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Biomedical subjects

R A Dixon

Publications and source records attributed to R A Dixon.

At least 19 recordsLinked to original sources
PubMed2006

Engineering isoflavone metabolism with an artificial bifunctional enzyme.

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.

Artificial Gene Fusion↗
PubMed2004

Permeabilizing effects of sub-inhibitory concentrations of microcystin on the growth of Escherichia coli.

Microcystin, a cyanotoxin produced by Microcystis aeruginosa, lacks potent antibacterial activity. When tested in combination, in vitro, inhibitory values for a range of hydrophobic antibiotics were significantly reduced in the presence of at least 1/20 x the minimum inhibitory concentration of microcystin. The degree of inhibition was equivalent to that of a well-characterised permeabilizing agent, polymyxin B nonapeptide. The permeabilizing ability of sub-inhibitory concentrations of microcystin to affect the envelope of Escherichia coli was demonstrated by a rapid and sustained reduction in absorbance values of lysozyme-treated cells and by enhanced uptake of crystal violet in microcystin-treated cultures. Direct effects of appropriate concentrations of microcystin on the integrity of bacterial outer and inner membranes were measured by release of specific enzyme markers. Although the exact mechanism for permeabilizing E. coli with microcystin has not been elucidated, the effects were consistent with permeability changes to the enterobacterial outer membrane caused by polymyxin B nonapeptide.

Anti-Bacterial Agents↗
PubMed2003

A study to detect Helicobacter pylori in fresh and archival specimens from patients with interstitial cystitis, using amplification methods.

OBJECTIVES: To detect Helicobacter pylori DNA in fresh and paraffin-embedded bladder biopsy specimens, and thus determine any possible role in interstitial cystitis (IC). MATERIALS AND METHODS: Thirty-three bladder biopsy samples were examined from patients with IC (29 paraffin-embedded and four freshly frozen) diagnosed according to National Institute of Diabetes, Digestive and Kidney Disease criteria. The positive control was a gastric biopsy sample from a patient with a known gastric ulcer caused by H. pylori infection. RESULTS: The anticipated polymerase chain reaction product size of 109 base pairs was obtained with the positive control, whereas none of the other biopsy samples (paraffin embedded or fresh) showed positive amplification specific for H. pylori. CONCLUSIONS: As there was no H. pylori DNA in any of the samples from patients with IC, it is an unlikely candidate in the pathogenesis of IC.

Biopsy↗
PubMed2003

Implementing a new health management information system in Uganda.

This paper reports on research investigating the health management information system (HMIS) implementation process in Uganda, utilizing the diffusion of innovation and dynamic equilibrium organizational change models. Neither perspective guided the HMIS development process. Instead, technological issues, rather than wider organizational issues, dominated the planned change. The need to consider the organizational context when changing information systems arises because the process is more complex than some practitioners have realized, when attempting to understand the causes of information management problems and developing HMIS in low-income countries. In particular, information system developers had not acknowledged that they were promoting an informational approach to management when they promoted a change from a centralized reporting system to a HMIS supporting use of information at the level of collection. Strategies to facilitate this approach were not advocated. Organizational theory can contribute to the diffusion of innovation framework. It has yielded an integration of Rogers's diffusion of innovation framework and Leavitt's concept of organizational forces in equilibrium. The diffusion framework describes the process, but the organizational model has given the context and reason for aspects of the process. The diffusion model does not predict what needs to change within the organization when a particular innovation is introduced, or how much. The addition of the organizational model has helped. These frameworks can facilitate the introduction of future information management innovations and allow practitioners to perceive their introduction as a staged process needing to be managed. Implications for practice are identified.

Diffusion of Innovation↗
PubMed2002

Rejection of an innovation: health information management training materials in east Africa.

A shift towards decentralization in many low-income countries has meant more skills are demanded of primary health care managers, including data and information handling at all levels of the health care system. Ministries of Health are changing their central reporting health information systems to health management information systems with emphasis on managers utilizing information at the point of collection. This paper reports on a research study to investigate the introduction of new information management strategies intended to promote an informational approach to management at the operational health service level in low-income countries. It aims to understand the process taking place when externally developed training materials (PHC MAP), which are intended to strengthen health management information systems, are introduced to potential users in an east African country. A case study has been undertaken and this research has demonstrated that the dynamic equilibrium approach to organizational change is applicable to the introduction of new information management strategies and management approaches in low-income countries. Although PHC MAP developers envisaged a technical innovation needing implementation, potential users saw the situation as one of organizational change. Contributions to theory have been made and many implications for introducing new information systems or the informational approach to management are identified. This theoretical framework could also facilitate the introduction of future information management innovations and would allow practitioners to perceive the introduction of information management innovations as one of organizational change that needs to be managed. Consequently, issues that may facilitate or inhibit adoption could be identified in advance.

