Biomedical subjects

D E Stafford

Publications and source records attributed to D E Stafford.

5 recordsLinked to original sources

PubMed2001

Metabolic engineering of indene bioconversion in Rhodococcus sp.

We have applied the methodology of metabolic engineering in the investigation of the enzymatic bioreaction network in Rhodococcus sp. that catalyzes the bioconversion of indene to (2R)-indandiol suitable for the synthesis of cis-1-amino-2-indanol, a precursor of the HIV protease inhibitor, Crixivan. A chemostat with a novel indene air delivery system was developed to facilitate the study of steady state physiology of Rhodococcus sp. 124. Prolonged cultivation of this organism in a continuous flow system led to the evolution of a mutant strain, designated KY1, with improved bioconversion properties, in particular a twofold increase in yield of (2R)-indandiol relative to 124. Induction studies with both strains indicated that KY1 lacked a toluene-inducible dioxygenase activity present in 124 and responsible for the formation of undesired byproducts. Flux analysis of indene bioconversion in KY1 performed using steady state metabolite balancing and labeling with [14C]-tracers revealed that at least 94% of the indene is oxidized by a monooxygenase to indan oxide that is subsequently hydrolyzed to trans-(1R,2R)-indandiol and cis-(1S,2R)-indandiol. This analysis identified several targets in KY1 for increasing (2R)-indandiol product yield. Most promising among them is the selective hydrolysis of indan oxide to trans-(1R,2R)-indandiol through expression of an epoxide hydrolase or modification of culture conditions.

Catalysis↗

PubMed2001

Metabolic engineering as an integrating platform for strain development.

Integration of the analytical framework and experimental tools of metabolic engineering with emerging technologies such as DNA microarrays and directed evolution stands to dramatically improve the approaches by which strain improvement and biocatalyst design are pursued in the future. Progress in genomics and applied molecular biology, together with increasing emphasis on renewable resource utilization for chemical production, has advanced metabolic engineering to the forefront of biotechnological interest.

Industrial Microbiology↗

PubMed2001

Application of radiolabeled tracers to biocatalytic flux analysis.

Radiolabeled tracers can provide valuable information about the structure of and flux distributions in biocatalytic reaction networks. This method derives from prior studies of glucose metabolism in mammalian systems and is implemented by pulsing a culture with a radiolabeled metabolite that can be transported into the cells and subsequently measuring the radioactivity of all network metabolites following separation by liquid chromatography. Intracellular fluxes can be directly determined from the transient radioactivity count data by tracking the depletion of the radiolabeled metabolite and/or the accompanying accumulation of any products formed. This technique differs from previous methods in that it is applied within a systems approach to the problem of flux determination. It has been used for the investigation of the indene bioconversion network expressed in Rhodococcus sp. KY1. Flux estimates obtained by radioactive tracers were confirmed by macroscopic metabolite balancing and showed that indene oxidation in steady state chemostat cultures proceeds primarily through a monooxygenase activity forming (1S,2R)-indan oxide, with no dehydrogenation of trans-(1R,2R)-indandiol. These results confirmed the significance of indan oxide formation and identified the hydrolysis of indan oxide as a key step in maximizing the production of (2R)-indandiol, a chiral precursor of the HIV protease inhibitor, Crixivan.

Carbon Radioisotopes↗

PubMed2000

Calcium-stimulated insulin secretion in diffuse and focal forms of congenital hyperinsulinism.

OBJECTIVES: To identify infants with hyperinsulinism caused by defects of the beta-cell adenosine triphosphate-dependent potassium channel complex and to distinguish focal and diffuse forms of hyperinsulinism caused by these mutations. STUDY DESIGN: The acute insulin response to intravenous calcium stimulation (CaAIR) was determined in 9 patients <20 years with diffuse hyperinsulinism caused by defective beta-cell sulfonylurea receptor (SUR1(-/-)), 3 patients with focal congenital hyperinsulinism (6 weeks to 18 months), a 10-year-old with insulinoma, 5 with hyperinsulinism/hyperammonemia syndrome caused by defective glutamate dehydrogenase (6 months to 28 years), 4 SUR1(+/-) heterozygotes with no symptoms, and 9 normal adults. Three infants with congenital focal disease, 1 with diffuse hyperinsulinism, and the child with insulinoma underwent selective pancreatic intra-arterial calcium stimulation with hepatic venous sampling. RESULTS: Children with diffuse SUR1(-/-) disease and infants with congenital focal hyperinsulinism responded to CaAIR, whereas the normal control group, patients with hyperinsulinism/hyperammonemia syndrome, and SUR1(+/-) carriers did not. Selective arterial calcium stimulation of the pancreas with hepatic venous sampling revealed selective, significant step-ups in insulin secretion that correlated anatomically with the location of solitary lesions confirmed surgically in 2 of 3 infants with congenital focal disease and in the child with insulinoma. Selective arterial calcium stimulation of the pancreas with hepatic venous sampling demonstrated markedly elevated baseline insulin levels throughout the pancreas of the infant with diffuse hyperinsulinism. CONCLUSIONS: The intravenous CaAIR is a safe and simple test for identifying infants with diffuse SUR1(-/-) hyperinsulinism or with focal congenital hyperinsulinism. Preoperative selective arterial calcium stimulation of the pancreas with hepatic venous sampling can localize focal lesions causing hyperinsulinism in children. The combination of these calcium stimulation tests may help distinguish focal lesions suitable for cure by local surgical resection.

ATP-Binding Cassette Transporters↗

PubMed1981

Interactions between fatiguing and nonfatiguing isometric contractions.

Isometric contractions of the handgrip muscles were exerted by eight male subjects (age range 19-24 yr) to determine the relationship between fatiguing and nonfatiguing isometric contractions. In a first series of experiments, subjects exerted fatiguing isometric contractions at tensions of 25, 40, or 70% of the maximum strength of the subjects (MVC) following a contraction at a nonfatiguing tension of 5 or 10% MVC for 3, 7, or 20 min. In a second series of experiments, subjects exerted pairs of fatiguing isometric contractions at the same tensions; however, during the 3-, 7-, or 20-min interval between the contractions the subjects either rested or exerted a contraction at 5 or 10% MVC. Nonfatiguing isometric contractions exerted prior to fatiguing contractions had little influence on the endurance for the fatiguing static effort. In contrast, when contractions at nonfatiguing tensions were exerted in the recovery interval following an isometric contraction at a fatiguing tension, the recovery of endurance was dramatically reduced.

Adult↗