Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.     

Enhancement of the Division of Equisetum arvense Protoplasts in Culture by Activated Charcoal and Their Further Development

Protoplasts were isolated from subcultured gametophytes of Equisetumarvense by treatment with Driselase and then cultured in vitro.Addition of activated charcoal (AC) to the culture medium enhancedthe rate of cell division, as well as the survival of both protoplastsand regenerated protoplasts. However, subsequent division ofcells was not observed after one or two cycles of replicationin cultures supplemented with AC. When regenerated protoplastswere transferred to fresh medium without AC 3 to 5 weeks afterthe first plating, the transferred cells formed rhizoids anddeveloped into small, young gametophytes without the prior formationof cell clusters or calluses. Furthermore, sprophytic shootsdifferentiated from the protoplast-derived gametophytes whenthey were cultured on medium supplemented with 6-benzylaminopurine(BA). (Received April 5, 1990; Accepted July 30, 1990)     

Interleukin-4 enhances in vitro mineralization in human osteoblast-like cells.

The effect of human interleukin-4 (hIL-4) on mineralization in human osteoblast-like cells was investigated. Confluent cells were incubated with hIL-4 for 16 or 30 days in the presence or absence, respectively, of alpha-glycerophosphate (alpha-GP), which accelerates the mineralization process. hIL-4 (0.3 ng/ml) induced mineralization with 1.9-, 26- and 37-fold increases of hydroxyproline, calcium, and osteocalcin content, respectively, in the presence of alpha-GP. Mineralization was not induced with other cytokines, hIL-1, hIL-2, hIL-6, or mIL-4. hIL-4 also induced mineralization in the absence of alpha-GP in a manner different from that of 1 alpha, 25(OH)2 vitaminD3 (1,25(OH)2VD3). These findings suggest that IL-4 may play an important role in bone formation.     

A 1H-NMR study of the solution conformation of cyclo(GRGDSPA): conformational effects on the physiological activity.

Solution conformations of cyclo(GRGDSPA) have been analyzed by the use of two-dimensional proton nuclear magnetic resonance spectroscopy and the dynamical simulated annealing calculation. It has been shown that the RGDS segment in cyclo(GRGDSPA) takes a beta-turn conformation. We have concluded that this beta-turn conformation is essential for the physiological activity of cyclo(GRGDSPA).     

Prosaposin Facilitates Sciatic Nerve Regeneration In Vivo

Abstract: Prosaposin, a multifunctional protein, is the precursor of saposins, which activate sphingolipid hydrolases. In addition to acting as a precursor for saposins, prosaposin has been shown to rescue hippocampal CA1 neurons from lethal ischemic damage in vivo and to promote neurite extension of neuroblastoma cells in vitro. Here we show that prosaposin, when added to a collagen-filled nerve guide after sciatic nerve transection in guinea pigs, increased dramatically the number of regenerating nerve fibers within the guide. To identify the target neurons of prosaposin during peripheral nerve regeneration, we determined the degree of atrophy and chromatolysis of neurons in the spinal anterior horn and dorsal root ganglia on the prosaposin-treated and untreated side. The effect of prosaposin on large spinal neurons and small neurons of the dorsal root ganglion was more conspicuous. Subsequent immunohistochemistry demonstrated that the atrophy of cholinergic large neurons in the anterior horn is prevented to significant extent by prosaposin treatment. These findings suggest that prosaposin promotes peripheral nerve regeneration by acting on α-motor neurons in the anterior horn and on small sensory neurons in the dorsal root ganglion. The present study raises the possibility of using prosaposin as a tool for the treatment of peripheral nerve injuries.     

Responses to urine odor in the tufted capuchin (Cebus apella)

When the tufted capuchin urinates, it frequently performs “urine-washing”. Previous studies have proposed several hypotheses about this behavior. This study investigated 1) whether the tufted capuchin can distinguish the urine odor of conspecific individuals from other groups, and 2) whether the capuchins can distiguish conspcific urine odor of from that of other species. When an odor bar that had been prepared in conspecific other group's cage was presented, the response (sniffing, licking, or biting) was significantly greater than that to any other odors, including that of the home group, 5 other species, or a neutral odor. This tendency was stronger in males than in females. and suggests that the tufited capuchin is able to discriminate its home group's urine odor from that of the other groups. This monkey may also discriminate its own species from others by urine odor. The sex difference of the response to the odor bar may be the result of differences in social role between males and females. These results support the idea that the tufted capuchin may use olfaction for social communication.     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Fusarium poae and Fusarium crookwellense, Fungi Responsible for the Natural Occurrence of Nivalenol in Hokkaido

To determine the reasons for the natural occurrence of nivalenol in the northernmost area of Japan, scabby wheat was harvested from 19 crop fields in Hokkaido. Mycological surveys and analysis for mycotoxin contamination were performed. Among 13 wheat grain samples harvested in seven locations, 9, 2, and 6 samples were positive for deoxynivalenol, nivalenol, and zearalenone, respectively, at levels ranging from 0.03 to 1.28 μg/g, 0.04 to 1.22 μg/g, and 2 to 25 ng/g, respectively. The predominant Fusarium species of the scabby wheat examined were F. sporotrichioides, F. avenaceum, F. poae, and F. crookwellense. Fifteen of 48 F. poae isolates and all four F. crookwellense isolates were screened for the production of seven derivatives of trichothecenes and zearalenone respectively, on rice culture. One isolate of F. poae produced diacetoxyscirpenol alone (4.3 μg/g); seven produced nivalenol (1.3 to 23.8 μg/g), 4-acetylnivalenol (0.1 to 4.6 μg/g), and diacetoxyscirpenol (0.9 to 99.5 μg/g); and five produced nivalenol alone (0.4 to 3.5 μg/g). The remaining two isolates produced no trichothecenes. Zearalenone production was not found in any isolate of F. poae tested. All isolates of F. crookwellense produced nivalenol (0.9 to 22.5 μg/g), 4-acetylnivalenol (0.5 to 25.0 μg/g), and zearalenone (1.4 to 162.5 μg/g). From these results, it is apparent that deoxynivalenol and zearalenone, and occasionally nivalenol, occur naturally throughout Hokkaido, and it is suggested that nivalenol-producing F. poae and F. crookwellense strains are responsible for the natural contamination with nivalenol found in the northernmost area of Japan. Furthermore, it was found for the first time that several isolates of F. poae distributed in Hokkaido possessed the ability to produce both type A and type B trichothecenes.     

Gene structure and expression of the MboI restriction--modification system.

The genes from Moraxella bovis encoding the MboI restriction--modification system were cloned and expressed in Escherichia coli. Three open reading frames were found in the sequence containing the genes. These genes, which we named mboA, mboB, and mboC, had the same orientation in the genome. Genes mboA and mboC encoded MboI methyltransferases (named M.MboA and M.MboC) with 294 and 273 amino acid residues, respectively. The mboB gene coded for MboI restriction endonuclease (R.MboI) with 280 amino acid residues. Recombinant E.coli-MBOI, which contained the whole MboI system, overproduced R.MboI. R.MboI activity from E.coli-MBOI was 480-fold that of M.bovis. The amino acid sequences deduced from these genes were compared with those of other restriction--modification systems. The protein sequences of the MboI system had 38-49% homology with those of the DpnII system.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.