Administrative Personnel↗
PubMed2002

A selectin inhibitor decreases neutrophil infiltration during acute Mannheimia haemolytica pneumonia.

The degree to which the selectin inhibitor TBC1269 reduces neutrophil infiltration in specific microscopic locations of the lung during acute pneumonia of neonates was determined. Neonatal calves were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 2 and 6 hours postinoculation (PI). One 6-hour group inoculated with M. haemolytica received TBC1269 intravenously before and after inoculation with M. haemolytica. Infiltrates of neutrophils were significantly higher in the alveolar lumen and septae but lower in the bronchial lumen and epithelium at 6 hours PI than at 2 hours PI. Significantly fewer neutrophils (P < 0.05) were present in the alveolar lumen and septae, and the bronchiolar lumen and lamina propria in the lungs of TBC1269-treated calves compared with untreated calves at 6 hours PI. TBC1269 did not alter the infiltration into bronchi and blood vessels or the expression of the selectin-independent adhesion molecule, ICAM-1. This work suggests that during acute pneumonia of neonates 1) neutrophil infiltrates progressively increase in the alveolar lumens and septae but decrease in the bronchial lumen and epithelium with time, 2) TBC1269 reduces neutrophil infiltration into specific regions of alveoli and bronchioles rather than uniformly throughout the lung, and 3) selectin inhibition does not affect the location and intensity of ICAM-1 expression.

Animals↗
PubMed2001

Natural products and plant disease resistance.

Plants elaborate a vast array of natural products, many of which have evolved to confer selective advantage against microbial attack. Recent advances in molecular technology, aided by the enormous power of large-scale genomics initiatives, are leading to a more complete understanding of the enzymatic machinery that underlies the often complex pathways of plant natural product biosynthesis. Meanwhile, genetic and reverse genetic approaches are providing evidence for the importance of natural products in host defence. Metabolic engineering of natural product pathways is now a feasible strategy for enhancement of plant disease resistance.

Biological Products↗
PubMed2001

Acyl substitution at the ortho position of anilides enhances oral bioavailability of thiophene sulfonamides: TBC3214, an ETA selective endothelin antagonist.

Sitaxsentan (3, TBC11251) (Wu et al. J. Med. Chem. 1997, 40, 1690) is an orally active ET(A) selective endothelin antagonist that attenuates pulmonary vascular hypertension and cardiac hypertrophy in rats (Tilton et al. Pulm. Pharmacol. Ther. 2000, 13, 87). It has demonstrated efficacy in a phase II clinical trial for congestive heart failure (Givertz et al. Circulation 2000, 101, 2922). During the discovery of 3, we observed several structure-oral bioavailability relationships. To investigate whether there is any generality in these trends, we synthesized some similar pairs of compounds in the latest series (Wu et al. J. Med. Chem. 1999, 42, 4485) and evaluated their oral properties. In both series, an acyl group at the 2-position of the anilide of these thiophene sulfonamides improved oral bioavailability. As a result of this exercise, TBC3214 (17) was identified as a sitaxsentan follow-on candidate. It is very potent (IC(50) for ET(A) = 40 pM) and highly selective for ET(A) vs ET(B) receptors (400 000-fold), with a half-life of >4 h and oral bioavailability of 25% in rats, 42% in cats, and 70% in dogs.

Administration, Oral↗
PubMed2001

The Medicago Genome Initiative: a model legume database.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

Computational Biology↗
PubMed2001

The biosynthesis of monolignols: a "metabolic grid", or independent pathways to guaiacyl and syringyl units?

Lignin is a complex polymer formed by the oxidative polymerization of hydroxycinnamyl alcohol derivatives termed monolignols. The major monolignols in dicotyledonous angiosperm lignin are monomethylated guaiacyl (G) units derived from coniferyl alcohol, and dimethylated syringyl (S) units derived from sinapyl alcohol. The biochemical pathways leading to the formation of monolignols feature successive hydroxylation and O-methylation of the aromatic ring and conversion of the side chain carboxyl to an alcohol function. The current view of the monolignol biosynthetic pathway envisages a metabolic grid leading to G and S units, through which the successive hydroxylation and O-methylation reactions may occur at different levels of side chain oxidation. The present article assesses biochemical and genetic evidence for and against such a model, including recent data on the methylation reactions of monolignol biosynthesis in alfalfa. We draw attention to portions of the currently accepted monolignol pathway that may require revision, and suggest an alternative model in which metabolic channeling allows for independent pathways to G and S lignin.

