Changes in quinolinic acid production and its related enzymes following D-galactosamine and lipopolysaccharide-induced hepatic injury

Increases in quinolinic acid (QUIN), a neurotoxic L-tryptophan metabolite, have been observed in human serum and cerebrospinal fluid and in animal models of severe hepatic injury. The aim of this study was to evaluate the changes in QUIN accumulation and its related enzymes after acute hepatic injury induced by D-galactosamine and endotoxin. Gerbils were given an intraperitoneal injection of pyrogen-free saline alone as control, lipopolysaccharide (LPS) alone (150 ng/kg), D-galactosamine alone (500 mg/kg) or a combination of D-galactosamine with LPS. Concentrations of QUIN, its related metabolites, and related enzyme activities were determined. D-Galactosamine treatment significantly decreased activities of hepatic aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) resulting in increased QUIN concentrations in serum and tissues. The magnitude of QUIN responses was markedly increased by endotoxin due to the increased availability of L-kynurenine, a rate-limiting substrate for QUIN synthesis. Further, infiltration of monocytes/macrophages, which is a possible major source of QUIN production in the liver, was shown by immunohistochemistry after hepatic injury induced by D-galactosamine and endotoxin. Increased serum QUIN concentrations are probably due to the increased substrate availability and the decreased activity of aminocarboxymuconate-semialdehyde decarboxylase in the liver, accompanying the increased monocyte/macrophage infiltration into the liver after hepatic injury.     

The twisted abdomen phenotype of Drosophila POMT1 and POMT2 mutants coincides with their heterophilic protein O-mannosyltransferase activity

Walker-Warburg syndrome, caused by mutations in protein O-mannosyltransferase-1 (POMT1), is an autosomal recessive disorder characterized by severe brain malformation, muscular dystrophy, and structural eye abnormalities. As humans have a second POMT, POMT2, we cloned each Drosophila ortholog of the human POMT genes and carried out RNA interference (RNAi) knock-down to investigate the function of these proteins in vivo. Drosophila POMT2 (dPOMT2) RNAi mutant flies showed a "twisted abdomen phenotype," in which the abdomen is twisted 30-60 degrees , similar to the dPOMT1 mutant. Moreover, dPOMT2 interacted genetically with dPOMT1, suggesting that the dPOMTs function in collaboration with each other in vivo. We expressed dPOMTs in Sf21 cells and measured POMT activity. dPOMT2 transferred a mannose to the dystroglycan protein only when it was coexpressed with dPOMT1. Likewise, dPOMT1 showed POMT activity only when coexpressed with dPOMT2, and neither dPOMT showed any activity by itself. Each dPOMT RNAi fly totally reduced POMT activity, despite the specific reduction in the level of each dPOMT mRNA. The expression pattern of dPOMT2 mRNA was found to be similar to that of dPOMT1 mRNA using whole mount in situ hybridization. These results demonstrate that the two dPOMTs function as a protein O-mannosyltransferase in association with each other, in vitro and in vivo, to generate and maintain normal muscle development.     

In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and our genetic studies suggest that GNBP1 acts upstream of the Toll ligand Sp?tzle. Altogether, our results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1.     

The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1)

DNA topoisomerase I and II have been shown to be modified with a ubiquitin-like protein SUMO in response to their specific inhibitors called 'poisons'. These drugs also damage DNA by stabilizing the enzyme-DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIbeta, but not its isoform IIalpha, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF-193 which does not damage DNA. The beta isoform immunoprecipitated from ICRF-treated cells was modified by multiple modifiers, SUMO-2/3, SUMO-1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF-induced degradation of topoisomerase IIbeta did not occur, suggesting that the SUMO modification pathway is essential for the degradation.     

Association of cathepsin E deficiency with development of atopic dermatitis

Atopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogen-free conditions. These mice showed the increase in the ratio of CD4+/CD8+ T cells, the strong polarization of na?ve T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1beta accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1beta were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1beta, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice.     

Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS)

Understanding the role of ‘epigenetic’ changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development.     

Role of 5-aminolevulinic acid (ALA) on active oxygen-scavenging system in NaCl-treated spinach (Spinacia oleracea)

ALA is a key precursor in the biosynthesis of porphyrins such as chlorophyll and heme, and was found to induce temporary elevations in the photosynthesis rate, APX, and CAT; furthermore, treatment with ALA at a low concentration might be correlated to the increase of NaCl tolerance of spinach plants. The photosynthetic rate and the levels of active oxygen-scavenging system in the 3rd leaf of spinach (Spinacia oleracea) plants grown by foliar treatment with 0, 0.18, 0.60 and 1.80 mmol/L 5-aminolevulinic acid under 50 and 100 mmol/L NaCl were analyzed. Plants treated with 0.60 and 1.80 mmol/L ALA showed significant increases in the photosynthetic rate at 50 and 100 mmol/L NaCl, while that of 0.18 mmol/L ALA did not show any changes at 50 mmol/L NaCl and a gradual decrease at 100 mmol/L NaCl. In contrast, the rate with 0 mmol/L ALA showed reduction at both concentrations of NaCl. The increase of hydrogen peroxide content by treatment with 0.60 and 1.80 mmol/L ALA were more controlled than that of 0 mmol/L ALA under both NaCl conditions. These ALA-treated spinach leaves also exhibited a lower oxidized/reduced ascorbate acid ratio and a higher reduced/oxidized glutathione ratio than the 0 mmol/L-treated spinach leaves when grown at both NaCl conditions. With regard to the antioxidant enzyme activities in the leaves, ascorbate peroxidase, catalase, and glutathione reductase activities were enhanced remarkably, most notably at day 3, by treatment with 0.60 and 1.80 mmol/L ALA under both NaCl conditions in comparison to that of 0 and 0.18 mmol/L ALA. These data indicate that the protection against oxidative damage by higher levels of antioxidants and enzyme activities, and by a more active ascorbate-glutathione cycle related to the increase of the photosynthesis rate, could be involved in the increased salt tolerance observed in spinach by treatment with 0.60 to 1.80 mmol/L ALA with NaCl.     

