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Biomedical subjects

R Butler

Publications and source records attributed to R Butler.

At least 19 recordsLinked to original sources
PubMed1992

Role of Th-1 lymphocytes in the development of protective immunity against Mycobacterium leprae. Analysis of lymphocyte function by polymerase chain reaction detection of cytokine messenger RNA.

The patterns of lymphokine mRNA expression during the development of protective immunity to Mycobacterium leprae after intradermal vaccination of mice with killed M. leprae were studied. Using a polymerase chain reaction-based technique for detecting mRNA expression in small numbers of cells, we observed changes in the mRNA expression of a number of cytokine genes in the lymph nodes draining the site of vaccination. In particular, IL-1 (-alpha and -beta), IL-2, TNF (-alpha and -beta), and IFN-gamma mRNA were readily detected, whereas IL-3, IL-4, IL-5, IL-6, IL-7, and granulocyte-macrophage colony-stimulating factor mRNA were not detected, or were detectable only at very low levels. This is consistent with the selective activation of Th-1 Th cells. The effect of in vitro exposure of these cells to the immunizing Ag was also investigated; again, IL-1, IL-2, TNF, and IFN-gamma mRNA were abundant, but in addition, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor mRNA were greatly increased, suggesting an important role in the recall response.

Animals↗
PubMed1992

Increased proliferation of activated T-lymphocytes in response to a monoclonal antibody (anti-p68).

A series of monoclonal antibodies was produced by immunization of mice with cells of the human promonocytic cell line CM-S; one of these recognized a membrane antigen (MW 68,000) constitutively expressed by these cells. Antigen p68 was also found to be expressed on all granulocytic cells and most mononuclear leukocytes from normal human peripheral blood, but not on hemopoietic precursor cells from bone marrow. Various types of leukemic cells also expressed antigen p68 as did various transformed human cell lines whether derived from hemopoietic cells or from other tissues. Antigen p68 is involved in T-lymphocyte regulation. In fact, the antibody anti-p68 has a strong synergistic effect increasing the proliferative response of peripheral blood T-lymphocytes both in the mixed lymphocyte reaction and when the lymphocytes are stimulated by suboptimal doses of lectin (phytohemagglutinin), tumor promoter phorbol esters, or tetanus toxoid. The anti-p68 antibody synergizes with the active metabolite of vitamin D3, 1,25-dehydroxyvitamin D3, to induce monocyte to macrophage maturation and enhances the function of mature granulocytes stimulated with the granulocyte-macrophage colony-stimulating factor in vitro.

Antibodies, Monoclonal↗
PubMed1992

Characterization of a YAC and cosmid contig containing markers tightly linked to the myotonic dystrophy locus on chromosome 19.

Myotonic dystrophy (DM) is caused by a defect in an unknown gene that maps to 19q13.3, flanked by the tightly linked markers ERCC1 on the proximal side and D19S51 on the distal side. We report the isolation and characterization of overlapping YAC and cosmid clones around D19S51 for the construction of a physical map around this locus. The resulting contig contains the markers D19S51 and D19S62 (another new marker tightly linked to the DM locus) and the distal breakpoint of a radiation hybrid cell line used in the physical mapping of the DM region. We have compared the restriction maps of the YACs and cosmids with that of the genome to investigate the fidelity of these clones.

Base Sequence↗
PubMed1992

Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16.

Chromosome 21 has often been used as a model system for the development of genome mapping and cloning strategies in humans. In this report methods for systematic chromosome walking, cloning, and mapping are exemplified in the construction of a 1.5-Mb yeast artificial chromosome (YAC) contig encompassing and extending 400 kb beyond each of the genetic loci D21S13 and D21S16. Isolation of insert-terminal sequences from YACs in this contig provides a set of closely spaced physical markers. These have been used to generate a long-range genomic restriction map.

Base Sequence↗
PubMed1992

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.

Base Sequence↗
PubMed1992

Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.

Chromosomes, Fungal↗
PubMed1992

Equilibrium and kinetic studies of the interactions of salmeterol with membrane bilayers.

The interaction of salmeterol with model membranes has been studied with regard to equilibrium and kinetic behavior, including determination of the membrane-based partition coefficient, the rate of dissociation of salmeterol from membranes, and the rate of association. These data were obtained in various membrane preparations and under various conditions (e.g., temperature, cholesterol content). The compound is very lipophilic, compared with other beta 2 agonists such as salbutamol, and has a rapid association rate and a moderate dissociation rate. The equilibrium data support the assertion that the salmeterol action measured in perfused tissue involves an exo-site for nonspecific binding that may be identified with or related to the lipid bilayer. The kinetic data in unilamellar and multilamellar liposomes of synthetic lipids further suggest that the approach to the exo-site and the active site may involve components in the native system other than the lipid bilayer in which the beta 2 receptor is located. These additional components may explain the slow onset and the extraordinarily long duration of action.

Albuterol↗
PubMed1991

The antibody repertoire to proteins of Mycobacterium leprae. Genetic influences at the antigen and epitope level.

