Mika Jikumaru

Effect of oxidative stress during repeated ovulation on the structure and functions of the ovary, oocytes, and their mitochondria

We previously reported that superoxide generated in the ovary induces apoptosis of granulosa cells to break down follicular walls, thereby supporting ovulation in rodents, and suggested that oxidative stress underlies the mechanism of ovarian aging. To test this hypothesis, we successfully induced ovulation repeatedly in mice by sequentially administrating pregnant mare serum gonadotropin, human chorionic gonadotropin, and prostaglandin F2alpha. Kinetic analysis revealed that the number of ovulated oocytes decreased significantly with repeated cycles of ovulation with a concomitant decrease in the gene expression of mitochondrial transcription factor A and nuclear respiratory factor 1 and an increase in oocytes having abnormally distributed mitochondria. Repeated ovulation decreased the amounts of mitochondrial DNA and increased 8-hydroxydeoxyguanosine in oocytes. Cell culture analysis of the in vivo fertilized oocytes revealed that their maturation from two cells to blastocyst was inhibited significantly by repeated ovulation. All these events induced by repeated ovulation were suppressed by oral administration of L-carnitine. These results suggest that oxidative stress associated with ovulation underlies the mechanism of ovarian aging and that L-carnitine may have therapeutic potential in patients with infertility and increased incidence of aneuploidy and to suppress impaired maturation of zygotes frequently observed in childbearing at an advanced age.

Mitochondria Determine the Efficacy of Anticancer Agents that Interact with DNA but Not the Cytoskeleton

Although chemotherapy is an important method for the treatment of patients with cancer, its efficacy is limited because of different sensitivities of tumor cells to anticancer agents and/or side effects on normal tissues. The present work demonstrates that mitochondria play a crucial role in the apoptosis of cancer cells induced by anticancer agents that interact with DNA but not with the cytoskeleton. Agents that interact with DNA selectively enhanced generation of reactive oxygen species (ROS) in mitochondria, released cytochrome c, and activated caspase-9 and caspase-3 to induce apoptosis of mesothelioma H2052 cells but not their ρ0 cells, which lack mitochondrial DNA (mtDNA). The sensitivity of a variety of cells to the agents showed positive correlation with the amounts of their mitochondria. In contrast, agents that selectively affect the cytoskeleton activated caspase-8 and caspase-3 and equally induced apoptosis of both H2052 and their ρ0 cells by a mitochondria-independent mechanism. The results suggest that mtDNA is a potential target for the anticancer agents that interact with DNA to induce ROS-dependent apoptosis of cancer cells, whereas agents that affect the cytoskeleton induce cell death by a mitochondria- and ROS-independent mechanism. The present observation is important for the selection of medicine for chemotherapy of patients with cancer.

Ultraviolet‐A Irradiation to the Eye Modulates Intestinal Mucosal Functions and Properties of Mast Cells in the Mouse

We previously reported that topical irradiation of the eye by ultraviolet‐B (UVB) activated hypothalamo‐pituitary‐adrenal axis (HPA‐A) of the mouse to increase 3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocytes in the skin by an inducible nitric oxide synthase (iNOS)‐dependent mechanism. This work demonstrates that irradiation of the eye by ultraviolet‐A (UVA) specifically increased DOPA‐positive cells in the mucosa of the jejunum and colon of C57BL/6J mice by some HPA‐ and iNOS‐independent mechanism. UVA‐induced increase in DOPA‐positive cells in the intestine was inhibited by the administration of hexamethonium or prazosin plus propranolol, blockers for the sympathetic nervous system. UVA irradiation of the eye increased DOPA‐ and histidine decarboxylase (HDC)‐positive cells in the intestinal mucosa of both C57BL/6J and WBB6F1/J mice but not in the mutant strain W/Wv of the latter that lack mast cells. UVA irradiation of the eye suppressed the intestinal peristalsis of control, hypophysectomized or iNOS−/− C57BL/6J mice by the mechanism that was inhibited by hexamethonium or prazosin plus propranolol. These observations suggest that UVA irradiation of the eye stimulated the sympathetic nervous system to increase the mucosal DOPA‐ and HDC‐positive mast cells and suppressed the peristalsis of the small intestine of the mouse.

Dynamic aspects of ascorbic acid metabolism in the circulation: analysis by ascorbate oxidase with a prolonged in vivo half-life.

