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W R Jacobs

Publications and source records attributed to W R Jacobs.

At least 73 records · Page 4Linked to original sources
PubMed1996

Transesophageal echocardiographic assessment of the contribution of intrinsic tissue thickness to the appearance of a thick mitral valve in patients with mitral valve prolapse.

OBJECTIVES: This prospective, blinded transesophageal echocardiographic study was performed to determine the relative contributions of leaflet redundancy and overlap versus intrinsic tissue thickening as mechanisms for the apparent increase in diastolic thickness of the mitral valve. BACKGROUND: Increased diastolic thickness of the mitral valve has been identified as an echocardiographic feature that predicts subsequent adverse sequelae in patients with mitral valve prolapse (MVP). METHODS: Eleven patients with clinical and transthoracic echocardiographic evidence of MVP and 11 age-matched control subjects underwent protocol transesophageal echocardiography to image the mitral valve in two orthogonal planes and to measure its thickness in systole and diastole. RESULTS: Maximal diastolic width of the slack, unloaded anterior leaflet was significantly greater in patients with MVP than in control subjects (mean +/- SD: 0.64 +/- 0.20 cm vs. 0.30 +/- 0.04 cm, p < 0.001). Similarly, diastolic posterior leaflet width was greater in patients with MVP (0.67 +/- 0.39 cm vs. 0.31 +/- 0.06 cm, p < 0.01). In contrast, minimal systolic width of the distended pressure-loaded mitral valve was not significantly different between patients with MVP and control subjects for either the anterior (0.22 +/- 0.05 cm vs. 0.20 +/- 0.04 cm, p = NS) or the posterior (0.25 +/- 0.07 cm vs. 0.24 +/- 0.05 cm, p = NS) leaflets. The percent change in leaflet width from diastole to systole (% delta W), an index of the contribution of dynamic factors (e.g., leaflet redundancy and overlap) to the apparent increase in diastolic leaflet thickness, was significantly greater in patients with MVP than in control subjects for both the anterior (% delta W 62 +/- 13% vs. 34 +/- 16%, p < 0.001) and the posterior (% delta W 54 +/- 19% vs. 22 +/- 21%, p < 0.005) leaflets. CONCLUSIONS: The apparent increase in diastolic mitral leaflet thickness in patients with MVP versus control subjects is largely attributable to dynamic factors such as leaflet redundancy, overlap and deformation. During diastole, when the mitral leaflets are slack and unstressed, the leaflets appear markedly thickened in patients with MVP. In contrast, during systole, when developed intraventricular pressure distends the leaflets, causing them to stretch and balloon into the left atrium, the intrinsic tissue thickness is much less than that measured in diastole. These findings have important implications for the morphologic criteria used to diagnose MVP and the potential pathophysiologic mechanisms for adverse sequelae in this syndrome.

Adult↗
PubMed1996

Auxotrophic vaccines for tuberculosis.

Tuberculosis is responsible for the deaths of more people each year than any other single infectious disease, with greater than 7 million new cases and 2 million deaths annually. It remains the largest attributable cause of death in HIV-infected individuals, responsible for 32% of deaths of HIV-infected individuals in Africa. The only currently available vaccine for tuberculosis, bacille Calmette-Guerin (BCG) is the most widely used vaccine in the world, being administered to approximately 100 million children each year. Although untoward effects were not seen in several studies of HIV-seropositive children, the safety of live attenuated BCG vaccine in HIV-positive adults remains unknown and a matter of some concern. To obviate potential adverse affects of BCG vaccines in immunodeficient individuals, we have studied five auxotrophic strains of BCG produced by insertional mutagenesis for safety in administration to mice with severe combined immunodeficiency disease (SCID), and for protection in a susceptible strain of mice. The results indicate that viable BCG could no longer be detected in mice receiving the auxotrophs after 16-32 weeks, and that infected SCID mice survived for at least 230 days. In contrast, all SCID mice succumbed within eight weeks to conventional BCG vaccine. When susceptible BALB/c mice were immunized with auxotrophs and subsequently challenged with virulent Mycobacterium tuberculosis, several of the auxotrophs produced comparable protection against intravenous and intratracheal challenge with M. tuberculosis relative to conventional BCG. These results suggest that auxotrophic strains of BCG represent a potentially safe and useful vaccine against tuberculosis for populations at risk for HIV.

