Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.     

Characterization of mouse cysteinyl leukotriene receptors mCysLT1 and mCysLT2: differential pharmacological properties and tissue distribution

Cysteinyl leukotrienes (LTs) are important proinflammatory mediators. Their precise roles in mice need to be elucidated to interpret mouse models of inflammatory diseases. For this purpose, we cloned and characterized mouse receptors for cysteinyl LTs, mCysLT(1) and mCysLT(2). mCysLT(1) and mCysLT(2) were composed of 339 amino acids with 87.3% identity and 309 amino acids with 73.4% identity to human orthologues, respectively. A pharmacological difference was noted between mouse and human CysLT(2). Pranlukast, a specific inhibitor for human CysLT(1), antagonized mCysLT(2) responses as determined by Ca(2+) elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT(1) mRNA was expressed mainly in skin, lung, and small intestine. mCysLT(2) was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT(1) and mCysLT(2) were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT(2) between human and mouse and the different distributions of CysLTs between mouse strains suggest that careful choice and interpretation are necessary for a study of CysLTs using animal models.     

Fourier transform IR study of aggregational behavior of N-acetyl-L- and N-butyloxycarbonyl-L-glutamic acid oligomeric benzyl esters in dioxane and benzene: beta-turn --> antiparallel beta-sheet transition

Resins from several different genera are studied using Fourier transform (FT)-Raman spectroscopy. Tree resins can be broadly divided into those that contain diterpenoid components and those that contain triterpenoid components. The diterpenoid resins analyzed are from the genera Pinus, Cedrus, and Agathis (kauri resin) and the triterpenoid resins examined are samples from Pistacia, Boswellia (frankincense), and Commiphora (myrrh) genera. A protocol is developed to nondestructively distinguish diterpenoid and triterpenoid resins and to differentiate the genera within the two types. The effects of oxidation on the discrimination of the FT-Raman spectra are considered.     

Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.     

A plant growth retardant related to chlamydocin and its proposed mechanism of action

A comparison of the plant growth retardant activity of the chlamydocin analogues, compound 1, six derivatives from 1 and 2, and two synthetic analogues revealed that there are two types of retardant in chlamydocin analogues. One, for example in compound 1, requires an oxygen atom at C-8 of the 2-aminodecanoic acid moiety to show retardant activity. The other, for example in compound 8, requires no oxygen atom at C-8 but requires a specific alkyl group chain length for activity. To determine the differences in mode of action of both types of retardant, rice seedlings were separately treated with compounds 1 and 8, and after appearance of dwarfism, their endogenous ABA and GA(1) levels were determined and compared to those of the control. Treatment with 1 (10 nmol/plant) increased ABA levels 4 times higher than that of the control and decreased GA(1) levels to 20% of that of the control. Treatment with 8 (30 nmol/plant) did not affect the ABA level but decreased GA(1) content to 5% of that of the control.     

cDNA cloning of an alternative splicing variant of protein kinase C delta (PKC deltaIII), a new truncated form of PKCdelta, in rats

Recently, an alternative splicing variant of mouse protein kinase C delta (PKC deltaII, GenBank Accession No. AB011812) has been reported which has a 78 bp (26 amino acid) insertion at the caspase-3 recognition sequence in the V3 region of PKC delta (PKC deltaI). We isolated a cDNA encoding a new variant of PKC delta (PKC deltaIII, AF219629), which has a 83 bp insertion at the same site in the V3 region, by RT-PCR using rat testis RNA as a template. In rats, the 83 bp insertion causes inframe termination, and rat PKC deltaIII protein is expressed as a truncated form, having only the regulatory domain without a catalytic domain. Genomic DNA analysis revealed that the difference between mouse PKC deltaII and rat PKC deltaIII is derived from the different sequence at the 5'-splicing donor sites. To investigate the potential functions of the truncated form of PKC delta, rat PKC deltaIII fused to green fluorescent protein (GFP) was expressed in CHO-K1 cells. PKC deltaIII-GFP was localized in the cytoplasm with dot-like accumulation and highly expressed on the plasma membrane, whereas PKC deltaI-GFP is localized homogeneously throughout the cytoplasm, including the nucleoplasm. Stimulation by phorbol ester caused weak translocation of deltaIII-GFP from the cytosol to the plasma membrane. These results suggest that PKC deltaIII may show a dominant negative effect against PKC deltaI, and that the modulation of signal transduction by alternative splicing variant may play a crucial role in the physiological and/or pathological conditions, and the pathogenesis of disease.     

Limited myogenic response to a single bout of weight-lifting exercise in old rats

The purpose of the present study was to compare themyogenic response of hindlimb muscles in young (14-20 wk of age)and old (>120 wk of age) rats with a single exhaustive bout of heavyresistance weight lifting. [³H]thymidine and[¹⁴C]leucine labeling were monitored for up to2 wk after the exercise bout to estimate serial changes in mitoticactivity and the level of amino acid uptake and myosin synthesis.Histological, histochemical, and immunohistochemical[anti-5-bromo-2'-deoxyuridine and myogenic determinationgenes (MyoD)] analyses of whole muscles and analysis ofmuscle-specific gene expression (MyoD) using Western blotting andRT-PCR were performed. Old rats showed significant muscle atrophy and alower exercise capacity than young rats. Exercise-induced muscledamage, as assessed in histological sections, and increases in serumcreatine kinase activity were evident in both young and old exercisedgroups. Mitotic activity was increased in young, but not old, rats 2 days after exercise. There was a biphasic increase in[¹⁴C]leucine uptake during the 14 dayspostexercise (peaks at 1-4 and 10 days) in young rats: only thefirst peak was observed in old rats. There was a lower uptake of[¹⁴C]leucine in the myosin fraction and animpaired expression of MyoD at the protein (immunohistochemistry andWestern blotting) and mRNA (RT-PCR) levels in old rats throughout thepostexercise period. These results demonstrate a reduced reparativecapability of muscle in response to a single bout of exercise in oldcompared with young rats.

     

Molecular cloning of cDNA encoding alpha-N-acetylgalactosaminidase from Acremonium sp. and its expression in yeast

Alpha-N-acetylgalactosaminidase (alpha-GalNAc-ase; EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine in various sugar chains. The cDNA, nagA, encoding alpha-GalNAc-ase from Acremonium sp. was cloned, sequenced, and expressed in yeast Saccharomyces cerevisiae. The nagA contains an open reading frame which encodes for 547 amino acid residues including 21 residues of a signal peptide in its N-terminal. The calculated molecular mass of mature protein from the deduced amino acid sequence of nagA is 57260 Da, which corresponds to the value obtained from SDS-PAGE of native and recombinant enzymes treated with endo-beta-N-acetylglucosaminidase H. The amino acid sequence of NagA showed significant similarity to those of eukaryotic alpha-GalNAc-ases and alpha-galactosidases (alpha-Gal-ases), particularly alpha-Gal-ase A (AglA) from Aspergillus niger. Phylogenetic analysis revealed that NagA does not belong to the cluster of vertebrate alpha-GalNAc-ase and alpha-Gal-ase but forms another cluster with AglA and yeast alpha-Gal-ases. Thus, the evolutionary origin of the fungal alpha-GalNAc-ase is suggested to be different from that of vertebrate alpha-GalNAc-ase. This is the first report of a microbial alpha-GalNAc-ase gene.