Effects of endurance training on three superoxide dismutase isoenzymes in human plasma

The effects of endurance training and acute exhaustive exercise on plasma levels of three superoxidedismutase (SOD) isoenzymes and the ability of superoxide generation in neutrophils were studied. Eighteen healthy male students, aged 17-22 years, who volunteered for this study, underwent three months of endurance training in swimming or running. Before and after the training course, they performed acute exercise and blood samples were collected before and after this exercise. The endurance training significantly increased maximal oxygen uptake (VO2max) in all subjects. Neither the endurance training nor the acute exercise affected the plasma CuZn-SOD level. Acute exercise after the training, but not before the training, increased both the plasma Mn-SOD and extracellular SOD (EC-SOD) levels by 33.6 and 33.5%, respectively. The training decreased the EC-SOD level at rest by 22.2%. Acute exercise after the training, but not before the training, increased the plasma lipid peroxide level, suggesting higher oxidative stress in trained subjects during exhaustive exercise. The ability of neutrophils to generate superoxide was increased by the acute exercise, but induction of the superoxide was suppressed after training. These results indicate that EC-SOD levels were changed in a different manner from the CuZn-SOD and Mn-SOD: it was decreased by training but was increased by acute exercise, suggesting that endurance training increases the reserve of EC-SOD in tissues. The results also suggest the possibility of plasma EC-SOD assay as a new index of endurance training.     

Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron

Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependent dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72+/-0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99+/-0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1+/-6.6 and 3.2+/-1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1+/-5.8, 2.6+/-0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure.     

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.     

The large linear plasmid pSLA2-L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism

The complete nucleotide sequence of the large linear plasmid pSLA2-L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2-L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames. It is especially noteworthy that three-quarters of the pSLA2-L DNA is occupied by secondary metabolism-related genes, namely two type I polyketide synthase (PKS) gene clusters for lankacidin and lankamycin, a mithramycin synthase-like type II PKS gene cluster, a carotenoid biosynthetic gene cluster and many regulatory genes. In particular, the lankacidin PKS is unique, because it may be a mixture of modular- and iterative-type PKSs and carries a fusion protein of non-ribosomal peptide synthetase and PKS. It is also interesting that all the homologues of the afsA, arpA, adpA and strR genes in the A-factor regulatory cascade in Streptomyces griseus were found on pSLA2-L, and disruption of the afsA homologue caused non-production of both lankacidin and lankamycin. These results, together with the finding of three possible replication origins at 50-63 kb from the right end, suggest that the present form of pSLA2-L might have been generated by a series of insertions of the biosynthetic gene clusters into the left side of the original plasmid.     

alpha-Selective glycosylation with 5-thioglucopyranosyl donors; synthesis of an isomaltotetraoside mimic composed of 5-thioglucopyranose units

A general method for alpha-selective glycosylation with 5-thioglucopyranosyl donors followed by efficient deprotection of the resulting products was developed. This methodology was utilized in the synthesis of an isomaltotetraoside analogue.     

A Marked Increase in Free Copper Levels in the Plasma and Liver of LEC Rats: An Animal Model for Wilson Disease and Liver Cancer

Most of copper present in rat plasma and liver binds to caeruloplasmin and metallothionein, respectively, and is not redox active. However, free forms of copper including loosely bound forms to other molecules are redox active. We assessed the free copper in Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease and liver cancer. Compared to those of control rats, the liver and plasma of LEC rats showed a marked elevation of free copper, especially at the stage of acute hepatitis, in parallel with an increase of total copper levels in the livers and a decrease of plasma caeruloplasmin (ferroxidase I) activity. At the onset of jaundice, the total copper levels, however, decreased in liver, but increased in plasma, while free copper levels in both liver and plasma remained higher. Free iron levels in both liver and plasma were also determined and did not change significantly, except for the case of plasma in jaundiced rats. The data are consistent with a proposal in which increased levels of redox active free copper in the liver of LEC rats catalyze Fenton-type reactions, producing a large flux of hydroxyl radicals that would play an important role in the observed liver dysfunction, leading to acute hepatitis, and, finally, hepatocarcinoma. This is the first demonstration that the free copper may participate in the pathophysiology of the LEC rats and Wilson disease.     

Sensitivity of the surface coat of the halotolerant green alga Dunaliella parva (Volvocales, Chlorophyceae) to lysozyme

The surface coat of Dunaliella parva Lerche was investigated using several techniques. Degradation by several cell lytic enzymes and ultrastructural observation revealed that D. parva has a specialized cell surface structure containing a glycoprotein that is sensitive not only to proteinases but also to lysozyme. This sensitivity was also demonstrated by electrophoresis of the cells and measurement of released glycerol after enzyme treatment. Immunochemical labeling indicated that the surface glycoprotein of D. parva is analogous to pepti-doglycan.