Inhibition of oxidative hemolysis in erythrocytes by mitochondria-targeted antioxidants of SkQ series

In the present work we studied the effect of antioxidants of the SkQ1 family (10-(6′-plastoquinonyl)decyltriphenylphosphonium) on the oxidative hemolysis of erythrocytes induced by a lipophilic free radical initiator 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and a water-soluble free radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). SkQ1 was found to protect erythrocytes from hemolysis, 2 μM being the optimal concentration. Both the oxidized and reduced SkQ1 forms exhibited protective properties. Both forms of SkQ1 also inhibited lipid peroxidation in erythrocytes induced by the lipophilic free radical initiator AMVN as detected by accumulation of malondialdehyde. However, in the case of induction of erythrocyte oxidation by AAPH, the accumulation of malondialdehyde was not inhibited by SkQ1. In the case of AAPH-induced hemolysis, the rhodamine-containing analog SkQR1 exerted a comparable protective effect at the concentration of 0.2 μM. At higher SkQ1 and SkQR1 concentrations, the protective effect was smaller, which was attributed to the ability of these compounds to facilitate hemolysis in the absence of oxidative stress. We found that plastoquinone in the oxidized form of SkQ1 could be reduced by erythrocytes, which apparently accounted for its protective action. Thus, the protective effect of SkQ in erythrocytes, which lack mitochondria, proceeded at concentrations that are two to three orders of magnitude higher than those that were active in isolated mitochondria.     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.     

Enhanced production of pigment-free pullulan by a morphogenetically arrested Aureobasidium pullulans (ATCC 42023) in a two-stage fermentation with shift from soy bean oil to sucrose

A two-stage fermentation process was established for the production of pigment-free pullulan by the yeast-like fungus Aureobasidium pullulans (ATCC 42023). In the first stage, starting at pH 4.5 with soy bean oil as the carbon source and glutamate as the nitrogen source, a cell mass of about 15 g l^–1 dry cell weight was obtained, the population being restricted mainly to the yeast form of the microorganism (yeast form more than 90% of total cells) and the formation of pigment in the culture being prevented. Small amounts of pullulan (less than 2 g l^–1) are produced at this phase, and the viscosity remained low throughout the entire growth stage. When the oil and glutamate source were nearly exhausted (below 5% of initial amounts), the cells were shifted to a production stage with sucrose as the carbon source with continued nitrogen depletion. Production of pullulan started immediately with no lag period. During 50 h of the production phase more than 35 g l^–1 of pullulan was produced (productivity approx. 0.7 g l^–1), resulting in a large increase in the viscosity of the broth. The production yield of pollulan on the sugar was about 0.6 g g^–1. Morphogenesis from the yeast form of the microorganism to chlamydospores was still restrained and no pigment was formed in the culture during the production stage. A pigment-free polysaccharide, with a molecular mass in the range of 600–750 kDa, was recovered from the supernatant of the broth after solvent precipitation.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.     

Kinetics of Escherichia coli destruction by microwave irradiation.

The kinetics of destruction of Escherichia coli cells suspended in a solution by microwave irradiation with a microwave oven were studied. During radiation at several powers, the temperature of 0.01 M phosphate buffer (PB), pH 7.0, in a glass beaker increased linearly at a rate of A (degrees Centigrade per second) according to the exposure time. When E. coli cells suspended in PB were exposed in the same beaker, the number of viable cells decreased according to the exposure time and the power used. The survival curve was approximated to a set of three linear parts. For each part, a rate constant of destruction (k) and an extrapolated starting temperature (T0) at several powers were estimated. Thereafter, the relationships between A and k and between A and T0 were studied. When a flat petri dish was used, the A value of exposed PB was lower and bacterial destruction was inhibited; the survival curve was similar to a curve predicted from the A value by using the relationships between the parameters. As the concentration of salt in the solution increased (from 0 to 1.35 M), the A value decreased and bacterial destruction was more suppressed. No remarkable difference between the destruction profiles for microwave exposure and conventional heating, which had the potential to generate an equal A value, was detected. These results showed that the parameter A of an irradiated solution is essential when kinetics of bacterial destruction by microwave exposure are studied and that the destruction profile can be interpreted mostly by means of thermal effects.