Adrenodoxin reductase and adrenodoxin. Mechanisms of reduction of ferricyanide and cytochrome c.

Adrenodoxin reductase, the flavoprotein moiety of the adrenal cortex mitochondrial steroid hydroxylating system, participates in adrenodoxin-dependent cytochrome c and adrenodoxin-independent ferricyanide reduction, with NADPH as electron donor for both of these 1-electron reductions. For ferricyanide reduction, adrenodoxin reductase cycles between oxidized and 2-electron-reduced forms, reoxidation proceeding via the neutral flavin (FAD) semiquinone form (Fig. 9). Addition of adrenodoxin has no effect upon the kinetic parameters of flavoprotein-catalyzed ferricyanide reduction. For cytochrome c reduction, the adrenodoxin reductase-adrenodoxin 1:1 complex has been shown to be the catalytically active species (Lambeth, J. D., McCaslin, D. R., and Kamin, H. (1976) J. Biol. Chem. 251, 7545-7550). Present studies, using stopped flow techniques, have shown that the 2-electron-reduced form of the complex (produced by reaction with 1 eq of NADPH) reacts rapidly with 1 eq of cytochrome c (k approximately or equal to 4.6 s-1), but only slowly with a second cytochrome c (k = 0.1 to 0.3 s-1). However, when a second NADPH is included, two more equivalents of cytochrome are reduced rapidly. Thus, the adrenodoxin reductase-adrenodoxin complex appears to cycle between 1- and 3-electron reduced states, via an intermediate 2-electron-containing form produced by reoxidation by cytochrome (Fig. 10). For ferricyanide reduction by adrenodoxin reductase, the fully reduced and semiquinone forms of flavin each transfer 1 electron at oxidation-reduction potentials which differ by approximately 130 mV. However, adrenodoxin in a complex with adrenodoxin reductase allows electrons of constant potential to be delivered from flavin to cytochrome c via the iron sulfur center...     

Point mutations in the proline-rich region of p22phox are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation

The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the membrane-associated gp91phox (also known as Nox2). p22phox associates with gp91phox and, through its proline-rich C terminus, provides a binding site for the tandem Src homology 3 domains of the activating subunit p47phox. Whereas p22phox is expressed ubiquitously, its participation in regulating the activity of other Nox enzymes is less clear. This study investigates the requirement of p22phox for Nox enzyme activity and explores the role of its proline-rich region (PRR) for regulating activity. Coexpression of specific Nox catalytic subunits (Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen. Small interfering RNAs decreased endogenous p22phox expression and inhibited reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5. Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly, p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, potently inhibited reactive oxygen production from Nox1 and Nox2 but not from Nox4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3 activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q and P160Q mutations of p22phox showed selective inhibition of Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit (p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress the importance of p22phox for the function of Nox1, Nox2, Nox3, and Nox4, and emphasize the key role of the PRR for regulating Nox proteins whose activity is dependent upon p47phox or NOXO1.     

Evaluation of two anti-gp91phox antibodies as immunoprobes for Nox family proteins: mAb 54.1 recognizes recombinant full-length Nox2, Nox3 and the C-terminal domains of Nox1-4 and cross-reacts with GRP 58

Progress in the study of Nox protein expression has been impeded because of the paucity of immunological probes. The large subunit of human phagocyte flavocytochrome b558 (Cytb), gp91phox, is also the prototype member of the recently discovered family of NADPH oxidase (Nox) proteins. In this study, we have evaluated the use of two anti-gp91phox monoclonal antibodies, 54.1 and CL5, as immunoprobes for Nox family proteins. Sequence alignment of gp91phox with Nox1, Nox3 and Nox4 identified regions of the Nox proteins that correspond to the gp91phox epitopes recognized by mAb 54.1 and CL5. Antibody 54.1 produced positive immunoblots of recombinant C-terminal fragments of these homologous proteins expressed in E. coli. Furthermore, only mAb 54.1 recognized full-length murine and human Nox3 expressed in HEK-293 cells, in immunoblots of alkali-stripped or detergent-solubilized membranes. 54.1 recognized Nox3 expression-specific proteins with Mr 30,000, 50,000, 65,000 and 88,000 for the murine protein and Mr of 38,000-58,000, 90,000, 100,000-130,000 and a broad species of higher than 160,000 for the human protein. We conclude that mAb 54.1 can serve as a probe of Nox3 and possibly other Nox proteins, if precautions are taken to remove GRP 58 and other crossreactive membrane-associated or detergent-insoluble proteins from the sample to be probed.     

