Anti-fibrotic effects of L-2-oxothiazolidine-4-carboxylic acid via modulation of nuclear factor erythroid 2-related factor 2 in rats

L-2-Oxothiazolidine-4-carboxylic acid (OTC) is a cysteine prodrug that maintains glutathione in tissues. The present study was designed to investigate anti-fibrotic and anti-oxidative effects of OTC via modulation of nuclear factor erythroid 2-related factor 2 (Nrf2) in an in vivo thioacetamide (TAA)-induced hepatic fibrosis model. Treatment with OTC (80 or 160 mg/kg) improved serum liver function parameters and significantly ameliorated liver fibrosis. The OTC treatment groups exhibited significantly lower expression of α-smooth muscle actin, transforming growth factor-β 1, and collagen α 1 mRNA than that in the TAA model group. Furthermore, the OTC treatment groups showed a significant decrease in hepatic malondialdehyde level compared to that in the TAA model group. Nrf2 and heme oxygenase-1 expression increased significantly in the OTC treatment groups compared with that in the TAA model group. Taken together, these results suggest that OTC restores the anti- oxidative system by upregulating Nrf2; thus, ameliorating liver injury and a fibrotic reaction.     

Antidiabetic effect of a prodrug of cysteine, L-2-oxothiazolidine-4-carboxylic acid, through CD38 dimerization and internalization.

CD38 is a bifunctional enzyme synthesizing (ADP-ribosyl cyclase) and degrading (cyclic ADP-ribose (cADPR) hydrolase) cADPR, a potent Ca(2+) mobilizer from intracellular pools. CD38 internalization has been proposed as a mechanism by which the ectoenzyme produced intracellular cADPR, and thiol compounds have been shown to induce the internalization of CD38. Here, we show that the disulfide bond between Cys-119 and Cys-201 in CD38 may be involved in CD38 dimerization and internalization. We tested the effect of a reducing agent, l-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, on CD38 internalization in pancreatic islets. OTC enhanced insulin release from isolated islets as well as CD38 internalization and cytoplasmic Ca(2+) level. Furthermore, islet cells treated with antisense CD38 oligonucleotide showed inhibition of OTC-induced insulin secretion. Intake of OTC in db/db mice ameliorated glucose tolerance, insulin secretion, and morphology of islets when compared with control mice. These data indicate that OTC improves glucose tolerance by enhancing insulin secretion via CD38/cADPR/Ca(2+) signaling machinery. Thus, OTC may represent a novel class of antidiabetic drug.     

Effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the metabolism and toxicity of bromobenzene: an acute study

The effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on certain aspects of the metabolism and toxicity of bromobenzene administered acutely to mice was investigated by (i) characterizing the influence of OTCA on the metabolic profile of low and high bromobenzene dose at 0-6, 6-12, and 12-24 h, (ii) determining the effective doses range and administration time for OTCA, as well as the optimum period for urine sampling; and (iii) measuring the efficacy of OTCA for protection against bromobenzene induced toxicity. Coadministration of OTCA and bromobenzene enhanced the urinary excretion of mercapturic acid and phenolic metabolites, during 6-12 h, by approximately 152 and 193%, respectively. Maximum efficacy was observed when OTCA (16.0 mmol/kg) was administered concomitantly with bromobenzene (4.0 mmol/kg). Finally, OTCA administration was found to afford substantial protection against elevation of plasma transaminases used as indices of bromobenzene-induced hepatotoxicity. N-acetylcysteine, another cysteine prodrug, had essentially similar effects on the metabolism and toxicity of bromobenzene. Thus, administration of cysteine prodrugs enhances the urinary excretion of several metabolites of bromobenzene and affords protection against bromobenzene-induced hepatotoxicity.     

