Protection against oxidative stress-induced hepatic injury by intracellular type II platelet-activating factor acetylhydrolase by metabolism of oxidized phospholipids in vivo

Membrane phospholipids are susceptible to oxidation, which is involved in various pathological processes such as inflammation, atherogenesis, neurodegeneration, and aging. One enzyme that may help to remove oxidized phospholipids from cells is intracellular type II platelet-activating factor acetylhydrolase (PAF-AH (II)), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids. Overexpression of PAF-AH (II) in cells or tissues was previously shown to suppress oxidative stress-induced cell death. In this study we investigated the functions of PAF-AH (II) by generating PAF-AH (II)-deficient (Pafah2(-/-)) mice. PAF-AH (II) was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium, and hepatocytes. Although PAF-AH activity was almost abolished in the liver and kidney of Pafah2(-/-) mice, Pafah2(-/-) mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2(-/-) mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl(4)) was injected into mice, Pafah2(-/-) mice showed a delay in hepatic injury recovery. Moreover, after CCl(4) administration, liver levels of the esterified form of 8-iso-PGF(2alpha), a known in vitro substrate of PAF-AH (II), were higher in Pafah2(-/-) mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-isoprostaglandin F(2alpha) and protects tissue from oxidative stress-induced injury.     

Autonomous differentiation of Drosophila spermatogonia in vitro

Spermatogenesis is a complex process that produces functional sperm by establishing male germline stem cells (mGSCs) in adult testes. To study Drosophila spermatogenesis in vitro , we examined various culture conditions of spermatogonia. Spermatogonia from larval testes began to differentiate soon after culture, whereas mGSCs did not undergo self-renewal division. Strikingly, 16-cell spermatogonia from early and late larval testes differentiated into motile spermatids autonomously. Furthermore, individual spermatogonia developed into motile spermatids even after mechanical dissociation from encapsulating cyst cells. This is the first study to report that spermatogonia in larval testes retain the ability to differentiate into spermatids in the absence of gonadal tissue. Our in vitro system should provide an excellent opportunity to study spermatogenesis in detail and apply genetic manipulation.     

Multi-phenotype behavioral characterization of inbred strains derived from wild stocks of Mus musculus

Many aspects of mouse behavior have been studied by using only a relatively small sample of available laboratory strains. These laboratory mice were derived from the so-called ``fancy mouse' and in most cases underwent extensive domestication before inbreeding. Thus, the behavioral repertoire of the laboratory mouse may be very different from that exhibited by stocks that have not been deliberately domesticated. Another inherent problem in analyzing mouse behavior is that genetic diversity is limited among currently available strains. In this respect, the use of strains that are derived from a variety of wild mice should provide a means to identifying novel behavioral phenotypes. We have investigated several behavioral phenotypes, using females of a number of mouse strains derived from wild mice of different subspecies, BFM/2, NJL, BLG2, HMI, CAST/Ei, KJR, SWN and MSM; a strain derived from fancy mice, JF1; and two laboratory strains, C57BL/6 and DBA/1. In this report, tests for locomotor activity, light-dark transitions, passive and active avoidance, and nociception were conducted. The results show great diversity of behavioral patterns between strains in contrast to less within-strain variability. We also found that two strains, KJR and SWN, both have good learning ability, whereas BLG2 mice exhibit impairment in both passive and active avoidance learning. Received: 11 January 2000 / Accepted: 27 March 2000     

Synthesis of 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl analogues of 5-fluorouracil, N-benzoyl adenine, uracil, thymine, N-benzoyl cytosine and evaluation of their antitumor activities

The synthesis of the unsaturated 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl nucleosides of 5-fluorouracil (6a), N⁶-benzoyl adenine (6b), uracil (6c), thymine (6d) and N⁴-benzoyl cytosine (6e), is described. Monoiodination of compounds 1a,b, followed by acetylation, catalytic hydrogenation and finally regioselective 2′-O-deacylation afforded the partially acetylated dideoxynucleoside analogues of 5-fluorouracil (5a) and N⁶-benzoyl adenine (5b), respectively. Direct oxidation of the free hydroxyl group at the 2′-position of 5a,b, with simultaneous elimination reaction of the β-acetoxyl group, afforded the desired unsaturated 4,6-dideoxy-3-fluoro-2-keto-β-d-glucopyranosyl derivatives 6a,b. Compounds 1c-e were used as starting materials for the synthesis of the dideoxy unsaturated carbonyl nucleosides of uracil (6c), thymine (6d) and N⁴-benzoyl cytosine (6e). Similarly a protection-selective deprotection sequence followed by oxidation of the free hydroxyl group at the 2′-position of the dideoxy benzoylated analogues 9c-e with simultaneous elimination reaction of the β-benzoyl group, gave the desired nucleosides 6c-e. None of the compounds was inhibitory to a broad spectrum of DNA and RNA viruses at subtoxic concentrations. The 5-fluorouracil derivative 6a was more cytostatic (50% inhibitory concentration ranging between 0.2 and 12 μM) than the other compounds.     

