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Choosing an optimal procedure to stabilize the chest wall for multiple rib fractures can be challenging for surgeons. Stabilization with rib plates or intramedullary devices requires exposure of fractured bones, whereas chest stabilization with intercostal Z-suture and mesh does not, yet it offers the chest wall a sufficient level of stability. Here, we report a case of a flail chest due to multiple rib fractures for which a simple chest wall fixation using intercostal Z-suture and mesh was efficient. This method could be a treatment option for multiple rib fractures.

Low expression of antioxidant enzymes makes pancreatic beta-cells susceptible to cell damage by oxidative stress. Pancreatic beta-cell loss caused by endoplasmic reticulum stress is associated with the onset of diabetes mellitus. The present studies were undertaken to investigate a possible involvement of proapoptotic gene CHOP in pancreatic beta-cells damage by oxidative stress. The induction of CHOP messenger RNA and apoptosis were investigated in betaHC-9 cells after the oxidative stress by hydrogen peroxide and ribose. Latter was examined after the suppression of CHOP by small interfering RNA. For in vivo study, the pancreatic beta-cells were examined in CHOP-knockout (KO) mice after multiple low-dose streptozotocin (MLDS) administration. In betaHC-9 cells, both hydrogen peroxide and ribose obviously increased apoptotic cells, accompanied with enhanced CHOP messenger RNA expression. However, the number of apoptotic cells by those stimulations was significantly reduced by the addition of small interfering RNA against CHOP. In vivo study also showed that CHOP-KO mice were less susceptible to diabetes after MLDS administration. Although the oxidative stress marker level was similar to that of MLDS-treated wild type, the pancreatic beta-cell area was maintained in CHOP-KO mice. The present studies showed that CHOP should be important in pancreatic beta-cell injury by oxidative stress and indicate that CHOP may play a role in the development of pancreatic beta-cell damage on the onset of diabetes mellitus.

Anti-apoptotic Bcl-2 localizes in the membranes of mitochondria and endoplasmic reticulum (ER) and resists a broad range of apoptotic stimuli. However, the precise function of Bcl-2 in ER is still unclear. We herein examined the anti-apoptotic potencies of Bcl-2 in mitochondria and ER in vitro. The mitochondria isolated from HeLa cells, which have little or practically no Bcl-2, were apoptosis-competent. That is, membrane-bound Bax was activated and cytochrome c was released when the isolated mitochondria were incubated at 35 degrees C. Cytochrome c release from the apoptosis-competent mitochondria was suppressed by co-incubation with the mitochondria with overexpressed Bcl-2 (Bcl-2 mitochondria), suggesting that Bcl-2 anchored in one mitochondrion can suppress cytochrome c release from another mitochondrion. Similar results were obtained when microsomes with overexpressed Bcl-2 (Bcl-2 microsomes) were co-incubated with apoptosis-competent mitochondria. A quantitative titration analysis showed that Bcl-2 in the ER suppresses cytochrome c release as efficiently as that in the mitochondria. An immunoprecipitation assay showed that Bcl-2 in both mitochondria and ER binds to Bax at almost the same degree. However, in the presence of tBid, co-incubation of apoptosis-competent mitochondria with Bcl-2 microsomes, but not with Bcl-2 mitochondria, diminished the Bax-binding to Bcl-2 significantly, suggesting that Bcl-2 in ER is readily inactivated by tBid. Co-incubation assay further confirmed that Bcl-2 in the ER, but not Bcl-2 in the mitochondria, is potentially inactivated by tBid. Our quantitative in vitro studies indicate that Bcl-2 in mitochondria and ER are similarly potent in inhibiting Bax-associated apoptosis of other mitochondria, but are regulated by tBid differently.

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.

Nitric oxide (NO) is synthesized from arginine by NO synthase (NOS), and the availability of arginine is one of the rate-limiting factors in cellular NO production. Citrulline that is formed as a by-product of the NOS reaction can be recycled to arginine by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), forming the citrulline-NO cycle. AS and sometimes AL have been shown to be coinduced with inducible NOS (iNOS) in various cell types including activated macrophages, microglia, vascular smooth muscle cells, glial cells, neuronal PC12 cells, retinal pigment epithelial cells, and pancreatic beta-cells. Coinduction of endothelial NOS (eNOS), AS, and AL are observed in human umbilical vein endothelial cells. In contrast, arginase can downregulate NO production by decreasing intracellular arginine concentrations. iNOS and arginase activities are regulated reciprocally in macrophages by cytokines, and this may guarantee the efficient production of NO. In contrast, iNOS and arginase isoforms (type I and/or II) are coinduced in immunostimulated macrophages, but not in PC12 cells and glial cells. These results indicate that NO production is modulated by the recycling and degradation of arginine. Arginase also plays an important role in regulation of polyamine and proline synthesis.

