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D C Cameron

Publications and source records attributed to D C Cameron.

At least 19 recordsLinked to original sources
PubMed1999

Metabolic engineering of a 1,2-propanediol pathway in Escherichia coli.

1,2-Propanediol (1,2-PD) is a major commodity chemical that is currently derived from propylene, a nonrenewable resource. A goal of our research is to develop fermentation routes to 1,2-PD from renewable resources. Here we report the production of enantiomerically pure R-1,2-PD from glucose in Escherichia coli expressing NADH-linked glycerol dehydrogenase genes (E. coli gldA or Klebsiella pneumoniae dhaD). We also show that E. coli overexpressing the E. coli methylglyoxal synthase gene (mgs) produced 1,2-PD. The expression of either glycerol dehydrogenase or methylglyoxal synthase resulted in the anaerobic production of approximately 0.25 g of 1,2-PD per liter. R-1,2-PD production was further improved to 0.7 g of 1,2-PD per liter when methylglyoxal synthase and glycerol dehydrogenase (gldA) were coexpressed. In vitro studies indicated that the route to R-1,2-PD involved the reduction of methylglyoxal to R-lactaldehyde by the recombinant glycerol dehydrogenase and the reduction of R-lactaldehyde to R-1, 2-PD by a native E. coli activity. We expect that R-1,2-PD production can be significantly improved through further metabolic and bioprocess engineering.

Anaerobiosis↗
PubMed1998

Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid.

We developed a stoichiometric model of Bacillus subtilis metabolism for quantitative analysis of theoretical growth and biochemicals production capacity. This work concentrated on biochemicals that are derived from the purine biosynthesis pathway; inosine, guanosine, riboflavin, and folic acid. These are examples of commercially relevant biochemicals for which Bacillus species are commonly used production hosts. Two previously unrecognized, but highly desirable properties of good producers of purine pathway-related biochemicals have been identified for optimally engineered product biosynthesis; high capacity for reoxidation of NADPH and high bioenergetic efficiency. Reoxidation of NADPH, through the transhydrogenase or otherwise, appears to be particularly important for growth on glucose, as deduced from the corresponding optimal carbon flux distribution. The importance of cellular energetics on optimal performance was quantitatively assessed by including a bioenergetic efficiency parameter as an unrestricted, ATP dissipating flux in the simulations. An estimate for the bioenergetic efficiency was generated by fitting the model to experimentally determined growth yields. The results show that the maximum theoretical yields of all products studied are limited by pathway stoichiometry at high bioenergetic efficiencies. Simulations with the estimated bioenergetic efficiency of B. subtilis, growing under glucose-limiting conditions, indicate that the yield of these biochemicals is primarily limited by energy and thus is very sensitive to the process conditions. The maximum yields that can reasonably be expected with B. subtilis on glucose were estimated to be 0.343, 0.160, and 0.161 (mol product/mol glucose) for purine nucleosides, riboflavin, and folic acid, respectively. Potential strategies for improving these maximum yields are discussed.

Bacillus↗
PubMed1998

Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells.

Methylglyoxal is an alpha-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes in vivo, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present in vivo is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 microM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 microM, 0.02% of cellular amino acid residues could be modified. As few as one or two "hits" with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5-10% of cellular proteins may be modified to physiologically significant levels.

Animals↗
PubMed1998

Purification and characterization of a Bacillus licheniformis phosphatase specific for D-alpha-glycerophosphate.