Expressions of adrenomedullin mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Adrenomedullin (AM) has diuretic, natriuretic, and hypotensive effects. To study the possible effects of HHE on the AM synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of a 5,500-m altitude level for 21 days. After 14 days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased (compared with control rats). The plasma AM protein level was significantly increased on day 21 of exposure to HHE. In the right ventricle (RV), right atrium, and left atrium of the heart, the expressions of AM mRNA and protein were increased in the middle to late phase (5-21 days) of HHE, whereas in the brain and lung they were increased much earlier (0.5-5 days). In situ hybridization and immunohistochemistry showed AM mRNA and protein staining to be more intense in the RV in animals in the middle to late phase of HHE exposure than in the controls. During HHE, these changes in AM synthesis, which occurred strongly in the RV, occurred alongside the increase in PAP. Conceivably, AM may play a role in modulating pulmonary hypertension in HHE.     

Variability of biochemical and clinical phenotype in X-linked liver glycogenosis with mutations in the phosphorylase kinase PHKA2 gene

X-linked liver glycogenosis (XLG) resulting from phosphorylase kinase (Phk) deficiency is one of the most common forms of glycogen storage disease. It is caused by mutations in the gene encoding the liver isoform of the Phk α subunit (PHKA2). In the present study, we address the issue of phenotypic and allelic heterogeneity in XLG. We have identified mutations in seven male patients. One of these patients represents the variant biochemical phenotype, XLG subtype 2 (XLG2), where Phk activity is low in liver but normal or even elevated in erythrocytes. He carries a K189E missense mutation, which adds to the emerging evidence that XLG2 is associated with missense mutations clustering at a few sites. Two patients display clinical phenotypes unusual for liver Phk deficiency, with dysfunction of the kidneys (proximal renal tubular acidosis) or of the nervous system (seizures, delayed cognitive and speech abilities, peripheral sensory neuropathy), respectively, in addition to liver glycogenosis. In the patient with kidney involvement, we have identified a missense mutation (P399S) and a trinucleotide deletion (2858del3) leading to the replacement of two amino acids by one new residue (N953/L954I), and a missense mutation has also been found in the patient with neurological symptoms (G1207W). These two cases demonstrate that PHKA2 mutations can also be associated with uncommon clinical phenotypes. Finally, in four typical XLG cases, we have identified three truncating mutations (70insT, R352X, 567del22) and an in-frame deletion of eight well-conserved amino acids (2452del24). Together, this study adds eight new mutations to the previously known complement of sixteen PHKA2 mutations. All known PHKA2 mutations but one are distinct, indicating pronounced allelic heterogeneity of X-linked liver glycogenosis with mutations in the PHKA2 gene. Received: 17 October 1997 / Accepted: 23 December 1997     

A novel synthesis of 2-deoxy-α-glycosides

The key step of the synthesis involves the reaction of glycals [3,4,6-tri-O-acetyl-d-glucal (1), the new glycal derivative 4-O-acetyl-1,5-anhydro-2,6-dideoxy-3-C-methyl-3-O-methyl-l-ribo-hex-l-enitol (2), and 3-acetamido-4,6,-di-O-acetyl-1,5-anhydro-2,3-dideoxy-d-arabino-hex-l-enitol (3)] with 1.5 molar equivalents of several alcohols in the presence ofN-bromosuccinimide in acetonitrile to give mainly the corresponding 2-bromo-2-deoxy-α-glycopyranosides (4-21). The glycopyranosides (4-8 and16-21) from1 and3 have the α-d-manno configuration and those (10-15) from2 have the α-l-altro configuration. The yields are high from1, virtually quantitative from2, and moderate from3. Debromination of the 2-bromo-2-deoxy compounds with tributylstannane and a radical initiator gives the corresponding 2-deoxy-α-glycopyranosides (22-38) in quantitative yields. In particular, the branched-chain glycal2 reacts with alcohols to give exclusively the corresponding α-glycopyranosides (27-32) of cladinose in strikingly high overall yields. The stereoselectivity and regiospecificity of the bromination reaction are described. 1,3-Dibromo-5,5-dimethylhydantoin andN-bromoacetamide are also found to be useful for the reaction.