Nitric oxide (NO) has been implicated in many physiological and pathological conditions in the eyes. The induction of inducible NO synthase (iNOS) and NO production have been noted in immunostimulated retinal pigment epithelial (RPE) cells. Cellular NO production depends on the availability of arginine, a substrate for NOS. Arginine can be regenerated from citrulline, another product of the NOS reaction, by argininosuccinate synthetase and argininosuccinate lyase, forming the citrulline-NO cycle. When rat RPE-J cells were treated with interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha) and lipopolysaccharide (LPS), and expression of the citrulline-NO cycle enzymes and related enzymes was analyzed, iNOS and argininosuccinate synthetase were highly induced at both mRNA and protein levels. On the other hand, argininosuccinate lyase was not induced. Among other related enzymes and transporters, mRNA for cationic amino acid transporter (CAT)-1 was weakly induced, whereas those for CAT-2, arginase I and II, ornithine aminotransferase and ornithine decarboxylase remained little changed. NO was produced by cells after stimulation with TNFalpha, IFNgamma and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. Our findings indicate that in activated RPE-J cells citrulline-arginine recycling is important for NO production.

The regulation of expression of the arginine-recycling enzymes and arginase isoforms in association with inducible nitric oxide synthase (iNOS) in the eye of endotoxin-induced uveitis (EIU) rats is investigated. An animal model of EIU was created in Wistar rats by intravitreal injection of lipopolysaccharide (LPS). mRNAs for argininosuccinate synthase (AS) and arginase I as well as for iNOS, measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), were induced in the eye of EIU rats. iNOS mRNA increased markedly 3 hr after injection, reached a maximum at 6-12 hr, and then decreased at 24 hr. AS mRNA remained little change at 3 hr and increased maximally at 6 hr (by about 3.3-fold), whereas arginase I mRNA increased later and reached a maximum at 12 hr (by about 4.2-fold). iNOS, AS, and arginase I proteins were also induced. AL and arginase II mRNAs remained little changed. In immunohistochemical analysis, iNOS, AS and arginase I were almost colocalized in infiltrated inflammatory cells in the vitreous, iris, ciliary body and inner layers of the retina. In conclusion, AS and arginase I are coinduced with iNOS in infiltrated inflammatory cells in the eyes of EIU rats, and may regulate NO production by changing intracellular concentration of arginine.

Hepatocyte nuclear factor 4alpha (HNF4alpha) regulates the expression of many genes preferentially expressed in liver. HNF4alpha-null mice die during embryogenesis precluding the analysis of its function in the adult. To circumvent this problem, liver-specific HNF4alpha-null mice were produced. Mice lacking hepatic HNF4alpha expression exhibited increased serum ammonia and reduced serum urea. This disruption in ureagenesis may be explained by a marked decrease in expression and activity of hepatic ornithine transcarbamylase (OTC). To determine the molecular mechanisms involved in transcriptional regulation of the mouse OTC gene, the OTC promoter region was analyzed. Sequence analysis revealed the presence of two putative HNF4alpha-binding sites in the mouse OTC promoter region. By using transient transfection analysis, it was established that high levels of promoter activity were dependent on both HNF4alpha-binding sites and the expression of HNF4alpha. Furthermore, the proximal HNF4alpha-binding site was found to be more important than the distal one for transactivating OTC promoter. These data demonstrate that HNF4alpha is critical for urea homeostasis by direct regulation of the OTC gene in vivo.

Both arginase isoforms (AI and AII) regulate high-level NO production by the inducible NOS, but whether the arginase isoforms also regulate low-level NO production by neuronal NOS (nNOS) is not known. In this study, 293 cells that stably overexpress nNOS gene (293nNOS cells) were transfected with rat AI (pEGFP-AI) or AII (pcDNA-AII) plasmids, and nitrite production was measured with or without supplemental l -arginine. Transfection with pEGFP-AI increased AI expression and activity 10-fold and decreased intracellular l -arginine by 50%. Nitrite production was inhibited by >80% when no l -arginine was supplemented but not when 1 mM l -arginine was present. The inhibition was reversed by an arginase inhibitor, Nω-hydroxy- l -arginine. Transfection with pcDNA-AII increased AII expression and activity but had little effect on nitrite production even if no l -arginine was added. These results suggest that, in 293nNOS cells, AI was more effective in regulating NO production by nNOS, most likely by competing for l -arginine.

