regions where the nucleotide sequences of UK isolates had been determined, there was no simple relationship between numbers of nucleotide differences and SSCP patterns: differences of only 2 or 3 nucleotides (nt) gave different SSCP patterns, whereas differences of as many as 29 nt did not. Although SSCP analysis has some potential as a rapid and sensitive tool for distinguishing virus isolates, differences detected do not necessarily relate to biological properties and the results are highly dependent on gel conditions.

Acrylic Resins↗
PubMed1996

ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex.

A gene conferring low-level isoniazid (INH) resistance on Mycobacterium smegmatis was isolated from a cosmid library of the genome of an INH-resistant Mycobacterium bovis strain. The gene had good homology with ahpC, the product of which is a subunit of alkyl hydroperoxide reductase, and also with a family of thiol-specific antioxidant enzymes. A mutation was found in the promoter upon comparison with the equivalent DNA sequence from the INH-sensitive parent strain. Promoter sequences from other INH-sensitive and INH-resistant M. bovis and Mycobacterium tuberculosis strains were sequenced and the mutation was found only in the INH-resistant strains. An INH-resistant M. tuberculosis strain also had an additional mutation in the promoter region. The wild-type promoter and promoters with one and two mutations were ligated into a reporter plasmid containing the lacZ gene. The presence of the first mutation resulted in a sixfold induction of beta-galactosidase activity, and the presence of both mutations caused a 10-fold induction. Increased expression of AhpC may account for some of the INH resistance of strains of the M. tuberculosis complex.

Amino Acid Sequence↗
PubMed1996

Idiopathic urethritis in the adolescent male.

OBJECTIVE: To study adolescent males presenting with protracted blood spotting and dysuria in an attempt to further understand the precise pathology and endoscopic findings in these boys. METHODS: Over a 3-year period the 7 patients had video endoscopies and tests to exclude infective and immunological disease. RESULTS: All boys were found to have a similar well-defined, circumferential length of inflammation in the bulbar urethra. No specific aetiology was illucidated, but 2 cases had a urethral stricture at the time of presentation, and two further cases subsequently required urethral dilatation. Two boys had a borderline low IgG, of probably little significance. CONCLUSION: Boys presenting with prolonged symptoms of dysuria and haematuria should be endoscoped, and detailed investigations looking for fastidious organisms and immune deficiency seem appropriate in those with significant urethral ulceration.

Adolescent↗
PubMed1995

Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme.

The multifunctional mammalian apurinic/apyrimidinic endonuclease (APE) is responsible for the repair of apurinic/apyrimidinic sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA-binding capability of JUN and FOS, as well as numerous other transcription factors through the alteration of the transcription factor redox state. Biochemical studies of organ homogenates have shown that APE is present in the different tissues studied at similar concentrations. The present study examines the immunohistochemical distribution of APE in several organs and demonstrates new and unexpected patterns of cellular and subcellular localization of this enzyme. In the hippocampus, the APE protein was highly expressed in neurons of the dentate gyrus and regions CA3 and CA4, and unexpectedly, the staining was primarily cytoplasmic. AP endonuclease immunoreactivity in the cerebellum was found in the granule and Purkinje cells, both cytoplasmic and nuclear. APE staining of the hypoglossal nucleus of the brainstem, where motor neurons that control tongue movement reside, showed reactivity in the cytoplasmic Nissl substance. Skin, liver, and duodenum demonstrated nuclear staining; however, in the duodenum, only the enterocyte nuclei of the proximal villus and the crypts of Lieberkuhn were stained, with no staining of the distal villus. These results suggest that APE has different regulatory and functional roles in different cells and organs of the body. This study shows the importance of correlating in vitro findings in tissue culture cells with the organism as a whole. The cytoplasmic staining seen in parts of the brain and in liver suggests that there may be additional functions for the APE yet to be described.

Autopsy↗
PubMed1995

Differential cellular expression of the human MSH2 repair enzyme in small and large intestine.

