Demonstration of ignition radiation temperatures in indirect-drive inertial confinement fusion hohlraums.

We demonstrate the hohlraum radiation temperature and symmetry required for ignition-scale inertial confinement fusion capsule implosions. Cryogenic gas-filled hohlraums with 2.2 mm-diameter capsules are heated with unprecedented laser energies of 1.2 MJ delivered by 192 ultraviolet laser beams on the National Ignition Facility. Laser backscatter measurements show that these hohlraums absorb 87% to 91% of the incident laser power resulting in peak radiation temperatures of T(RAD)=300 eV and a symmetric implosion to a 100 µm diameter hot core.

Artemisinin enhances heme-catalysed oxidation of lipid membranes.

Artemisinin, a sesquiterpene endoperoxide derived from a traditional Chinese herbal remedy for fevers, is a promising new antimalarial drug, particularly useful against multidrug resistant strains of P. falciparum. Despite widespread clinical use, its mode of action remains uncertain. We investigated whether its antimalarial properties could be explained by an ability to enhance the redox activity of heme, formed in the parasite food vacuole from digested hemoglobin. Artemisinin caused a sustained threefold increase, followed by a gradual decline, in the peroxidase activity of heme. It also enhanced the ability of heme to oxidize membrane lipids about sixfold. An unexpected finding was the potentiation of heme-catalysed membrane lipid oxidation by Vitamin E. The changes in redox-catalytic activity induced by artemisinin were paralleled by major changes in the absorption spectrum of heme, culminating in loss of the Soret band. We propose a model in which artemisinin binds irreversibly to heme in the parasite food vacuole, preventing its polymerization to chemically inert hemozoin, and promoting heme-catalysed oxidation of the vacuolar membrane by molecular oxygen, which leads, ultimately, to vacuole rupture and parasite autodigestion.

Quinoline anti-malarial drugs inhibit spontaneous formation of beta-haematin (malaria pigment).

Polymerisation of haematin to beta-haematin (haemozoin or malaria pigment) in acidic acetate solutions was studied using infrared spectroscopy. The reaction was found to occur spontaneously between 6 and 65 degrees C, in 0.1-4.5 M acetate and pH 4.2-5.0. The anti-malarial drugs quinine, chloroquine and amodiaquin were found to block spontaneous beta-haematin formation, while the anti-malarially inactive 9-epiquinine and 8-hydroxyquinoline had no effect on the reaction, as did primaquine, a drug which is active only against exo-erythrocytic stages of infection. It is argued that the intra-erythrocytically active anti-malarial agents act by binding to haematin, blocking beta-haematin formation and leaving toxic haematin in the parasite food vacuoles.

The high grade match kidney sharing algorithm of the South-Eastern Organ Procurement Foundation (SEOPF): altering recipient demographics through improved matching.

BACKGROUND: Studies of kidneys shared through the South-Eastern Organ Procurement Foundation (SEOPF) have shown that regional organ procurement (ROP) trays can predict negative crossmatch in highly sensitized patients when the HLA match is of a high grade. In an attempt to offer more well-matched kidneys to highly sensitized patients, SEOPF organized the High Grade Match (HGM) Program. METHODS: This United Network for Organ Sharing (UNOS)-approved allocation variance requires mandatory sharing of all kidneys by participating centers after UNOS mandatory sharing requirements have been met. The HGM levels of sharing are: (1) 0 A,B mismatch (MM); panel-reactive antibody (PRA) > or = 40%; negative ROP crossmatch; (2) 0 B,DR MM with > or = 40% PRA; negative ROP crossmatch; (3) 0 B,DR MM with PRA < 40%. Non-HGM cadaveric transplants at the same participating centers--locally or distally procured--serve as the control group. RESULTS: During the first 18 months of this program, the 23 participating centers shared 124 kidneys of the 1592 that were available. Well-matched kidneys (two mismatches or less) accounted for 91.1% in the HGM group, but only 19% of the controls (P<0.0001). Highly sensitized patients (PRA > or = 40%) represented 13.8% of the HGM group, but only 3.3% of the non-HGM group (P<.0001). With HGM kidneys, there was a shift in recipient demographics. Patients with blood group O, female patients, older patients, and retransplanted patients all accounted for significantly larger percentages of the HGM group compared with the non-HGM control group. The racial composition of the recipients of high-grade matches was, however, no different than that of the control recipients at the same centers. CONCLUSION: The HGM Program resulted in longer ischemia times, but graft survival was not affected. The 1-year actuarial graft survival rate (Kaplan-Meier) for HGM kidneys was not different from the control cadaveric graft survival rate. By sharing kidneys based on improved HLA matches with consideration for high PRA, the HGM Program offered more transplant opportunities to women, blood group O recipients, retransplants, and older patients.