NOXO1, regulation of lipid binding, localization, and activation of Nox1 by the Phox homology (PX) domain

NOXO1 (Nox organizing protein 1) and NOXA1 (Nox activating protein 1) are homologs of p47phox and p67phox. p47phox functions in phagocytes as an essential organizing protein mediating the binding of other regulatory proteins during activation of the phagocyte oxidase, and its translocation to the membrane is triggered upon cell activation by hyperphosphorylation, which relieves autoinhibition of SH3 and PX domains. NOXO1 lacks an autoinhibitory region and phosphorylation sites that are present in p47phox. Co-transfection of Nox1, NOXO1, and NOXA1 reconstitutes ROS (reactive oxygen species) generation in HEK293 cells in the absence of cell stimulation. NOXO1 binds to the phosphatidylinositol (PtdIns) lipids PtdIns 3,5-P2, PtdIns 5-P, and PtdIns 4-P. Unlike p47phox, which is located in the cytosol of resting cells and translocates to the plasma membrane where gp91phox is located, NOXO1 co-localizes with Nox1 in the membranes of resting cells. This localization of NOXO1 is dictated by its PX domain, since this domain but not the remainder of the molecule localizes to membranes. A point mutation in the PX domain of holo-NOXO1 decreases lipid binding resulting in cytosolic localization and also inhibits NOXO1-activation of Nox1. Thus, in transfected HEK293 cells, NOXO1 and NOXA1 activate Nox1 without the need for agonist activation, and this is mediated in part by binding of the NOXO1 PX domain to membrane lipids.     

Identification of adult mouse neurovirulence determinants of the Sindbis virus strain AR86

Sindbis virus infection of mice has provided valuable insight into viral and host factors that contribute to virus-induced neurologic disease. In an effort to further define the viral genetic elements that contribute to adult mouse neurovirulence, the neurovirulent Sindbis virus strain AR86 was compared to the closely related (22 single amino acid coding changes and the presence or absence of an 18-amino-acid sequence in nsP3 [positions 386 to 403]) but avirulent Girdwood strain. Initial studies using chimeric viruses demonstrated that genetic elements within the nonstructural and structural coding regions contributed to AR86 neurovirulence. Detailed mapping studies identified three major determinants in the nonstructural region, at nsP1 538 (Ile to Thr; avirulent to virulent), an 18-amino-acid deletion in nsP3 (positions 386 to 403), and nsP3 537 (opal to Cys; avirulent to virulent), as well as a single determinant in the structural genes at E2 243 (Leu to Ser; avirulent to virulent), which were essential for AR86 adult mouse neurovirulence. Replacing these codons in AR86 with those found in Girdwood resulted in the attenuation of AR86, while the four corresponding AR86 changes in the Girdwood genetic background increased virulence to the level of wild-type AR86. The attenuating mutations did not adversely affect viral replication in vitro, and the attenuated viruses established infection in the brain and spinal cord as efficiently as the virulent viruses. However, the virus containing the four virulence determinants grew to higher levels in the spinal cord at late times postinfection, suggesting that the virus containing the four attenuating determinants either failed to spread or was cleared more efficiently than the wild-type virus.     

Cholesterol sulfate inhibits adrenal mitochondrial cholesterol side chain cleavage at a site distinct from cytochrome P-450scc. Evidence for an intramitochondrial cholesterol translocator

Cholesterol sulfate inhibits (K1/2, 6 microM) the side chain cleavage of exogenous cholesterol in intact rat adrenal mitochondria. Inhibition is at a site other than cytochrome P-450scc: the spin state of the hemoprotein is not perturbed, and its activity is unaffected as judged by the failure to inhibit the metabolism both of 25-hydroxycholesterol and of endogenous cholesterol in a mitochondrial "steroidogenic pool." In contrast, 25-hydroxycholesterol, known to interact with the cytochrome, prevented the cleavage of both endogenous and exogenous cholesterol and produced the expected optical changes in the hemoprotein. Inhibition was specific, since a variety of related compounds including pregnenolone sulfate were not effective. Metabolic conversion to other species was insufficient to account for inhibition, indicating that cholesterol sulfate is the effective molecule. A hallmark of an inhibitor of a transport system is that disruption of the barrier to transport eliminates inhibition. Sonic disruption of mitochondria abated by 70% the effect of cholesterol sulfate, but did not affect inhibition by 25-hydroxycholesterol. Thus, the cholesterol sulfate appears to inhibit an intramitochondrial cholesterol translocation system that functions to move cholesterol into a steroidogenic pool. The high content of cholesterol sulfate in adrenal cortex (Drayer, N.M., Roberts, K.D., Bandi, L., and Lieberman, S. (1964) J. Biol. Chem. 239, 3112-3114) suggests a possible regulatory role for this molecule.     