The human MSH2 (hMSH2) protein is responsible for the initial recognition of mismatched nucleotides during the postreplication mismatch repair process. Loss of hMSH2 function has been demonstrated to lead to the accumulation of replication errors, resulting in a mutator phenotype, which may be responsible for the multiple mutations required for multi-stage carcinogenesis. Alterations of the hMSH2 gene has been linked to approximately 60% of hereditary nonpolyposis colon cancer cases. Colon tumors in hereditary nonpolyposis colon cancer patients originate within benign preneoplastic adenomas and display replication errors in the form of microsatellite instability. The aim of this study was to investigate the cellular expression of the hMSH2 protein in cells of the large and small intestines. Using antibody specific for hMSH2, we have determined that this protein is highly expressed in cells of the crypts of Lieberkühn that are undergoing rapid renewal in both the ileum and colon. Proliferative perifibroblasts in the colon also showed significant presence of the hMSH2 protein. These results confirm the hypothesis that hMSH2 is expressed in highly proliferative cells of the gut, and mutations in this gene could, therefore, be expected to expedite the progression of adenoma to carcinoma in this tissue.

Animals↗
PubMed1995

An analysis of spontaneous deletion sites in soil-borne wheat mosaic virus RNA2.

Plants mechanically inoculated with soil-borne wheat mosaic virus (SBWMV, Oklahoma isolate) generated several deleted forms of RNA2, the smaller genomic RNA (3593 nt). Four naturally deleted forms were cloned by RT-PCR methods and the sequences around each deletion site were compared. SBWMV RNA2 molecules were deleted for 519, 759, 964, or 1030 nt, respectively, each within the coat protein-readthrough domain. No common sequences were found flanking the 5'- and 3'-sites of deletion; however, the 5'-site of each deletion lay in the region between genome coordinates 1417-1465. The delta 759nt clone isolated from virus-infected plants after a single mechanical passage was identical to a clone described previously (J. Chen, S. A. MacFarlane, and T. M. A. Wilson, 1994, Virology 202, 921-929) which became the dominant, stable form of SBWMV RNA2 after five or more serial mechanical transfers. In this study, a total of 33 independent, RT-PCR clones with an apparent deletion of 759 nt, isolated after one to seven mechanical passages, were also sequenced and their precise deletion sites were compared. Twenty-six clones contained the original "stable," 759-nt deletion, whereas 7 clones, which were found only during the first four passages, had deletions at one of four alternative sites. After the fifth mechanical passage the stable 759-nt deletion dominated. The results also show that, in general, the smaller deletions are not intermediates in the larger deletion process. The complete nucleotide sequence of SBWMV RNA2 (Oklahoma) is also reported.

Base Sequence↗
PubMed1995

A conserved, precise RNA encapsidation pattern in Tobamovirus particles.

The bidirectional RNA encapsidation pathway in nine sequenced Type 1 Tobamovirus genomes will result in RNA-coat protein assembly, up to and including the first transcribed G, adjacent to the 5'-cap structure (m7 Gppp). This precision is highly conserved, despite wide interstrain variations in the absolute position of the phase-determining core of the origin-of-assembly sequence (Gxx)n and in overall genome length (6311-6507 nts). A Type 2 Tobamovirus genome did not comply with this pattern. All genomes had a statistically significant bias for G at every third (or 3n) position, resulting in a preponderance of GNN codons and hence a high Val, Ala, Gly, Asp, Glu content, at least in the large (126/183 kDa) and amino-coterminal replicase protein genes. Contrary to predictions from the X-ray fibre diffraction structure of tobacco mosaic virus (TMV, U1 strain), only one (pepper mild mottle virus) of the nine Type 1 Tobamoviruses positioned the preferred G-repeat in the most favourable (5') position of the trinucleotide binding site on each coat protein (CP) subunit. In all but one of the eight remaining Type 1 Tobamovirus genomes, G would predominate in the CP 3'-site. The significance of these observations for TMV particle assembly, disassembly and host cell interactions are discussed.

Base Composition↗
PubMed1995

Molecular characterisation of UK isolates of barley yellow mosaic bymovirus.

Several isolates of barley yellow mosaic virus (BaYMV) from different sites in the UK, including some that were virulent on European resistant winter barley cultivars (resistance-breaking strain: BaYMV-2) and some that were not, were examined by RT-PCR, restriction mapping and sequencing of selected parts of the virus genome. Nucleotide and predicted amino acid sequences were determined for the 5'-terminal region, part of the NIa coding region and the coat protein coding region on RNA 1 and an area at the N-terminus of the 70-kDa protein coding region on RNA 2. The sequences differed from those previously reported for a BaYMV isolate from Japan and for two German isolates, one of which was of the BaYMV-2 strain. There were no strain-specific amino acid differences and the few, non-consecutive, nucleotide differences detected were probably not significant and were insufficient to develop a rapid diagnostic test to distinguish BaYMV-2 from other isolates. Restriction mapping of RNA 2 cDNA again showed no consistent strain-related differences. The differences previously reported between the two German isolates are probably not strain-related.

