Glucose-6-phosphate dehydrogenase contributes to the regulation of glucose uptake in skeletal muscle

The development of skeletal muscle insulin resistance is an early physiological defect, yet the intracellular mechanisms accounting for this metabolic defect remained unresolved. Here, we have examined the role of glucose-6-phosphate dehydrogenase (G6PDH) activity in the pathogenesis of insulin resistance in skeletal muscle. Methods Multiple mouse disease states exhibiting insulin resistance and glucose intolerance, as well as obese humans defined as insulin-sensitive, insulin-resistant, or pre-diabetic, were examined. Results We identified increased glucose-6-phosphate dehydrogenase (G6PDH) activity as a common intracellular adaptation that occurs in parallel with the induction of insulin resistance in skeletal muscle and is present across animal and human disease states with an underlying pathology of insulin resistance and glucose intolerance. We observed an inverse association between G6PDH activity and nitric oxide synthase (NOS) activity and show that increasing NOS activity via the skeletal muscle specific neuronal (n)NOS&mu; partially suppresses G6PDH activity in skeletal muscle cells. Furthermore, attenuation of G6PDH activity in skeletal muscle cells via (a) increased nNOS&mu;/NOS activity, (b) pharmacological G6PDH inhibition, or (c) genetic G6PDH inhibition increases insulin-independent glucose uptake. Conclusions We have identified a novel, previously unrecognized role for G6PDH in the regulation of skeletal muscle glucose metabolism. <br /

Improving the iMM904 S. cerevisiae metabolic model using essentiality and synthetic lethality data

<p>Abstract</p> <p>Background</p> <p><it>Saccharomyces cerevisiae </it>is the first eukaryotic organism for which a multi-compartment genome-scale metabolic model was constructed. Since then a sequence of improved metabolic reconstructions for yeast has been introduced. These metabolic models have been extensively used to elucidate the organizational principles of yeast metabolism and drive yeast strain engineering strategies for targeted overproductions. They have also served as a starting point and a benchmark for the reconstruction of genome-scale metabolic models for other eukaryotic organisms. In spite of the successive improvements in the details of the described metabolic processes, even the recent yeast model (i.e., <it>i</it>MM904) remains significantly less predictive than the latest <it>E. coli </it>model (i.e., <it>i</it>AF1260). This is manifested by its significantly lower specificity in predicting the outcome of grow/no grow experiments in comparison to the <it>E. coli </it>model.</p> <p>Results</p> <p>In this paper we make use of the automated GrowMatch procedure for restoring consistency with single gene deletion experiments in yeast and extend the procedure to make use of synthetic lethality data using the genome-scale model <it>i</it>MM904 as a basis. We identified and vetted using literature sources 120 distinct model modifications including various regulatory constraints for minimal and YP media. The incorporation of the suggested modifications led to a substantial increase in the fraction of correctly predicted lethal knockouts (i.e., specificity) from 38.84% (87 out of 224) to 53.57% (120 out of 224) for the minimal medium and from 24.73% (45 out of 182) to 40.11% (73 out of 182) for the YP medium. Synthetic lethality predictions improved from 12.03% (16 out of 133) to 23.31% (31 out of 133) for the minimal medium and from 6.96% (8 out of 115) to 13.04% (15 out of 115) for the YP medium.</p> <p>Conclusions</p> <p>Overall, this study provides a roadmap for the computationally driven correction of multi-compartment genome-scale metabolic models and demonstrates the value of synthetic lethals as curation agents.</p

Cost-Effectiveness Analysis of Diagnostic Options for Pneumocystis Pneumonia (PCP)

Diagnosis of Pneumocystis jirovecii pneumonia (PCP) is challenging, particularly in developing countries. Highly sensitive diagnostic methods are costly, while less expensive methods often lack sensitivity or specificity. Cost-effectiveness comparisons of the various diagnostic options have not been presented.We compared cost-effectiveness, as measured by cost per life-years gained and proportion of patients successfully diagnosed and treated, of 33 PCP diagnostic options, involving combinations of specimen collection methods [oral washes, induced and expectorated sputum, and bronchoalveolar lavage (BAL)] and laboratory diagnostic procedures [various staining procedures or polymerase chain reactions (PCR)], or clinical diagnosis with chest x-ray alone. Our analyses were conducted from the perspective of the government payer among ambulatory, HIV-infected patients with symptoms of pneumonia presenting to HIV clinics and hospitals in South Africa. Costing data were obtained from the National Institutes of Communicable Diseases in South Africa. At 50% disease prevalence, diagnostic procedures involving expectorated sputum with any PCR method, or induced sputum with nested or real-time PCR, were all highly cost-effective, successfully treating 77-90% of patients at $26-51 per life-year gained. Procedures using BAL specimens were significantly more expensive without added benefit, successfully treating 68-90$189-232 per life-year gained. A relatively cost-effective diagnostic procedure that did not require PCR was Toluidine Blue O staining of induced sputum ($25 per life-year gained, successfully treating 68$109 per life-year gained) compared with several molecular diagnostic options.For diagnosis of PCP, use of PCR technologies, when combined with less-invasive patient specimens such as expectorated or induced sputum, represent more cost-effective options than any diagnostic procedure using BAL, or chest x-ray alone