Fabaceae↗
PubMed2001

Chemical syntheses of caffeoyl and 5-OH coniferyl aldehydes and alcohols and determination of lignin O-methyltransferase activities in dicot and monocot species.

To investigate the substrate preferences of O-methyltransferases in the monolignol biosynthetic pathways, caffeoyl and 5-hydroxy coniferyl aldehydes were synthesized by a new procedure involving a Wittig reaction with the corresponding hydroxybenzaldehydes. The same procedure can also be used to synthesize caffeoyl and 5-hydroxyconiferyl alcohols. Relative O-methyltransferase activities against these substrates were determined using crude extracts and recombinant caffeic acid O-methyltransferase from alfalfa (Medicago sativa), and crude extracts from the model legume Medicago truncatula, tobacco, wheat and tall fescue. Extracts from all these species catalyzed methylation of the various monolignol aldehydes and alcohols more effectively than the corresponding hydroxycinnamic acids.

Alcohols↗
PubMed2001

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases.

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.

Animals↗
PubMed2001

Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases.

Chalcone O-methyltransferase (ChOMT) and isoflavone O-methyltransferase (IOMT) are S-adenosyl-l-methionine (SAM) dependent plant natural product methyltransferases involved in secondary metabolism in Medicago sativa (alfalfa). Here we report the crystal structure of ChOMT in complex with the product S-adenosyl-l-homocysteine and the substrate isoliquiritigenin (4,2',4'-trihydroxychalcone) refined to 1.8 A as well as the crystal structure of IOMT in complex with the products S-adenosyl-l-homocysteine and isoformononetin (4'-hydroxy-7-methoxyisoflavone) refined to 1.4 A. These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection. As such, this work provides a structural basis for understanding the substrate specificity of the diverse family of plant OMTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.

Amino Acid Sequence↗
PubMed2001

Structure and mechanism of chalcone synthase-like polyketide synthases.

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure-function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity.

Acyltransferases↗
PubMed2001

Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols.

Measurement of relative O-methyltransferase activities against all potential substrates in the monolignol pathway in developing alfalfa stem extracts revealed activities in the order: caffeoyl CoA > caffeoyl alcohol > 5-hydroxyferulic acid > caffeoyl aldehyde > 5-hydroxyconiferyl alcohol > 5-hydroxyferuloyl CoA > 5-hydroxyconiferaldehyde > caffeic acid. Maxima for all activities occurred in the seventh internode. In stem extracts from transgenic alfalfa with antisense downregulated caffeoyl CoA O-methyltransferase (CCoAOMT), activities with all substrates except for the two coenzyme A esters were unaffected. In contrast, downregulation of caffeic acid O-methyltransferase (COMT) reduced activities against the non-esterifed substrates in the order: 5-hydroxyconiferyl alcohol > 5-hydroxyferulic acid and caffeoyl alcohol > caffeoyl aldehyde > caffeic acid > 5-hydroxyconiferaldehyde. Recombinant COMT expressed in Escherichia coli exhibited the highest V(max)/K(m) values with 5-hydroxyconiferaldehyde and caffeoyl aldehyde, and the lowest with caffeic acid. These results indicate that COMT is unlikely to methylate caffeic acid during lignin biosynthesis in vivo, and provide enzymatic evidence for an alternative pathway to monolignols involving methylation of caffeoyl aldehyde and/or caffeoyl alcohol by COMT. The concept of independent pathways to guaiacyl and syringyl monolignols is discussed.

Catechol O-Methyltransferase↗
PubMed2001

A study to detect Gardnerella vaginalis DNA in interstitial cystitis.

OBJECTIVE: To investigate the possible role of Gardnerella vaginalis in interstitial cystitis (IC), using molecular methods to avoid difficulties with the culture and recovery of viable organisms, and the problems associated with the recovery of low numbers of culturable organisms. MATERIALS AND METHODS: Thirty-three bladder biopsy samples (29 paraffin-embedded and four freshly frozen) from patients with IC, diagnosed according to National Institute of Diabetes, Digestive and Kidney Diseases criteria, were assessed. Biopsy samples were used as urine samples may be contaminated by normal vaginal flora. A positive control comprised a 'normal' biopsy sample from a patient with a previous bladder tumour, seeded with G. vaginalis NCTC 10915. Microbial DNA was extracted from all paraffin-embedded and fresh specimens, and subjected to in vitro amplification by polymerase chain reaction (PCR) with G. vaginalis-specific primers. RESULTS: The anticipated PCR product of 333 base pairs was obtained with the positive control, whereas none of the other biopsy samples showed positive amplification specific for G. vaginalis. CONCLUSION: As there was no G. vaginalis DNA in any of the samples from patients with IC, it is an unlikely candidate in the pathogenesis of IC.

Cystitis, Interstitial↗