Polyamines--putrescine, spermidine, and spermine--are a group of positively charged organic molecules that are present in all living cells. They are important regulators of cell growth and differentiation, but the precise mechanism of their action is not known. Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines. Recent studies demonstrated that down-regulation of polyamine biosynthesis by irreversible inhibition of ODC with difluoromethylornithine (DFMO0 is a novel therapeutic approach for the treatment of murine lupus in autoimmune MRL-lpr/lpr mice. Since murine lupus in this strain is associated with a major alteration in thymic T cell subopulations, we questioned whether abnormal polyamine biosynthesis contributes to aberrant T cell maturation in the thymus of MRL-lpr/lpr mice. Thymocytes were analyzed for cell surface markers, CD4 and CD8 by 2-color flow cytometry using their respective monoclonal antibodies. The proportion of thymocyte subsets in disease-free mice (8-10 week of age) was approximately 72% double positive (DP; CD4+CD8+) cells, 5-7% double negative (DN; CD4-CD8-) cells, 11-16% CD4+ cells and 7-8% CD8+ cells. At 14 weeks of age, a stage of clinical disease expression, thymocytes were marked by the presence of approximately 40% DN cells and approximately 25% DP cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Transmembrane Ca2+ influx is recognized as a universal second messenger that transduces T-cell activation signals to cytoplasm and nucleus, thereby stimulating transcription and cell division. To examine the role of endogenous factors that regulate mitogenic Ca2+ signalling of T-cells, we measured the concanavalin (Con) A-induced increase in cytoplasmic free calcium ([Ca2+]i) in spleen cells of BALB/c mice, using flow cytometry with an indicator dye, Indo-1 acetoxymethyl ester (Indo-1/AM). Con A is a polyclonal activator of T-cells. Unstimulated splenocytes had a [Ca2+]i of 100 nM. [Ca2+]i increased with Con A in a dose-dependent manner up to a concentration of 50 micrograms/ml. In the presence of 50 micrograms/ml Con A, [Ca2+]i was 350 nM. Natural polyamines (putrescine, spermidine and spermine) inhibited Con-A-induced Ca2+ influx in a dose-dependent manner. Putrescine was the most effective polyamine in desensitizing the Ca2+ signal, and decreased [Ca2+]i from 350 nM in the absence of putrescine to 250 nM in the presence of 100 microM putrescine. This effect was not mimicked by structurally related homologues or inorganic cations, suggesting a specific structural effect of the polyamine. H.p.l.c. analysis showed that polyamines were internalized during incubation of cells in vitro. In experiments using monoclonal anti-CD4 and anti-CD8 antibodies, we found a differential effect of putrescine on Ca2+ influx in CD4 and CD8 subpopulations of T cells. For CD4+ cells, [Ca2+]i decreased from 625 nM to 420 nM in the presence of 500 microM putrescine, whereas [Ca2+]i was not affected by putrescine in CD8+ cells. These data suggest that natural polyamines have cell-specific effects on mitogen-stimulated Ca(2+)-influx in T-cell subsets.
Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of cellular polyamines, putrescine, spermidine and spermine. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ODC and thereby depletes putrescine and spermidine levels in vivo and in vitro. Previous studies in lupus-prone MRL-lpr/lpr mice treated with 1% DFMO in drinking water have been associated with improved lifespan, and reduced anti-DNA antibody production, lymphadenopathy, and splenic polyamine levels. Since glomerulonephritis is a major cause of morbidity and mortality in lupus, we studied the effect of DFMO on renal histology of MRL-lpr/lpr mice. Female BALB/c and MRL-(+)/+ mice were used as controls. Dose response studies revealed that 1.5% DFMO in drinking water had maximum therapeutic efficacy and produced a significant 79% increase in the median lifespan of a group of 20 mice compared to an equal number of controls (P less than 0.001). Renal histologic studies were performed on kidney sections from four to five mice each from DFMO-treated and untreated groups at 12, 16, 20, 24 and 29 weeks of age. Sections were read blinded to duration and treatment and scored by four major histologic criteria (glomerulonephritis, interstitial inflammation, perivascular inflammation, and vasculitis) and showed significant reduction in all these parameters in DFMO-treated mice when compared to age- and sex-matched untreated mice of the same strain. DFMO treatment had no significant effect on pulmonary histologic findings on these mice. DFMO treatment reduced ODC activity and polyamine concentrations in treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Polyamines--putrescine, spermidine, and spermine--are small organic cations that are present in all living cells. Recent studies revealed that polyamines could provoke a left-handed Z-DNA conformation in poly(dA-dC).poly(dG-dT) and related alternating purine-pyrimidine sequences. In order to examine whether polyamine-induced Z-DNA conformation of poly(dA-dC).poly(dG-dT) is capable of eliciting anti-Z-DNA antibodies, we immunized rabbits with poly(dA-dC).poly(dG-dT) in the presence and absence of spermidine and spermine. Rabbits immunized with the polynucleotide alone produced antibodies reacting toward poly(dA-dC).poly(dG-dT) and heat-denatured calf thymus DNA (ssDNA). In contrast, immunization with poly(dA-dC).poly(dG-dT) complexed with spermidine or spermine produced antibodies reacting with Z-DNA in addition to those binding toward poly(dA-dC).poly(dG-dT) and ssDNA. Antibodies elicited by polynucleotide.polyamine complexes had no reactivity toward polyamines. Solution inhibition studies suggested that anti-poly(dA-dC).poly(dG-dT), anti-ssDNA and anti-Z-DNA antibodies are distinct populations that favor each one of these antigens. Our results suggest that natural polyamines are capable of altering the immunogenicity of polynucleotides by mechanisms involving the stabilization of Z-DNA conformation. This result may have implications in the recent findings of high levels of polyamines and anti-Z-DNA antibodies in the sera of lupus patients and autoimmune mice.
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