Adult↗
PubMed1996

Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70.

The Mycobacterium bovis antigens MPB70 and MPB83 are homologous cross-reactive proteins. It has been reported previously that MPB83 is glycosylated and exists in two forms with apparent molecular masses of 23kDa and 25kDa, whereas the apparent molecular mass of MPB70 is 22kDa. Using a monoclonal antibody, SB10, which recognizes an epitope common to both MPB70 and MPB83, we compared the expression of these proteins in M. bovis BCG, virulent M. bovis and virulent Mycobacterium tuberculosis by Western blotting of bacterial lysates. The previously described pattern of high and low producing substrains of BCG for MPB70 was also applicable for MPB83. Virulent M. bovis was found to express high levels of MPB70 and MPB83. Immunoblotting experiments using sera from Balb/c mice infected with live M. tuberculosis H37Rv revealed that although the MPB83 homologue of M. tuberculosis, MPT83, is expressed at low levels in M. tuberculosis when grown in vitro, the protein is highly immunogenic during infection with live bacteria. A clone from a mycobacterial shuttle cosmid library of M. tuberculosis H37Rv was isolated which expressed both MPT70 and MPT83. Genetic analysis of this cosmid revealed that MPT70 and MPT83 were encoded by separate genes with the gene encoding MPT83 situated 2.4kb upstream of mpt70. Both genes are transcribed in the same direction. The gene encoding MPT83 was cloned and DNA sequencing revealed an open reading frame of 660bp encoding a protein with a predicted molecular mass of 22kDa. Recombinant MPT83 was expressed in Escherichia coli from the native AUG initiation codon by translational coupling. In E. coli MPT83 was expressed as a 23kDa antigen whereas in the rapid growing mycobacterium Mycobacterium smegmatis the protein was expressed as a 25kDa protein indicating post-translational modification of the protein by M. smegmatis. In recombinant M. smegmatis MPT83 was predominantly cell associated whereas MPT70 was secreted into the culture medium. Amino acid sequence comparison between MPT83 and MPT70 revealed a 61% identity between the proteins, although little homology was apparent at the amino terminus. In MPT83 this region contained a typical lipoprotein signal peptide cleavage motif and a putative signal motif for O glycosylation. Both these motifs were absent from the amino acid sequence of MPT70.

Amino Acid Sequence↗
PubMed1996

Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages.

The ability of slow-growing mycobacteria to replicate within host mononuclear phagocytes is thought to be central to the pathogenesis of mycobacterial infection. However, because of the lack of a mycobacterial mutant defective for intracellular replication, it has not been possible to test this hypothesis directly. Previously, we showed that a BCG leucine auxotroph with a transposon disruption of the leuD gene is unable to grow in mice. Here we demonstrate that this mutant is also incapable of replicating within cultured macrophages in vitro. Complementation of the leuD mutation with the leuCD genes of Escherichia coli restored wild-type levels of growth in macrophages, establishing that the defect for intracellular replication was due to leucine auxotrophy per se and not to a polar effect of the transposon insertion on an adjacent gene. These results suggest that the inability of the leucine auxotroph to grow in mice was due to its sequestration, after phagocytosis, in an intracellular compartment from which it could not obtain leucine.

Animals↗
PubMed1996

Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates.

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.

Alleles↗
PubMed1996

Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis.