The effects of both H-2 and non-H-2 genes on the antibody response to the proteins of Mycobacterium leprae were investigated. Using a B10 series of congenic mice we found that the repertoire of antibody responses was under H-2 gene control, and that non-H2 genes were also involved. By Western blotting, differences in the number and m.w. of proteins recognised by mice of different genetic background were apparent. Such differences were also reflected in the total antibody response to a soluble extract of M. leprae (M. leprae sonicate), as measured by ELISA. Concentrating on one particular Ag, the 65-kDa heat shock protein, we found that all strains of mice developed antibodies following immunization with the purified recombinant protein, although there was a continuous distribution in the titer of antibodies obtained, with differences between individual strains indicating both H-2 and non-H-2 effects. Using a library of overlapping peptides based on the amino acid sequence of this protein, we have mapped the B cell epitopes in the different strains of mice. H-2 genes had no effect on the structure and number of epitopes recognized, although this was influenced by non-H-2 genes. There was a high level of concordance between actual epitopes recognized and those predicted by calculations of antigenic index, and B cell epitopes were located in similar positions to previously determined T cell epitopes.

Amino Acid Sequence↗
PubMed1991

Identification of the ancestral haplotype for apolipoprotein B suggests an African origin of Homo sapiens sapiens and traces their subsequent migration to Europe and the Pacific.

The probable ancestral haplotype for human apolipoprotein B (apoB) has been identified through immunological analysis of chimpanzee and gorilla serum and sequence analysis of their DNA. Moreover, the frequency of this ancestral apoB haplotype among different human populations provides strong support for the African origin of Homo sapiens sapiens and their subsequent migration from Africa to Europe and to the Pacific. The approach used here for the identification of the ancestral human apoB haplotype is likely to be applicable to many other genes.

Africa↗
PubMed1991

A yeast artificial chromosome contig encompassing the cystic fibrosis locus.

The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.

Base Sequence↗
PubMed1991

Association of a genetic polymorphism in human apolipoprotein B-100 with intermediate density lipoprotein concentrations.

Immunochemical techniques have been used to identify five antigenic (Ag) sites on apolipoprotein B-100 (apoB), the major protein constituent of very low density (VLDL), intermediate density (IDL), and low density lipoproteins (LDL). Each Ag site results from allelic variation at a specific locus of the apoB gene. In the present study, we assessed whether variations in the five Ag loci were associated with concentrations of plasma lipids or lipoprotein fractions measured by analytical ultracentrifugation in a group of 44 healthy men. Pair-wise analyses of the Ag markers revealed that Ag(a1/d), in association with either Ag(x/y) or Ag(t/z), is significantly related to plasma IDL-mass concentrations. In this cohort we detected no significant associations of the Ag alleles (singly or in combination) with plasma total cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, or mass of total VLDL or LDL. These results suggest that genetic variations in the apoB molecule may predispose to variations in concentrations of IDL that could have consequences for atherosclerotic risk.

Adult↗
PubMed1991

Lung edema formation following inhalation injury: role of the bronchial blood flow.

We investigated the contribution of the bronchial blood flow to the lung lymph flow (QL) and lung edema formation after inhalation injury in sheep (n = 18). The animals were equally divided into three groups and chronically prepared by implantation of cardiopulmonary catheters and a flow probe on the common bronchial artery. Groups 1 and 2 sheep were insufflated with 48 breaths of cotton smoke while group 3 received only room air. Just before injury, the bronchial artery of group 2 animals was occluded. The occlusion was maintained for the duration of the 24-h study period. At the end of the investigation, samples of lung were taken for determination of blood-free wet weight-to-dry weight ratio (W/D). Inhalation injury induced a sevenfold increase in QL in group 1 (7 +/- 1 to 50 +/- 9 ml/h; P less than 0.05) but only a threefold increase in group 2 (10 +/- 2 to 28 +/- 7 ml/h; P less than 0.05). The mean W/D value of group 1 animals was 23% higher than that of group 2 (5.1 +/- 0.4 vs. 3.9 +/- 0.2; P less than 0.05). Our data suggest that the bronchial circulation contributes to edema formation in the lung that is often seen after the acute lung injury with smoke inhalation.

Animals↗
PubMed1991

Bacterial sampling by absorbent paper points. An in vitro study.

This in vitro study was designed to test the hypothesis that sampling of bacterial suspensions by absorbent paper points would accurately reflect the proportion of the bacterial species whether they are homogeneously or non-homogeneously distributed in the sample site. The non-homogeneously distributed populations consisted of equal numbers of colony forming units (CFU) of two periodontal bacterial species present as separate cultures, one layered on top of the other. The species in the top layer accounted for greater than 90% of the total number of CFU detected by paper point sampling, regardless of which species formed the top layer. In contrast, when the layers were mixed prior to sampling to give a homogeneous bacterial distribution, the paper points detected equal numbers of CFU of each species. Thus the hypothesis was not supported and the proportions yielded by paper point sampling are not the same when the bacteria are non-homogeneously distributed as when they are homogeneously distributed. Ultrastructural observations have documented a non-homogeneous distribution of bacterial species in periodontal pockets. If the specific microbiota in the apical portion of deep periodontal pockets are important to the periodontal disease process, these data suggest that bacterial samples obtained by absorbent paper points misrepresent the presence of such bacterial species.

Absorption↗
PubMed1990

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.

Aldehyde Reductase↗
PubMed1990

A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.

Base Sequence↗