A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway

Analysis in Escherichia coli of Plasmodium falciparum dihydropteroate synthase (DHPS) alleles implicated in resistance to sulfadoxine

Mutations in Plasmodium falciparum dihydropteroate synthase have been linked to resistance to the antimalarial drug, sulfadoxine, which competes with the dihydropteroate synthase substrate, p-aminobenzoate. In an effort to evaluate the role of these mutations in a simple model system, we have expressed six relevant alleles of the P. falciparum dihydropteroate synthase gene in Escherichia coli. When each construct was produced in a dihydropteroate synthase disrupted E. coli strain that required thymidine, the thymidine requirement was lost, indicating heterologous complementation had occurred. In the presence of sulfadoxine, the growth of the strain with the wild-type dihydropteroate synthase allele was inhibited while those containing each of the five mutant alleles grew, indicating that these mutations can confer sulfadoxine resistance in E. coli. When tested against twelve additional &#039;sulfa&#039; drugs a variety of responses were obtained. All strains were resistant to sulfadiazine, but the wild-type allele conferred sensitivity to all other sulfa drugs. Three alleles conferred resistance to dapsone, a drug that is to be targetted for a new regime of malaria treatment in Africa. All mutant alleles remained sensitive to sulfachloropyridazine and sulfacetamide. These results suggest new drugs that could be tried for effective malaria treatment

Folate biosynthesis - Reappraisal of old and novel targets in the search for new antimicrobials

Folate biosynthesis remains a key target for antimicrobial therapy. Folate is an essential vitamin (vitamin B9) that is required for many one-carbon transfer reactions and is a critical precursor for the biosynthesis of purines, pyrimidines, and amino acids. Unlike higher eukaryotes that scavenge preformed folates, prokaryotic and lower eukaryotic microorganisms are dependent on several enzymes for the de novo biosynthesis of folate. One of these enzymes, dihydropteroate synthase (DHPS), is the target of the first chemically-synthesized antimicrobial agents, the sulfadrugs, which date back to the 1940s. Others are essential enzymes that remain to be explored as drug targets. Resistance to the sulfadrugs rapidly emerges due to the ability of the microbe to alter its susceptibility to the drug by various means. Recently a number of new structures of the enzymes in the pathway has become available. We review the recent literature relating to these targets (the enzymes: GTP cyclohydrolase (GTP-CH); 7,8-dihydroneopterin aldolase (DHNA), 6-hydroxymethyl- 7,8-dihydropterin pyrophosphokinase (HPPK), dihydropteroate synthase (DHPS), dihydrofolate synthase (DHFS)), their mode of action and how current drugs may modulate this on a structural level. Furthermore, these data advance our understanding of the emergence of drug resistance and may aid efforts and play a major role in the design of new, more effective compounds as antimicrobial agents. To this end we also review the recent literature in the development of inhibitors of these enzymes. Future progress in this key area has the potential to benefit the war against devastating organisms such as drug-resistant Staphylococcus aureus and Plasmodium falciparum

The three-dimensional structure of the bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/dihydropteroate synthase of Saccharomyces cerevisiae

In Saccharomyces cerevisiae and other fungi, the enzymes dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) are encoded by a polycistronic gene that is translated into a single polypeptide having all three functions. These enzymatic functions are essential to both prokaryotes and lower eukaryotes, and catalyse sequential reactions in folate biosynthesis. Deletion or disruption of either function leads to cell death. These enzymes are absent from mammals and thus make ideal antimicrobial targets. DHPS is currently the target of antifolate therapy for a number of infectious diseases, and its activity is inhibited by sulfonamides and sulfones. These drugs are typically used as part of a synergistic cocktail with the 2,4-diaminopyrimidines that inhibit dihydrofolate reductase. A gene encoding the S.cerevisiae HPPK and DHPS enzymes has been cloned and expressed in Escherichia coli. A complex of the purified bifunctional polypeptide with a pterin monophosphate substrate analogue has been crystallized, and its structure solved by molecular replacement and refined to 2.3A resolution. The polypeptide consists of two structural domains, each of which closely resembles its respective monofunctional bacterial HPPK and DHPS counterpart. The mode of ligand binding is similar to that observed in the bacterial enzymes. The association between the domains within the polypeptide as well as the quaternary association of the polypeptide via its constituent DHPS domains provide insight into the assembly of the trifunctional enzyme in S.cerevisiae and probably other fungal species