Effects of tacrolimus on hyperlipidemia after successful renal transplantation: a Southeastern Organ Procurement Foundation multicenter clinical study.

BACKGROUND: Tacrolimus has been shown to have a less adverse effect on the lipid profiles of transplant patients when the drug is started as induction therapy. In order to determine the effect tacrolimus has on lipid profiles in stable cyclosporine-treated renal transplant patients with established hyperlipidemia, a randomized prospective study was undertaken by the Southeastern Organ Procurement Foundation. METHODS: Patients of the 13 transplant centers, with cholesterol of 240 mg/dl or greater, who were at least 1 year posttransplant with stable renal function, were randomly assigned to remain on cyclosporine (control) or converted to tacrolimus. Patients converted to tacrolimus were maintained at a level of 5-15 ng/ml, and control patients remained at their previous levels of cyclosporine. Concurrent immunosuppressants were not changed. Levels of total cholesterol, triglycerides, total high-density lipoprotein, low-density lipoprotein (LDL), very-low-density lipoprotein, and apoproteins A and B were monitored before conversion and at months 1, 3, and 6. Renal function and glucose control were evaluated at the beginning and end of the study (month 6). RESULTS: A total of 65 patients were enrolled; 12 patients failed to complete the study. None were removed as a result of acute rejection or graft failure. Fifty-three patients were available for analysis (27 in the tacrolimus group and 26 controls). Demographics were not different between groups. In patients converted to tacrolimus treatment, there was a -55 mg/dl (-16%) (P=0.0031) change in cholesterol, a -48 mg/dl (-25%) (P=0.0014) change in LDL cholesterol, and a -36 mg/dl (-23%) (P=0.034) change in apolipoprotein B. There was no change in renal function, glycemic control, or incidence of new onset diabetes mellitus in the tacrolimus group. CONCLUSION: Conversion from cyclosporine to tacrolimus can be safely done after successful transplantation. Introduction of tacrolimus to a stable renal patient does not effect renal function or glycemic control. Tacrolimus can lower cholesterol, LDL, and apolipoprotein B. Conversion to tacrolimus from cyclosporine should be considered in the treatment of posttransplant hyperlipidemia.

1-Chloro-2,4-dinitrobenzene-mediated irreversible inactivation of acidic glutathione S-transferases. Inactivation mechanism--a saturation-type or simple second-order kinetic process?

A critical analysis of the inactivation kinetics exhibited by the acidic human glutathione S-transferase (GST) enzymes is presented. Data on the 1-chloro-2,4-dinitrobenzene (CDNB)-facilitated inactivation of human placental GST pi have been utilized in conjunction with published inactivation data from the literature to answer the following two questions: (a) do the inactivation kinetics deviate significantly from a simple pseudo first-order model? (b) What is the kinetic mechanism of irreversible electrophilic co-substrate-mediated inactivation of human acidic GSTs? Inactivation of human placental GST pi in the presence of 7-aminocephalosporanic acid, a non-electrophilic non-substrate ligand, is characterized and shown to occur via a process analogous to the second mechanism proposed for CDNB inactivation of the enzyme, namely: pH- and [ligand]-independent solvational inactivation.

Factors affecting the inactivation of human placental glutathione S-transferase pi. The kinetic mechanism and pH-dependence of solvational and 1-chloro-2,4-dinitrobenzene-mediated inactivation of the enzyme.

