Physiological and pathological implications of semicarbazide-sensitive amine oxidase

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the deamination of primary amines. Such deamination has been shown capable of regulating glucose transport in adipose cells. It has been independently discovered that the primary structure of vascular adhesion protein-1 (VAP-1) is identical to SSAO. VAP-1 regulates leukocyte migration and is related to inflammation. Increased serum SSAO activities have been found in patients with diabetic mellitus, vascular disorders and Alzheimer's disease. The SSAO-catalyzed deamination of endogenous substrates, that is, methylamine and aminoacetone, led to production of toxic formaldehyde and methylglyoxal, hydrogen peroxide and ammonia, respectively. These highly reactive aldehydes have been shown to initiate protein cross-linkage, exacerbate advanced glycation of proteins and cause endothelial injury. Hydrogen peroxide contributes to oxidative stress. 14C-methylamine is converted to 14C-formaldehyde, which then forms labeled long-lasting protein adduct in rodents. Chronic methylamine treatment increased the excretion of malondialdehyde and microalbuminuria, and enhanced the formation of fatty streaks in C57BL/6 mice fed with an atherogenic diet. Treatment with selective SSAO inhibitor reduces atherogenesis in KKAy diabetic mice fed with high-cholesterol diet. Aminoguanidine, which blocks advanced glycation and reduces nephropathy in animals, is in fact more potent at inhibiting SSAO than its effect on glycation. It suggests that SSAO is involved in vascular disorders under certain pathological conditions. Although SSAO has been known for several decades, its physiological and pathological implications are just beginning to be recognized.     

The NADPH oxidase of phagocytic cells is an electron pump that alkalinises the phagocytic vacuole

Summary Phagocytic cells of the immune system contain an oxidase that is important for the killing and digestion of engulfed microbes. This is an electron transport chain that transfers electrons from NADPH in the cytosol to oxygen to form superoxide and hydrogen peroxide in the phagocytic vacuole. Absence or abnormality of this oxidase results in the syndrome of CGD, characterised by a profound predisposition to infection. The electron transport chain consists of a flavocytochrome b located in the plasma membrane and membrane of the specific granules. It is composed of a and b-subunits, with apparent molecular masses of 23 kDa and 76–92 kDa, respectively. The b-subunit is a member of the FNR family of reductases with FAD and NADPH binding sites. Based upon the crystal structure of FNR we have constructed a model of the more hydrophilic C terminal half of this b-subunit, which acts as a guide to the organisation of the molecule, and provides a template on which to map mutations in CGD. The location of the heme is uncertain. Electron transport is dependent upon an activation complex of cytosolic proteins including p40 ^phox , p47 ^phox , and p67 ^phox , and the small GTP binding protein, p21 ^rac . This oxidase system is important for the killing and digestion of bacteria and fungi. This might be accomplished in a number of ways. The oxidase produces superoxide and hydrogen which might be toxic themselves. The hydrogen peroxide can act as substrate for myeloperoxidase which can oxidise chloride and iodide to chlorine and iodine and their hypohalous acids. The proteins contained within the cytoplasmic granules are also very important in the killing process. These are neutral proteinases that require a neutral or slightly alkaline pH for optimal activity. The oxidase transports electrons, unaccompanied by protons, across the wall of the phagocytic vacuole, resulting in an elevation of the vacuolar pH, thereby optimising conditions for killing and digestion of engulfed organisms by these neutral proteinases.     

NADPH binding component of neutrophil superoxide-generating oxidase

The 2',3'-dialdehyde derivative of NADPH was used as an affinity labeling reagent of a solubilized NADPH-dependent superoxide-generating oxidase preparation of pig neutrophils. The analogue served as both an electron donor and a competitive inhibitor of the NADPH oxidase against NADPH. The apparent Michaelis constant (Km) for the derivative (31 microM) was essentially the same as that for NADPH (33 microM). The activity of the superoxide formation in the presence of 2',3'-dialdehyde NADPH was about a half of that in the presence of NADPH. Incubation of the enzyme with the derivative inactivated the superoxide-generating activity and the inactivation was prevented by the addition of NADPH. We performed the labeling of the oxidase preparation with 2',3'-dialdehyde NADPH and sodium cyanoboro[3H]hydride, based on the above results. A protein of 66,000 daltons was selectively labeled among more than 20 bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were visualized with Coomassie Brilliant Blue. The protein was not labeled when the oxidase preparation was pretreated with p-chloromercuribenzoate or it was labeled in the presence of excess NADPH. The protein is suggested to be the NADPH binding component of the neutrophil superoxide-generating oxidase system.     

NOX3, a superoxide-generating NADPH oxidase of the inner ear

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed (e.g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47(phox) and p67(phox), but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs.     

The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.     

The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel.

The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.     

Physiological and molecular aspects of salt stress in plants

The study of salt stress mechanisms in plants has become an important issue for the modern agricultural development, climate change, and global food crisis. The plant response to high salt concentrations is complex and comprehensive; it includes many different processes, which should be correctly coordinated. The effect of excessive salt concentrations on plants results in osmotic stress and creates an ionic inbalance due to the accumulation of toxic ions, such as Cl^? and, especially, Na⁺. Salt stress also has negative impact on mineral homeostasis, in particular Ca²⁺ and K⁺. The progress in transcryptomics, genomics, and molecular biology revealed a new gene families that participate in the formation of salt stress response in plants. This review describes the fundamental principles and mechanisms of plant salt tolerance, maintenance of ion homeostasis. In this paper the detailed analysis of the maine transport membrane systems responsible for the transport of ions and their role in plant salt stress were conducted. The perspectives and directions for the further biotechnological and genetic improvement of salt tolerance in plants are underlied.     

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.     

Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.     

Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages.

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the participation of both membrane and cytosolic components. We reported that ammonium sulfate fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143, 4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of cytosol in two fractions (sigma 1 and sigma 2) that support O2- production by solubilized membrane synergistically. We now describe the purification of sigma 1 to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction sigma 2. Sigma 1 was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether sigma 1 was added to cell-free systems composed of: (a) solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b) purified cytochrome b559, incorporated in liposomes, and purified sigma 2. Sigma 1 was purified by a sequence comprising ammonium sulfate fractionation, hydrophobic chromatography on phenyl-Superose, absorption with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sigma 1 of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the presence of both 24- and 22-kDa bands. We suggest that, in its native form, sigma 1 might represent a complex of the 24- and 22-kDa proteins. The specific roles of each molecule in NADPH oxidase function remain to be determined.     

Physiological aspects of surface-immobilized Catharanthus roseus cells

Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O₂ consumption and CO₂ production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures. Offprint requests to: Denis Rho