Effect of Atm disruption on spontaneously arising and radiation-induced deletion mutations in mouse liver

Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse "gpt-delta" system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi- (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm+/+ genotype showed a dose-dependent increase in the Spi- mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8x10(-6). The livers from Atm-/- mice yielded a virtually identical dose-response curve for X rays with a background fraction of 2.4x10(-6). Structural analyses revealed no significant difference in the proportion of -1 frameshifts and larger deletions between Atm+/+ and Atm-/- mice, although larger deletions prevailed in X-ray-induced Spi- mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.     

Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane

Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane. By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells. The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.     

Selective cleavage of 10-methyl benzo[b]naphtho[2,1-d]thiophene by recombinant Mycobacterium sp. strain

Recombinant Mycobacterium sp. strain MR65 carrying dszABCD genes was used for desulfurization of 10-methylbenzo[b]naphtho[2,1-d]thiophene (10-methyl BNT) in the hexadecane phase. The specific activity was 25% of that of dibenzothiophene (DBT). One of two major metabolites of 10-methyl BNT produced by strain MR65 was identified as 1-methoxy-2-(3-methylphenyl)naphthalene by ¹H and ¹³C NMR. The other major metabolite and two minor metabolites were determined as 1-hydroxy-2-(3-methylphenyl)naphthalene, 2-(2-methoxy-3-methylphenyl)naphthalene and 2-(2-hydroxy-3-methylphenyl)naphthalene, respectively, by HPLC and GC-MS. The production ratio of the two desulfurization metabolite isomers was 0.99:0.01, calculated on the basis of peak GC areas. These results indicated that the C-S bond adjacent to the naphthalene skeleton was selectively cleaved to form the two major compounds.     

Effect of dietary fiber on the lipid metabolism and immune function of aged Sprague-Dawley rats

Eight-month-old Sprague-Dawley rats were fed on diets containing dietary fiber at the 5% level for 3 weeks to examine the effect on the lipid metabolism and immune function. Among cellulose, guar gum, partially hydrolyzed guar gum (PHGG), glucomannan and highly methoxylated pectin, guar gum induced a significant decrease in the food intake and weight gain, as well as a significant increase in the liver weight. In addition, the epidydimal adipose tissue weight of the rats fed on PHGG was significantly higher than that of the rats fed on cellulose. There was no significant effect on the serum lipid levels, but the serum IgG level of the rats fed on guar gum was significantly lower than that of the rats fed on cellulose. The IgA and IgG productivity in mesenteric lymph node (MLN) lymphocytes was significantly higher in the rats fed on guar gum, glucomannan and pectin than in those fed on cellulose, while the effect on Ig productivity in spleen lymphocytes was not as marked. In addition, only guar gum induced a significant increase of IgM productivity in MLN lymphocytes when compared to the cellulose group. These results suggest that enhancement of the immune function by dietary fiber is mainly expressed in the gut immune system.     

Purification and characterization of α-D-galactosidase produced by ADG cell line established from abalon digestive gland

ADG cell line was established from an abalonedigestive gland and previously characterized. ADGcells have the potential to grow in protein-freeculture and secrete l3 types of glycosidases. Inthis article, we determined the origin of ADG cell line,using electron microscopy, and purified a glycosidasesecreted by these cells. The electron microscopicanalysis showed that ADG cell line contains severalnuclei, which suggests that they may be derived fromprotist cells. Moreover, -D-galactosidasethat hydrolyzes p-nitorophenyl galactopyranosidewas purified 130-fold from the spent culture medium ofADG cells. The molecular weight of the enzyme,determined by sodium dodecyl sulfate polyacrylamidegel electrophoresis and gel filtration analysis, wasshown to be 43 and 42 kDa, respectively, and itappeared to consist of a single polypeptide chain. The purified enzyme preparation was practically freefrom other glycosidases secreted from the cells. Catalytic activity was optimal at pH 5.5 and at atemperature of 37 °C. The enzyme was also the most stable at pH 5.5.     

Role of cardiac renin-angiotensin system in sarcoplasmic reticulum function and gene expression in the ischemic-reperfused heart

The aim of this study was to explore the possible participation of cardiac renin-angiotensin system (RAS) in the ischemia-reperfusion induced changes in heart function as well as Ca²⁺-handling activities and gene expression of cardiac sarcoplasmic reticulum (SR) proteins. The isolated rat hearts, treated for 10 min without and with 30 M captopril or 100 M losartan, were subjected to 30 min ischemia followed by reperfusion for 60 min and processed for the measurement of SR function and gene expression. Attenuated recovery of the left ventricular developed pressure (LVDP) upon reperfusion of the ischemic heart was accompanied by a marked reduction in SR Ca²⁺-pump ATPase, Ca²⁺-uptake and Ca²⁺-release activities. Northern blot analysis revealed that mRNA levels for SR Ca²⁺-handling proteins such as Ca²⁺-pump ATPase (SERCA2a), ryanodine receptor, calsequestrin and phospholamban were decreased in the ischemia-reperfused heart as compared with the non-ischemic control. Treatment with captopril improved the recovery of LVDP as well as SR Ca²⁺-pump ATPase and Ca²⁺-uptake activities in the postischemic hearts but had no effect on changes in Ca²⁺-release activity due to ischemic-reperfusion. Losartan neither affected the changes in contractile function nor modified alterations in SR Ca²⁺-handling activities. The ischemia-reperfusion induced decrease in mRNA levels for SR Ca²⁺-handling proteins were not affected by treatment with captopril or losartan. The results suggest that the improvement of cardiac function in the ischemic-reperfused heart by captopril is associated with the preservation of SR Ca²⁺-pump activities; however, it is unlikely that this action of captopril is mediated through the modification of cardiac RAS. Furthermore, cardiac RAS does not appear to contribute towards the ischemia-reperfusion induced changes in gene expression for SR Ca²⁺-handling proteins.     

