Streptozotocin is not a spontaneous NO donor.
The reaction of streptozotocin with oxymyoglobin was analyzed and compared with results using various compounds that spontaneously generate nitric oxide in solution.
Cysteine↗
Biomedical subjects
V Kolb-Bachofen
Publications and source records attributed to V Kolb-Bachofen.
The content of glutathione and glutathione S-transferases and the glutathione peroxidase activity in rat liver nuclei determined by a non-aqueous technique of cell fractionation.
Hepatocellular nuclei require glutathione, glutathione S-transferases (GSTs) and Se-dependent glutathione peroxidase (GPx) for intranuclear protection against damage from electrophiles or products of active oxygen. Data so far available from the literature on nuclei isolated in aqueous systems range from glutathione, GSTs and GPx either being absent altogether to being present in quantities in excess of those in the cytoplasm. This paper describes a small-scale preparation of a nuclear fraction from rat liver by a non-aqueous technique, designed to retain nuclear water-soluble molecules in situ, since low-molecular-mass compounds can diffuse freely into other compartments during aqueous separation. This non-aqueous procedure shows the nucleus to contain glutathione at 8.4 mM and soluble GSTs at 38 micrograms/mg of protein, the enrichment over the homogenate being 1.2-1.4-fold. Se-dependent GPx activity was also present in the nucleus (56 m-units/mg), although with slightly lower activity than in the homogenate (0.7-fold).
Animals↗
Nitric oxide induces apoptosis in mouse thymocytes.
Nitric oxide (NO) produced at high concentrations by the inducible NO synthase is an important effector molecule involved in immune regulation and defense. We have examined whether NO represents a signal for triggering apoptosis in thymocytes. Freshly isolated thymocytes were incubated with different chemical NO donors for various intervals. Apoptosis was determined by detection of DNA strand breaks with in situ nick translation. All NO donors induced thymocyte apoptosis with 30% positive thymocytes vs 10% in controls after 8 h. Apoptosis was prevented by addition of ZnSO4. Short-term pre-exposure to NO resulted in protection from apoptosis induced by glucocorticoids comparable with the protective effect of heat shock. Flow cytometry revealed that NO treatment as well as heat shock or dexamethasone incubation is accompanied by reduction in the CD4+ CD8+ thymocyte subpopulation. Apoptosis induction was accompanied by increased expression of p53, as detected by PCR analysis 2 h after NO donor addition. In vivo treatment of mice with endotoxin results in increased thymic apoptosis. Focal apoptosis was found to occur in close proximity to blood vessels 18 h after LPS treatment. Capillary endothelium and dendritic cells adjacent to apoptotic foci were found to stain strongly for inducible NO synthase expression. Furthermore, in an in vitro experiment using cocultures of thymocytes with LPS/cytokine-activated endothelial cells expressing inducible NO synthase, a significantly increased rate of thymocyte apoptosis was found, and this could be prevented completely by inhibiting NO production. Addition of dexamethasone to these cocultures did not lead to a further increase in the percentage of apoptotic thymocytes, underlining the protective effect of NO on dexamethasone-induced apoptosis.
Animals↗
Insulitis and islet-cell antibody formation in rats with experimentally reduced beta-cell mass.
We studied the effect of severe reduction of beta-cell mass by 90% pancreatectomy on the immune tolerance to the endocrine pancreas. Four months after subtotal pancreatectomy all LEW.Han rats had developed mononuclear infiltration of islets and 9 of 14 rats were positive for islet-cell antibodies. Electron microscopy revealed lymphocytic invasion of endocrine tissue, lysis of beta cells and phagocytotic macrophages. None of these changes were seen 2 weeks after 90% pancreatectomy or 4 months after 10% pancreatectomy. Weekly substitution of islet antigens in the form of a homogenate of 100 islets into 90% pancreatectomized LEW.Han rats almost completely prevented the development of insulitis and autoantibodies. The dependence of insulitis on T cells was shown when 90% pancreatectomy in LEW.rnu rats (i.e., the congenic athymic nude strain), did not result in islet infiltration. The exocrine tissue remained normal in all experimental groups. During the observation period insulitis was not associated with overt diabetes but was accompanied by substantial enlargement of islets and of beta-cell mass, as shown by morphometry. Suppression of islet inflammation by injection of islet antigens abolished beta-cell regeneration, despite continuing metabolic stress in rats with 90% pancreatectomy. The findings indicate induction of islet autoimmunity in response to 90% but not to 10% pancreatectomy. We conclude that severe reduction of the islet-antigen mass allows the development of T-cell-dependent islet autoimmunity which indicates a loss of immune tolerance. In addition, the data suggest the existence of islet-antigen autoreactive immune cells in rats not genetically predisposed to autoimmune diabetes. Finally, we conclude that selective beta-cell regeneration occurs in association with insulitis.
Animals↗
Expression of membrane-associated C-reactive protein by human monocytes: indications for a selectin-like activity participating in adhesion.
