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Background: Substance use during adolescence can have a number of negative consequences and interfere with normal brain development. Given limited time and resources, brief group- and school-based prevention programs are an efficient strategy for educating youth about the effects of substance use on health outcomes., Objectives: To determine if a science-based, interactive substance prevention program could improve student knowledge and influence students' attitudes toward future substance use behaviors., Methods: The Just Say Know program was given to 1,594 middle and high school students. The facilitator engaged students in an interactive, hour-long session covering brain basics and effects of substance use. Students completed an eight-item pre- and post-knowledge-based test to measure learning outcomes along with feedback questions about youths' attitudes toward substance use and the program., Results: After the program, 94% of students reported that it provided helpful information; 92% reported it may influence their approach to substance use, with 76% specifying that they would delay or cut back on substance use. Knowledge-based test performance increased by 78%, with high schoolers displaying significantly higher scores than middle schoolers, but both showing similar improvements in scores. Students who reported higher levels of friends' substance use had smaller improvements from pre- to posttest., Conclusion: Results suggest Just Say Know , a scientifically-based prevention program, is effective in increasing adolescents' program based-knowledge, has the potential to affect youths' attitudes toward substance use, and is well-received. These findings provide preliminary evidence that a cost-effective, neuroscience-informed group prevention program might reduce or delay adolescents' future substance use.

Insulin resistance is driven, in part, by activation of the innate immune system. We have discussed the evidence linking nucleotide-binding oligomerization domain (NOD)1, an intracellular pattern recognition receptor, to the onset and progression of obesity-induced insulin resistance. On a molecular level, crosstalk between downstream NOD1 effectors and the insulin receptor pathway inhibits insulin signaling, potentially through reduced insulin receptor substrate action. In vivo studies have demonstrated that NOD1 activation induces peripheral, hepatic, and whole-body insulin resistance. Also, NOD1-deficient models are protected from high-fat diet (HFD)-induced insulin resistance. Moreover, hematopoietic NOD1 deficiency prevented HFD-induced changes in proinflammatory macrophage polarization status, thus protecting against the development of metabolic inflammation and insulin resistance. Serum from HFD-fed mice activated NOD1 signaling ex vivo; however, the molecular identity of the activating factors remains unclear. Many have proposed that an HFD changes the gut permeability, resulting in increased translocation of bacterial fragments and increased circulating NOD1 ligands. In contrast, others have suggested that NOD1 ligands are endogenous and potentially lipid-derived metabolites produced during states of nutrient overload. Nevertheless, that NOD1 contributes to the development of insulin resistance, and that NOD1-based therapy might provide benefit, is an exciting advancement in metabolic research., (Copyright © 2019 Endocrine Society.)

The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.

The product of the Rv2276 gene of Mycobacterium tuberculosis is a cytochrome P450 (P450 MT2, CYP121) which has been shown to bind tightly to a range of azole-based antifungal drugs (e.g. miconazole, clotrimazole). These drugs are potent inhibitors of mycobacterial growth, suggesting that P450 MT2 (CYP121) may be a potential drug target. The enzyme has been overexpressed in Escherichia coli and crystallized by the hanging-drop method. Crystals of P450 MT2 (CYP121) belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 78.3, c = 265.6 A. Native data have been collected to 1.6 A resolution and Hg-derivative data to 2.5 A resolution using a synchrotron-radiation source.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.

The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase. The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO. The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca(2+), whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40-50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca(2+) concentrations and which retains activity in the absence of CaM. In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the FMN and FAD cofactors of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain. The results indicate that CaM binding does not modulate the reduction potentials of the flavins, but appears to control electron transfer primarily via a large structural rearrangement. We also report the creation of chimaeric enzymes in which the reductase domains of nNOS and flavocytochrome P450 BM3 (Bacillus megaterium III) have been exchanged. Despite its very different flavin redox potentials, the BM3 reductase domain was able to support low levels of CaM-dependent NO synthesis, whereas the NOS reductase domain did not effectively substitute for that of cytochrome P450 BM3.

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.