Effect of L-2-oxothiazolidine-4-carboxylate administration on glutathione and cysteine concentrations in guinea pig liver and kidney

Effect of L-2-oxothiazolidine-4-carboxylate administration on the glutathione and cysteine concentrations in liver and kidney was studied in the guinea pig. Liver glutathione concentration increased significantly by 21 to 29% at one to three hours after the intraperitoneal injection of 5 mmol of sodium L-2-oxothiazolidine-4-carboxylate per kg of body weight. Cysteine concentration did not change significantly. In contrast to the liver, a significant increase in cysteine and also a significant decrease in glutathione concentrations were observed in the kidney. Incubation of L-2-oxothiazolidine-4-carboxylate with liver and kidney homogenates resulted in cysteine formation of 1.21 and 0.56 mumol per g of fresh tissue per 30 min, respectively. These results seem to indicate that, in the liver, L-2-oxothiazolidine-4-carboxylate administration resulted in the formation of cysteine, which was utilized for glutathione synthesis. In the kidney, it seems to be suggested that the administration of this compound accelerated glutathione turnover.     

Enzymatic synthesis oF L-tryptophan from D,L-2-amino-delta2-thiazoline-4-carboxylic acid and indole by Pseudomonas sp. TS1138 L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, S-carbamyl-L-cysteine amidohydrolase, and Escherichia coli L-tryptophanase

L-Tryptophan (L-Trp) is an essential amino acid. It is widely used in medical, health and food products, so a low-cost supply is needed. There are 4 methods for L-Trp production: chemical synthesis, extraction, enzymatic synthesis, and fermentation. In this study, we produced a recombinant bacterial strain pET-tnaA of Escherichia coli which has the L-tryptophanase gene. Using the pET-tnaA E. coli and the strain TS1138 of Pseudomonas sp., a one-pot enzymatic synthesis of L-Trp was developed. Pseudomonas sp. TS1138 was added to a solution of D,L-2-amino-delta2-thiazoline-4-carboxylic acid (DL-ATC) to convert it to L-cysteine (L-Cys). After concentration, E. coli BL21 (DE 3) cells including plasmid pET-tnaA, indole, and pyridoxal 5'-phosphate were added. At the optimum conditions, the conversion rates of DL-ATC and L-Cys were 95.4% and 92.1%, respectively. After purifying using macroporous resin S8 and NKA-II, 10.32 g of L-Trp of 98.3% purity was obtained. This study established methods for one-pot enzymatic synthesis and separation of L-Trp. This method of producing L-Trp is more environmentally sound than methods using chemical synthesis, and it lays the foundations for industrial production of L-Trp from DL-ATC and indole.     

Influence of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the urinary elimination of mercapturic acids of ethylene oxide, dibromoethane, and acrylonitrile: a dose-effect study

Metabolic disposition of ethylene oxide, dibromoethane, and acrylonitrile in rats after acute exposure was studied by examining the relationship between dose and urinary metabolites, and by establishing the influence of a glutathione precursor, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on the above relationship. Respective urinary metabolites, hydroxyethylmercapturic acid, cyanoethylmercapturic acid, thiocyanate, and ethylene glycol, were quantified to estimate the extent to which each compound was metabolized. The animals were given either ethylene oxide (0.34, 0.68, or 1.36 mmol/kg), dibromoethane (0.2, 0.4, or 0.6 mmol/kg), or acrylonitrile (0.10, 0.38, or 0.76 mmol/kg). Urine samples were collected at 24 h. The metabolic biotransformation of all three chemicals to their respective mercapturic acids was strongly indicative of saturable metabolism. Administration of OCTA (4-5 mmol/kg) enhanced gluthathione availability and increased excretion of urinary mercapturic acids at the higher doses of the chemicals. This study indicates that OTCA increases the capacity for detoxification via the glutathione pathway thereby partially correcting the nonlinearity between the administered dose of ethylene oxide, dibromoethane, and acrylonitrile and the amount of certain urinary metabolites.     