Chronic Cerebral Hypoperfusion Induces Transient Reversible Monoaminergic Changes in the Rat Brain

Chronic restriction of cerebral blood flow in hypoperfused Wistar rats has been proposed as a new model of cerebrovascular-type dementia. Using this model, we have investigated central monoaminergic neuronal systems that are closely related to higher brain function. Monoamine and monoamine-metabolite levels were determined, as relative monoaminergic markers, at 1 day and 1,3,6 and 12 weeks after the bilateral occlusion of common carotid arteries. Dopaminergic changes in the frontal cortex and striatum were observed in hypoperfused rats at 1–3 weeks following occlusion. Serotonergic changes were recognized at four brain regions examined (frontal cortex, hippocampus, striatum and thalamus+midbrain). In particular, the immediate enhancement of serotonin turnover in the striatum appeared to influence the reaction to the acute ischemic attack such as vasoconstriction produced by hypoperfusion. Our findings suggest that chronic cerebral hypoperfusion induces transient reversible changes in central monoaminergic neuronal function within three weeks of ligation of carotid arteries. This time interval seems to represent a turning point in the process of chronic cerebral hypoperfusion-induced progressive brain injury.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Induction of Differentiation and Apoptosis in Human Promyelocytic Leukemia HL60 Cell Line by a New Type of Steroids

Mer-NF8054X is a new type of steroid whose structure has been established as 11-oxo-18, 22-cycloergosta-6, 8(14)-diene-3β, 5β, 9β, 23S-tetraol (an 18, 22-cycloergostane), which has been reported to have antifungal activity againstAspergillus fumigatus.However, other biological activities are unknown. Herein, we reported that Mer-NF8054X inhibited cell growth of HL60 human leukemia cells, when used either singly or in combination with retinoic acid (RA). In addition, Mer-NF8054X alone induced differentiation and apoptosis of HL60 cells. The induction of differentiation of HL60 cells by Mer-NF8054X was synergistic in combination with RA. On the other hand, Emesterone A, an analogue of Mer-NF8054X which is missing a hydroxy residue from the third position, showed much lower activity than Mer-NF8054X on the inhibition of cell growth and the induction of cell differentiation and apoptosis. However, Emesterone B, an analogue of Emesterone A which is missing a hydroxy residue from the fifth position, showed higher activity than Emesterone A but lower activity than Mer-NF8054X when examined for the inhibition of cell growth and the induction of cell differentiation and apoptosis. These results suggested that Mer-NF8054X and its analogs may be a new type of differentiation inducing agent. The hydroxy residue at the third position or fifth position in Mer-NF8054X may be necessary, but not essential, for inhibition of growth and induction of both differentiation and apoptosis of HL60 cells. In addition, Mer-NF8054X enhanced the differentiation of HL60 cells induced by RA. Based on these results, Mer-NF8054X may have utility in the clinic in combination with RA for leukemia patients.     

Effect of ruthenium precursor on hydrogen-treated active carbon supported ruthenium catalysts for ammonia synthesis

Active carbon (A.C.), after treatment at 1123 K with hydrogen for more than 24 h, was found to be very effective as a support for ruthenium catalysis in ammonia synthesis. The activity of barium promoted Ru catalysts supported on this treated A.C. is affected remarkably by the Ru precursor. Ru₃(CO)₁₂ was found previously to be the most effective Ru precursor for oxide supports such as Al₂O₃, MgO, and CeO₂ in ammonia synthesis. However, in this study, the Ru---Bao/A.C. catalyst prepared from Ru(acac)₃ was found to yield the highest activity, while the catalyst prepared from Ru₃(CO)₁₂ resulted in the lowest activity among several precursors. Transmission electron microscopy and hydrogen chemisorption showed that the particle size of Ru obtained from the decomposition of Ru(acac)₃ on hydrogen-treated A.C. is smaller than the particle size of Ru obtained from the decomposition of Ru₃(CO)₁₂. Additionally, RuCl₃ was found to be an effective precursor for Ru/A.C. catalyst. It has been suggested that chlorine ions can be removed easily from A.C. by hydrogen reduction at 773 K, and that this results in the high activity.