l -Arginine is a substrate for the synthesis of proteins, nitric oxide (NO), creatine, urea, proline, glutamate, polyamines and agmatine. In the central nervous system (CNS), arginine is extracted from the blood and exchanged by cells through carriers called cationic amino acid transporters (CAT) and belonging to the so-called system y + . In order to better understand the arginine transport in the CNS, we studied in detail the regional distribution of the cells expressing the CAT2(B) transcript in the adult rat brain by non-radioisotopic in situ hybridization. We show that CAT2(B) is expressed in neurons and oligodendrocytes throughout the brain, but is not detected in astrocytes. The pattern of localization of CAT2(B) in the normal adult rat brain fits closely that of CRT1, a specific creatine transporter. Our study demonstrates that the in vivo expression of CAT2(B) differs from that reported in vitro, implying that local cellular interactions should be taken into account in studies of gene regulation of the CAT2(B) gene. Our work suggests that CAT2(B) may play a role in case of increased NO production as well as arginine or creatine deficiency in the brain.

Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.

DnaJ is an essential cochaperone of mammalian heat shock cognate 70 (hsc70) protein. We previously found that dj2 (HSDJ/hdj-2/rdj1), rather than dj1 (hsp40/hdj-1), is a partner DnaJ for the hsc70-based chaperone system. Here, we compared the distribution of dj1, dj2, and the newly found dj3 (cpr3/DNJ3/HIRIP4/rdj2) in cultured cells. Both dj3 as well as dj2 were farnesylated and were ubiquitously expressed. In immunocytochemical and subfractionation studies, these two proteins colocalized with hsc70 under normal conditions. However, dj1 and hsc70 apparently colocalized in the nucleoli after heat shock. Simultaneous depletion of dj2 and dj3 from rabbit reticulocyte lysate markedly reduced mitochondrial import of pre-ornithine transcarbamylase and refolding of guanidine-denatured luciferase. Re-addition of either dj2 or dj3 led to recovery of these reactions. In a reconstituted system, both hsc70-dj2 and hsc70-dj3 were effective in protein refolding. Anti-apoptotic protein bag-1 further stimulated ATP hydrolysis and protein refolding by both pairs. Thus, dj2 and dj3 are the partner DnaJs of hsc70 within the cell, functionally similar and much more efficient than dj1, and bag-1 is a positive cochaperone of the hsc70-dj2 and hsc70-dj3 systems.

Arginine is an intermediate of the urea cycle in the liver. It is synthesized by the first four enzymes of the cycle, carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinate lyase, and is hydrolyzed to urea and ornithine by arginase I, forming the cycle. In endotoxemia shock, inducible nitric oxide (NO) synthase (iNOS) is induced in hepatocytes and arginine is utilized for NO production. Regulation of the genes for iNOS and the urea cycle enzymes was studied using lipopolysaccharide (LPS)-treated rat livers. When rats were injected intraperitoneally with LPS, iNOS mRNA was markedly induced. Cationic amino acid transporter-2 and C/EBPbeta mRNAs were also highly increased. In contrast, mRNAs for all the urea cycle enzymes except ornithine transcarbamylase were gradually decreased and reached 16-28% of controls at 12 h. However, all these enzymes remained unchanged at protein level up to 24 h. In light of these results, we suggest that synthesis of urea cycle enzymes is downregulated and that the protein synthetic capacity is directed to synthesis of proteins required for defense against endotoxemia.

The objective of this study was to elucidate the mechanism by which cyclic AMP increases arginase activity in cultured human Caco-2 tumor cells. Caco-2 cells were incubated for 24 h in the presence of 8-bromo cyclic AMP or forskolin, and the cells were harvested, lysed, and assayed for total arginase activity. Both test agents increased arginase activity by twofold, and this was attributed to the induction of the arginase II isoform. Both arginase II mRNA and protein showed increased expression in response to 8-bromo cyclic AMP and forskolin, and these effects were inhibited by H-89 (protein kinase A inhibitor), enhanced by okadaic acid (phosphatase inhibitor), and enhanced by 1-methyl-3-isobutylxanthine (cyclic nucleotide phosphodiesterase inhibitor). Cyclic GMP did not appear to be involved in arginase II induction. These observations indicate that cyclic AMP stimulates arginase II gene expression by mechanisms involving activation of protein kinase A and consequent activation of appropriate transcription factors.

