Trp180 of endothelial NOS and Trp56 of bacterial saNOS modulate sigma bonding of the axial cysteine to the heme.

The proximal ligand of thiolate-coordinated heme proteins is crucial for the activation of the oxygen molecule and hydroxylation of substrates. In nitric oxide synthases (NOSs), the heme axial cysteine ligand forms a hydrogen bond to the side chain indole nitrogen of a tryptophan residue. Resonance Raman spectroscopy was used to probe W56F and W56Y variants of the NOS of Staphylococcus aureus (saNOS) and the analogous W180 variants of the endothelial NOS oxygenase domain (eNOSox). We show that the variants displayed lower nu(Fe-NO) and nu(Fe-CO) frequencies indicating that these mutations increased the electron density on the axial cysteine in their Fe(III)NO and Fe(II)CO complexes. We also show by UV-visible spectroscopy that the Fe(II)CO complexes of the variants displayed a red-shifted Soret optical transition in addition to the lower nu(Fe-CO) thus establishing that these properties are sensitive indicators of the modulation of the basicity of the axial cysteine. We infer, based on its spectroscopic properties, that ferrous eNOSox W180Y saturated with l-arginine and tetrahydrobiopterin forms a tyrosine-cysteine hydrogen bond when bound to CO. Evidence for such a hydrogen bond was not obtained for the Fe(III)NO protein nor for the analogous saNOS variant. These mutations reveal interesting differences in the response of NOS isotypes to analogous mutations at conserved residues and clearly show that the heme-Fe to cysteine sigma bond is modulated by the Cys-Trp hydrogen bond in NOSs. These studies serve as a basis to gain information on the role played by this hydrogen bond in oxygen activation in this class of enzymes.

Obese mice lacking inducible nitric oxide synthase are sensitized to the metabolic actions of peroxisome proliferator-activated receptor-gamma agonism.

OBJECTIVE: Synthetic ligands for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) improve insulin sensitivity in obesity, but it is still unclear whether inflammatory signals modulate their metabolic actions. In this study, we tested whether targeted disruption of inducible nitric oxide (NO) synthase (iNOS), a key inflammatory mediator in obesity, modulates the metabolic effects of rosiglitazone in obese mice. RESEARCH DESIGN AND METHODS: iNOS(-/-) and iNOS(+/+) were subjected to a high-fat diet or standard diet for 18 weeks and were then treated with rosiglitazone for 2 weeks. Whole-body insulin sensitivity and glucose tolerance were determined and metabolic tissues harvested to assess activation of insulin and AMP-activated protein kinase (AMPK) signaling pathways and the levels of inflammatory mediators. RESULTS: Rosiglitazone was found to similarly improve whole-body insulin sensitivity and insulin signaling to Akt/PKB in skeletal muscle of obese iNOS(-/-) and obese iNOS(+/+) mice. However, rosiglitazone further improved glucose tolerance and liver insulin signaling only in obese mice lacking iNOS. This genotype-specific effect of rosiglitazone on glucose tolerance was linked to a markedly increased ability of the drug to raise plasma adiponectin levels. Accordingly, rosiglitazone increased AMPK activation in muscle and liver only in obese iNOS(-/-) mice. PPAR-gamma transcriptional activity was increased in adipose tissue of iNOS(-/-) mice. Conversely, treatment of 3T3-L1 adipocytes with a NO donor blunted PPAR-gamma activity. CONCLUSIONS: Our results identify the iNOS/NO pathway as a critical modulator of PPAR-gamma activation and circulating adiponectin levels and show that invalidation of this key inflammatory mediator improves the efficacy of PPAR-gamma agonism in an animal model of obesity and insulin resistance.

Determinants of return behavior: a comparison of current and lapsed donors.

BACKGROUND: There is a need to identify factors explaining why some people stop donating blood. STUDY DESIGN AND METHODS: A random mail survey of first-time (FT) and repeat (RPT) current (donating within 6 months before survey) and lapsed (donating >2 years prior) donors was conducted. The self-administered questionnaire included questions on personal, social, and behavioral characteristics. RESULTS: Among 1280 current and 1672 lapsed donors with valid addresses, the participation rate was 66.8 and 39.2 percent, respectively. In FT donors, the odds of lapsing increased with education (odds ratio [OR], 2.18; 95% confidence interval [CI], 1.34-3.55 for college or higher vs. Grade 12 or less education). Lapsed FT donors were more often asked to donate (OR, 1.89; 95% CI, 1.32-2.70) and had less interest in incentives (p < 0.001) than current FT donors. In RPT donors, lapsed status was associated with being younger (p < 0.001) and female (OR, 1.19; 95% CI, 1.00-1.42). Lapsed status was inversely associated with satisfaction with the last donation experience in both FT (p = 0.043) and RPT (p < 0.001) donors. Lapsed and current donors did not differ in perceived need for blood, personal transfusion experience, or mean reported altruistic behavior score. CONCLUSION: A positive donation experience appears to be a major determinant of donor return behavior. Lapsed donors do not appear, on average, to engage in fewer altruistic behaviors than currently active donors. Retention marketing strategies that appeal solely to altruistic values need to be further evaluated for their effectiveness.

Comparative real-time characterization of frother bubble thin films.

Methylisobutyl carbinol (MIBC) and methoxy polypropylene glycol (Dowfroth 250) bubble thin films were characterized by composition and thickness determinations, using FT-IR spectroscopy and UV-visible spectrophotometry, respectively. The infrared investigation indicates that the role of frother molecules in creating bubble thin films is based on their effectiveness in inducing H-bonding reorganization of the surrounding free water molecules into a coherent network. From UV-visible interference patterns, it is found that MIBC forms films with a measured critical end thickness of less than 160 nm, compared to 612+/-30 nm for Dowfroth 250. The observed values of the free water layer thickness are comparable at 5.65+/-0.28 microm for MIBC and 6.75+/-0.34 microm for Dowfroth 250. A higher stability of an MIBC bubble over a Dowfroth 250 bubble is related to the different polarization and orientation of the molecules sustaining the boundary surface water network. The findings are used to explain the different action and associated froth characteristics of the two common industrial frothing agents.

Declining value of preoperative autologous donation.

BACKGROUND: Preoperative autologous blood donation (PABD) has been shown to decrease allogeneic blood transfusion requirements in major elective surgery. Changes in transfusion practice motivated an examination of blood use from 1993 to 2000 of patients participating in the Héma-Québec PABD program. STUDY DESIGN AND METHODS: Blood donation and transfusion, type of surgery, and demographic characteristics were prospectively entered into a computer database for patients participating in the Héma-Québec PABD program. RESULTS: Autologous donations represented from 0.8 to 2 percent of total blood collections and have declined by 26 percent after peaking in 1995. The mean number of units collected per patient declined, as did the number of units transfused per patient and the utilization rate. For radical prostatectomy, knee replacement surgery, hip replacement surgery, and scoliosis, utilization rates were 72, 60, 83, and 78 percent in 1993 compared with 50, 50, 58, and 58 percent in 2000, respectively. In 2000, 18 percent of patients were receiving a 1-unit autologous transfusion. Depending on the surgical procedure, 85 to 95 percent of patients avoided allogeneic transfusion; this did not change significantly from 1993 to 2000. CONCLUSION: Patients participating in the PABD program successfully avoided allogeneic transfusion in over 85 percent of cases. However, declining utilization rates and frequent 1-unit transfusions demonstrate the decreasing utility of PABD over time.