The YAP1 and YAP2 genes encode yeast transcription factors of the c-jun family. We show that yeast mutants deleted for either the YAP1 or the YAP2 genes are hypersensitive to oxidants, particularly H2O2, and that these genes play a role in regulating the induction of the H2O2 adaptive stress response in Saccharomyces cerevisiae. They do not significantly affect the regulation of the superoxide adaptive stress response. The intrinsic resistance of stationary-phase and respiring yeast cells towards superoxide anions is unaffected by deletion of the YAP1 and YAP2 genes. However, resistance towards H2O2 under these conditions is significantly reduced. We show that expression of the yeast GSH1 gene (encoding gamma-glutamylcysteine synthetase) and the SSA1 gene (encoding an HSP70 isoform) are induced by oxidants. Unlike the SSA1 and thioredoxin (TRX2) genes, expression of the GSH1 gene is more strongly induced by superoxide anions than by H2O2. In the absence of added oxidants, transcription of the GSH1 gene is reduced in strains carrying the yap1 deletion. However, we show that Yap1 is not required for the superoxide anion-mediated induction of GSH1 gene expression. Furthermore, while the H2O2-mediated induction of SSA1 expression is shown to by YAP1 dependent, the heat-shock-mediated induction of the SSA1 gene does not require YAP1. We also present evidence to show that the YAP2 gene does not regulate the expression of the TRX2, SSA1 or GSH1 genes.

Exponentially growing Saccharomyces cerevisiae cells are more sensitive to oxidants such as hydrogen peroxide and superoxides than stationary phase cells. Using disruption mutations in the genes encoding the two S. cerevisiae superoxide dismutases, we show that the principal mechanism of toxicity of redox-cycling compounds, such as menadione and plumbagin, is via the production of superoxide anions. Using two-dimensional polyacrylamide gel electrophoresis we have compared the pattern of protein expression in cells labelled with L-[35S]methionine and stressed with either H2O2 or menadione. Three groups of proteins were evident: those whose levels are elevated by both H2O2 and menadione, and those specifically induced by either H2O2 or menadione. Experiments with promoter fusions demonstrated that one of the heat inducible forms of HSP70 (SSA1) was inducible with H2O2. Furthermore, induction of the yeast H2O2-responsive TRX2 promoter by menadione required the metabolism of menadione.

A 3.2 kb chromosomal DNA fragment which complements the defects in a series of twelve moa::Mucts insertion mutants has been sequenced. Five open reading frames (ORFs) were identified and these are arranged in a manner consistent with their forming an operon. The encoded proteins (MoaA-MoaE) have predicted molecular weights of 37,346, 18,665, 17,234, 8843 and 16,981 respectively. Examination of subclones of the whole locus in an expression system demonstrated the predicted products. N-terminal amino acid sequences for the moaA, B, C and E products confirmed the translational starts. Genetic analysis distinguished four classes of moa mutants corresponding to genes moaA, C, D and E. Potential promoter sequences upstream of moaA and a possible transcription termination signal have been identified. Genetic analysis of the chlA1 and chlM mutants, which have been biochemically characterized as defective in molybdopterin biosynthesis, indicates that these carry lesions in moaA and moaD respectively. The moa locus is orientated clockwise at 17.7 minutes in the chromosome.

The Southern Arizona Regional Blood Program of the American Red Cross in cooperation with Tucson hospitals conducted a pilot program of the provision of intraoperative autologous transfusion services. The service provided instruments, staff, and disposables to salvage and wash red cells shed during scheduled and emergency surgical procedures. The lower cost and other efficiencies of providing this service through a regional blood center suggest that it may be more appropriate to offer this service on a regional basis than exclusively within a hospital. Hospitals and regional blood centers should consider offering this advanced form of hemotherapy to their communities.