Nitric oxide (NO) is involved in many physiological and pathological processes in the brain. NO is synthesized from arginine by nitric oxide synthase (NOS), with citrulline generated as a by-product of the reaction. Thus, citrulline can by recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) via the citrulline-NO cycle. Rat astroglioma C6 cells were treated with bacterial lipopolysaccharide (LPS), interferon-gamma (IFNgamma) and tumor necrosis factor-alpha, and the expression of the enzymes of the citrulline-NO cycle was investigated by RNA blot and immunoblot analyses. NO production from arginine and citrulline was also assessed. iNOS mRNA and protein were induced 6-12 h after stimulation with LPS and cytokines and decreased at 24 h. AS mRNA increased up to 12 h and decreased at 24 h. AS protein increased gradually up to 48 h. On the other hand, AL mRNA remained unchanged by stimulation. NO production from arginine was enhanced by the treatment with LPS and cytokines. NO production was also observed when arginine was replaced by citrulline. These results indicate that NO production is enhanced in LPS- and cytokine-stimulated C6 cells due to induction of iNOS and that the citrulline-arginine recycling is important for NO production.

The mitochondrial import receptor translocase of the outer membrane of mitochondria (Tom20) consists of five segments, an N-terminal membrane-anchor segment, a linker segment rich in charged amino acids, a tetratricopeptide repeat motif, a glutamine-rich segment, and a C-terminal segment. To assess the role of each segment, four C-terminally truncated mutants of the human receptor (hTom20) were constructed, and the effect of their overexpression in COS-7 cells was analyzed. Expression of a mutant lacking the tetratricopeptide repeat motif inhibited preornithine transcarbamylase (pOTC) import to the same extent as the wild-type receptor. Thus, overexpression of the membrane-anchor and the linker segments is sufficient for the inhibition of import. Expression of either the wild-type receptor or a mutant lacking the C-terminal end of 20 amino acid residues stimulated import of pOTC-green fluorescent protein (GFP), a fusion protein in which the presequene of pOTC was fused to green fluorescent protein. On the other hand, expression of mutants lacking either the glutamine-rich segment or larger deletions inhibited pOTC-GFP import. In vitro import of pOTC was inhibited by the wild-type hTom20 and the mutant lacking the C-terminal end, but much less strongly by the mutant lacking the glutamine-rich segment. On the other hand, import of pOTC-GFP was little affected by any of the forms of hTom20. In binding assays, pOTC binding to hTom20 was only moderately decreased by the deletion of the glutamine-rich segment, whereas pOTC-GFP binding was completely lost by this deletion. Binding of pOTCN-GFP a construct that contains an additional 58 N-terminal residues of mature OTC, resembled that of pOTC. All of these results indicate that the region 106-125 containing the glutamine-rich segment of hTom20 is essential for binding and import stimulation in vivo of pOTC-GFP and for inhibition of in vitro import of pOTC. The results also indicate that this region is important for mitochondrial aggregation. The different behaviors of pOTC and the pOTC-GFP chimera toward hTom20 mutants is explicable on the basis of the conformation of the precursor proteins.

Ammonia produced by amino acid metabolism is detoxified through conversion into urea by the ornithine cycle in the liver, whereas carbon skeletons of amino acids are converted to glucose by gluconeogenic enzymes. Promoter and enhancer sequences of several genes for ornithine cycle enzymes interact with members of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family. Disruption of the C/EBPalpha gene in mice causes hypoglycemia associated with the impaired expression of gluconeogenic enzymes. Here we examined the expression of ornithine cycle enzyme genes in the livers of C/EBPalpha-deficient mice. mRNA levels for the first, third, fourth, and fifth enzymes of five enzymes in the cycle were decreased in C/EBPalpha-deficient mice. Protein levels for the first, second, fourth, and fifth enzymes were also decreased. In situ hybridization analysis revealed that the enzyme mRNAs were distributed normally in the periportal region but were disordered in C/EBPalpha-deficient mice with relatively higher mRNA levels in the midlobular region. Blood ammonia concentrations in the mutant mice were severalfold higher than in wild-type mice. Thus, C/EBPalpha is crucial for ammonia detoxification by ornithine cycle enzymes and for coordination of gluconeogenesis and urea synthesis.