PNAS4 knockout does not induce obviously neurocytes apoptosis and abnormal development in mice brain

Apoptosis is one kind of programmed cell death and contributes to development of a variety of organs such as brain. PNAS4 has been reported to be a novel apoptosis-related gene. Overexpression and knocking down of PNAS4 would cause zebrafish and Xenopus lavis developmental abnormalities. But its function and apoptotic mechanism in mammals are still unknown. Here, we first reported that established PNAS4 CKO (conditional knock out) mice using recombineering technology. We prepared its polyclonal antibodies which recognized both myc-PNAS4 overexpression protein and WT and CKO mice brain tissue and MEFS cells with high titre and specificity. Further we detected that PNAS4 was highly expressed in the embryonic period. However, we observed neither neural structural abnormality nor apoptosis signal in PNAS4 CKO mice brain. Our data suggested that PNAS4 was not involved in mice brain development and apoptosis.     

Purification and characterization of 5-oxo-l-prolinase from Paecilomyces varioti F-1, an ATP-dependent hydrolase active with l-2-oxothiazolidine-4-carboxylic acid

An enzyme cleaving l-2-oxothiazolidine-4-carboxylic acid to l-cysteine was purified 75-fold with 8% recovery to near homogeneity from crude extracts of Paecilomyces varioti F-1, which had been isolated as a fungus able to assimilate l-2-oxothiazolidine-4-carboxylic acid. The molecular mass was estimated to be 260 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 140 kDa upon SDS-PAGE. The maximum activity was observed at a range of pH 7.0–8.0 and at 50 °C. The enzyme activity was completely inhibited by SH-blocking reagents such as AgNO₃, p-chloromercuribenzoic acid, N-ethylmaleimide, and N-bromosuccinimide. The enzyme required ATP, Mg²⁺, and KCl for the cleavage of l-2-oxothiazolidine-4-carboxylic acid. The enzyme also cleaved 5-oxo-l-proline to l-glutamic acid and is considered to be 5-oxo-l-prolinase. Received: 23 March 1999 / Accepted: 22 June 1999     

Il-4 and IL-13, but not IL-10, protect human synoviocytes from apoptosis

Interleukin-4, which has been contemplated for the treatment of rheumatoid arthritis and/or osteoarthritis because of its anticatabolic properties, has also been shown to modulate apoptosis. Because inadequate apoptosis is thought to contribute to synovial hyperplasia, we have investigated the ability of IL-4 and other Th2 cytokines to protect human synovial cells from apoptosis. Human synoviocytes or synovial explants were pretreated with IL-4, IL-10, and IL-13 before exposure to NO donor sodium-nitro-prusside (SNP). Apoptosis was evaluated by microscopy, annexin V-FITC, 3-(4,5-dimethylthiazol-2-gl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl-2H: tetrazolium inner salt (MTS) test, pulse field gel electrophoresis, and a method proposed in this study based on (32)P Klenow end labeling of high m.w. DNA. Pretreatment by IL-4 or IL-13, but not IL-10, protected human synoviocytes from apoptosis induced by SNP. Even at doses as high as 2 mM SNP, up to 86% and 56% protection was achieved, after IL-4 and IL-13 treatment, respectively. Cell survival was dependent on IL concentration. IL-4 and IL-13 also had antiapoptotic effects on SNP-treated human synovial explants. Effects of IL-4 and IL-13 varied in the presence of phosphatidylinositol-3 kinase and protein kinase C inhibitors, implying the involvement of these pathways in antiapoptotic signaling. Antiapoptotic effects were dramatically inhibited by LY294002, and partially by the protein kinase C inhibitor G? 6976, while insulin-like growth factor increased synoviocyte survival. The possibility that IL-4 and IL-13 may enhance synovial expansion in vivo by their antiapoptotic effects is discussed.     