Bacillus licheniformis ("Ford's type") was found to contain a novel enzyme, D-alpha-glycerophosphatase. The enzyme is highly specific for D-alpha-glycerophosphate, effecting little or no hydrolysis of L-alpha- or beta-glycerophosphate or other similar compounds. All other known alpha-glycerophosphatases preferentially hydrolyze the L isomer. The products of the D-alpha-glycerophosphatase reaction were identified as glycerol and inorganic phosphate. The enzyme is a monomer with an apparent molecular mass of approximately 25 kDa. As with most phosphatases, it requires divalent magnesium for activity, but unlike the nonspecific acid and alkaline phosphatases, its optimum pH is around neutral. Its K(m) for D-alpha-glycerophosphate in the presence of 1 mM Mg2+ was found to be 4.3 mM. D-alpha-glycerophosphatase was produced in B. licheniformis fermentations whether or not high levels of phosphate were present; the same was true of glycerol formation. D-alpha-glycerophosphatase is not strongly inhibited by inorganic phosphate and would therefore be capable of catalyzing the formation of glycerol in the presence of high levels of phosphate. The D-alpha-glycerophosphatase of B. licheniformis is similar in characteristics to L-alpha-glycerophosphatases known to synthesize glycerol in vivo, suggesting that D-alpha-glycerophosphatase may be the final enzyme in the fermentative glycerol formation pathway of B. licheniformis.

Amino Acid Sequence↗
PubMed1998

Construction and characterization of a 1,3-propanediol operon.

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose. Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B12, which must be provided in E. coli fermentations. However, the amount of coenzyme B12 needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.

Acetates↗
PubMed1996

Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture.

Methylglyoxal is present at low levels in most cells as a by-product of glycolysis and a product of lipid and amino acid catabolism. The most widely accepted method for measurement of methylglyoxal involves the derivatization of methylglyoxal with 1,2-diaminobenzene derivatives, such as o-phenylenediamine, followed by quantification of the resulting quinoxaline with high-performance liquid chromatography (HPLC). Here we describe the modification of this procedure for the measurement of free intra- and extracellular methylglyoxal in animal cells grown in culture. Cell harvest and sample volume measurement techniques were developed. Solid-phase extraction prior to methylglyoxal derivatization reduced interferences unique to cell culture, such as the phenol red indicator dye used in most cell culture media, and extended the useful life of the HPLC column. In addition, this extraction step significantly lessened the interference represented by oxidative degradation of nucleic acids to methylglyoxal by perchloric acid under assay conditions. The concentration of free intracellular methylglyoxal in Chinese hamster ovary (CHO) cells grown in culture ranged from 0.7 +/- 0.3 microM (mean +/- 2 standard deviations; n = 4) to 1.2 +/- 0.3 microM (mean +/- 2 standard deviations; n = 7). The concentration of free extracellular methylglyoxal in the growth medium was 0.07 +/- 0.02 microM (mean +/- 2 standard deviations; n = 4), severalfold less than that found inside the cell. A possible explanation for the difference between measured free intracellular and extracellular methylglyoxal levels is that the assay for free intracellular methylglyoxal also measures some reversibly bound methylglyoxal.

Animals↗
PubMed1996

Detection of methylglyoxal as a degradation product of DNA and nucleic acid components treated with strong acid.

The 1,2-diaminobenzene derivation assay for methylglyoxal in biological systems involves the use of perchloric acid, both as a deproteinizing agent and to prevent the spontaneous formation of methylglyoxal from glycolytic pathway intermediates. However, while using a modification of the standard literature assay to measure methylglyoxal in Chinese hamster ovary cells, we found that oxidation of nucleic acids and related compounds by perchloric or trichloroacetic acid results in the formation of methylglyoxal. Compounds containing 2-deoxyribose gave higher levels of methylglyoxal than those containing ribose; purine nucleotides and deoxynucleotides gave more methylglyoxal than did the pyrimidines. Nucleic acids were the most susceptible to degradation, with 12-fold more methylglyoxal being formed from DNA than RNA. Oxidation of nucleic acids increased with higher temperatures and with decreasing nucleic acid fragment size. Another product of nucleic acid oxidation was 2,3-butanedione, the 1,2-diaminobenzene derivative of which is sometimes used as an internal standard during methylglyoxal measurement. Unless accounted for during the assay procedure, the generation of methylglyoxal and 2,3-butanedione due to the oxidation of nucleic acids may lead to substantial errors in the determination of methylglyoxal concentrations in biological systems.