A new sesquiterpene, (1 S ,2 S ,6 R ,8 R )-1-isopropyl-5-(hydroxymethyl)-8-methyltricyclo[4.4.0.0 2,R ]dec-4-ene ( 1 ), was isolated and identified from the leaf surface lipids of Nicotiana umbratica Burbidge. The structure of compound 1 , including the absolute stereochemistry, was established on the basis of its spectral data and X-ray diffraction analysis of 15- p -bromobenzoate ( 1a ).

Nitric oxide is synthesized by nitric-oxide synthase from arginine, a common substrate of arginase. Rat peritoneal macrophages were cultured in the presence of bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of nitric-oxide synthase (iNOS) and liver-type arginase (arginase I) was analyzed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased to a near maximum at 8-12 h. On the other hand, arginase I mRNA that was undetectable prior to the treatment began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. mRNA for arginase II, an arginase isozyme, was not detected in the LPS-activated peritoneal cells. mRNA for CCAAT/enhancer-binding protein beta (C/EBPbeta), a transactivator of the arginase I gene, was also induced, and the induction was more rapid than that of arginase I mRNA. Changes in iNOS and arginase I mRNAs were also examined in LPS-injected rats in vivo. iNOS mRNA increased rapidly in the lung and spleen, reached a maximum 2-6 h after the LPS treatment, and decreased thereafter. Arginase I mRNA was induced markedly and more slowly in both tissues, reaching a maximum in 12 h. Thus, arginase I appears to have an important role in down-regulating nitric oxide synthesis in murine macrophages by decreasing the availability of arginine, and the induction of arginase I is mediated by C/EBPbeta.

The gene for liver-type arginase, an ornithine cycle enzyme, is induced by glucocorticoids in a delayed secondary manner. An enhancer element located around intron 7 of the rat arginase gene shows delayed glucocorticoid responsiveness, and it harbors two sites binding with members of the CCAAT/enhancer binding protein (C/EBP) family. Here, we investigate the role of these C/EBP binding sites in glucocorticoid response of the arginase gene. When inserted in front of the herpes simplex virus thymidine kinase promoter, these C/EBP sites exhibited glucocorticoid responsiveness in reporter transfection assay using rat hepatoma H4IIE cells. In footprint analysis using nuclear extracts of H4IIE cells, profiles of the protected areas of the two C/EBP sites changed when cells were treated with dexamethasone. In gel shift analysis, the complex formation for the two C/EBP sites was augmented in response to dexamethasone. Antibody supershift/inhibition analysis demonstrated that a major portion of the binding proteins induced by dexamethasone is C/EBPβ. Induction of arginase mRNA by dexamethasone was preceded by augmentation of the C/EBP site-binding activities, which followed increase in C/EBPβ mRNA. These results were consistent with the notion that the glucocorticoid response of the arginase gene is mediated by C/EBPβ.

Mouse embryonic stem cells can differentiate into various cell types within cell aggregates called embryoid bodies (EBs). This structure consists of ectodermal, mesodermal, and endodermal tissues, which resemble the embryo of egg-cylinder stage. After 8-10 days in culture, about half of the EBs expand into large cystic structures homologous to visceral yolk sac of postimplantation embryos. To study endoderm differentiation at molecular level, we examined expression of endoderm marker genes during the processes of EB development. alpha-Fetoprotein (AFP) and transthyretin (TTR) transcripts increased at the stage when embryoid bodies began to form yolk-sac-like structures and were expressed strongly thereafter. Expression of hepatocyte nuclear factor (HNF) 4, a variant form of HNF1 (also called HNF1beta), and HNF3beta started before the onset of AFP and TTR expression. HNF1 (also called HNF1alpha) expression began a few days after the onset of the expression of the transcription factors described above. Serum albumin (ALB) transcript was only found in late large cystic EBs. Also, AFP gene expression preceded ALB gene expression. These results suggest that the patterns of endoderm gene expression during EB development reflect the order found during mouse development in vivo, and EB formation may serve as an in vitro system to study the differentiation process.

Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.