The kinetics of the inactivation of human placental GSH S-transferase pi has been studied at 25 degrees in the pH range 6.5 less than or equal to pH less than or equal to 9. At pH values less than or equal to 7.0 the inactivation of GSH S-transferase pi incubated in the absence of GSH and (i) in the absence or (ii) in the presence of CDNB (0-1.5 X 10(-3) mol/dm3) exhibited pseudo first-order kinetics with kobs for (i) and (ii) approximately equal (approximately 0.002 sec-1). The extent of inactivation in (i) approached a limiting value of 50% at infinite dilution of the enzyme; while in the presence of CDNB the extent of inactivation approached 100%. At any given pH such that 7 less than pH less than or equal to 9 the pseudo first-order inactivation rate constant, kobs, exhibits a linear dependence on [CDNB] (Eqn 1): kobs = k1 + k2 [CDNB] (1) where k1 is invariant with pH and approximately equal to 0.002 sec-1. The first-(k1) and second-(k2)-order components of kobs suggest at least two mechanisms for the inactivation of GST by CDNB, these are: (i) a pH-invariant facilitation of solvational inactivation and (ii) a pH-dependent nucleophilic reaction of a thiol group (pKa = 8.85 +/- 0.08) at or spatially close to the active site of the enzyme. A mechanistic rationale for the enzyme functioning as a dimer is discussed in detail.

Ferriprotoporphyrin IX mediated oxygen activation/insertion reactions: the anerobic reduction of the aniline-Fe3+-microperoxidase-8 complex by NADH.

A spectrophotometric study of the reduction of the Fe3+ microperoxidase-8-aniline (Fe3+-MP-8-An) complex has been carried out. Addition of NADH to a solution of Fe3+-MP-8-An under strictly anerobic conditions results in the formation of a species with lambda max = 414 nm (Fe3+-MP-8-An lambda max 407 nm). The kinetics of formation of this species show an induction period (tau) which follows saturation kinetics with respect to [aniline] with Km(app) = 2.2 x 10(-3) mol dm-3, i.e., close to that obtained in the preceding paper from O2 consumption kinetics mediated by MP-8. Addition of an anerobic solution of the NADH reduced MP-8-An complex, to a saturated O2 solution at pH 12 in the presence of 0.5 mM NADH and aniline 10 mM results in the virtual elimination of the induction phase, which has previously characterized O2 consumption kinetics in ferriprotoporphyrin IX oxygen activation systems. The Arrhenius activation energy for the reduction of the Fe3+-MP-8-An complex is close to that observed for the first reductive step in the cyt P-450 O2 activation cycle. Anerobic reduction of Fe3+-MP-8 by sodium dithionite in 20% MeOH/Aq at pH 8 followed by anerobic titration of the Fe2+-MP-8 (lambda max 420.5 nm) with aniline at pH 12 gives rise to a species lambda max 415 with KD for the process = 4.4 x 10(-3) mol dm-3 (+/- 1.2 x 10(-3) mol dm-3).

Haem-peptide-protein interactions: Part 5. The haem undecapeptide microperoxidase-11 (Fe3+MP-11)/human serum albumin (HSA) reaction in aqueous methanolic solution. A simple system demonstrating the effect of hydrophobicity on ligand release from a ligand-protein complex.

Previous studies of the interaction of the haem undecapeptide (MP-11) with lipidated human serum albumin in aqueous solution have been extended to a range of MeOH/H2O solution compositions. It is demonstrated that the kinetic mechanism for the interaction does not change from a simple second- and first-order reversible scheme as XMeOH is increased, however while k1--the association rate constant is essentially invariant with XMeOH, K-1 increases some 600-fold over the range studied. The result is interpreted in terms of increased solvational stabilization of MP-11 and transition state as XMeOH increases and it is noted that the system provides a simple demonstration of the effect of hydrophobicity on facilitating transported ligand release from ligand/carrier protein molecules.

A comparative study of pH/activity profiles for the anaerobic H2O2 and alkyl hydroperoxide supported N demethylation of N methylaniline catalyzed by alkaline haematin and microsomal cytochrome P-450.

A comparative study of the effect of pH on haemin-mediated H2O2 and alkyl hydroperoxide-supported N-demethylation of N-methylaniline and the corresponding cytochrome P-450 mediated process has been carried out. This extends previous studies and provides quantitative evidence that the same active oxygen species are involved in both the enzymic and metalloporphyrin mediated processes. Furthermore, this evidence clearly indicates that the environment at the point of catalysis in the enzyme active site differs considerably from that of the bulk solution.