Diaminopimelate (DAP) is a unique metabolite used for both the biosynthesis of lysine in bacteria and the construction of the peptidoglycan of many species of bacteria, including mycobacteria. DAP is synthesized by bacteria as part of the aspartate amino acid family, which includes methionine, threonine, isoleucine, and lysine. Aspartokinase, the first enzyme in this pathway, is encoded by the ask gene in mycobacteria. Previous attempts to disrupt this gene in Mycobacterium smegmatis were unsuccessful, even when the cells were supplied with all the members of the aspartate family, suggesting that unlike other bacteria, mycobacteria may have an absolute requirement for this pathway even when growing in rich medium containing DAP. The purpose of this study was to determine if the ask gene and the aspartate pathway are essential to M. smegmatis. This study describes a test for gene essentiality in mycobacteria, utilizing a counterselectable marker (streptomycin resistance) in conjunction with a specially constructed merodiploid strain. We have used this system to show that the ask gene could not be disrupted in wild-type M. smegmatis, using selective rich medium supplemented with DAP unless there was an extra copy of ask provided elsewhere in the chromosome. Disruption of ask was also possible in a lysine auxotroph incapable of converting DAP to lysine. The ask mutant, mc21278 (ask1::aph), exhibits multiple auxotrophy (Met-, Thr-, DAP-, and Lys-) and is complemented by the ask gene. This is the first description of DAP auxotrophy in mycobacteria. The ask mutant lyses when deprived of DAP in culture, a characteristic which can be exploited for the reproducible preparation of protoplasts and mycobacterial extracts. The evidence presented here indicates that the aspartate pathway is essential to M. smegmatis and that DAP is the essential product of this pathway.

Aspartate Kinase↗
PubMed1995

Mutation of the principal sigma factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex.

Tuberculosis continues to be responsible for the deaths of millions of people, yet the virulence factors of the causative pathogens remain unknown. Genetic complementation experiments with strains of the Mycobacterium tuberculosis complex have identified a gene from a virulent strain that restores virulence to an attenuated strain. The gene, designated rpoV, has a high degree of homology with principal transcription or sigma factors from other bacteria, particularly Mycobacterium smegmatis and Streptomyces griseus. The homologous rpoV gene of the attenuated strain has a point mutation causing an arginine-->histidine change in a domain known to interact with promoters. To our knowledge, association of loss of bacterial virulence with a mutation in the principal sigma factor has not been previously reported. The results indicate either that tuberculosis organisms have an alternative principal sigma factor that promotes virulence genes or, more probably, that this particular mutant principal sigma factor is unable to promote expression of one or more genes required for virulence. Study of genes and proteins differentially regulated by the mutant transcription factor should facilitate identification of further virulence factors.

Amino Acid Sequence↗
PubMed1995

Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis.

The inhA gene has been recently shown to encode a common protein target for isoniazid and ethionamide action in Mycobacterium tuberculosis. In this paper, we demonstrate that the M. tuberculosis InhA protein catalyzes the NADH-specific reduction of 2-trans-enoyl-ACP, essential for fatty acid elongation. This enzyme preferentially reduces long-chain substrates (12-24 carbons), consistent with its involvement in mycolic acid biosynthesis. Steady-state kinetic studies showed that the two substrates bind to InhA via a sequential kinetic mechanism, with the preferred ordered addition of NADH and the enoyl substrate. The chemical mechanism involves stereospecific hydride transfer of the 4S hydrogen of NADH to the C3 position of the 2-trans-enoyl substrate, followed by protonation at C2 of an enzyme-stabilized enolate intermediate. Kinetic and microcalorimetric analysis demonstrates that the binding of NADH to the S94A mutant InhA, known to confer resistance to both isoniazid and ethionamide, is altered. This difference can account for the isoniazid-resistance phenotype, with the formation of a binary InhA-NADH complex required for drug binding. Isoniazid binding to either the wild-type or S94A mutant InhA could not be detected by titration microcalorimetry, suggesting that this compound is a prodrug, which must be converted to its active form.

Acyl Coenzyme A↗
PubMed1995

Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis.

Resistance to isoniazid in Mycobacterium tuberculosis can be mediated by substitution of alanine for serine 94 in the InhA protein, the drug's primary target. InhA was shown to catalyze the beta-nicotinamide adenine dinucleotide (NADH)-specific reduction of 2-trans-enoyl-acyl carrier protein, an essential step in fatty acid elongation. Kinetic analyses suggested that isoniazid resistance is due to a decreased affinity of the mutant protein for NADH. The three-dimensional structures of wild-type and mutant InhA, refined to 2.2 and 2.7 angstroms, respectively, revealed that drug resistance is directly related to a perturbation in the hydrogen-bonding network that stabilizes NADH binding.