We report the effect of the ornithine transcarbamylase (OTC) transgene composed of 1.3 kb of the 5' flanking region of the rat OTC gene fused to rat OTC cDNA on urinary orotic acid excretion in OTC-deficient spf-ash (sparse-fur with abnormal skin and hair) mice during overnight-starvation and nitrogen loading. During starvation, spf-ash mice with about 6% and 2% of control levels of OTC activity in the liver and small intestine excreted a large amount of orotic acid in the urine. Transgenic spf-ash mice with about 10% and 30% of the control OTC activities in the liver and small intestine did not excrete more than the normal level of orotic acid. Accidental parasitization of transgenic spf-ash mice with ticks (Myocoptes musculinus) resulted in decrease of the OTC activities in the liver and small intestine to the levels in spf-ash mice, and increased excretion of orotic acid. During extermination of the ticks, the mice showed varied levels of OTC activity and orotic acid excretion. On nitrogen loading, transgenic spf-ash mice as well as spf-ash mice excreted larger amounts of orotic acid, while control mice showed no increase in its excretion. The levels of urinary orotic acid were inversely correlated to the logarithms of the OTC activities in the liver and small intestine, the correlation being significantly higher with intestinal OTC than with hepatic OTC activity. These results suggest that the level of OTC activity in the small intestine is important for production of orotic acid.

The sparse fur-abnormal skin and hair (SPF-ASH) mouse is a model for the human X-linked hereditary disease, ornithine transcarbamylase (OTC) deficiency. This condition is characterized by abnormal skin and delayed hair growth, hyperammonemia, orotic aciduria and low levels of serum citrulline and arginine. Murakami et al. [1] established a line of transgenic mice, by introducing the recombinant rat OTC (rOTC) gene into fertilized C57BL mouse eggs. We introduced the rOTC gene into SPF-ASH mice by mating SPF-ASH heterozygotes and transgenic mice, which carried this gene. The hemizygous SPF-ASH mice bearing the rOTC gene showed normal hair growth without sparse fur, normal urinary orotic acid excretion and normal serum citrulline and arginine levels. These mice showed OTC activities 2 and 6 times higher in the liver and small intestine, respectively, than the SPF-ASH mice but about 12% and 27% those of the controls [2].

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and hepatocyte nuclear factor-4 (HNF-4) are orphan members of the steroid/thyroid receptor superfamily and exhibit ubiquitous and liver-enriched tissue distribution, respectively. The gene for rat ornithine transcarbamylase (OTC), an ornithine cycle enzyme, is mainly expressed in the liver and is under the control of the promoter and the 11-kilobase upstream enhancer, both of which are liver-selective. Two sites of the promoter region and two sites of the enhancer region of the OTC gene, as well as the ovalbumin promoter site, were recognized by both HNF-4 and COUP-TF, showing that these two factors have closely related binding specificities. Since HNF-4 activated expression from the OTC promoter in cotransfection analysis, this factor appears to participate in liver-selective activation of the OTC gene. On the other hand, COUP-TF repressed the expression from the OTC promoter, whereas it activated expression from several other promoters. Therefore, COUP-TF plays a dual regulatory role depending on the promoter context. Repression of a tissue-specific promoter by a ubiquitous transactivator and derepression by a related tissue-enriched transactivator is potentially an important mechanism for tissue-specific activation of a gene.

Electroantennogram and single sensillum recordings were performed on the male cigarette beetle, Lasioderma serricorne , antennae. Strong antennal responses were elicited by not only natural sex pheromone component (4S,5S,7S)-serricornin (SSS-serricornin) but also non-natural behavioural inhibitor, (4S,5S,7R)-serricornin (SSR-serricornin). In the case of mixture application of the SSS- and SSR-serricornin, integrated electroantennogram responses were observed. Single sensillum recording from male flagella sensilla showed that the majority of basiconic type sensilla responded to the behavioural inhibitor as well as to the sex pheromone. However, a few of them responded only to the inhibitor. The behavioural inhibitory action of SSR-serricornin toward the SSS-serricornin results from central integration of the information from separate receptor cells rather than blockage at the peripheral receptors.

Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.

The synthesis of a simple analog of periplanone-B with high biological activity is described mainly on the examination of the conformational property of the key intermediate 10-t-butoxy-6-methyldeca-2,6-dienone by molecular mechanics calculation and dynamic NMR spectroscopy

The absolute stereochemistry of serricornin was determined to be (4S,6S,7S) by the synthesis of its (4S,6R,7R)-isomer using a carbohydrate synthon.

