Carrier-mediated hepatobiliary transport of a novel antifolate, N-[4-[(2,4-dianninopteridine-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]ca rbonyl-L-homoglutamic acid, in rats. (9/119)

The hepatic uptake and biliary excretion of a novel methotrexate derivative, N-[4-[(2,4-diamminopteridine-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]ca rbonyl-L-homoglutamic acid (MX-68), were examined in rats in vitro using isolated hepatocytes and bile canalicular membrane vesicles (CMVs), respectively. The uptake of MX-68 by isolated rat hepatocytes showed a saturable component (Km = 2.15 microM and Vmax = 2.34 pmol/min/mg of protein) and was inhibited by ATP-depletors and anionic compounds such as taurocholate and probenecid. [3H]MX-68 uptake was also inhibited by folate analogs such as methotrexate and 5CH3-tetrahydrofolate, but the effect of these compounds was slightly less than that of unlabeled MX-68. On replacing Na+ with choline, MX-68 uptake remained unchanged, whereas the methotrexate uptake was reduced. Uptake of MX-68 increased as the extracellular pH fell from 7.5 to 5.5. These results suggest that MX-68 is taken up via active transport systems. The uptake of MX-68 by CMVs prepared from normal rats exhibited clear ATP dependence, whereas ATP had only a minimal effect on the uptake by CMVs from Eisai-hyperbilirubinemic rats with a hereditary deficiency in canalicular multispecific organic anion transporter (cMOAT). The initial uptake rate of ATP-dependent MX-68 transport showed saturation with kinetic parameters similar to those of methotrexate. MX-68 inhibited the ATP-dependent transport of 2,4-dinitrophenyl-S-glutathione, a typical substrate for cMOAT, the inhibition constant (162 microM) being comparable with the Km of ATP-dependent MX-68 transport. These results suggest that the biliary excretion of MX-68 via the bile canalicular membrane is mediated mainly by cMOAT. In conclusion, active transport systems are involved in membrane penetration of MX-68 both at sinusoidal and canalicular sides in the liver, the latter being mainly involved with methotrexate (MTX) whereas the former differs partially from that for MTX.  (+info)

Two new elastin cross-links having pyridine skeleton. Implication of ammonia in elastin cross-linking in vivo. (10/119)

Isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. The structures of these amino acids were determined to have 3,4,5- and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (C(18)H(28)N(4)0(6)) identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine. We have named these pyridine cross-links desmopyridine (DESP) and isodesmopyridine (IDP), respectively. Structure analysis of these pyridine cross-links implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. The elastin incubated with ammonium chloride showed that DESP and IDP levels increased as the allysine content decreased. DESP and IDP were measured by high pressure liquid chromatography (HPLC) with UV detection and were found in a variety of bovine tissues. The DESP/desmosine (DES) and IDP/isodesmosine (IDE) ratios in aorta elastin were higher than in other tissues. DESP and IDP contents in human aorta elastin were found to be gradually increased with age. The concentration of IDP was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride (mean +/- S.D.; 11.1 +/- 0.9 nmol/mg elastin) when compared with normal rats (5.9 +/- 1.5 nmol/mg elastin). Although DESP and IDP are present at only trace concentrations in the tissue elastin, these pyridine cross-links may be useful biomarkers for the aortic elastin damaged by ammonia.  (+info)

Functional and evolutionary relationship between arginine biosynthesis and prokaryotic lysine biosynthesis through alpha-aminoadipate. (11/119)

Our previous studies revealed that lysine is synthesized through alpha-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from alpha-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an argD homolog of T. thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the argD-like gene, now termed lysJ, in T. thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the epsilon-amino group of N(2)-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When N(2)-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the delta-amino group of N(2)-acetylornithine to 2-oxoglutarate 16 times more efficiently than when N(2)-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in T. thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis.  (+info)

Effect of methionine on cephalosporin C and penicillin N production by a mutant of Cephalosporium acremonium. (12/119)

A mutant with enhanced potential to utilize sulfate for cephalosporin C production was isolated from a strain of Cephalosporium acremonium. The mutant displayed potency levels more than twofold that of the parent in the presence of sulfate but its productivity was severely inhibited by more than 0.5% of methionine which gave high cephalosporin C production with the parent. In a complex medium norleucine stimulated cephalosporin C production by the mutant in the presence of sulfate, whereas it showed no effect on the parent. In an incubation system with sulfur-starved cells of the mutant, L-methionine, but not the D-isomer, gave lower cephalosporin C production and a delayed production of penicillin N. However, it exhibited a stimulatory effect in the presence of valine or alpha-aminoadipic acid, the constituent amino acids of the antibiotic. Norleucine showed a similar effect to that of L-methionine in the presence of sulfate. On the basis of these results, characteristics of the mutant are discussed in connection with the effect of methionine.  (+info)

