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I Charles

Publications and source records attributed to I Charles.

At least 19 recordsLinked to original sources
PubMed2004

Comparison of ligand-induced conformational changes and domain closure mechanisms, between prokaryotic and eukaryotic dehydroquinate synthases.

Dehydroquinate synthase (DHQS) is a potential target for the development of novel broad-spectrum antimicrobial drugs, active against both prokaryotes and lower eukaryotes. Structures have been reported for Aspergillus nidulans DHQS (AnDHQS) in complexes with a range of ligands. Analysis of these AnDHQS structures showed that a large-scale domain movement occurs during the normal catalytic cycle, with a complex series of structural elements propagating substrate binding-induced conformational changes away from the active site to distal locations. Compared to corresponding fungal enzymes, DHQS from bacterial species are both mono-functional and significantly smaller. We have therefore determined the structure of Staphylococcus aureus DHQS (SaDHQS) in five liganded states, allowing comparison of ligand-induced conformational changes and mechanisms of domain closure between fungal and bacterial enzymes. This comparative analysis shows that substrate binding initiates a large-scale domain closure in both species' DHQS and that the active site stereochemistry, of the catalytically competent closed-form enzyme thus produced, is also highly conserved. However, comparison of AnDHQS and SaDHQS open-form structures, and analysis of the putative dynamic processes by which the transition to the closed-form states are made, shows a far lower degree of similarity, indicating a significant structural divergence. As a result, both the nature of the propagation of conformational change and the mechanical systems involved in this propagation are quite different between the DHQSs from the two species.

Amino Acid Sequence↗
PubMed2003

Neomycin binding to Watson-Hoogsteen (W-H) DNA triplex groove: a model.

Neomycin is the most effective aminoglycoside (groove binder) in stabilizing a DNA triple helix. It stabilizes TAT, as well as mixed base DNA triplexes, better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (in the presence of salt), without any effect on the DNA duplex. (1) Triplex stabilization by neomycin is salt dependent (increased KCl and MgCl(2) concentrations decrease neomycin's effectiveness, at a fixed drug concentration). (2) Triplex stabilization by neomycin is pH dependent (increased pH decreases neomycin's effectiveness, at a fixed drug concentration). (3) CD binding studies indicate approximately 5-7 base triplets/drug apparent binding site, depending upon the structure/sequence of the triplex. (4) Neomycin shows nonintercalative groove binding to the DNA triplex, as evident from viscometric studies. (5) Neomycin shows a preference for stabilization of TAT triplets but can also accommodate CGC(+) triplets. (6) Isothermal titration calorimetry (ITC) studies reveal an association constant of approximately 2 x 10(5) M(-)(1) between neomycin and an intramolecular triplex and a higher K(a) for polydA.2polydT. (7) Binding/modeling studies show a marked preference for neomycin binding to the larger W-H groove. Ring I/II amino groups and ring IV amines are proposed to be involved in the recognition process. (8) The novel selectivity of neomycin is suggested to be a function of its charge and shape complementarity to the triplex W-H groove, making neomycin the first molecule that selectively recognizes a triplex groove over a duplex groove.

Anti-Bacterial Agents↗
PubMed2002

Synthesis of aminoglycoside-DNA conjugates.

Development of aminoglycoside-nucleic acid conjugates is presented. Synthesis of a DNA dimer covalently linked to kanamycin and neomycin isothiocyanates has been carried out. The development of such conjugates will help couple the sequence specificity of nucleic acids to the electrostatic/shape complementarity of aminoglycoside antibiotics in binding nucleic acid targets.

Aminoglycosides↗
PubMed1999

Tissue distribution of the human soluble guanylate cyclases.

Soluble guanylate cyclase (sGC) is an important component of the NO signaling pathway. Human sGC isoforms alpha(1), alpha(2), and beta(1) show differential mRNA tissue distributions. alpha(1) and beta(1) are expressed in most tissues; however, the alpha(2) isoform shows a more restricted expression pattern with high levels in brain, placenta, spleen, and uterus only. Both alpha subunits exist as multiple transcripts whereas beta(1) exists as a single message. This study reports for the first time the tissue distribution of human sGC message and demonstrates that sGC isoforms are nonuniformly expressed which may be useful if the enzyme is to be exploited as a therapeutic target.

