Nonenzymatic reduction of nitrosobenzene to phenylhydroxylamine by NAD(P)H

The nonenzymatic reduction of nitrosobenzene by NADPH and NADH in aqueous buffer solution at 25°C is described. Both reactants quantitatively convert nitrosobenzene to phenylhydroxylamine. Rate constants for reduction (k_r) were determined spectrophotometrically and found to be identical at pH 5.7 and 7.4 and independent of buffer concentration. The values of k_NADH (124–149 M^?1 sec^?1) and k_NADPH (131–170 M^?1 sec^?1) are essentially identical. The reaction is not subject to general catalysis or specific salt effects. The oxidation of phenylhydroxylamine by NAD(P) to nitrosobenzene is only stimulated by a factor of 1.2 over oxidation in its absence (when the ratio of NADP: phenylhydroxylamine was 8:1).     

Lymphokine and phorbol (PMA) regulation of complement (C2) synthesis using U937

Human monocytes synthesize large amounts of the second complement component (C2) after incubation with a T-lymphocyte product called monocyte complement stimulator (MCS). The human monocyte-like cell line, U937, also synthesizes C2 and can be stimulated to increase this synthesis by lymphokine-rich culture supernates. Additionally, phorbol myristate acetate (PMA), an agent which induces maturational changes in other macrophage-like cell lines, also stimulates C2 synthesis by U937 cells. Lymphokine and PMA stimulation of C2 secretion by U937 are both reversibly inhibitable by cycloheximide. At optimal concentrations for stimulation of C2 synthesis, PMA inhibits [³H]thymidine incorporation by U937 indicating that increased C2 is not due to increased numbers of U937 cells.     

Hepatic low-level chemiluminescence during redox cycling of menadione and the menadione-glutathione conjugate: relation to glutathione and NAD(P)H:quinone reductase (DT-diaphorase) activity

Formation of excited species such as singlet molecular oxygen during redox cycling (one-electron reduction-oxidation) was detected by low-level chemiluminescence emitted from perfused rat liver and isolated hepatocytes supplemented with the quinone, menadione (vitamin K3). Chemiluminescence was augmented when the two-electron reduction of the quinone catalyzed by NAD(P)H:quinone reductase was inhibited by dicoumarol, thus underlining the protective function of this enzyme also known as DT-diaphorase. Interference with NADPH supply by inhibition of energy-linked transhydrogenase by rhein or of mitochondrial electron transfer by antimycin A led to a depression in the level of photoemission. Unexpectedly, glutathione depletion of the liver led to a lowering of chemiluminescence elicited by menadione, whereas conversely the depletion of glutathione led to increased chemiluminescence levels when a hydroperoxide was added instead of the quinone. As the GSH conjugate of menadione, 2-methyl-3-glutathionyl-1,4-naphthoquinone, studied with microsomes, was shown also to be capable of redox cycling, we conclude that menadione-induced chemiluminescence of the perfused rat liver does not only arise from menadione itself but from the menadione-GSH conjugate as well. Therefore, the conjugation of the quinone with glutathione is not in itself of protective nature and does not abolish semiquinone formation. A biologically useful aspect of conjugate formation resides in the facilitation of biliary elimination from the liver. Nonenzymatic formation of the conjugate from menadione and GSH in vitro was found to be accompanied by the formation of aggressive oxygen species.     

Phorbol diester-induced H2O2 production by peritoneal macrophages. Different H2O2 production by macrophages from normal and BCG-infected mice despite comparable phorbol diester receptors

