Short-term ascorbic acid deficiency induced oxidative stress in the retinas of young guinea pigs

We examined whether short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs. Four-week-old guinea pigs were given a scorbutic diet (20 g/animal/day) with and without adequate ascorbic acid (400 mg/animal/day) in drinking water for 3 weeks. The serum concentrations of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 14.1 and 4.1%, respectively, of those in the adequate group. The retinal contents of the reduced form of ascorbic acid and the oxidized form of ascorbic acid in the deficient group were 6.4 and 27.3%, respectively, of those in the adequate group. The retinal content of thiobarbituric acid-reactive substances, an index of lipid peroxidation, was 1.9-fold higher in the deficient group than in the adequate group. Retinal reduced glutathione and vitamin E contents in the deficient group were 70.1 and 69.4%, respectively, of those in the adequate group. This ascorbic acid deficiency did not affect serum thiobarbituric acid-reactive substances and reduced glutathione concentrations but increased serum vitamin E concentration. These results indicate that short-term ascorbic acid deficiency induces oxidative stress in the retinas of young guinea pigs without disrupting systemic antioxidant status.     

Effect of centrifuge-induced artificial gravity and ergometric exercise on cardiovascular deconditioning, myatrophy, and osteoporosis induced by a -6 degrees head-down bedrest

We have reported that centrifuge-induced artificial gravity with ergometric exercise could reduce developing cardiovascular deconditioning in humans. In the present study, we examined this load could prevent the myatrophy and osteoporosis induced by head-down bedrest for 20 days. Subjects were ten healthy male volunteers with informed consent. They were requested to lie down at -6 degrees for 20 days, and evaluation for cardiovascular deconditioning, myatrophy, and osteoporosis. As the result, high G-load with low intensity exercise suppressed the orthostatic intolerance and increase in serum osteoporotic marker, whereas low G-load with high intensity ergometric exercise maintained the maximal oxygen intake, heart dimension, and prevented myatrophy. The combination of high/low G-load with low/high intensity exercise will determine the optimal protocol for prevention of cardiovascular deconditioning, myatrophy, and osteoporosis.     

Oligoamine-acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex

Oligoamines (spermidine, dipropylenetriamine and propylenediamine) were covalently attached to acridine via a hexamethylene linker. These oligoamine–acridine conjugates were efficiently bound to gap sites in substrate DNA, and promoted the DNA hydrolysis by a homogeneous Ce(IV)/ethylenediamine-N,N,N′,N′-tetraacetate (EDTA) complex at these sites. In contrast, the hydrolysis of the double-stranded portion in the DNA was little affected by these conjugates, although they were strongly bound thereto by the intercalation of their acridine moieties. As a result, the gap site was selectively and efficiently hydrolyzed by combining the Ce(IV)/EDTA complex with the oligoamine– acridine conjugate. Either the oligoamine or the acridine was only poorly active for the purpose, substantiating the essential role of cooperation between them. The promotion of gap-selective DNA hydrolysis by the conjugates has been ascribed to electrostatic stabilization of a negatively charged transition state by their positive charges.     

Increased dopamine and its metabolites in SH-SY5Y neuroblastoma cells that express tyrosinase

Oxidized metabolites of dopamine, known as dopamine quinone derivatives, are thought to play a pivotal role in the degeneration of dopaminergic neurons. Although such quinone derivatives are usually produced via the autoxidation of catecholamines, tyrosinase, which is a key enzyme in melanin biosynthesis via the production of DOPA and subsequent molecules, may potentially accelerate the induction of catecholamine quinone derivatives by its oxidase activity. In the present study, we developed neuronal cell lines in which the expression of human tyrosinase was inducible. Overexpression of tyrosinase in cultured cell lines resulted in (i) increased intracellular dopamine content; (ii) induction of oxidase activity not only for DOPA but also for dopamine; (iii) formation of melanin pigments in cell soma; and (iv) increased intracellular reactive oxygen species. Interestingly, the expressed tyrosinase protein was initially distributed in the entire cytoplasm and then accumulated to form catecholamine-positive granular structures by 3 days after the induction. The granular structures consisted of numerous rounded, dark bodies of melanin pigments and were largely coincident with the distribution of lysosomes. This cellular model that exhibits increased dopamine production will provide a useful tool for detailed analyses of the potentially noxious effects of oxidized catecholamine metabolites.     