Synthesis of 3-fluoro-6-S-(2-S-pyridyl) nucleosides as potential lead cytostatic agents

The 3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-β-d-glucopyranosyl nucleoside analogs 7 were prepared via two facile synthetic routes. Their precursors, 3-fluoro-6-thio-glucopyranosyl nucleosides 5a-e, were obtained by the sequence of deacetylation of 3-deoxy-3-fluoro-β-d-glucopyranosyl nucleosides 2a-e, selective tosylation of the primary OH of 3 and finally treatment with potassium thioacetate. The desired thiolpyridine protected analogs 7a-c,f,g were obtained by the sequence of deacetylation of 5a-c followed by thiopyridinylation and/or condensation of the corresponding heterocyclic bases with the newly synthesized peracetylated 6-S-(2-S-pyridyl) sugar precursor 13, which was obtained via a novel synthetic route from glycosyl donor 12. None of the compounds 6 and 7 showed antiviral activity, but the 5-fluorouracil derivative 7c and particularly the uracil derivative 7b were endowed with an interesting and selective cytostatic action against a variety of murine and human tumor cell cultures.     

Wingless signaling initiates mitosis of primordial germ cells during development in Drosophila

The germline cells of Drosophila are derived from pole cells, which form at the posterior pole of the blastoderm and become primordial germ cells (PGCs). To elucidate the signal transduction pathways for the development of embryonic PGCs, we examined the effects of various growth factors on the proliferation of PGCs. Up- and down-regulation of Wingless (Wg) in both of soma and PGCs caused an increase and a decrease in the number of PGCs, respectively. The Wg/β-catenin signaling pathway began to occur in PGCs at the same time as the PGCs began to divide during the embryonic stage in both sexes. In addition, PGCs were found to produce wg mRNA as they begin to divide. Thus, Wg functions as an autocrine factor to initiate mitosis in embryonic PGCs. Decapentaplegic affected the growth of PGCs from the end of the embryonic stage. The results indicate that these growth factors regulate the division of embryonic PGCs in a stage-specific manner.     

Antioxidant capacity of BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, and uric acid as evaluated by ORAC method and inhibition of lipid peroxidation

The role of radical-scavenging antioxidant against oxidative stress has received much attention. The antioxidant capacity has been assessed by various methods. Above all, oxygen radical absorbance capacity (ORAC) has been frequently employed [Prior et.al., J. Agric. Food Chem.2005, 53, 4290]. In the present study, the antioxidant capacity of 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653) and uric acid was assessed by ORAC method using pyranine as a reference probe and compared with that against lipid peroxidation of human plasma. It was found that BO-653 was assessed to be much less potent than uric acid by ORAC method, whereas BO-653 exerted much higher antioxidant activity than uric acid against plasma lipid peroxidation. The reason for such discrepancy is discussed. The results suggest that ORAC method is suitable for the assessment of free radical scavenging capacity, but not for the assessment of antioxidant capacity against lipid peroxidation in plasma.     

Characterization of novel furan compounds on the basis of their radical scavenging activity and cytoprotective effects against glutamate- and lipopolysaccharide-induced insults

There is increasing evidence indicating that free radicals and oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) are related to the onset and development of neurodegenerative diseases. In order to prevent and/or delay these diseases, the use of radical-scavenging antioxidants and inhibitors against oxygenases has received much attention. In the present study, we examined the radical-scavenging activity and cytoprotective effects of some novel furan compounds with potent inhibitory activity against oxygenases such as COX-1, COX-2, and 5-LOX. The radical-scavenging activity was assessed by studying the bleaching of beta-carotene by free radicals generated from an azo initiator. In this assay system, the rate constants for scavenging peroxyl radicals by furan S and furan L was estimated to be 2 x 10(4) and 3 x 10(4) M(-1) s(-1), respectively. We also investigated the cytoprotective effects of these compounds against cell death induced by several toxins. We found that the furan compounds exhibited cytoprotective effects against PC12 cell death induced by linoleic acid hydroperoxide, primary neuronal cell death induced by glutamate, and cell death induced by lipopolysaccharide. These results suggest the beneficial effects of the furan compounds against disorders related to glutamate and lipopolysaccharide.