High throughput and quantitative enzymology in the genomic era.

Accurate predictions from models based on physical principles are the ultimate metric of our biophysical understanding. Although there has been stunning progress toward structure prediction, quantitative prediction of enzyme function has remained challenging. Realizing this goal will require large numbers of quantitative measurements of rate and binding constants and the use of these ground-truth data sets to guide the development and testing of these quantitative models. Ground truth data more closely linked to the underlying physical forces are also desired. Here, we describe technological advances that enable both types of ground truth measurements. These advances allow classic models to be tested, provide novel mechanistic insights, and place us on the path toward a predictive understanding of enzyme structure and function.

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics.

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5'-phosphatase interaction with c-Abl.

AIMS/HYPOTHESIS: It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. METHODS: Human EndoC-ßH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-ßH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-ßH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. RESULTS: Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total ß-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. CONCLUSIONS/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

[Practice for insulin infusion in preterm infants]. / Modalités d'administration de l'insuline par voie intraveineuse chez le nouveau-né prématuré.

UNLABELLED: Transient neonatal hyperglycemia is commonly observed during the first week of life in the preterm infants less than 30 weeks of gestational age. Continuous insulin infusion is an effective treatment in this situation. OBJECTIVE: To ascertain how insulin is administered in different french neonatal intensive care units. MATERIAL AND METHODS: We surveyed 49 neonatal intensive care units with a questionnaire. Response rate was 77.5% (38/49). RESULTS: Thirty four of 38 neonatal intensive care units reported the use of insulin infusions in this setting. Glucose level indicating insulin therapy and the initial insulin doses were quite variable according to the different units (respectively 7-16.5 mmol/l and 0.01-0.1 U/kg/h). A range of minimal insulin concentrations was used (0.01-0.1 U/ml), 57% utilizing concentration between 0.05 and 0.2 U/ml. Flow rates below 0.3 ml/h were used at time by 76%. Albumin was rarely added. Fifty seven percent of the neonatal intensive care units took counter-measures such as preconditioning and flushing the tubing to control insulin loss due to adsorption. The counter-measures were differently applied. Despite these measures, hyperglycemia and insulin resistance were frequently observed (respectively 30% and 47%). The different practices are discussed according to the literature. CONCLUSION: In order to deliver insulin reliably, we suggest an insulin delivery method for the preterm infants.