Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

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.

Control of electron transfer in neuronal NO synthase.

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.

Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase.

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 role of the YAP1 and YAP2 genes in the regulation of the adaptive oxidative stress responses of Saccharomyces cerevisiae.

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.

Analysis of Saccharomyces cerevisiae proteins induced by peroxide and superoxide stress.

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.

Significance of isolated hepatitis B core antibody in blood donors.

BACKGROUND: About 25% of blood donors who test positive for antibody to hepatitis B core antigen (anti-HBc) have no other positive hepatitis B serologic results. Because of the potential importance and diagnostic uncertainty of this test result, we studied its significance by assessing the serologic response to hepatitis B vaccine in donors with an isolated anti-HBc pattern. METHODS: Specimens from 300 blood donors that were positive for anti-HBc by enzyme immunoassay were tested for anti-HBc by radioimmunoassay and for antibody to hepatitis B surface antigen (anti-HBs). A subgroup of 37 were further studied after administration of hepatitis B vaccine and compared with 34 similarly vaccinated age- and sex-matched seronegative controls. Measurements of anti-HBs were made at vaccination and 1, 2, 4, 8, 25, and 30 weeks after initial vaccination. RESULTS: Among 300 donors who tested positive for anti-HBc by enzyme immunoassay, the radioimmunoassay for anti-HBc was negative in 76 (25.3%) and the test for anti-HBs was negative in 104 (34.7%). Significant differences were observed for radioimmunoassay anti-HBc and anti-HBs titers, alanine aminotransferase, and male-female ratios between four distinct serogroups (A through D) defined by the combination (positive/negative) of radioimmunoassay anti-HBc and anti-HBs results. No significant differences between the study and control groups were observed in the magnitude of anti-HBs responses at any of the six postvaccine testing periods. CONCLUSIONS: Isolated anti-HBc in US blood donors is usually a false-positive result, regardless of the titer.

Molecular genetic analysis of the moa operon of Escherichia coli K-12 required for molybdenum cofactor biosynthesis.

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.

An autologous blood program coordinated by a regional blood center: a 5-year experience.

The Southern Arizona Regional Red Cross blood program offers preoperative autologous blood deposit to all patients and intraoperative autotransfusion services to all hospitals in the region. During a 5-year period, the amount of preoperatively deposited autologous blood and intraoperatively salvaged red cells available increased from 0.3 to 19.6 percent of the community's total collections. Further increases in the availability and use of autologous blood may be achieved by community-wide integration of services.

Intraoperative autotransfusion: a community program.

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.

Determinants of homologous blood usage utilizing autologous platelet-rich plasma in cardiac operations.

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.

Autologous platelet-rich plasma in cardiac surgery: effect on intraoperative and postoperative transfusion requirements.

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.

Intraoperative autotransfusion in cardiac operations. Effect on intraoperative and postoperative transfusion requirements.

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.

Red blood cell zinc protoporphyrin to evaluate anemia risk in deferred blood donors.

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.

Predonation hematocrit measurement by whole blood electrical conductivity assay.

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.

RBC zinc protoporphyrin to screen blood donors for iron deficiency anemia.

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.