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Biomedical subjects

W Bishai

Publications and source records attributed to W Bishai.

15 recordsLinked to original sources
PubMed2005

Bactericidal activity of the nitroimidazopyran PA-824 in a murine model of tuberculosis.

The nitroimidazopyran PA-824 has potent in vitro activity against Mycobacterium tuberculosis, a narrow spectrum of activity limited primarily to the M. tuberculosis complex, and no demonstrable cross-resistance to a variety of antituberculosis drugs. In a series of experiments, we sequentially characterized the activity of PA-824 in an experimental murine model of tuberculosis. The minimal effective dose was 12.5 mg/kg of body weight/day. The minimal bactericidal dose (MBD) was 100 mg/kg/day. When PA-824 was used as monotherapy at the MBD, it exhibited promising bactericidal activity during the initial intensive phase of therapy that was similar to that of the equipotent dose of isoniazid in humans. In combination with isoniazid, PA-824 prevented the selection of isoniazid-resistant mutants. Perhaps more importantly, PA-824 also demonstrated potent activity during the continuation phase of therapy, during which it targeted bacilli that had persisted through an initial 2-month intensive phase of treatment with rifampin, isoniazid, and pyrazinamide. Together, these data strongly support further evaluation of PA-824 in combination with first- or second-line antituberculosis drugs to determine its potential contribution to the treatment of drug-susceptible or multidrug-resistant tuberculosis, respectively.

Animals↗
PubMed2002

Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

Bacterial Proteins↗
PubMed2000

Is drug resistance affecting treatment outcomes?

Antimicrobial drug resistance is a growing concern for the medical community. Although cases of drug resistance to Streptococcus pneumoniae are on the increase, there has been no change in mortality rates. The fact that the medical literature shows no proven correlation between drug-resistant S pneumoniae and clinical failure suggests that concerns about drug resistance in S pneumoniae may be overstated. Therefore, in treating community-acquired pneumonia, physicians should also weigh other important considerations such as pharmacology, safety, tolerability, and dosing convenience.

Anti-Bacterial Agents↗
PubMed1999

Solid-phase sequence scanning for drug resistance detection in tuberculosis.

DNA chip arrays hold considerable promise for diagnostic sequencing of polymerase chain reaction (PCR) products. To date, however, arrays have been relatively expensive, complex to use and difficult to interpret, preventing their adaptation to the clinical lab. A moderate density array method has been developed that enables efficient, easy-to-interpret and robust solid-phase PCR product sequencing. Here, the results of Mycobacterium tuberculosis rifampin resistance mutation detection by primer-extension-based sequence scanning of the rpo B gene of M. tuberculosis are presented. Rifampin resistant clinical isolates were identified in as little as 1 h post PCR amplification with visual results detection.

Bacterial Proteins↗
PubMed1993

New vectors for direct cloning of PCR products.

We describe the construction of two new vectors for direct cloning of polymerase chain reaction (PCR) products. This was done by inserting a synthetic DNA fragment containing two adjacent XcmI sites between the Asp718 and BamHI sites of the M13mp18 and M13mp19 phages. Cleavage of these M13 derivatives with XcmI will result in a linearized vector with a single thymidine nucleotide at the 3' ends. Thus, these vectors would be very useful for direct cloning of PCR-generated products with high efficiency.

Bacteriophage M13↗
PubMed1988

Cell receptor specific targeted toxins: genetic construction and characterization of an interleukin 2 diphtheria toxin-related fusion protein.

We have genetically replaced the diphtheria toxin receptor binding domain with a DNA insert encoding the T-cell growth factor interleukin-2 (IL-2). The toxin-related IL-2 fusion gene encodes a 70,586 dalton protein, pro-IL-2-toxin. The mature form of IL-2-toxin is exported to the periplasmic space of recombinant Escherichia coli and has a molecular weight of 68,086. IL-2-toxin has been partially purified from periplasmic extracts of recombinant E. coli, and it is shown to contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components. The chimeric toxin is targeted toward IL-2 receptor bearing T-cells in vitro.