Mouse peritoneal macrophages respond to environmental stimuli in different ways depending on their state of differentiation. Macrophages from mice with bacillus Calmette--Guerin (BCG) infection produced large amounts of H2O2 in response to phorbol diesters (PDEs), while those from noninfected mice produced little or no H2O2. The effects of PDEs on cells are mediated by specific cellular receptors for these ligands. The purpose of this study was to determine if the varying responses of macrophages from different groups of mice were caused by differences in their receptors for the PDE ligands. By all parameters studied, the binding of [20-3H]phorbol 12,13-dibutyrate ( [3H]PDBu) was similar in all macrophages irrespective of their ability to produce H2O2 in response to PDEs. Binding of [3H]PDBu was rapid at 23 degrees C reaching a maximum at 10-20 min with a subsequent decline to 50-60% of maximum by 30-60 min. Binding was slower at 0 degrees C reaching a maximum at 90-120 min. The binding was reversible, with dissociation kinetics paralleling association kinetics. The binding was saturable; the Kd's (45 to 91 nM) and number of binding sites (about 7-14 X 10(5)/cell or 11-12 pmol/mg protein) were essentially the same for the different classes of macrophages. The binding was specific, and analogs of PDBu inhibited [3H]PDBu binding to macrophages with potencies comparable to their potencies in causing in vivo tumor promotion and elicitation of other cellular responses in vitro. The ligands [3H]PDBu and [3H]PMA were degraded to comparable degrees by macrophages from normal or BCG-infected mice. Macrophages from C3H/HeJ and C3H/HeN mice, although known to differ in their abilities to respond to stimuli such as lymphokines and LPS, did not differ in their ability to produce H2O2 in response to PDEs or in their receptors for PDEs. Results of this study suggest that in vivo "activation" of macrophages in mice infected with BCG is not associated with a change in the cells' receptors for PDEs, but may be associated with "postreceptor" changes such as linkage of the PDE receptor with NAD(P)H oxidase, a change in NAD(P)H oxidase, or induction of synthesis of NAD(P)H oxidase.     

Vanadate and molybdate stimulate the oxidation of NADH by superoxide radical

Vanadate or molybdate strongly accelerate the cooxidation of NADH, or of reduced nicotinamide mononucleotide, by the xanthine oxidase plus xanthine reaction. Superoxide dismutase eliminated the effect of vanadate or molybdate, while catalase was without effect. It follows that vanadate or molybdate accelerate the oxidation of dihydropyridines by O-2. A stoichiometry of 4 NADH oxidized per O-2 introduced suggests a chain reaction for which a mechanism is proposed. These results provide an explanation for the reported stimulation, by vanadate, of NADH oxidation by biological membranes.     

Hypoxanthine and xanthine levels determined by high-performance liquid chromatography in plasma, erythrocyte, and urine samples from healthy subjects: the problem of hypoxanthine level evolution as a function of time

The levels of hypoxanthine and xanthine are determined in plasma, erythrocyte, and urine samples by a reverse-phase high-performance liquid chromatographic (HPLC) method. The hypoxanthine concentration increases in erythrocyte and plasma samples when whole blood is stored at room temperature between sampling and centrifugation. Furthermore, the hypoxanthine concentration increases in erythrocyte samples when they are kept apart at room temperature before analysis, whereas the plasma hypoxanthine level remains constant. This result proves an endogenous formation of hypoxanthine in erythrocytes with time, at room temperature. These studies show the necessity of rigorous conditions for the collection, transport, and treatment of blood samples. In order to achieve accurate results, the blood must be centrifuged immediately after collection. The erythrocyte and plasma samples must be stored frozen until deproteinization and HPLC analysis. Under these conditions, the concentrations of hypoxanthine and xanthine in plasma are 2.5 +/- 1 and 1.4 +/- 0.7 microM, respectively. In erythrocyte samples, hypoxanthine concentration reaches 8.0 +/- 6.2 microM.     

Effect of chromium(III) on poly(dG-dC) conformation

The interaction of chromium(III) with poly(dG-dC) inhibits the B to Z transition and results in the condensation of the polymer at high Cr/nucleotide ratios. At low Cr/nucleotide ratios chromium(III) enhanced the ability of ethanol to induce the B to Z transition of poly(dG-dC). The effects of chromium(III) on the conformation of DNA may be related to the carcinogenicity of chromium compounds.     