Hemin-mediated para-hydroxylation of aniline: a potential model for oxygen activation and insertion reactions of mixed function oxidases.

The activation of molecular oxygen by alkaline hemin (ferriprotoporphyrin IX) has been studied. In the presence of reductant nicotineamide adenine dinucleotide (NADH) or nicotineamide adenine dinucleotide phosphate (NADPH) and organic substrate, aniline, hemin activates oxygen to the hydroperoxide anion (HO2-) and subsequently mediates insertion of active oxygen into the benzene ring of the substrate to form p-aminophenol, with a high degree of regiospecificity. Oxygen activation does not occur in the absence of aniline. Stoichiometry of the reaction indicates that two electrons are required per molecule of oxygen activated or atom of oxygen inserted into the substrate aromatic ring system. Direct measurement of H2O2 and of the pKa for maximum rate of p-aminophenol formation (11.7 +/- 0.1) indicate participation of the hydroperoxide anion as the active oxygen species in the rate-determining step of the insertion reaction. Powerful scavengers of the hydroxyl radical (OH) have little effect on the formation of H2O2 or p-aminophenol by the system. Superoxide dismutase (10(-7) mol dm-3) inhibited both p-aminophenol and H2O2 formation, when added to the system immediately prior to initiation of the reaction. Studies involving N-phenylhydroxylamine indicate that aromatic ring hydroxylation is occurring directly and not by rearrangement of an N-hydroxylated intermediate. Implications of hemin-mediated hydroxylation reactions for those of enzymatic mixed function oxidase activity are discussed.

Superoxide dismutase inhibition of alkaline hemin-mediated O2 activation.

Inhibition of hemin-mediated O2 activation by bovine superoxide dismutase and the copper tetrammine complex has been examined. It is shown that the inhibitory effect of these species is not due to metabolism of O2-., but to a nonspecific inhibition of the process by the Cu2+ ion. We propose that the inhibition occurs at the level of electron transfer from NADH to iron in an analogous manner to that proposed for the Cu2+ inhibition of microsomal electron transfer.

Haem peptide/protein interaction. Part 6: The kinetic mechanisms of the interactions with, and inhibition of enzymic activity of the human erythrocyte glutathione S-transferase isoenzyme rho (p), by haem octa-, nona-, and undecapeptides MP-8/-9/-11.

The binding of the cytochrome-c derived haem peptides microperoxidase-8, -9, and -11 (MP-8, -9, and -11) to the human erythrocyte glutathione S-transferase rho (GST-p) enzyme is demonstrated. Inhibition by the haem peptides of the enzymic conjugation of glutathione (GSH) with the electrophilic cosubstrate 1-chloro-2, 4-dinitrobenzene (CDNB) is mixed-type with respect to CDNB, and Ki, the inhibition constant, increases with increasing length of the peptide chain. The results obtained here for the GST-p are compared to those published recently for the previously-supposed identical isoenzyme human placental GST-pi.

Heme-peptide/protein interactions: the binding of heme octa and undecapeptides, and microperoxidase-8 and -11, to human serum albumin.

The interaction of the heme octa (MP-8) and undeca (MP-11) peptides derived from cytochrome c with lipidated human serum albumin (HSA) has been investigated in aqueous solution. It is demonstrated that complex formation occurs in each case with a 1:1 stoichiometry. CN- binding has been used to investigate the accessibility of the heme in each complex by comparison with CN- interaction with methemalbumin. A preliminary study of the kinetics of the Fe3+MP-8/11 human serumalbumin (HSA) interaction demonstrates a clear ligand-size-related effect on mechanism of interaction--an ad hoc explanation of which is given in terms of HSA existing as two nonconverting conformers in solution.

The nature of heme-aniline interactions during hemin-mediated oxygen activation and insertion reactions.

The interaction of aniline with hemin during oxygen activation and insertion mediated by the metalloporphyrin has been studied by a combined theoretical, semiempirical, and experimental approach. Results indicate that association is via a planar pi bonding interaction between the aromatic pi electrons of the amine and tetrapyrrole ring system. The effect of very high concentrations of hydroxyl radical scavengers on the system is discussed.