Blotting, Northern↗
PubMed1997

Nitric oxide synthase is expressed in human macrophages during foreign body inflammation.

Although nitric oxide (NO) is a well documented effector molecule in rodent macrophages, its significance in human mononuclear phagocytic cells has been controversial. The foreign body inflammatory reaction around loosened joint replacement implants leads to formation of an osteolytic granulomatous pseudo-synovial membrane rich in activated macrophages. We studied 13 specimens of interface membrane tissue collected from revision surgery of aseptically loosened hip and knee prostheses for the presence of inducible NO synthase (iNOS). The presence of iNOS was demonstrated immunohistochemically in 10 of these specimens. Within the tissue this enzyme was confined to macrophages and vascular endothelial cells. iNOS activity was demonstrated biochemically by measuring the calcium-independent generation of citrulline from L-arginine, and the presence of iNOS mRNA was demonstrated using reverse transcriptase polymerase chain reaction. NO synthesis in the interface tissue may be an important factor in the maintenance of the inflammatory and osteolytic processes.

Adult↗
PubMed1996

Identification of the domains of neuronal nitric oxide synthase by limited proteolysis.

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.

Amino Acid Sequence↗
PubMed1996

Role of the Bordetella pertussis P.69/pertactin protein and the P.69/pertactin RGD motif in the adherence to and invasion of mammalian cells.

The role of the Bordetella pertussis P.69/pertactin protein in mammalian cell adhesion and invasion was investigated. Salmonella strains expressing surface-associated P.69/pertactin from a chromosomally located prn gene were significantly more invasive than isogenic parental strains. This effect was most pronounced in the poorly invasive, semi-rough S. typhimurium strain LB5010. Escherichia coli K-12 strain HB101 harbouring the plasmid p41869D, which encodes the full-length prn gene under the control of the tac promoter on the broad-host-range plasmid pMMB66EH, was significantly more adhesive to HEp-2 and Chinese Hamster Ovary (CHO) cells growing in culture than E. coli HB101(pMMB66EH). However, the ability of E. coli to invade mammalian cells was not affected by P.69/pertactin expression. P.69/pertactin-mediated adhesiveness of HB101 to HEp-2 and CHO cells was not influenced by the viability of the bacterial cells. However, adherence was markedly reduced when assays were performed for less than 3 h, at 4 degrees C or in the presence of cycloheximide, suggesting the active participation of the eukaryotic cell in bacterial adhesion. Site-directed mutagenesis was used to mutate Asp to Glu in an Arg-Gly-Asp (RGD-->RGE) sequence present in mature P.69/pertactin and the mutated gene was cloned in the same broad-host-range vector (plasmid p41869E). This mutation had no detectable influence on the ability of P.69/pertactin to mediate adhesion of HB101 to HEp-2 or CHO cells. Plasmids p41869D and p41869E were introduced into the bvg-negative B. pertussis strain BP347. Expression of P.69RGD or P.69RGE did not enhance the adhesiveness of BP347 for epithelial (HEp-2 and CHO) cells.

Animals↗
PubMed1995

Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase.

We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 micrograms/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol.min-1.mg-1, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol.min-1.mg-1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol.min-1.mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis.

Amino Acid Oxidoreductases↗
PubMed1994

Expression of the Bordetella pertussis P.69 pertactin adhesin in Escherichia coli: fate of the carboxy-terminal domain.

The mature pertactin protein (P.69) of Bordetella pertussis can be isolated from the bacterial cell surface as a polypeptide with an apparent molecular mass of 69,000 Da as determined by sodium dodecyl sulphate gel electrophoresis. However the open reading frame of prn, the pertactin gene, encodes a polypeptide with a predicted molecular mass of 93,478 Da, referred to as P.93. Expression of the prn gene in Escherichia coli leads to the synthesis of the full-length P.93 polypeptide, which is rapidly processed to the mature P.69 protein located at the cell surface. The P.93 precursor polypeptide is processed at both termini. A 34 amino acid long signal sequence is removed from the amino-terminus and a polypeptide sequence of about 30,000 Da (P.30) is cleaved from the carboxy-terminus. Deletion of the 3' region of prn, encoding P.30, results in the expression of an intracellular form of P.69. Antiserum which recognizes P.30 was raised using synthetic peptides based on the primary amino acid sequence of the region. This anti-P.30 serum was used in a Western blot analysis of fractionated cells of B. pertussis and E. coli harbouring the intact prn gene. The P.30 polypeptide was readily detected in outer membrane fractions prepared from both of these bacterial species, although it could not be shown to be exposed at the cell surface.