Conformational comparison of human pituitary growth hormone and human chorionic somatomammotropin (human placental lactogen) by second-order absorption spectroscopy

Second-order absorption spectra strongly suggest the presence of a hydrogen bond between the single Trp of human pituitary growth hormone (hGH) and a carboxylate ion. This hydrogen-bonded complex is buried within the hydrophobic interior of the hGH molecule. Although the homologous Trp in human chorionic somatomammotropin [human placental lactogen, HCS(hPL)] is also buried within the hydrophobic interior of the molecule, there is no evidence that it is hydrogen bonded in the native protein. However, during the early stages of thermolysin digestion of HCS(hPL), both difference and second-order absorption spectra do indicate the transient presence of a similar hydrogen-bonded Trp-carboxylate complex. The molar extinction coefficients of hGH and HCS(hPL) have been refined.     

Chelated iron-catalyzed OH. formation from paraquat radicals and H2O2: mechanism of formate oxidation

Traces of iron, when complexed with either EDTA or diethylenetriaminepentaacetic acid (DTPA), catalyze an OH.-producing reaction between H2O2 and paraquat radical (PQ+.): H2O2 + PQ+.----PQ++ + OH. + OH-.[1]. Kinetic studies show that oxidation of formate induced by this reaction occurs by a Fenton-type mechanism, analagous to that assumed in the metal-catalyzed Haber-Weiss reaction, in which the rate determining step is H2O2 + Fe2+ (chelator)----Fe3+(chelator) + OH. + OH-,[7]; with k7 = 7 X 10(3) M-1 s-1 for EDTA and 8 X 10(2) M-1 s-1 for DTPA at pH 7.4. PQ+. rapidly reduces both Fe3+ (EDTA) and Fe3+ (DTPA), and hence allows both agents to catalyze [1] with comparable efficiency, in contrast to the much lower efficiency reported for the latter as a catalyst for the Haber-Weiss reaction. The catalytic properties of these chelating agents is attributed to their lowering of E0 (Fe3+/Fe2+) by 0.65 V, thus making [7] thermodynamically possible at pH 7. Approximately 2.5% of the OH. produced is consumed by internal or "cage" reactions, which decompose the chelator and produce CO2; however, the majority (97%) diffuses into the bulk solution and participates in competitive reactions with OH. scavengers.     

Crystal and molecular structure of [Cu(5'-inosine monophosphate) (dipyridylamine) (H2O)]2.4H2O.

The ternary complex [Cu(5′-IMP)(dpa)(H₂O)]₂ has been prepared and its structure analyzed by x-ray diffraction. It has a dimeric structure in which the 5′-IMP ligands coordinate solely through their phosphate groups. This geometry is in marked contrast to that of another $\text{Cu}\text{5′-}\text{IMP}$ ternary complex, [Cu(5′-IMPH)(bipy)(H₂O)₂]⁺, which shows metal binding through the purine base rather than the phosphate group.     

Effect of aging on neurotransmitter receptor binding in rat and human brain.

β-Adrenergic and GABA receptor binding were measured in brain areas of rats 3 to 24 months of age. While GABA receptor binding was not significantly different across age in any area, β-adrenergic receptor binding was significantly reduced in the cerebellum and brain stem, but not cerebral cortex, of 24-month-old animals. The loss in β-adrenergic receptor binding does not correlate in a temporal fashion with the reported decrease in norepinephrine-stimulated cyclic AMP accumulation in the cerebellum which occurs as early as 12 months of age. An age-related reduction in β-adrenergic binding was also noted in human cerebellar tissue obtained at autopsy, suggesting that the cerebellar dysfunction seen with aging may be related to a loss of cerebellar neurons which receive noradrenergic input.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.     