Bacterial Proteins↗
PubMed1995

Does glycemic control affect growth velocity in children with insulin-dependent diabetes mellitus.

To determine the effect of glycemic control on linear growth in children with insulin-dependent diabetes mellitus type 1, we studied 82 patients (40 male, 42 female) over a 6-year period. The mean +/-SD for age of onset and duration of IDDM were 7.3 +/- 3.9 years and 4.8 +/- 3.5 years, respectively. At each clinic visit, glycemic control was assessed by measuring glycosylated hemoglobin (GHb). For a total of 751 clinic visits, the mean +/- SD for chronologic age and GHb were 11.5 +/- 3.8 years and 10.2% +/- 2.3%, respectively. Good glycemic control was correlated with more frequent clinic visits (r= 0.219, P < 0.05). Growth was assessed by determining both weight and height, which were normalized for age and sex by calculating Z scores for weight and height and GHb. Moreover, regression analysis revealed no significant correlation between GHb levels and delta Z for either weight or height. While a significant correlation was observed between delta Z for weight and height (r = 0.30, P < 0.01), the relationship was not affected by glycemic control. Therefore, these data demonstrate that weight gain and growth rate do not seem to be significantly affected by glycemic control. This study also confirms that linear growth velocity is dependent on weight gain and suggests that in type 1 children, weight gain and level of growth-producing hormones such as insulin-like growth factor-1 (IGF-1) are more important regulators of linear growth than glycemic control.

Blood Glucose↗
PubMed1995

Effect of an inelastic aortic synthetic vascular graft on exercise hemodynamics.

This study compared aortic input impedance characteristics between patients with aortic interposition Dacron grafts placed for traumatic aortic injury and normal age-matched control subjects. All subjects were examined at rest and after treadmill exercise. Magnetic resonance imaging was conducted to rule out anatomic (stenosis) effects. Exercise increased characteristic impedance (ie, reduced aortic distensibility) by 29% and decreased total systemic arterial compliance by 21% in the patient group, whereas the normal control group showed insignificant change in these variables after exercise. Peripheral pressure wave reflection was reduced substantially with exercise (27%) in the control group, with much less reduction observed in the patient group. These abnormal vascular hemodynamics were associated with significantly high cardiac energetic costs in the patient group. A plausible explanation for the observed differences lies in the exaggerated vascular impedance mismatch between compliant aorta and inelastic graft, when cardiac output increases dramatically.

Adult↗
PubMed1995

Bacterial vaccine vectors and bacillus Calmette-Guérin.

Recent advances in biotechnology now allow a more modern approach to the development of vaccines, particularly that of recombinant vaccines. Bacterial vaccine vectors have the advantage over viral vectors in that the former have the ability to express a greater number of antigens in different forms. Although no recombinant bacterial vaccines are currently in use, bacillus Calmette-Guérin (BCG), Salmonella species, and Escherichia coli are being developed as vaccine vectors. We review plasmid systems and mutant strains developed for the expression of foreign antigens, with particular emphasis on those developed for BCG. We describe the development of antigen expression systems as well as the immune response elicited by recombinant BCG vaccine strains to bacterial and human immunodeficiency virus (HIV) antigens. A modified recombinant BCG carrier with selection for the stable maintenance of rDNA is proposed.

Administration, Oral↗
PubMed1995

L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria.

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug-sensitive and drug-resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.

Bacterial Typing Techniques↗
PubMed1995

In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.

Animals↗
PubMed1995

Rapid Mycobacterium species assignment and unambiguous identification of mutations associated with antimicrobial resistance in Mycobacterium tuberculosis by automated DNA sequencing.

