Molecular characteristics of tissue-bound semicarbazide-sensitive amine oxidase (SSAO) in guinea pig tissues

Various mammalian tissues contain a tissue-bound amine oxidizing enzyme distinct from mitochondrial outer membrane enzyme, monoamine oxidase (MAO, EC 1.4.3.4), termed semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6). An increase in SSAO activity was found in patients suffering from vascular disorders such as diabetes and diabetic complications. It has previously been shown that 2-bromoethylamine (2-BEA) is a potent, and selective suicidal inhibitor of tissue-bound SSAO. The aim of this study was to investigate the interaction of this suicidal SSAO inhibitor with the tissue-bound enzyme in guinea pig lung, kidney, stomach, and heart homogenates. The conditions necessary for this inhibitor to titrate the concentrations of this enzyme were also determined. 2-BEA appears to interact with SSAO, as reported previously for this enzyme from different sources, in a manner consistent with an irreversible, "suicide" reaction. Because of this property, 2-BEA could be used to titrate the concentrations of SSAO active centers in these tissues under the appropriate conditions employed. Although some possible non-specific binding of the inhibitor to sites other than the active center of the enzyme, metabolism of this inhibitor and/or presence of enzyme subtypes was hypothesized, the molecular characteristics of SSAO in these tissues (Km, Vmax values, enzyme efficiencies, approximate enzyme concentrations, and molecular turnover numbers) towards the substrate kynuramine (0.1 mM) at pH 7.4 and 37 degrees C have been estimated.     

Mutation rate at swine microsatellite loci

During genotyping of 38 microsatellites for QTL (quantitative trait loci) mapping in three F₂ swine populations, five mutant alleles were detected in a total of 66,436 parent-offspring transfers of microsatellite alleles, which gives an overall mutation rate of 7.52×10^–5 per locus per generation. No significant (P<0.05) association between mutation rates and other factors (i.e., GC contents in the flanking regions, heterozygosity, and repeat number) was revealed. Detailed sequencing showed that four out of five mutant alleles were caused by insertions of one to five repeats, respectively. The other mutant allele was produced by either an insertion of three repeats or a change of 30 base pairs (a deletion of 16 CT repeats and an insertion of one CA repeat). An insertion of one base pair in the flanking region of a microsatellite was also detected. Together, these data indicate that expansions are more common than contractions among microsatellites and that the mutation processes are very complicated, do not fit with the strict stepwise mutation model and may vary from locus to locus.     

Role of antioxidant defences in the species-specific response of isolated atria to menadione

In previous works we demonstrated that 2-methyl-1,4-naphthoquinone (menadione) causes a marked increase in the force of contraction of guinea pig and rat isolated atria. This inotropic effect was significantly higher in the guinea pig than in the rat and was strictly related to the amount of superoxide anion (O(2)(*-)), generated as a consequence of cardiac menadione metabolism through mitochondrial NADH-ubiquinone oxidoreductase. The present study was designed to further elucidate the basis of these quantitatively different positive inotropic responses. To this purpose, we measured O(2)(*-) and hydrogen peroxide (H(2)O(2)) produced by mitochondria isolated from guinea pig and rat hearts in the presence of 20 microM menadione. Moreover, we evaluated the menadione detoxification activity (DT-diaphorase) and the antioxidant defences of guinea pig and rat hearts, namely their GSH/GSSG content, Cu/Zn- and Mn-dependent superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx) activities. Our results indicate that DT-diaphorase activity and glutathione levels were similar in both animal species. By contrast, guinea pig mitochondria produced greater amounts of O(2)(*-) and H(2)O(2) than those of rat heart. This is probably due to both the higher Mn-SOD activity (2.93 +/- 0.02 vs. 1.95 +/- 0.06 units/mg protein; P < 0.05) and to the lower Gpx activity (10.09 +/- 0.30 vs. 32.67 +/- 1.02 units/mg protein; P < 0.001) of guinea pig mitochondria. A lower CAT activity was also observed in guinea pig mitochondria (2.40 +/- 0.80 vs. 6.13 +/- 0.20 units/mg protein; P < 0.01). Taken together, these data provide a rational explanation for the greater susceptibility of guinea pig heart to the toxic effect of menadione: because of the greater amount of O(2)(*-) generated by the quinone and the higher mitochondrial Mn-SOD activity, guinea pig heart is exposed to more elevated concentrations of H(2)O(2) that is less efficiently detoxified, because of lower Gpx and CAT levels of mitochondria.     