Recombinant clones containing the promoter region of the human insulin receptor gene were isolated from genomic libraries derived from nondiabetic persons. A 1.5-kilobase pair fragment of the 5'-flanking region was sequenced. One transcriptional start site, located at 203 bases upstream from the start of translation was identified by nuclease S1 mapping and the primer extension experiment using the human insulin receptor mRNA. The bacterial chloramphenicol acetyltransferase assay revealed that a 573-base pair fragment immediately preceding the ATG has promoter activity and that the transcript initiates from the normal start site of the insulin receptor gene in the COS cells. The promoter region contains neither a "TATA box" nor a "CAAT box," has an extremely high G + C content, and contains seven central components of potential Sp 1 binding sites (GGGCGG or CCGCCC). These features are common to those found in the regulatory regions of a class of constitutively expressed "housekeeping" genes. A comparison between the promoter sequence of the human insulin receptor and those of other "housekeeping" genes revealed the presence of homologous sequences among these genes, in addition to the potential Sp 1 binding sites.

Three enzymes of peroxisomal β-oxidation of rat liver were synthesized in a cell-free protein-synthesizing system derived from rabbit reticulocyte lysate. The invitro products of acyl-CoA oxidase and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase multifunctional protein were similar in size to or slightly larger than the subunit of the respective mature enzymes. The invitro product of peroxisomal 3-ketoacyl-CoA thiolase was about 3,000 daltons larger than the mature subunit. The hepatic levels of translatable mRNAs coding for these three enzymes were about 10 times higher in rats fed a di(2-ethylhexyl)phthalate-containing diet than in control animals.

Orinithine carbamoyltransferase [EC 2.1.3.3] of rat liver, a mitochondrial matrix enzyme, is synthesized in a larger precursor form in cell-free protein-synthesizing systems. In cell-free translation programmed with polysomes or polysomal RNAs from rat liver, activity of mRNA coding for the precursor of the enzyme was seven to nine times higher in free polysomes than in membrane-bound polysomes. In rat liver slices, the pulse-labeled precursor of the enzyme was found to be present in a cytosol fraction and to rapidly disappear from the cytosol in pulse-chase experiments. These results indicate that ornithine carbamoyltransferase is initially synthesized on free polysomes as a larger precursor, which is then imported rapidly into mitochondria where it is processed to form the mature enzyme.

Levoglucosenone ( 1 ) was used as the starting material for short and stereoselective routes to chiral synthons for (—)-δ-multistriatin ( 2 ) and (+)-Prelog-Djerassi lactonic acid ( 3 ). The stereochemistry of these synthons is discussed on the basis of high-resolution 1 H-n.m.r. data.

The total synthesis of (−)-periplanone-B, the sex excitant pheromone of the American cockroach, Periplaneta americana is accomplished starting from (+)-limonene.

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.

A polysaccharide containing d -xylose, l -arabinose, d -mannose, d -galactose and d -glucose residues in the molar ratio of 0.07:0.16:1.

Rat 3-oxoacyl-CoA thiolase, an enzyme of the fatty acid beta-oxidation cycle, is located in the mitochondrial matrix. Unlike most mitochondrial matrix proteins, the thiolase is synthesized with no transient presequence and possesses information for mitochondrial targeting and import in the mature protein of 397 amino acid residues. cDNA sequences encoding various portions of the thiolase were fused in frame to the cDNA encoding the mature portion of rat ornithine transcarbamylase (lacking its own presequence). The fusion genes were transfected into COS cells, and subcellular localization of the fusion proteins was analyzed by cell fractionation with digitonin. When the mature portion of ornithine transcarbamylase was expressed, it was recovered in the soluble fraction. On the other hand, the fusion proteins containing the NH2-terminal 392, 161, or 61 amino acid residues of the thiolase were recovered in the particulate fraction, whereas the fusion protein containing the COOH-terminal 331 residues (residues 62-392) was recovered in the soluble fraction. Enzyme immunocytochemical and immunoelectron microscopic analyses using an anti-ornithine transcarbamylase antibody showed mitochondrial localization of the fusion proteins containing the NH2-terminal portions of the thiolase. These results indicate that the NH2-terminal 61 amino acids of rat 3-oxoacyl-CoA thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. Pulse-chase experiments showed that the ornithine transcarbamylase precursor and the thiolase traveled from the cytosol to the mitochondria with half-lives of less than 5 min, whereas the three fusion proteins traveled with half-lives of 10-15 min. Interestingly, in the cells expressing the fusion proteins, the mitochondria showed abnormal shapes and were filled with immunogold-positive crystalloid structures.

