Selenium (Se) is an essential trace element. Se is found as selenocysteine (Sec) in Se-proteins. Sec is the 21st amino acid, because Sec has its tRNA, the codon UGA and those components in its translational machinery. Sec UGA codon shares with major stop codon UGA. We purified Sec synthesizing enzymes, such as seryl-tRNA synthetase (SerRS), Sec synthetase (SecS) and selenophosphate synthetase (SePS). I described the procedures to prepare Sec tRNA, SerRS, SecS, SePS and [75Se]H2Se in detail. We clarified that SecS composed of two proteins, SecSα and SecSβ. Sec synthesizing and incorporating systems present in Monela, Animalia and Protoctista but not in Plantae and Fungi. We showed that protozoa had Sec tRNA on which Sec was synthesized from Ser-tRNA by bovine and protozoa SecS. Some worms, such as Caenorhabditis elegans and Fasiola gigantica, also had Sec tRNA on which Sec was synthesized by bovine liver SecS or C. elegans enzymes. We showed recognition sites of mammalian Sec tRNA by SecS. The identitiy units of Sec tRNA are 9 bp aminoacyl- and 6 bp D-stems. This recognition is not the base-specific manner but the length-specific manner. From comparison of the phylogeny trees of Sec synthesizing system and translation system, we concluded that the evolution of Sec synthesizing system is older than that of the translation system.
Two efficient methods to construct the indazole nucleus have been developed, both of which utilize palladium-catalyzed intramolecular carbon-nitrogen bond formation. One is based on intramolecular Buchwald-Hartwig amination reaction of 2-halobenzophenone tosylhydrazones. The catalyst system we developed for this reaction allows the cyclization to proceed under very mild conditions and thus could be applied to a wide range of substrates with acid- or base-sensitive functional groups. Furthermore, this methodology could be applied for the construction of benzoisoxazole ring system. In addition, catalytic C—H activation with palladium followed by intramolecular amination of benzophenone tosylhydrazones was also accomplished with the aid of the catalyst system such as Pd(OAc)2/Cu(OAc)2/AgOCOCF3, which gave another route to indazoles. Using this combination, indazoles with various functional groups could be obtained in good to high yields, especially in the case of substrates having electron donating group such as methoxy group on benzene ring. Interesting chemo- and regioselectivity were also observed in this reaction.
The placenta plays numerous important roles to support fetal development such as gas exchange, nutrient supply, and hormone production. Placental defects underlie many aspects of pregnancy losses and complications; thus understanding and regulating gene function during placentation is of high clinical relevance. However, the lack of a facile and efficient method for placenta-specific gene manipulation has hampered study of the placenta. We have previously shown that transduction of fertilized mouse eggs with lentiviral (LV) vectors efficiently generates transgenic animals; however, transgene expression occurred in both the fetus and the placenta. In the present study, we transduced zona-free blastocysts with LV vectors expecting placenta-specific gene expression, since most placental cells differentiate from trophoblast cells that form the outermost layer of the blastocyst. Transgene expression was observed in trophoblast cells from preimplantation stages and in the placenta throughout gestation. All the analyzed placentas carried the transgene, while none of the fetuses became transgenic. By applying this method, embryonic lethality caused by placental defects in several knockout animal models was substantially rescued. This technology provides a powerful system for gene manipulation exclusively in placental organogenesis with implications for the treatment of placental dysfunction.
Macro- and microvascular disease states currently represent the principal causes of morbidity and mortality in patients with type I or type II diabetes mellitus. Abnormal vasomotor responses and impaired endothelium-dependent vasodilation have been demonstrated in various beds in different animal models of diabetes and in humans with type I or type II diabetes. The principal mediators of diabetes-associated vascular dysfunction are increases in glucose, oxidized low-density lipoprotein, endothelin-1, angiotensin II, insulin, or growth factors. An accumulating body of evidence indicates that abnormal production of oxidative stress may be one of several factors contributing to vascular dysfunction in diabetes. It is possible that in diabetic states, hyperinsulinemia initiates oxidant stress, leading to vascular dysfunction at a later stage. We and others have demonstrated that in models of hyperinsulinemia and hyperglycemia, · NO production and/or · NO responsiveness are impaired in aortic strips. Several recent studies have shown that the formation of nitrotyrosine and/or peroxynitrite impairs vascular · NO responsiveness and · NO production. Our findings suggest that the coexistence of a high insulin level and an established diabetic state may lead to the excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in sarcoendo plasmic reticulum Ca2+ ATPase function. This review summarizes the results of our recent studies on the involvement of insulin and oxidative stress in the blood vessels of diabetic animals.