Genistein inhibits the inward rectifying potassium current in guinea pig ventricular myocytes

Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. Previous studies have shown that genistein has additional effects, among which the direct blocking effects on various ionic channels have recently been disclosed. Using whole-cell voltage clamp and current clamp techniques, we demonstrate that micromolar concentrations of genistein dose-dependently and reversibly inhibit the inward rectifying K(+) current, and depolarize the resting membrane potential, resulting in abnormal automaticity in guinea pig ventricular myocytes. Interestingly, another potent tyrosine kinase inhibitor, tyrphostin 51, did not produce the same inhibitory effect, while the inactive analogue of genistein, daidzein, had a similar blocking effect. We suggest that genistein directly blocks the inward rectifying K(+) current in ventricular myocytes, and one should be cautious of its pro-arrhythmic effect in clinical use.     

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.     

Cloning of the pig renal organic anion transporter 1 (pOAT1)

A pig kidney cDNA library was screened for the porcine ortholog of the multispecific organic anion transporter 1 (pOAT1). Several positive clones were isolated resulting in two alternatively spliced cDNA clones of pOAT1 (pOAT1 and pOAT1A). pOAT1-cDNAs consist of 2126 or 1895 base pairs (EMBL Acc. No. AJ308234 and AJ308235) encoding 547 or 533 amino acid residue proteins with 89, 87, 83 and 81% homology to the human, rabbit, rat, and mouse OAT1, respectively. Heterologous expression of pOAT1 in Xenopus laevis oocytes revealed an apparent K(m) for [3H]PAH of 3.75 +/- 1.6 microM. [3H]PAH uptake mediated by pOAT1 was abolished by 0.5 mM glutarate or 1 mM probenecid. Functional characterization of pOAT1A did not show any affinity for [3H]PAH. In summary, we cloned two alternative splice variants of the pig ortholog of organic anion transporter 1. One splice form (pOAT1) showed typical functional characteristics of organic anion transporter 1, whereas the second form appears not to transport PAH.     

Protein molecular dynamics with electrostatic force entirely determined by a single Poisson-Boltzmann calculation

The electrostatic force including the intramolecular Coulombic interactions and the electrostatic contribution of solvation effect were entirely calculated by using the finite difference Poisson-Boltzmann method (FDPB), which was incorporated into the GROMOS96 force field to complete a new finite difference stochastic dynamics procedure (FDSD). Simulations were performed on an insulin dimer. Different relative dielectric constants were successively assigned to the protein interior; a value of 17 was selected as optimal for our system. The simulation data were analyzed and compared with those obtained from 500-ps molecular dynamics (MD) simulation with explicit water and a 500-ps conventional stochastic dynamics (SD) simulation without the mean solvent force. The results indicate that the FDSD method with GROMOS96 force field is suitable to study the dynamics and structure of proteins in solution if used with the optimal protein dielectric constant.     

Caprellids (Crustacea: Amphipoda) from Papua New Guinea,with the description of a new species

This is the first study of caprellid amphipods from the coast of Papua New Guinea. Several collections from Madang Lagoon (north) and Bootless Bay (south) have been studied. Seven species in seven genera are recorded, of which Pseudoproto papua sp. nov. is described as new to science. The genus Pseudoproto Mayer, 1903 has consisted, so far, of only one species, Pseudoproto fallax Mayer, 1903. Although only a single male has been found of Pseudoproto papua sp. nov., differences in antennae, mouthparts, gnathopod 2 and pereopods 3 and 4 have revealed that it represents a new species of Pseudoproto. Lateral view figures of all species, together with a key to species level for the Caprellidea from Papua New Guinea are also included. Communicated by H.-D. Franke     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Purification,characterization and developmental expression of pig liver PSP

We have isolated a perchloric acid-soluble protein designated as PL-PSP from the post-mitochondria supernatant fraction of pig liver. It is soluble in 5% perchloric acid and purified by ammonium sulfate fractionation and CM-Sephadex chromatography. The PL-PSP showed approximately 80–90% homology with PSP isolated from rat liver (RL-PSP) with its partial amino acid sequences. The protein has a molecular mass of approximately 14 kDa which was slightly higher than that of RL-PSP. It inhibited protein synthesis in a rabbit reticulocyte lysate system. The expression of PL-PSP was predominant in liver, kidney and duodenum, and was also expressed in stomach, lung and brain. PL-PSP expression in liver increased from the 1st day to the 1st month. Thus, our findings are the first report on the presence of a PSP in porcine tissues which may be involved in the regulation of cellular growth and differentiation.