Glycolytic inhibitor, 2-deoxy-D-glucose, does not enhance radiation-induced apoptosis in mouse thymocytes and splenocytes in vitro

Earlier studies have shown that 2-deoxy-D-glucose (2-DG), a glucose analogue and inhibitor of glycolytic ATP production selectively enhances radiation-induced damage in cancer cells by inhibiting the energy (ATP) dependent postirradiation DNA and cellular repair processes. A reduction in radiation induced cytogenetic damage has been reported in normal cells viz., peripheral blood lymphocytes and bone marrow cells. Since induction of apoptosis plays a major role in determining the radiosensitivity of some most sensitive normal cells including splenocytes and thymocytes, we investigated the effects of 2-DG on radiation induced apo tosis in these cells in vitro. Thymocytes and splenocytes isolated from normal Swiss albino mouse were irradiated with Co60 gamma-rays and analyzed for apoptosis at various post-irradiation times. 2-DG added at the time of irradiation was present till the termination of cultures. A time dependent, spontaneous apoptosis was evident in both the cell systems, with nearly 40% of the cells undergoing apoptosis at 12 hr of incubation. The dose response of radiation-induced apoptosis was essentially similar in both the cell systems and was dependent on the incubation time. More than 70% of the splenocytes and 60% of the thymocytes were apoptotic by 12 hr following an absorbed dose of 2 Gy. Presence of 2-DG marginally reduced the fraction of splenocytes undergoing apoptosis at all absorbed doses, while no change was observed in thymocytes. Presence of 2-DG did not significantly alter either the level or the rate of induction of spontaneous apoptosis in both these cell systems. These results are consistent with the earlier findings on radiation-induced cytogenetic damage in human PBL in vitro and mouse bone marrow cells and lend further support to the proposition that 2-DG does not enhance radiation damage in normal cells, while radiosensitizing the tumors and hence is an ideal adjuvant in the radiotherapy of tumors.     

Benzothiadiazole and l-2-oxothiazolidine-4-carboxylic acid reduce the severity of Sharka symptoms in pea leaves: effect on antioxidative metabolism at the subcellular level

The effect of treatment with benzothiadiazole (BTH) or l -2-oxothiazolidine-4-carboxylic acid (OTC), and their interaction with Plum pox virus (PPV) infection, on antioxidative metabolism of pea plants was studied at the subcellular level. PPV infection produced a 20% reduction in plant growth. Pre-treatment of pea plants with OTC or BTH afforded partial protection against PPV infection, measured as the percentage of leaves showing symptoms, but neither BTH nor OTC significantly reduced the virus content. PPV infection caused oxidative stress, as monitored by increases in lipid peroxidation and protein oxidation in soluble and chloroplastic fractions. In leaves of non-infected plants, OTC increased the content of reduced glutathione (GSH) and total glutathione; accordingly, an increase in the redox state of glutathione was observed. An increase in oxidized glutathione (GSSG) was found in symptomatic leaves from infected plants. A similar increase in GSSG was also observed in asymptomatic leaves from infected, untreated plants. However, no changes in GSSG occurred in asymptomatic leaves from infected plants treated with BTH and OTC and, accordingly, a higher redox state of GSH was recorded in those leaves, which could have had a role in the reduction of symptoms, as observed in asymptomatic leaves from infected plants treated with BTH or OTC. Treatment with BTH or OTC had some effect on antioxidant enzymes in soluble and chloroplastic fractions from infected pea leaves. An increase in antioxidative mechanisms, such as GSH-related enzymes (DHAR, GR and G6PDH), as well as APX and POX, at the subcellular level was observed, which could play a role in reducing the severity of cellular damage induced by Sharka in pea leaves.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

TLR4 and Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta, but not MyD88, regulate Escherichia coli-induced dendritic cell maturation and apoptosis in vivo

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2(-/-), TLR4(-/-) and TIR domain-containing adapter-inducing IFN-beta (TRIF)(-/-) mice. In contrast, DC apoptosis is reduced only in TLR4(-/-) and TRIF(-/-) mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.     