Radiocontrast nephropathy (RCN) is a major complication after radiographical examination with iodinated contrast media (CM). Although little is known about the mechanism of RCN, a direct toxic action on renal cells and/or decrease in renal blood flow are considered to be implicated in the pathogenesis of the disease/the condition, A large number of vasodilatory agents, including endothelin antagonists, adenosine antagonists, atrial natriuretic peptide, calcium channel blockers, dopamine, dopamine D1 receptor agonist fenoldopam, and prostaglandin E1 have been tried clinically to prevent RCN, however, most of them have failed. Although prophylactic effects of antioxidant N-acetylcysteine have recently been reported by several investigators, only hydration is a universally accepted protocol to prevent it. In our recent in vitro and in vivo study, we have elucidated that CM induced apoptosis of renal tubular cells through the reduction in Bcl-2 expression and the subsequent activation of caspase-9 and caspase-3. Moreover, we found that CM caused an increase in ceramide content in renal tubular cells, which leads to apoptosis by inhibiting the phosphorylation of Akt and cAMP responsive element binding protein (CREB) and the subsequent reduction in Bcl-2 expression. The inhibitor of ceramide synthase, fumonisin B1, reversed both the elevation of ceramide content and renal cell injury induced by CM. On the other hand, a prostacyclin analog beraprost prevented RCN in mice by the increase of endogenous cAMP and subsequent CREB phosphorylation resulted in enhancement of Bcl-2 expression. These findings suggest that ceramide synthesis inhibitor or beraprost is potentially useful for the prophylaxis of RCN.
Sensitive and selective methods, based on chemiluminescence reactions, were introduced for determination of reactive oxygen species (ROS) and their applications to biological samples and health foods. First, a sensitive method for determination of H2O2 by peroxyoxalate chemiluminescence (PO-CL) was developed. This method could be applied to determine small amounts of H2O2 in cola drinks and bacterial contamination of food items. Secondly, the combination of immobilized enzyme column reactor, or ultraviolet irradiation system, with the PO-CL detection method was able to determine clinical substrates (i.e. choline-containing phospholipids, polyamines and D-amino acids) and organic peroxides. Also, an evaluation method of the quenching effect of luminol chemiluminescence against ROS was developed. The sensitive, rapid and precise measurement of the quenching effect against ROS such as superoxide, singlet oxygen, hydroxyl radical, peroxynitrite and hypochlorous ion was achieved. The proposed method could be applied to rosemary extracts, natural colorants and grape seed extracts.
Since lipid oxidation is involved in the deterioration of hypercholesterolemia-related atherosclerosis, ingestion of drinks and foods with antioxidant actions is useful for preventing lipid oxidation. Goishi-tea is a post-fermented-tea manufactured by a unique method in Japan, and may be useful for preventing various disorders. However, there is no scientific evidence. In this study, we compared the radical scavenging activity of goishi-tea with that of other teas, and administered this tea to a rabbit model of hypercholesteremia to evaluate its usefulness in the inhibition of hypercholesteremia and atherosclerosis. The radical scavenging activity of goishi-tea was similar to that of green-tea, and was higher than that of other types of fermented-teas. On the other hand, some difference of components was found between goishi-tea and green-tea. In cholesterol-fed rabbits, low-density lipoprotein (LDL)-cholesterol level in the goishi-tea-group was lower than that in the green-tea-group. Plasma lipidperoxide value was also lower in the goishi-tea-group than in the green-tea and tap-water-groups. On aortic endothelial staining, fat area in the goishi-tea-group was lower than that in the tap-water-group. Furthermore, fat accumulation in the aortic intima in the goishi-tea-group was very low. Goishi-tea has higher antioxidant activities than the other fermented-teas tested, which were generally low, and decreased serum lipid levels, suggesting that goishitea is a very peculiar fermented-tea with usefulness in the prevention of hypercholesterolemia and atherosclerosis.
Clinical pharmacy training III (bedside training) in the School of Pharmaceutical Sciences, Kyushu University of Health and Welfare is intended to train pharmacists who can also cope with medical emergencies. Therefore we produced original scenarios that provide experience of various medical emergencies using emergency care simulators. As a result, these simulators enabled students to experience dealing with various medical emergencies such as cardiopulmonary resuscitation, automated external defibrillation (AED), adrenalin administration, and oxygen inhalation. In addition, a survey on the necessity for and the degree of the understanding of training contents associated with emergency care simulators was performed before and at the end of clinical training. After clinical training, the necessity for and the degree of the understanding of these training contents significantly increased (p<0.01). The introduction of emergency care simulators into clinical pharmacy training provides experience of not only cardiopulmonary resuscitation but also the treatment psocedures as well as observation of improvement in the pathological condition after drug administration, which increases pharmacists' awareness of patient needs in drug therapy. Therefore these simulators are helpful for pharmacy education aiming at improving pharmacists' pharmaceutical care ability.