Three peroxisomal enzymes of beta-oxidation from rat liver were synthesized in a cell-free protein-synthesizing system derived from a lysate of rabbit reticulocytes. The in vitro products of acyl-CoA oxidase (EC 1.3.99.3) and a bifunctional protein containing enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities were apparently the same in size and charge as the subunit of the respective mature enzymes; that of 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was about 3,000 Da larger and more basic than its mature subunit. The free polysome fraction of rat liver was 3.1-5.7 times more active than the membrane-bound polysome fraction in the synthesis of the three peroxisomal enzymes; these values were similar to those for cytosolic enzymes and differed from that for serum albumin. In isolated rat hepatocytes, radiolabeled acyl-CoA oxidase and bifunctional protein increased with time with no appreciable change in the subunit size. On the other hand, the labeled putative precursor of 3-ketoacyl-CoA thiolase, as well as the mature form of the enzyme, was detected in the hepatocytes. The radioactivity of the putative precursor reached a plateau in 30 min; that of the mature subunit appeared after a lag time of about 5 min and increased with time up to 90 min. In pulse-chase experiments, the putative precursor disappeared with an apparent half-life of several minutes. When the hepatocytes were fractionated into the cytosolic and the particulate fractions, one half of labeled acyl-CoA oxidase and 60% of the bifunctional protein were recovered in the cytosolic fraction after 10 min of labeling, whereas 70-80% of the labeled enzymes were recovered in the particulate fraction after 40-60 min of labeling. These results indicate that the three enzymes of peroxisomal beta-oxidation are synthesized on free polysomes, released into the cytosol, and then transported into peroxisomes. Our findings also indicate that 3-ketoacyl-CoA thiolase undergoes proteolytic processing during maturation. The temporal sequence of the proteolytic cleavage and intracellular transport of the thiolase remains to be determined.

The conformational difference of the title compound ( 1 ) in the solid state and in solution has been investigated by X-ray crystallography and high-field proton n.m.r. spectrometry. In the solid state, compound 1 adopts the 4 C 1 ( d ) conformation ( 1a ), whereas 1 exists preferentially in the 1 C 4 ( d ) conformation ( 1b ) in chloroform solution.

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of N- acetyl- L -glutamate , an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in N- acetyl- L -glutamate concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.

Total, free and membrane-bound polysomes from the liver of rats were assayed for mRNA activity in vitro. Controls (n = 3) and rats 1 (day 2, n = 3) or 3 days (day 4, n = 3) after a 25% body surface burn were used. The sedimentation profiles of total and bound polysomes shifted to a more aggregated state on day 2, and the peaks of monosomes and their sub-units in the free polysomal fraction decreased markedly. Significant changes in mRNA activity were observed in 23 and 17 polypeptides out of 170 on days 2 and 4, respectively. Most of the polypeptides with an increased activity were synthesised by bound polysomes. The mRNA activity for albumin was decreased by 60% on day 4. The activity for carbamoyl-phosphate synthetase I was increased two-fold on day 2. Although treatments of normal animals with counter-regulatory hormones produced characteristic patterns of in vitro products in each case, they mimicked only partially the effect of burn injury. These results indicate that burn injury induces marked changes in the level of translatable mRNAs associated with both free and membrane-bound polysomes in the liver, and suggest that other factors in addition to the counter-regulatory hormones are involved during the course of these changes.

To pursue the mechanism underlying dietary induction of ornithine decarboxylase (ODC) activity in rat liver, changes in free-polysome-associated messenger RNA (mRNA) activity for the enzyme were studied in rats fed diets containing either casein or zein. The enzyme activity increased 50- to 100-fold in 4 h after feeding a 50% casein diet. Changes in the amount of immunoreactive ODC protein roughly paralleled changes in the enzyme activity. Dietary casein was found to cause a several fold increase in polysomal mRNA activity coding for this enzyme, which preceded the increase in catalytic activity. Therefore, the induction of hepatic ODC by casein feeding is due partly to an increase in the activity of its mRNA associated with free polysomes and partly to some translational and/or posttranslational control. On the other hand, feeding the zein diet hardly increased ODC activity, immunoreactive ODC protein or free-polysomal ODC-mRNA activity. This indicates that adequate supply and balance of amino acids are required both for the increase in polysome-associated ODC-mRNA activity and for the translational and/or posttranslational mechanism(s) during the induction of hepatic ODC activity.

The total synthesis of (-)-periplanone-B 1, the major sex excitant and attractant pheromone of the American cockroach was accomplished starting from (+)-dihydrolimonene 2. Intramolecular alkylation of the substituted α-phenylthioacrylate 10 gave rise to the ( E )-cyclodecene 11. Second key reaction was the reductive elimination of vicinal phenylthiobenzoate 14 to provide the conjugated diene 15. Overall yield of 1 through 28 steps was 0.5 % .