Structures of two N(1)-bound platinum(II)-6-oxopurine complexes. Comparisons with complexes derived from platinum (II) anti-tumor agents

The preparation and molecular structure of [(diethylenetriamine) (7,9-dimethylhypoxanthine) platinum(II)] (PF₆)₂·1.5H₂O and [(ethylenediamine) (7,9-dimethylhypoxanthine)₂platinum(II)] (PF₆)₂, are reported. These complexes represent the first structurally characterized N(1)-bound Pt(II) 6-oxopurine complexes. In each case, the Pt(II)N(1) bond length [2.051(6)A in the diethylenetriamine complex and 2.021(8)A in the ethylenediamine complex] indicates a strong metal-to-base binding. Both complexes contain interligand hydrogen bonds, with the ammine ligand acting as the donor and the O(6) atom of the base acting as the acceptor. These N(1)-bound complexes are compared with N(7)-bound 6-oxopurine and N(3)-bound cytosine complexes of Pt(II) anti-tumor agents.     

Effect of antithrombin III, alpha 1-proteinase inhibitor and heparin on amidolytic activity of nerve growth factor (7S-NGF)

A series of potent inhibitors of angiotensin-converting enzyme (dipeptidyl carboxypeptidase, E.C. 3.4.15.1) derived from benzofused 1-carboxyalkyl-3-(1-carboxy-3-phenyl-propylamino) lactams (III) is described. In the most effective inhibitors (I50 2-4 X 10(-9)M) the lactam is 7 or 8 membered and the N-1 side chain is carboxymethyl or carboxyethyl. Conformational and steric factors pertinent to binding to the enzyme are discussed.     

Stimulation of prostaglandin E2(PGE2) secretion by benzylpenicilloyl (BPO) carrier conjugates in human peripheral blood lymphocytes

The conjugates of the main determinant of penicillin, benzylpenicilloyl (BPO), with various protein carriers can stimulate mononuclear cells from human peripheral blood lymphocytes (PBL) to increase PGE₂ secretion. Thus, BPO bound to either human γ-globulin (BPO-HGG), bovine γ-globulin (BPO-BGG), and keyhole limpet menocyanin (BPO-KLH) increased the PGE₂ level 8–20 times above the level produced by stimulation with the carriers alone. Since in previous studies it was shown that preincubation with BPO-HGG can suppress the thymidine uptake after subsequent stimulation with BPO-KLH, we investigated if the induction of this unresponsiveness could be caused by PGE₂. The results of this study show that: (a) although the amount of PGE₂ secreted by the stimulation of BPO-HGG was 20-fold greater than the basic level, this amount is not sufficient to suppress significantly the thymidine uptake; (b) preincubation of PBL with higher than physiological concentration of externally added PGE₂ caused significant suppression of thymidine uptake after stimulation with BPO-KLH, but the addition of physiological concentration of PGE₂ did not show the same effect; (c) preincubation with BPO-HGG had no effect on PGE₂ level after subsequent challenge with BPO-KLH; and (d) inhibitor of PGE fails to influence the suppression of BPO-KLH proliferation induced by BPO-KLH.     

Effect of ferrate, a site-specific phosphate analog, on human deoxyhemoglobin

Ferrate ion, a phosphate analog and a potent oxidizing agent, is known to inactivate a number of enzymes which interact with phosphoryl compounds. In contrast, enzymes which do not interact with phosphoryl compounds are not affected by comparable concentrations of ferrate. To further explore the specificity of ferrate as a reagent which is specific for phosphoryl binding sites, a study of its effect on human hemoglobin A was undertaken. In the deoxy form, this protein is known to interact with 2,3-bisphosphoglycerate, its natural allosteric inhibitor of cooperative binding of oxygen, while as oxyhemoglobin it does not interact with the inhibitor. Treatment with ferrate ion caused the loss of approximately three amino acid residues per beta chain of human deoxyhemoglobin, His-2, His-143, and Tyr-145, and one residue, presumably Tyr-42, per alpha chain. Oxyhemoglobin was not affected by the reagent. 2,3-Bisphosphoglycerate was found to protect deoxyhemoglobin from the action of ferrate. His-2 and His-143 are among the residues reported to be implicated in the binding of 2,3-bisphosphoglycerate by deoxyhemoglobin [A. Arnone (1972) Nature (London) 237, 146-148].