Animals↗
PubMed1996

A contrast technique to improve sampling in cavitating lung lesions.

Twelve patients with cavitating lung lesions of uncertain aetiology, who had percutaneous aspiration combined with the instillation of radiographic contrast media, are presented. A diagnosis was made in 11 of the 12 patients (92%). A positive culture was obtained in all patients already receiving treatment for lung abscesses. Cultures of lavage fluid were positive in 89% of infected cavities. The instillation of radiographic contrast media into the lung cavity assists biopsy of the cavity wall, can be used to lavage the cavity, and may facilitate expectoration of sputum.

Adult↗
PubMed1996

Intramural aortic dissection.

We present 10 cases of intramural aortic dissection. The cases are all characterized by the presence of intramural haematoma without the presence of a patent false lumen. The radiological features and possible aetiologies are discussed. The key radiological finding is the presence of a hyperdense rim in the aortic wall on a non-contrast-enhanced computed tomography (CT) scan. In one case, a delayed diagnosis was made using magnetic resonance imaging (MRI). In a further case, the delayed development of a large aortic ulcer was demonstrated. Intramural aortic dissection has only recently been described in the radiological literature. The aetiology of this condition remains controversial. The imaging findings may be subtle and the diagnosis is still frequently being overlooked. We believe CT to be the primary diagnostic test for this condition, and its advantages over MRI and transoesophageal echocardiography (TOE) are discussed.

Aged↗
PubMed1995

Computed tomography demonstration of mosaic oligaemia in pulmonary embolism.

Previous reports on computed tomography findings in pulmonary embolism have concentrated on describing large peripheral wedged-shaped opacities thought to represent infarcts, and filling defects in the central vessels presumed to represent emboli. Two cases are presented of mosaic oligaemia with areas of apparent increase in density initially though to represent infiltrates.

Adult↗
PubMed1995

The Hutson loop and prosthesis: clinical uses in hepato-biliary intervention.

A number of method of making a Roux-en-Y hepaticojejunostomy have been advocated to help the radiologist gain access to the biliary system. We have found markers and rings unsatisfactory and therefore designed and tested our own prosthesis. Reliable rapid access is easily achieved using this device with a much reduced radiation dose to both patient and radiologist. The prosthesis has been used for the past 3 years, and the last 23 patients are reviewed. Seven patients have required repeated interventions via the loop and we present the findings and our results.

Adult↗
PubMed1995

What's new in pulmonary radiology?

Research is still continuing to produce the ideal radiographic emulsion and the film plate will be in use for some time yet. Digital imaging will develop, but there are problems to overcome. Computerised tomography (CT) continues to improve despite early predictions to the contrary, and magnetic resonance imaging (MRI) will make an impact, especially with MRI angiography and fast-gated scanning techniques.

Humans↗
PubMed1993

The juxtaphrenic peak (Katten's sign) is produced by rotation of an inferior accessory fissure.

Tenting of the diaphragm (Katten's sign) is a common finding in upper lobe collapse or where there is loss of volume. Twenty-nine patients have been studied, 12 of these prospectively following upper lobectomy. Division of the inferior pulmonary ligament was carried out in these 12 patients. The computed tomography findings confirm that this sign is due to an accessory oblique fissure.

Diaphragm↗
PubMed1992

Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing Klebsiella pneumoniae dha regulon genes.

1,3-Propanediol (1,3-PD) is an intermediate in chemical and polymer synthesis. We have previously expressed the genes of a biochemical pathway responsible for 1,3-PD production, the dha regulon of Klebsiella pneumoniae, in Escherichia coli. An analysis of the maximum theoretical yield of 1,3-PD from glycerol indicates that the yield can be improved by the cofermentation of sugars, provided that kinetic constraints are overcome. The yield of 1,3-PD from glycerol was improved from 0.46 mol/mol with glycerol alone to 0.63 mol/mol with glucose cofermentation and 0.55 mol/mol with xylose cofermentation. The engineered E. coli also provides a model system for the study of metabolic pathway engineering.

Acetates↗