OBJECTIVE: To develop and demonstrate the utility of automated DNA sequencing strategies for rapid and unambiguous identification of Mycobacterium species and mutations associated with antimicrobial resistance in Mycobacterium tuberculosis. DESIGN AND SPECIMENS: A 360-base pair segment of the gene (hsp65) encoding a 65-kd heat shock protein was characterized from 91 isolates assigned to 24 Mycobacterium species by traditional biochemical techniques. Areas of seven genes recently shown to contain mutations associated with antimicrobial resistance in M tuberculosis strains were also sequenced in a sample of 128 resistant organisms. Early positive BACTEC 460 cultures and acid-fast, bacterium-positive sputum specimens from patients with tuberculosis were also studied. RESULTS: Automated DNA sequencing identified species-specific polymorphism in the target segment of hsp65, successfully identified organisms to the species level in smear-positive sputum samples, and unambiguously characterized seven genes associated with antimicrobial resistance in M tuberculosis. CONCLUSIONS: Rapid identification of M tuberculosis and other Mycobacterium species is possible by automated DNA sequencing of a portion of hsp65. The technique is also feasible for analysis of some smear-positive sputum specimens. Unambiguous characterization of target segments of genes harboring mutations associated with antimicrobial resistance in M tuberculosis is possible from primary patient specimens. Taken together, the data demonstrate the feasibility of mycobacterial species identification and potential to identify mutations associated with antimicrobial resistance in less than 48 hours.

Alleles↗
PubMed1994

Implications of multidrug resistance for the future of short-course chemotherapy of tuberculosis: a molecular study.

Tuberculosis-control programmes are compromised by the increased frequency of multidrug-resistant strains of Mycobacterium tuberculosis. We used the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis techniques to establish the molecular basis of resistance in 37 drug-resistant isolates of M tuberculosis, and correlated these findings with clinical and antibiotic-sensitivity data. Resistance to isoniazid was found in 36 strains, 16 of which were also resistant to ethionamide. Of the 36 isoniazid-resistant strains, 23 had mutations in the katG gene, and 5 of these also had mutations in the inhA gene. A further 5 strains had alterations in the inhA locus without the katG gene being mutated. Rifampicin resistance was less frequent (13 strains) and usually associated with isoniazid resistance (11 of 13 strains). Mutations in the rpoB gene were detected for all these rifampicin-resistant isolates. Mutations in the rpsL and rrs genes, associated with streptomycin resistance, were found in 13 of 25 and 2 of 25 streptomycin-resistant strains, respectively. The same chromosomal mutations, or combinations of mutations, were found in strains displaying single or multidrug resistance, from cases of both primary and secondary resistance, and from patients infected with human immunodeficiency virus. Thus, multidrug resistance is not due to a novel mechanism and tuberculosis chemotherapy is not subject to a new threat.

AIDS-Related Opportunistic Infections↗
PubMed1994

Cloning, sequencing, and expression of Rhodococcus L-phenylalanine dehydrogenase. Sequence comparisons to amino-acid dehydrogenases.

L-Phenylalanine dehydrogenase catalyzes the NAD(+)-dependent, reversible, oxidative deamination of L-phenylalanine to form ammonia, phenyl pyruvate, and NADH. The enzyme has been purified to homogeneity from Rhodococcus sp. M4, and a partial amino acid sequence was obtained. A cosmid library of Rhodococcus sp. M4 genomic DNA was prepared and used to isolate a 2.5-kilobase PstI fragment that contained the pdh gene. The open reading frame of 1068 nucleotides encodes a polypeptide of 356 amino acids, portions of which match the amino acid sequence determined for the purified enzyme. Expression of the Rhodococcus pdh gene in Escherichia coli, which does not contain a phenylalanine dehydrogenase activity, yields a soluble enzyme exhibiting phenylalanine dehydrogenase activity. Both the enzyme purified from Rhodococcus and the enzyme expressed in E. coli are post-translationally modified by removal of the amino-terminal methionine. The overall amino acid sequence is homologous to previously reported sequences of leucine and phenylalanine dehydrogenases as well as several glutamate dehydrogenases. The amino-terminal portion of the enzyme contains residues involved in L-amino acid binding and catalysis, while the carboxyl-terminal portion contains the presumptive dinucleotide-binding domain. A detailed sequence comparison of Rhodococcus phenylalanine dehydrogenase with leucine, phenylalanine, and glutamate dehydrogenases suggests residues involved in general amino acid binding and others that provide for amino acid discrimination.

Amino Acid Oxidoreductases↗