Dual roles for IFN-gamma,but not for IL-4, in spontaneous autoimmune thyroiditis in NOD.H-2h4 mice

Spontaneous autoimmune thyroiditis (SAT) is an organ-specific autoimmune disease characterized by chronic inflammation of the thyroid by T and B lymphocytes. To investigate the roles of Th1 and Th2 cytokines in the pathogenesis of SAT, IFN-gamma(-/-) and IL-4(-/-) NOD.H-2h4 mice were generated. IL-4(-/-) mice developed lymphocytic SAT (L-SAT) comparable to that of wild-type (WT) mice. They produced little anti-mouse thyroglobulin (MTg) IgG1, but had levels of anti-MTg IgG2b comparable to WT mice. Compared with WT mice, IFN-gamma(-/-) mice produced significantly less anti-MTg IgG1 and IgG2b. Absence of IFN-gamma resulted in abnormal proliferation of thyroid epithelial cells with minimal lymphocyte infiltration. Thyroids of IFN-gamma(-/-) mice had markedly reduced B lymphocyte chemoattractant expression, B cell and plasma cell infiltration, and decreased MHC class II expression on thyrocytes compared with WT mice. Adoptive transfer of WT splenocytes to IFN-gamma(-/-) mice restored the capacity to develop typical L-SAT, enhanced anti-MTg IgG1 and IgG2b production, up-regulated MHC class II expression on thyrocytes and decreased thyrocyte proliferation. These results suggest that IFN-gamma plays a dual role in the development of SAT. IFN-gamma is required for development of L-SAT, and it also functions to inhibit thyroid epithelial cell proliferation.     

In vivo and in vitro escape from neutralizing antibodies 2G12, 2F5, and 4E10

Recently, passive immunization of human immunodeficiency virus (HIV)-infected individuals with monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 provided evidence of the in vivo activity of 2G12 but raised concerns about the function of the two membrane-proximal external region (MPER)-specific MAbs (A. Trkola, H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard, Nat. Med. 11:615-622, 2005). In the light of MPER-targeting vaccines under development, we performed an in-depth analysis of the emergence of mutations conferring resistance to these three MAbs to further elucidate their activity. Clonal analysis of the MPER of plasma virus samples derived during antibody treatment confirmed that no changes in this region had occurred in vivo. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients during the trial supported the phenotypic evaluation, demonstrating that mutations in these sites are associated with resistance. In vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2G12 pressure. Notably, in vitro resistance mutations differed, in most cases, from those found in vivo. Importantly, in vitro selection with 2F5 and 4E10 demonstrated that resistance to these MAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity. This remarkable vulnerability of the virus to interference within the MPER calls for a further evaluation of the safety and efficacy of MPER-targeting therapeutic and vaccination strategies.     

Neurokinin-1 receptor agonists are involved in mediating neutrophil accumulation in the inflamed, but not normal, cutaneous microvasculature: an in vivo study using neurokinin-1 receptor knockout mice

We have used tachykinin neurokinin-1 receptor (NK1 receptor) knockout mice to learn of the link between NK1 receptors and neutrophil accumulation in normal naive skin, as compared with inflamed skin. Intradermal substance P (300 pmol) induced edema formation in wild-type mice, but not in NK1 knockout mice, as expected. However, in contrast to IL-1beta (0.3 pmol), substance P did not induce neutrophil accumulation in wild-type mice. IL-1beta-induced neutrophil accumulation was similar in wild-type and knockout mice, but a significant (p < 0.05) contributory effect of added NK1 agonists, which by themselves have no effect on neutrophil accumulation in normal skin, was observed. The results support the concept that NK1 agonists such as substance P cannot act on their own to mediate neutrophil accumulation in naive skin and provide direct evidence that in inflamed skin, under certain circumstances, the NK1 receptor can play a pivotal role in modulating neutrophil accumulation during the ongoing inflammatory process. We investigated responses to two inflammatory stimuli (carrageenin and zymosan). Neutrophil accumulation was significantly attenuated (p < 0.001) in carrageenin- but not zymosan-induced inflammation in NK1 knockout mice. The carrageenin (500 microg)-induced response was inhibited (p < 0.05) by a NK1 receptor antagonist, SR140333 (480 nmol/kg i.v. at -5 min), in the wild-type group. The bradykinin B1 and B2 receptor antagonists (desArg9[Leu8]bradykinin and HOE 140) each reduced neutrophil accumulation to carrageenin in wild-type animals (p < 0.05), but did not cause further reduction of the suppressed response of knockout mice. The results provide evidence that kinin receptors participate in NK1 receptor-dependent neutrophil accumulation in inflamed mouse skin.     