Base Sequence↗
PubMed1995

Expression in Escherichia coli of a rat cDNA encoding an apurinic/apyrimidinic endonuclease.

A rat cDNA (rAPEN) with 85% DNA identity to the major human apurinic/apyrimidinic (AP) endonuclease gene was used to construct a fusion between it and glutathione-S-transferase (GST). The GST-rAPEN fusion was subsequently overexpressed in Escherichia coli, purified on glutathione-agarose affinity columns, and the purified protein tested for AP endonuclease activity. DNA nicks were found to be specifically introduced into AP DNA in a reaction that was dependent upon the time of incubation and the amount of GST-rAPEN added. The DNA scissions produced by GST-rAPEN were determined to be adjacent and 5' to an AP site. The purified fusion protein was also able to efficiently remove 3'-(4 hydroxy-5-phospho-2-pentenal) residues, and to a lesser extent 3'-phosphoglycolate residues. The GST-rAPEN activity failed to exhibit any 3'-5' exonuclease activity, a characteristic shared by the major AP endonuclease in bovine and human.

Animals↗
PubMed1995

Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis.

Isoniazid (INH) resistance of the Mycobacterium tuberculosis Complex (MtbC) is associated with both loss of catalase activity and mutation of the inhA gene. However, the relative contributions of these changes to resistance and to the loss of virulence for guinea-pigs is unknown. In this study, a virulent strain of Mycobacterium bovis, a member of the MtbC, was exposed to increasing concentrations of INH. Two INH-resistant strains were produced which had lost catalase activity. Strain WAg405, which had a higher resistance to INH, also had a mutation in the inhA gene. This demonstrated that loss of catalase activity and mutation of inhA had a cumulative effect on INH resistance. When a functional katG gene was integrated into the genome of WAg405 the INH resistance was greatly reduced. This indicated that most of the resistance had been caused by loss of catalase activity. While the parent INH-sensitive strain was virulent for guinea-pigs, the INH-resistant strains were significantly less virulent. Integration of a functional katG gene into the most resistant strain restored full virulence. This clearly established that katG is a virulence factor for M. bovis and that mutation of the inhA gene has no effect on virulence.

Animals↗
PubMed1995

Coordinate gene expression of luteinizing hormone-releasing hormone (LHRH) and the LHRH-receptor after prolactin stimulation in the rat Nb2 T-cell line: implications for a role in immunomodulation and cell cycle gene expression.

PRL has been shown to induce a number of genes after the stimulation of quiescent Nb2 T-cells, including c-fos, c-myc, ornithine decarboxylase, interferon regulatory factor-1, and others. One of these genes, LHRH, has not previously been reported to respond in this manner, although we and others have reported its presence in rat and human T- and B-cells. Furthermore, recent evidence suggests that LHRH functions as an immunoregulator in a cytokine-like manner. Using the rat immature T-cell line Nb2, we present data showing for the first time that 1) the LHRH gene is regulated by PRL at various times during the cell cycle; 2) an alternatively spliced LHRH messenger RNA exists in Nb2 cells and may produce a new truncated GnRH-associated peptide (alternatively called PIF for PRL-inhibiting factor); 3) the LHRH receptor is expressed in lymphocytes in a manner similar to the LHRH gene after PRL addition, and its complementary DNA sequence is identical to that of the pituitary receptor; 5) the SH gene, found on the opposite strand of the LHRH gene, is expressed in lymphocytes at the same time and in the same manner as the LHRH gene; 6) the LHRH messenger RNA has a very short half-life in these cells; and 7) the lymphocyte LHRH transcription start site is essentially the same as the hypothalamic site. These data strengthen the relationship between PRL and LHRH expression in the immune system and further support our contention that LHRH is an important immunoregulator, on par with other known cytokines.

Animals↗
PubMed1994

Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli.

The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro. Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles. To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants. The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications. Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA. U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties. However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein). If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked. In vivo, E. coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants.

Base Sequence↗