Amino Acid Sequence↗
PubMed1994

The inducible form of nitric oxide synthase (NOS2) isolated from murine macrophages maps near the nude mutation on mouse chromosome 11.

Nitric oxide synthase has been shown to mediate streptozocin-induced diabetes and to act as an antimicrobial agent in murine macrophages. Using a cDNA probe for the inducible form of nitric oxide synthase (Nos2) isolated from murine macrophages we have determined that the gene maps within 1 cM of the nude mutation on mouse Chromosome 11. The position of Nos2 was also mapped relative to the markers 115, Evi2, Cchlbl (previously unmapped), and Gfap. This map location is discussed relative to map locations for disease susceptibility loci involved in mediating cutaneous leishmaniasis (ScII) and autoimmune type-I diabetes (Idd4).

Animals↗
PubMed1993

Repeated induction of nitric oxide synthase and leishmanicidal activity in murine macrophages.

Murine macrophages express high levels of nitric oxide (NO) synthase and produce large amounts of NO when stimulated with interferon-gamma plus lipopolysaccharide in vitro. The expression of NO synthase peaks at 12 h after stimulation and declines rapidly to the background level by 72 h. These macrophages can be repeatedly reactivated to express similar levels of NO synthase. The reactivation is not due to newly divided cells since peritoneal macrophages which do not divide in vitro and J774 cells cultured in the presence of colchicine can also be restimulated to express NO synthase. The reactivation is accompanied by re-expression of NO synthase mRNA, as assessed by polymerase chain reaction analysis. Furthermore, the reactivated macrophages are fully capable of killing the intracellular protozoan parasite Leishmania major.

Amino Acid Oxidoreductases↗
PubMed1992

Construction of a genetically defined Salmonella typhi Ty2 aroA, aroC mutant for the engineering of a candidate oral typhoid-tetanus vaccine.

The construction of a Salmonella typhi Ty2 strain harbouring defined deletions in both the aroA and aroC genes is described. These deletions have been fully defined at the molecular level by DNA sequencing and have been introduced in such a way that no foreign DNA remains in the S. typhi genome. This strain is attenuated in mice when given by the intraperitoneal route suspended in hog gastric mucin and is attenuated to a similar level to strains harbouring deletions in aroA or aroC alone indicating that both lesions are capable of attenuating independently. We have used this defined S. typhi aroA aroC strain to express stably a non-toxic 50 kDa fragment of tetanus toxin (fragment C) from a gene incorporated into the chromosome. This strain has the advantage of harbouring no antibiotic-resistance markers and we consider it to be a candidate bivalent oral typhoid-tetanus vaccine.

Administration, Oral↗
PubMed1992

Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis.

The P.69 Bordetella pertussis protective antigen was expressed by use of the trc promoter from the chromosome of a Salmonella typhimurium aro vaccine strain, BRD509, by integrating the prn gene, encoding the 93-kDa precursor of this protein, into the aroC locus. P.69 was detected on the cell surface of the S. typhimurium strain (BRD640) by agglutination and immunoelectron microscopy. BALB/c mice immunized orally or intravenously with BRD640 showed a significant level of protection against an aerosol challenge with virulent B. pertussis, compared with control animals. No anti-P.69 antibodies in the serum or anti-P.69 antibody-secreting cells in the lungs were detected in BRD640-vaccinated animals, although cells isolated from spleens showed a P.69-dependent cell proliferative response. In contrast, low levels of anti-P.69 antibodies in the serum and anti-P.69 antibody-secreting cells in the lungs were detected in immunized mice following a B. pertussis challenge.

Adhesins, Bacterial↗
PubMed1991

The Mycobacterium tuberculosis shikimate pathway genes: evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases.

The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi.

The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated Mr of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69% homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-related with its S. typhi counterpart.

Amino Acid Sequence↗