Because AA (L-ascorbic acid) scavenges various types of free radicals to form MDAA (monodehydroascorbic acid) and DAA (dehydroascorbic acid), its regeneration from the oxidized metabolites is critically important for humans and other animals that lack the ability to synthesize this antioxidant. To study the dynamic aspects of AA metabolism in the circulation, a long acting AOase (ascorbate oxidase) derivative was synthesized by covalently linking PEG [poly(ethylene glycol)] to the enzyme. Fairly low concentrations of the modified enzyme (PEG-AOase) rapidly decreased AA levels in isolated fresh plasma and blood samples with a concomitant increase in their levels of MDAA and DAA. In contrast, relatively high doses of PEG-AOase were required to decrease the circulating plasma AA levels of both normal rats and ODS (osteogenic disorder Shionogi) rats that lack the ability to synthesize AA. Administration of 50 units of PEG-AOase/kg of body weight rapidly decreased AA levels in plasma and the kidney without affecting the levels in other tissues, such as the liver, brain, lung, adrenal grand and skeletal muscles. PEG-AOase slightly, but significantly, decreased glutathione (GSH) levels in the liver without affecting those in other tissues. Suppression of hepatic synthesis of GSH by administration of BSO [L-buthionin-(S,R)-sulfoximine] enhanced the PEG-AOase-induced decrease in plasma AA levels. These and other results suggest that the circulating AA is reductively regenerated from MDAA extremely rapidly and that hepatic GSH plays important roles in the regeneration of this antioxidant.

Prevention of scattered light-induced asthenopia and fatigue by a polarized filter

Background: It has been well documented that a long‐time irradiation of the eye by a strong light elicits eyestrain and fatigue. To elucidate the mechanism for the induction of light‐induced fatigue and asthenopia, changes in the mouse were analyzed after white light‐irradiation to the eye.

Fasting differentially modulates the immunological system: its mechanism and sex difference.

The immunological properties and hormonal metabolism in rodents are affected by physical and psychological stress more strongly in males than in females. To elucidate the mechanism and physiological significance of the sex difference in the susceptibility of animal to stresses, changes in the immunological system in plasma and intestine and hormonal status in plasma were compared among 8-week-old male and female ICR mice before and after fasting. During the fasting of animals, the expression of immunoglobulin A in intestinal mucosa, and cortisol, interleukin-10 and interferon-γ in plasma increased. These changes occurred more apparently in males than in females. Under identical conditions, the plasma levels of testosterone decreased markedly with concomitant occurrence of apoptosis in the testis, while the plasma levels of estradiol decreased calmly, and no appreciable apoptosis occurred in the ovary. These results indicate that testosterone enhances the stress-induced modulation of the immune system by some mechanism that was antagonized by estradiol.

Role of hydrogen generation by Klebsiella pneumoniae in the oral cavity

Some gastrointestinal bacteria synthesize hydrogen (H2) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H2 or what role H2 might play in the oral cavity. It was found that a significant amount of H2 is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H2-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H2. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H2 in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe++ and H2O2 increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H2 generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H2 generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.

α-Melanocyte-Stimulating Hormone Plays an Important Role in the Onset of Pollinosis in a Pollen Allergy Mouse Model

Background: α-Melanocyte-stimulating hormone (α-MSH) is a neuropeptide that controls melanogenesis in pigmentary cells. In addition, its potent immunomodulatory activity has been recently described in cutaneous inflammatory disorders. However the mechanism of such pollen allergies remains to be elucidated. The purpose of this study was to investigate the role of α-MSH in a murine model of pollen allergy. Methods: Eight-week-old male BDF-1 mice were sensitized with Cry j I. After the last intranasal antigen, the number of sneezes was counted for 5 min. In addition, the serum levels of IgE and neuronal hormones were measured by ELISA. The expression of IgA, melanocortin receptor 1 (MC1R) and MC5R in the trachea were also observed by immunohistochemistry. Results: Both the concentration of α-MSH and adrenocorticotropin in plasma increase in pollen allergy model mice. Furthermore, MC5R increased in the trachea; however, MC1R did not increase in the trachea. In addition, the changes in sneezing and IgA expression in the pollen allergy model mice were suppressed by α-MSH antibody treatment, but they remained unchanged after MC1R antagonist (agouti) treatment. Conclusions: These results indicate that sneezing due to pollen allergy is associated with an increased concentration of α-MSH and the expression of MC5R.