Oxidative deamination of lysine residue in plasma protein of diabetic rats. Novel mechanism via the Maillard reaction. (13/119)

The levels of alpha-aminoadipic-delta-semialdehyde residue, the oxidative deamination product of lysine residue, in plasma protein from streptozotocin-induced diabetic rats were evaluated. alpha-Aminoadipic-delta-semialdehyde was converted to a bisphenol derivative by acid hydrolysis in the presence of phenol, and determined by high performance liquid chromatography. Analysis of plasma proteins revealed three times higher levels of alpha-aminoadipic-delta-semialdehyde in diabetic subjects compared with normal controls. Furthermore, we explored the oxidative deamination via the Maillard reaction and demonstrated that the lysine residue of bovine serum albumin is oxidatively deaminated during the incubation with various carbohydrates in the presence of Cu2+ at a physiological pH and temperature. This experiment showed that 3-deoxyglucosone and methylglyoxal are the most efficient oxidants of the lysine residue. When the reaction was initiated from glucose, a significant amount of alpha-aminoadipic-delta-semialdehyde was also formed in the presence of Cu2+. The reaction was significantly inhibited by deoxygenation, catalase, and a hydroxyl radical scavenger. The mechanism we propose for the oxidative deamination is the Strecker-type reaction and the reactive oxygen species-mediated oxidation. Based on these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction.  (+info)

Mutants of Saccharomycopsis lipolytica defective in lysine catabolism. (14/119)

Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests.  (+info)

Domain structure characterization of the multifunctional alpha-aminoadipate reductase from Penicillium chrysogenum by limited proteolysis. Activation of alpha-aminoadipate does not require the peptidyl carrier protein box or the reduction domain. (15/119)

The alpha-aminoadipate reductase (alpha-AAR) of Penicillium chrysogenum, an enzyme that activates the alpha-aminoadipic acid by forming an alpha-aminoadipyl adenylate and reduces the activated intermediate to alpha-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for alpha-aminoadipic acid, ATP, and NADPH were determined. Sequencing of the N-terminal end confirmed the 10 first amino acids deduced from the nucleotide sequence. Its domain structure has been investigated using limited proteolysis and active site labeling. Trypsin and elastase were used to cleave the multienzyme, and the location of fragments within the primary structure was established by N-terminal sequence analysis. Initial proteolysis generated two fragments: an N-terminal fragment housing the adenylation and the peptidyl carrier protein (PCP) domains (116 kDa) and a second fragment containing most of the reductive domain (28 kDa). Under harsher conditions the adenylation domain (about 64 kDa) and the PCP domain (30 kDa) become separated. Time-dependent acylation of alpha-AAR and of fragments containing the adenylation domain with tritiated alpha-aminoadipate occurred in vitro in the absence of NADPH. Addition of NADPH to the labeled alpha-AAR released most of the radioactive substrate. A fragment containing the adenylation domain was labeled even in absence of the PCP box. The labeling of this fragment (lacking PCP) was always weaker than that observed in the di-domain (adenylating and PCP) fragment suggesting that the PCP domain plays a role in the stability of the acyl intermediate. Low intensity direct acylation of the PCP box has also been observed. A domain structure of this multienzyme is proposed.  (+info)

Adenosine A2A or A3 receptors are required for inhibition of inflammation by methotrexate and its analog MX-68. (16/119)

OBJECTIVE: Low-dose weekly methotrexate therapy remains a mainstay in the treatment of inflammatory arthritis. Results of previous studies demonstrated that adenosine, acting at one or more of its receptors, mediates the antiinflammatory effects of methotrexate in animal models of both acute and chronic inflammation. We therefore sought to establish which receptor(s) is involved in the modulation of acute inflammation by methotrexate and its nonpolyglutamated analog MX-68 (N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]-c arbonyl]-L-homoglutamic acid). METHODS: We studied the effects of low-dose methotrexate (0.75 mg/kg intraperitoneally [IP] every week for 5 weeks), MX-68 (2 mg/kg IP 2 days and 1 hour before induction of inflammation), dexamethasone (1.5 mg/kg IP 1 hour before induction of inflammation), or vehicle control on acute inflammation in an air-pouch model in A(2A) and A(3) receptor knockout mice. RESULTS: Low-dose weekly methotrexate treatment increased the adenosine concentration in the exudates of all mice studied and reduced leukocyte and tumor necrosis factor alpha accumulation in the exudates of wild-type mice, but not in those of A(2A) or A(3) receptor knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was equally effective at suppressing leukocyte accumulation in A(2A) knockout, A(3) knockout, and wild-type mice, indicating that the lack of response was specific for methotrexate and MX-68. CONCLUSION: These findings confirm that adenosine, acting at A(2A) and A(3) receptors, is a potent regulator of inflammation. Moreover, these results provide strong evidence that adenosine, acting at either or both of these receptors, mediates the antiinflammatory effects of methotrexate and its analog MX-68.  (+info)