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

The implementation and initial evaluation of a physical therapy program for captive chimpanzees (Pan troglodytes)

Due to advances in captive nonhuman primate (NHP) medical care, the number of geriatric chimpanzees (≥35-years old) is growing. With old age comes a variety of physical conditions, including arthritis, stroke, and mobility impairments. Programs aimed at enhancing the welfare of geriatric chimpanzees are now quite common, but there are few published empirical evaluations of the efficacy of such programs. The current study aimed to create, implement, and evaluate the effects of participation in a physical therapy (PT) program on physical health, mobility, welfare, and behavior. Nine chimpanzees with mobility impairments participated in personalized PT routines (using positive reinforcement training) twice per week for 5 months. Additionally, nine control chimpanzees (non-mobility-impaired, matched with PT chimpanzees on age and gender) participated in body exam behavior sessions (also using positive reinforcement training) twice per week. All chimpanzees were rated on 14 health, well-being, and behavior items, as well as level of mobility throughout the PT program. Chimpanzees that participated in the PT program showed significant increases in ratings of physical health, well-being, and activity levels across phases of the program. Furthermore, compared to control chimpanzees, PT chimpanzees showed significant increases in ratings of ease of movement. Because raters were not blind to physical therapy treatment, our results represent an initial evaluation of the program that may suggest that participation in the PT program has physical, behavioral, and welfare benefits. Assessments of novel geriatric-focused care strategies and programs are essential to further enhance the welfare of the captive chimpanzee population, which is currently comprised of many geriatric animals, whose proportion of the captive population will only increase.     

Regulation of mitochondrial compartment volumes in rat adrenal cortex by ether stress

In vivo ether stress of rats causes release of pituitary adrenocorticotropin (ACTH) leading to activation of steroidogenesis in adrenal cortex mitochondria. The present studies show that this treatment also induces a decrease in the volume of the intermembrane space in isolated adrenal mitochondria. This decrease is accompanied by an increase in the volume of the matrix, thus leaving the total mitochondrial volume approximately constant. These effects are prevented by the protein synthesis inhibitor, cycloheximide, and are specific to the adrenal gland. The decrease in the intermembrane space (or increase in the matrix volume) is correlated with activation of the cholesterol side chain cleavage reaction (the regulated step in steroidogenesis). We propose as a working hypothesis that these changes reflect a hormonally regulated alteration in the relationship between the outer and inner mitochondrial membranes, which may facilitate the rate-limiting movement of cholesterol from the outer to the inner membrane where the side chain cleavage enzyme is located.     

Modulation of the free sphingosine levels in human neutrophils by phorbol esters and other factors

Because free long-chain bases have been recently found to have potent pharmacological effects when added to neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623) and other cell types, the levels in human neutrophils were measured by high-performance liquid chromatography. Sphingosine was the major free long-chain base in freshly isolated cells and ranged from 13 to 101 pmol/10(7) cells for different donors (mean +/- S.E. of 50 +/- 5, n = 17). Upon incubation at 37 degrees C, there was a time-dependent increase in free sphingosine (57 +/- 8% in 1 h, n = 17), but no change was seen at 4 or 25 degrees C. The sphingosine was apparently derived from more complex sphingolipids because little (less than 1%) could be accounted for by new synthesis from [14C]serine. Greater increases in free sphingosine were obtained when neutrophils were incubated with serum, plasma, or serum lipoproteins (about 2-fold higher than for cells incubated alone). In contrast, agonists such as phorbol 12-myristate 13-acetate, A23187, arachidonic acid, low concentrations (10 nM) of N-formyl-methionyl-leucyl-phenylalanine, and opsonized zymosan either decreased the amount of free sphingosine or blunted the time-dependent increase. This may be due to enhanced removal of free sphingosine because phorbol 12-myristate 13-acetate-treated cells exhibited an increased conversion of exogenously added [3H]sphinganine to ceramides. Endogenous sphingosine was approximately one-tenth the level found in neutrophils when exogenous long-chain bases were added to inhibit protein kinase C. Hence, depending on the subcellular localization of the endogenous versus exogenous long-chain bases, the amounts of free sphingosine in neutrophils might be sufficient to affect the function of these cells.