Cell Line↗
PubMed1987

Diphtheria toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphtheria toxin-related interleukin-2 fusion protein.

We have genetically replaced the diphtheria toxin receptor binding domain with a synthetic gene encoding interleukin-2 (IL-2) and a translational stop signal. The diphtheria toxin-related T-cell growth factor fusion gene encodes a 70 586-d polypeptide, pro-IL-2-toxin. The mature form of IL-2-toxin has a deduced mol. wt of 68,086 and is shown to be exported to the periplasmic compartment of Escherichia coli (pABI508), and contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components. IL-2-toxin has been purified from periplasmic extracts of recombinant strains of E. coli (pABI508) by immunoaffinity chromatography using immobilized anti-IL-2. The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL-2 receptor are resistant to IL-2-toxin action.

Bacterial Proteins↗
PubMed1987

Genetic assembly and selective toxicity of diphtheria-toxin-related polypeptide hormone fusion proteins.

The reports of Miyanohara et al. (1986) and Murphy et al. (1986) were the first to describe the genetic construction, expression, and receptor-specific selective toxicity of a chimaeric toxin. In the present report, we have extended these earlier observations and have shown that the fusion of a modified gene encoding IL-2 to a truncated diphtheria toxin gene also results in the expression of a biologically active chimaeric IL-2 toxin. In both instances we have used receptor-binding-domain substitution and have genetically coupled those portions of the diphtheria toxin structural gene that encode the ADP-ribosyl transferase activity of fragment A and lipid-associating domains of fragment B to modified genes which encode either the polypeptide hormone alpha-MSH or the T-cell growth factor IL-2. The chimaeric toxins expressed from these gene fusions have been shown to be selectively targeted to those eukaryotic cells that carry specific surface receptors for the ligand compounds of the hybrid. For example, in the case of the IL-2 toxin, it is clear that the selective action of this hybrid protein is based upon both its diphtheria-toxin and IL-2-related components. Following binding to the IL-2R on activated and/or malignant T-cell, IL-2 toxin is internalized by receptor-mediated endocytosis. Upon acidification of the endosome, diphtheria toxin fragment B portions of the chimaeric toxin facilitate the delivery of fragment A to the cytosol where it catalyses the ADP ribosylation of EF-2. The assembly of chimaeric toxins at the level of the gene offers several advantages over chemical linkage. Since chemical linkage of the toxophore and ligand components of the conjugate toxins requires activation of the epsilon-amino moiety of lysine residues with reagents that will allow for subsequent disulphide linkage, the precise site of coupling is generally not known. In addition, there has been considerable concern over the lability of the disulphide bond between the toxophore and ligand components in vivo due to the action of disulphide reductases. The assembly of chimaeric toxins at the level of the gene allows for precise linkage of the toxophore and ligand components. Since the linkage between the toxophore and ligand is a peptide bond, the chimaeric toxin should be stable in vivo. In addition, the genetic construction of chimaeric toxins also allows for further protein engineering through site-directed mutagenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

Genetic construction, expression, and melanoma-selective cytotoxicity of a diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion protein.

The structural gene for diphtheria toxin, tox, has been modified at its Sph I site by the introduction of an oligonucleotide linker encoding a unique Pst I restriction endonuclease site and a synthetic oligonucleotide encoding alpha-melanocyte-stimulating hormone (alpha-MSH). The resulting fusion gene directs the expression of a diphtheria toxin-related alpha-MSH hybrid protein in which the diphtheria toxin receptor-binding domain has been replaced with alpha-MSH sequences. The chimeric toxin has been partially purified from periplasmic extracts of recombinant Escherichia coli K-12 and has been found to be selectively toxic for alpha-MSH receptor-positive human malignant melanoma NEL-M1 cells in vitro.

ADP Ribose Transferases↗