The present investigation was aimed to evaluate the possibility of using different concentrations and polymeric grades of hydroxypropyl methylcellulose (K4M, K15M and K100M) for transdermal delivery of methotrexate, an immunosuppressant drug for rheumatoid arthritis. The matrix films were evaluated for their physicochemical characterization followed by in vitro and in vivo evaluation. Selected formulations were subjected for their in vivo studies on healthy rabbits following balanced incomplete block design. The relevance of difference in the in vitro dissolution rate profile and pharmacokinetic parameters (Cmax, tmax, AUC(s), t1/2, Kel, and MRT) were evaluated statistically. The thickness and weight of the patch increased with the increase in polymeric grade and content. Fourier transform infrared spectroscopy and differential scanning calorimetry results confirm that there is no interaction between drug and polymer used. X-ray diffraction study reveals an amorphous state of drug in the matrix films. The in vitro drug release followed Higuchi kinetics (r=0.972-997; p<0.001) as its coefficient of correlation values predominates over zero order and first order release kinetics. In vitro dissolution profiles and pharmacokinetic parameters showed a significant difference between test products (p<0.01), but not within test products. A quantitatively good correlation was found between per cent of drug absorbed from the transdermal patches and AUC(s). A significant in vitro/in vivo correlation was observed when per cent drug released was correlated with serum drug concentration. Out of the various formulations made, the selected formulations are better in their in vitro dissolution and pharmacokinetic characteristics and thus hold potential for transdermal delivery.
The promyelocytic leukemia (PML) gene is a tumor suppressor gene associated with cell apoptosis, cell proliferation, and senescence. However, the role of PML in the ethanol-induced apoptosis is not fully-known. In this study, using wild-type mouse embryo fibroblasts (MEF) and PML null MEF cells, we found that (1) ethanol (100 mM and 200 mM) could obviously induce apoptosis of wild-type MEF cells, whereas, in PML null MEF cells, the pro-apoptotic function of ethanol was partially blocked; (2) the expression levels of phosphorylated p53 and two of its target genes, p21 and Bax, could be significantly up-regulated by ethanol (200 mM) in wild-type MEF cells in a time-dependent manner, but not in PML null MEF cells. These results indicate that PML plays an important role in ethanol-induced apoptosis, and p53-dependent apoptotic pathway may be involved in this process.
The track records of the use of anti-methicillin-resistant Staphylococcus aureus agents (anti-MRSA agents) in a 5-year period (2001.4-2006.3) were collected, and cases in which anti-MRSA agents were used for >4 days were selected. In each case, the results of laboratory data and bacterial examination before and after administering the anti-MRSA agents were investigated retrospectively. In addition, it was also investigated in each case whether therapeutic drug monitoring (TDM) was carried out. It was observed that the number of patients treated with anti-MRSA agents and the total dose of anti-MRSA agents used tended to increase over time, except for arbekacin sulfate. It was, however, shown that treatment with anti-MRSA agents resulted in significant decreases in body temperature, C-reactive protein, and white blood cell counts. Bacterial examination was conducted in 75.6% of the patients treated with anti-MRSA agents, with MRSA being detected in 72.4% of the cases examined. On the other hand, TDM was also conducted in 60% of the cases, but this was at a lower percentage than that of the other examinations. Quantitative bacterial examination after treatment with anti-MRSA agents indicates that TDM can be considered important for the appropriate use of anti-MRSA agents.
Physiologically, anemia often occurs during pregnancy because of an increase in circulating plasma volume. Pregnancy anemia is found prenatally in 50-75% of women. Based on the present survey performed in our obstetrics and gynecology ward, 52% of women experienced anemia during their pregnancy. This suggests that normal physiological changes due to pregnancy alone are not the only factors contributing to pregnancy anemia. Therefore to study the influence of lifestyle on pregnancy anemia, we investigated prepregnancy lifestyles on the assumption that the accumulation of several factors over a long period is usually the cause of anemia. The present results suggest that (i) the probability of anemia is slight in late pregnancy, if a normal Hb concentration is maintained in early pregnancy; (ii) the menstrual cycle is involved in the onset of anemia during early pregnancy; (iii) the number of meals taken and the level of alcohol consumption influence Hb concentration in late pregnancy. We believe that these findings provide a useful information source for advising patients on avoiding pregnancy anemia, which we can also use as guidance for outpatients at puberty. In conclusion, to prevent pregnancy anemia it is important to keep a regular menstrual cycle before pregnancy, and to take 3 meals/day and abstain from alcohol before and during pregnancy.