In vitro and in vivo toxicity of 2',3'-dideoxycytidine in mice.

3T3 mouse embryo fibroblast cell growth was inhibited in a concentration dependent manner by 2',3'-dideoxycytidine (ddCyd), a strong inhibitor of human immunodeficiency virus. Cell growth inhibition was associated with an increased incorporation of ddCyd into cell DNA. In contrast SP2/0-Ag14 (a mouse myeloma) cell growth is not inhibited by 100 microM ddCyd both in the presence or absence of hypoxanthine and thymidine. Furthermore, in vitro spleen cell proliferation, upon phytohemagglutinin (PHA) addition, was much more affected by ddCyd in C57BL/6 mice than in Swiss albino mice. That indeed ddCyd affects spleen cell proliferation was confirmed by studies on splenocytes obtained from C57BL/6 mice that received ddCyd for 2 weeks in drinking water. These results suggest that ddCyd toxicity in mice is cell and strain dependent and that the toxicity mechanism is related to the incorporation of the drug in cell DNA.     

Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4(-/-)), IL-13 (IL-13(-/-)), or IL-4 and IL-13 combined (IL-4/13(-/-)). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4(-/-) mice developed fibrosis, whereas the IL-13(-/-) and IL-4/13(-/-) mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4(-/-), IL-13(-/-), and IL-4/13(-/-) mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13(-/-) mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-beta1 production was not reduced in IL-13(-/-) mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Ralpha1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.     

Differential effects of l-DOPA on monoamine metabolism, cell survival and glutathione production in midbrain neuronal-enriched cultures from parkin knockout and wild-type mice

l-DOPA is the most effective treatment for Parkinson's disease but in isolated neuronal cultures it is neurotoxic for dopamine (DA) neurones. Experiments in vivo and clinical studies have failed to show toxicity of l-DOPA in animals or patients but that does not exclude the possibility of a toxic effect of l-DOPA on patients with certain genetic risk factors. Mutations of the parkin gene are the most frequent cause of hereditary parkinsonism. Parkin null mice have a mild phenotype that could be modified by different neurotoxins. The aim of this study was to investigate whether the toxic effects of l-DOPA on DA neurones are amplified in parkin null mice. We have measured the effects of l-DOPA on cell viability, tyrosine hydroxylase (TH) expression, DA metabolism and glutathione levels of parkin knockout (PK-KO) midbrain cultures. Neuronal-enriched cultures from PK-KO mice have similar proportions of the different cell types with the exception of a significant increment of microglial cells. l-DOPA (400 microm for 24 h) reduced the number of TH-immunoreactive cells to 50% of baseline and increased twofold the percentage of apoptotic cells in cultures of wild-type (WT) animals. The PK-KO mice, however, are not only resistant to the l-DOPA-induced pro-apoptotic effects but they have an increased number of TH-immunoreactive neurones after treatment with l-DOPA, suggesting that l-DOPA is toxic for neurones of WT mice but not those of parkin null mice. MAPK and phosphatidylinositol-3 kinase signalling pathways are not involved in the differential l-DOPA effects in WT and PK-KO cultures. Intracellular levels of l-DOPA were not different in WT and parkin null mice but the intracellular and extracellular levels of DA and 3-4-dihydroxyphenylacetic acid, however, were significantly increased in parkin null animals. Furthermore, monoamine oxidase activity was significantly increased in parkin null mice, suggesting that these animals have an increased metabolism of DA. The levels of glutathione were further increased in parkin null mice than in controls both with and without treatment with l-DOPA, suggesting that a compensatory mechanism may protect DA neurones from neuronal death. This study opens new avenues for understanding the mechanisms of action of l-DOPA on DA neurones in patients with Park-2 mutations.