Metabolism of acetaldehyde to methyl and acetyl radicals: in vitro and in vivo electron paramagnetic resonance spin-trapping studies

Acetaldehyde oxidation by enzymes and cellular fractions has been previously shown to produce radicals that have been characterized as superoxide anion, hydroxyl, and acetyl radicals. Here, we report that acetaldehyde metabolism by xanthine oxidase, submitochondrial particles and whole rats produces both the acetyl and the methyl radical, although only the latter was unambiguously identified in vivo. Electron paramagnetic resonance (EPR) characterization of both radicals was possible by the use of two spin traps, 5,5-dimethyl 1-pyrroline N-oxide (DMPO) and alpha-(4-pyridyl 1-oxide)-N-t-butylnitrone (POBN), and of acetaldehyde labeled with (13)C. The POBN-acetyl radical adduct proved to be unstable, but POBN was employed to monitor acetaldehyde metabolism by Sprague-Dawley rats because previous studies have shown its usefulness for in vivo spin trapping. EPR analysis of the bile collected from treated and control rats showed the presence of the POBN-methyl and of an unidentified, biomolecule-derived, POBN adduct. Because decarbonylation of the acetyl radical is one of the routes for methyl radical formation from acetaldehyde, detection of the latter in bile provides strong evidence for the production of both radicals in vivo. The results may be relevant to understanding the toxic effects of acetaldehyde itself and of its more relevant biological precursor, ethanol.     

Multiple forms of α2-macroglobulin from a bony fish, the common carp (Cyprinus carpio): striking sequence diversity in functional sites

Unlike mammals, bony fish possess multiple genes encoding the complement component C3, a member of the alpha2-macroglobulin (alpha2M) protein family, presumably expanding the diversity of immune recognition. To examine whether the alpha2M gene has also duplicated and diverged in the bony fish lineage, cDNA cloning of alpha2M from a pseudotetraploid teleost, the common carp (Cyprinus carpio), was conducted and resulted in the isolation of three distinct alpha2M sequences from a single individual, indicating the presence of multiple alpha2M genes in this species. The deduced amino acid sequences contained a post-translational cleavage signal, predicting a C3-like two-chain structure, as in lamprey alpha2M. Two distinct alpha2M proteins were purified from carp serum; both proved to be Mr 380,000 dimers, the subunits of which are composed of disulfide-linked alpha chains (Mr 93,000) and beta chains (Mr 85,000), as reported for the alpha2M from plaice, another teleost species. The presence of an internal thioester in the alpha chain was demonstrated by its autolytic fragmentation and direct incorporation of [14C]methylamine. Interestingly, the three forms of carp alpha2M exhibited outstanding sequence diversity in the bait region which displays target sequences for various proteases, and in the C-terminal region of the alpha chain assigned as the receptor-binding domain, while an Asn residue at the position corresponding to the catalytic His in C3 was completely conserved in the carp alpha2Ms, as in most alpha2Ms of other animals. The possible functional significance of the sequence diversity is discussed.     

A study on the surface hydrophobicity of lipases

Surface properties, including surface net and local hydrophobicities, of bovine serum albumin, gamma-globulin, and six lipases of different origins were evaluated using the aqueous two-phase partitioning method. Each showed a specific and characteristic pattern of surface properties. Correlations between the protein surface hydrophobicities and the coverages of the proteins by lipid-coating with a synthetic detergent, dioleyl glucosyl L-glutamate, were discussed. The results indicated that the surface net hydrophobicity of each protein was indicative of the affinity of the protein for the coating detergent applied in lipid-coating.     

Dynamics of a hybrid system of a brain neural network and an artificial nonlinear oscillator

In the brain, many functional modules interact with each other to execute complex information processing. Understanding the nature of these interactions is necessary for understanding how the brain functions. In this study, to mimic interacting modules in the brain, we constructed a hybrid system mutually coupling a hippocampal CA3 network as an actual brain module and a radial isochron clock (RIC) simulated by a personal computer as an artificial module. Return map analysis of the CA3-RIC system's dynamics showed the mutual entrainment and complex dynamics dependent on the coupling modes. The phase response curve of CA3 was modeled regarding the CA3 as a nonlinear oscillator. Using the phase response curves of CA3 and RIC, we reconstructed return maps of the hybrid system's dynamics. Although the reconstructed return maps almost agreed with the experimental data, there were deviations dependent on the coupling mode. In particular, we noted that the deviation was smaller under the bidirectional coupling conditions than during the one-way coupling from RIC to CA3. These results suggest that brain modules may flexibly change their dynamical properties through interaction with other modules.     

Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney

Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.     

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-alpha and interleukin-1beta contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Insulin resistance and endothelial dysfunction in smokers: effects of vitamin C

Cigarette smoking impairs endothelial function and is one of the major risk factors for atherosclerosis and coronary heart disease. Insulin resistance is associated with major risk factors for atherosclerosis. We examined the effects of vitamin C on insulin sensitivity and endothelial function by measuring steady-state plasma glucose (SSPG) and flow-mediated dilation (FMD) of the brachial artery. We studied 16 current smokers with normal glucose tolerance, 15 nonsmokers with impaired glucose tolerance (IGT), and 17 nonsmokers with normal glucose tolerance as controls. Both SSPG and FMD were blunted in smokers and nonsmokers with IGT compared with controls. In smokers, vitamin C decreased SSPG (P < 0.01 by ANOVA) with decreasing plasma thiobarbituric acid-reactive substances (TBARS) (P < 0.05 by ANOVA) and improved FMD (P < 0.05 by ANOVA). Furthermore, vitamin C improved both SSPG (P < 0.005 by ANOVA) and FMD (P < 0.05 by ANOVA) in nonsmokers with IGT. SSPG, FMD, or TBARS in controls did not change after vitamin C infusion. There was a significant correlation between SSPG and FMD both in smokers and nonsmokers with IGT, whereas no correlation was observed in controls. In conclusion, both insulin sensitivity and endothelial function were impaired in smokers and nonsmokers with IGT and were improved by vitamin C. Thus increased reactive oxygen species play an important role in the pathogenesis of insulin resistance as well as endothelial dysfunction in smokers and nonsmokers with IGT.     

The role of LH pulse frequency in ACTH-induced ovarian follicular cysts in heifers

The aim of the present study was to induce ovarian cysts experimentally in cattle using ACTH and to closely examine the role of LH pulse frequency in ovarian cyst formation. Five regularly cycling Holstein-Friesian heifers (15-18-month-old) were used. Ovaries were scanned daily using an ultrasound scanner with a 7.5 MHz rectal transducer. Daily blood samples were obtained via tail venepuncture for hormone analyses. Additional blood samples (for FSH and LH pulses) were obtained through an indwelling jugular vein catheters every 15 min for 8 h on Days 2 (early luteal phase; ELP), 12 (mid-luteal phase; MLP) and 19 (follicular phase; FP) of control estrous cycle and on alternate days during follicular cyst (FC) formation and persistence. Cysts were induced using subcutaneous injections of ACTH (Cortrosyn) Z; 1 mg) every 12 h for 7 days beginning on Day 15 of the subsequent estrous cycle. Plasma concentrations of progesterone (P4), estradiol-17beta, FSH and LH were determined by double antibody radioimmunoassay while cortisol concentration was determined by enzyme immunoassay (EIA). Ovarian follicular and endocrine dynamics were normal during the control estrous cycles. Ovarian follicular cysts were induced in four of the five heifers. Mean maximum size of cysts was larger (P<0.05) than that of ovulatory follicles (26.78+/-3.65 versus 14.1+/-0.90 mm), respectively. Cortisol levels were increased during ACTH treatment. High concentrations of estradiol and low progesterone were observed after cyst formation. LH pulse frequency was significantly reduced (P<0.05) during cyst formation and persistence compared to ELP (7.5+/-0.75) and FP (6.5+/-0.58), but was not significantly (P=0.23) different from MLP (2.8+/-0.29) pulses. Mean LH pulse amplitude and concentrations were not different. Similarly, the mean pulse frequency, amplitude and concentration of FSH were not different between control study and cystic heifers. These results suggest that the LH pulse frequency observed following ACTH treatment may interact with high estradiol concentration to induce ovarian cyst formation in heifers.