Reduction of natural adenovirus tropism to the liver by both ablation of fiber-coxsackievirus and adenovirus receptor interaction and use of replaceable short fiber

The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and alphav integrin, both CAR binding ability and fiber shaft length played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.     

5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.     

Induction of structural and numerical changes of chromosome, centrosome abnormality, multipolar spindles and multipolar division in cultured Chinese hamster V79 cells by exposure to a trivalent dimethylarsenic compound

Dimethylarsine iodide (DMI) was used as a model compound of trivalent dimethylarsenicals [DMA(III)], and the biological effects were extensively investigated in cultured Chinese hamster V79 cells. When the cytotoxic effects of DMA(III) were compared with those of inorganic arsenite and dimethylarsinic acid [DMA(V)], DMA(III) was about 10,000 times more potent than DMA(V), and it was even 10 times more toxic than arsenite. Depletion of cell glutathione (GSH) did not influence the cytotoxic effects of DMA(III), whereas it enhanced the cytotoxicity of arsenite. Chromosome structural aberrations, such as gaps, breaks and pulverizations, and numerical changes, such as aneuploidy, hyper- and hypo-tetraploidy, were induced by DMA(III) in a concentration-dependent manner. Mitotic index increased 9-12h after the addition of DMA(III), and then declined. By contrast, the incidence of multinucleated cells increased conversely with the decrease in mitotic index at and after 24h of exposure. The mitotic cell-specific abnormality of centrosome integrity and multipolar spindles were induced by DMA(III) in a time- and concentration-dependent manner. Moreover, DMA(III) caused abnormal cytokinesis (multipolar division) at concentrations that were effective in causing centrosome abnormality, multipolar spindles and aneuploidy. These results showed that DMA(III) was genotoxic on cultured mammalian cells. Results also suggest that DMA(III)-induced multipolar spindles and multipolar division may be associated with the induction of aneuploidy. In addition, the centrosome may be a primary target for cell death via multinucleated cells.     

DNA damage by ethylbenzenehydroperoxide formed from carcinogenic ethylbenzene by sunlight irradiation

Ethylbenzene, widely used in human life, is a non-mutagenic carcinogen. Sunlight-irradiated ethylbenzene caused DNA damage in the presence of Cu2+, but unirradiated ethylbenzene did not. A Cu+ -specific chelator bathocuproine inhibited DNA damage and catalase showed a little inhibitory effect. The scopoletin assay revealed that peroxides and H(2)O(2) were formed in ethylbenzene exposed to sunlight. These results suggest that Cu+ and alkoxyl radical mainly participate in DNA damage, and H(2)O(2) partially does. When catalase was added, DNA damage at thymine and cytosine was inhibited. Ethylbenzenehydroperoxide, identified by GC/MS analysis, induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and caused DNA damage at consecutive guanines, as observed with cumenehydroperoxide. Equimolar concentrations of H(2)O(2) and acetophenone were produced by the sunlight-irradiation of 1-phenylethanol, a further degraded product of ethylbenzene. These results indicate a novel pathway that oxidative DNA damage induced by the peroxide and H(2)O(2) derived from sunlight-irradiated ethylbenzene may lead to expression of the carcinogenicity.     

Coordinate induction of AMP deaminase in human atrium with mitochondrial DNA deletion

Despite the heteroplasmic lower population of mitochondrial (mt) DNA deletion, mtDNA deletion is significantly related to the loss of atrial adenine nucleotides. To elucidate its mechanism, we examined the frequency of a 7.4-kb mtDNA deletion, the concentration of adenine nucleotides, and the activity of AMP catabolic enzymes in 10 human right atria obtained from cardiac surgery, using quantitative PCR, HPLC, and immunoprecipitations. The atrial concentrations of ATP, ADP, AMP, and the total adenine nucleotides were significantly lower in patients with deletion than those in patients without deletion, despite the lower frequency of their deletion. The activities of total AMP deaminase (AMPD), liver-type (AMPD 2), and heart-type isoform (AMPD 3) were significantly higher in patients with deletion than in patients without deletion, although there was no significant difference in the cytosolic 5(')-nucleotidase among them. In conclusion, mtDNA deletion coordinately induces AMP deaminase to contribute to the loss of atrial adenine nucleotides through degrading AMP excessively.