H2O2-and alkyl hydroperoxide-supported para hydroxylation of aniline by alkaline hematin.

Under alkaline conditions and in the presence of reductant, NADH or NADPH, and molecular oxygen, hemin catalyzes the regiospecific para-hydroxylation of aniline to form p-aminophenol [P. A. Adams, D. A. Baldwin, and M. C. Berman, J. Chem. Soc. (Lond) Chem. Commun. 856 (1979); P. A. Adams and M. C. Berman, J. Inorg. Biochem. 17, 1 (1982)]. The reaction has now been studied in the presence of H2O2 and alkyl hydroperoxides and in the absence of oxygen and reductant. Results indicate that the H2O2- and alkyl hydroperoxide-supported processes proceed via different mechanisms involving, on the one hand, the hydroperoxide anion (HO2-) and on the other, the undissociated alkyl hydroperoxide molecule (ROOH). The addition of superoxide dismutase to the reaction had no effect, unlike the NADH/O2 supported reaction where the enzyme completely inhibits reaction. Similarities between the hemin-mediated peroxide-supported reactions reported here, and the cytochrome P-450-mediated peroxide-supported reactions reinforce our earlier contentions that the alkaline hemin system appears to be a good model for the in vivo activation of oxygen by hemoproteins.

Oxygen activation and ligand binding by pure heme-octapeptide microperoxidase-8 (MP-8).

The rapid, efficient preparation of pure microperoxidase-8 (MP-8) is described. Ligand binding studies confirm that MP-8 is monomeric in alkaline solution. It is shown that the monomeric MP-8 activates oxygen in a similar manner to that already reported for alkaline hemin, establishing the octapeptide as a possible second generation model for the oxygen activation/insertion reactions of the cytochrome P-450.

Hemes and hemoproteins. 1: Preparation and analysis of the heme-containing octapeptide (microperoxidase-8) and identification of the monomeric form in aqueous solution.

The heme-octapeptide from cytochrome c, Microperoxidase-8 (MP-8), was prepared by peptic and tryptic digestion of horse heart cytochrome c and purified by gel permeation chromatography in about 50% yield. Conditions for the identification of MP-8 by TLC and analysis by HPLC are described. Study of the concentration-dependence of the absorption spectrum showed that at concentrations of less than or equal to 2.5 X 10(-5) M in aqueous solution at pH 7, 25 degrees C and mu = 0.1, MP-8 exists as an equilibrium mixture of monomers and dimers with KD = 1.17 +/- 0.02 X 10(5) M-1, decreasing to 1.21 +/- 0.02 X 10(4) M-1 and 2.16 +/- 0.21 X 10(3) M-1 in 20% and 50% (v/v) methanol:water mixtures, respectively. Comparison of the Soret region spectrum of monomeric MP-8 with other hemoproteins suggests that it is six-coordinate in aqueous solution with water and His as axial ligands.

Kinetics of heme octapeptide (microperoxidase-8; MP-8) formation studied by high-pressure liquid chromatography (HPLC) monitoring of the peptic and tryptic hydrolysis of horse heart cytochrome-c.

The kinetics of the sequential peptic and tryptic hydrolysis of cytochrome-c to give the heme-peptides microperoxidase-11 (MP-11) and -8 (MP-8), respectively, has been investigated by high performance liquid chromatography (HPLC), and we demonstrate that MP-8 can be prepared from cytochrome-c to the point of lyophilization within 4 hr.

The iron environment in heme and heme-antimalarial complexes of pharmacological interest.

Mössbauer spectroscopy has been utilized to probe the electronic environment of iron in a number of Ferriprotoporphyrin IX complexes of relevance to malaria. The markedly different iron environments found for the complexes of hemin with quinine, chloroquine, and the Chinese herbal antimalarial artesunate suggest that these compounds act by protecting the heme from polymerization to insoluble hemozoin, and by facilitating the transport of the protected heme to the food vacuole membrane where it is able to exercise its cytotoxic redox catalytic activity. Mössbauer parameters determined here for purified malaria pigment and synthetic beta-hematin confirm the chemical identical-ness of these species. The Mössbauer spectra of the complexes are discussed in light of the proposed structures of the complexes.