The present study evaluated (1) the influence of the collection of autologous platelet-rich plasma intraoperatively in addition to intraoperative autotransfusion on homologous blood usage and bleeding in cardiac operations; (2) the influence of age, sex, body surface area, type of operation, and reoperations on homologous blood usage and bleeding in cardiac operations utilizing intraoperative autotransfusion and autologous platelet-rich plasma collected intraoperatively; and (3) the influence of the preoperative administration of aspirin, Persantine (dipyridamole), heparin sodium, thrombolytic agents, Coumadin (crystalline warfarin sodium), and nonsteroid, antiinflammatory drugs on homologous blood usage and bleeding in cardiac operations utilizing intraoperative autotransfusion and autologous platelet-rich plasma collected intraoperatively. The results demonstrated a decrease in homologous blood use and bleeding when autologous platelet-rich plasma is collected in addition to the use of intraoperative autotransfusion. All of the patient and procedural variables influenced homologous blood usage and bleeding to some extent. Only the thrombolytic agents affected blood usage by increasing homologous plasma usage. All other drugs evaluated did not influence blood utilization or the amount of bleeding intraoperatively or postoperatively.

The Southern Arizona Red Cross Blood program, in conjunction with participating hospitals and cardiac surgeons, evaluated the effect of a program to harvest autologous platelet-rich plasma (PRP) from patients immediately prior to undergoing cardiopulmonary bypass surgery. The PRP was transfused back to the patient after heparin neutralization was achieved at the completion of cardiopulmonary bypass. The effect of this autologous PRP product on homologous plasma and platelet usage was examined. The study demonstrates a significant decrease in homologous plasma and platelet usage when autologous PRP is used in cardiac surgery.

We evaluated a rapid RBC zinc protoporphyrin (ZP) test in 1,147 male and 615 female blood donors to study its value in screening for evolving iron deficiency anemia. Fifteen men (1.8%) and 32 women (7.9%) who returned to donate were found to be anemic. A matched sample analysis between anemic and nonanemic donors demonstrated significant differences in serum ferritin levels, percent iron saturation, and the RBC ZP level from samples collected during the initial evaluation. Red cell ZP correlated well with the natural logarithm of serum ferritin in both men and women who later became anemic. The predictive value of RBC ZP levels compared favorably with that of the serum ferritin level. We also observed a strong association between the yearly donation frequency and RBC ZP concentration. These findings indicate that predonation RBC ZP testing may be useful in screening for iron depletion and potential risk of anemia in blood donors.

The whole blood electrical conductivity method for predonation hematocrit determinations was studied. Forty capillary and venous blood samples were tested concurrently with electrical conductivity and centrifugation methods. The electrical conductivity method demonstrated acceptable accuracy but was less precise than the spun microhematocrit technique. Results from capillary samples were uniformly less precise and significantly lower than determinations from venous specimens. This study and previous reports suggest that capillary hematocrit values by any method may not accurately reflect the blood donor's venous hematocrit level. Blood centers should independently validate their anemia screening methods to avoid unnecessary deferrals and protect the anemic donor.

The Southern Arizona Regional Red Cross Blood Program, in cooperation with two cardiac surgery groups, examined the effect of intraoperative autotransfusion on red cell, plasma, and platelet usage during and after cardiac operations. The study evaluated whether intraoperative autotransfusion influenced intraoperative or postoperative blood usage and whether regular use was more effective than selective use. The study demonstrated that intraoperative autotransfusion reduces intraoperative and postoperative blood use and that regular use of intraoperative autotransfusion is more effective than selective use.

A large proportion of potential blood donors who are deferred are inappropriate to be donors because of unreliable predonation anemia screening methods. In this study, venous hemoglobin concentrations were within acceptable limits in 71% of 275 anemia deferrals. Red blood cell zinc protoporphyrin (RBC ZP) was evaluated as a screening test to improve the accuracy of detecting anemia in prospective blood donors. The frequency of abnormally low venous hemoglobin concentrations in anemia deferrals having fingerstick capillary microhematocrit (MH) values within 3% of the minimum requirement, together with normal RBC ZP levels (less than 53 micrograms/dL [0.943 mumol/L] RBC), was 2%, and not significantly different from the prevalence of venous anemia observed in eligible blood donors. Anemia deferrals with elevated RBC ZP results had a significantly increased rate of iron depletion and anemia. Capillary RBC ZP measurements in combination with the MH test have the potential to safely decrease inappropriate anemia deferrals.