We have shown previously that rat liver macrophages (Kupffer cells) express a membrane-bound form of C-reactive protein (mCRP) on their surface which is identical to a galactose-specific particle receptor activity. We now establish the presence of mCRP on human monocyte-macrophages using immunocytochemistry with an anti-neoCRP specific monoclonal antibody and RNA-RNA in situ hybridization to demonstrate the presence of CRP-specific mRNA. Concomitant with mCRP expression, cells exhibit galactose-dependent uptake of particles coated with lactosylated bovine serum albumin. Adhesion experiments on fibronectin-coated surfaces that mCRP on human blood monocytes may act as a selectin-like adhesion molecule, mediating initial carbohydrate-specific contacts which are followed by peptide-specific recognition via integrin receptors.
Amino Acid Sequence↗
Autoimmune versus inflammatory type I diabetes: a controversy?
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Animals↗
Nitric oxide generation during cellular metabolization of the diabetogenic N-methyl-N-nitroso-urea streptozotozin contributes to islet cell DNA damage.
The N-methyl-N-nitroso-urea streptozotocin is an antibiotic with diabetogenic, carcinogenic and antitumor activity thought to act via alkylation of DNA and proteins. Evidence points to a release of bioactive nitric oxide (NO) from streptozotocin as an additional cytotoxic activity of this drug. Here we show by EPR spectroscopy, that NO is not generated during spontaneous decay of streptozotocin but that its metabolization in rat hepatocytes and pancreatic islet cells yields NO. This NO formation is not due to a NO synthase (NOS) activity since NO formation in hepatocytes in the presence of streptozotocin is not blocked by the NOS inhibitor NG-methyl-L-arginine. By iNOS-specific RT-PCR no positive signal for specific mRNA presence was obtained in streptozotocin-treated cells, proving that iNOS activity was not induced during cell isolation procedures and did not account for the NO release. Furthermore, early DNA-strand breaks induced either by SZ or by the NO donor nitroprusside were both significantly reduced in the presence of an intracellular NO scavenger. In contrast, DNA damage found after incubation with the purely alkylating agent methylmethanesulfonate was not inhibited by the NO trap. These results prove that intracellular formation of NO occurs during degradation of SZ within cells. This NO appears to contribute significantly to streptozotocin-induced cytotoxicity.
Alkylating Agents↗
Inducible nitric oxide synthase and its product nitric oxide, a small molecule with complex biological activities.
More and more attention is paid to the radical nitric oxide which is now known to be part of the mammalian physiology and immune system. Nitric oxide is synthesized by one of the most complicated and fascinating enzyme families identified so far. Inducible nitric oxide synthesis after appropriate stimuli has regulatory, cytostatic and/or toxic consequences and may play an important role in the pathophysiology of several diseases.
Animals↗
Independent modulation of galactose-specific receptor expression in rat liver cells.
The expression of galactose-specific receptors on liver cells from rats at the end of pregnancy and from estrogen-treated animals was studied. The number and distribution of binding sites were estimated on hepatocytes and Kupffer and endothelial cells in vitro as well as in situ by means of protein-gold complexes. Hepatocytes and endothelial cells from pregnant rats showed an increased binding activity of at least three times for hepatocytes and one and a half times for endothelial cells with respect to normal rat livers. The increase in the hepatocyte receptor expression was paralleled by an increase in the level of its specific messenger RNA (mRNA). On Kupffer cells, a decreased number of binding sites, at least three times less than control values, was measured. The correlation between the altered hormonal level during pregnancy and the expression of galactose binding sites was examined in hepatocytes and Kupffer cells isolated from virgin rats treated with the synthetic estrogen diethylstilbesterol. In estrogen-treated rats both the binding sites and the specific mRNA of hepatocytes increased as compared with vehicle-treated or untreated animals. In contrast, in Kupffer cells both the estrogen treatment as well as vehicle-only injection led to a significant reduction in the expression of binding sites as compared with virgin untreated animals. To establish whether the decrease of galactose binding sites in Kupffer cells was related to the activation of macrophages or to the removal of plasma membrane caused by enhanced nonspecific phagocytosis, in situ binding experiments were performed after lipopolysaccharide (LPS)-stimulation or latex-bead phagocytosis. Nonspecific phagocytosis does not affect the binding activity, which instead appears strongly reduced after LPS injection. These findings suggest an independent response of galactose-specific receptor expression systems in the different types of liver cells to modulating agents.
Animals↗
Glucagon effect on intracellular proteolysis and pericanalicular location of hepatocyte lysosomes in isolated perfused guinea pig livers.
In guinea pigs, glucagon choleresis is accompanied by a significant, but transient, stimulation of biliary protein secretion, which can be accounted for mainly by biliary discharge of lysosomal enzymes. To clarify whether intracellular proteolysis--a process regulated by glucagon and taking place predominantly in the lysosomes--may interact with biliary protein secretion, we determined hepatic proteolytic activity and bile secretory function during substrate deprivation, amino acid supplementation, and glucagon administration in isolated perfused guinea pig livers. To further elucidate the nature of transient lysosomal enzyme release into bile during glucagon infusion, we analyzed pericanalicular distribution of lysosomes by quantitative electron microscopy. The results demonstrate that intracellular proteolysis is accompanied by biliary excretion of lysosomal enzymes. Glucagon-induced secretion of these enzymes as well as labeled proteins into bile occurs independent of protein breakdown and cannot be modulated by addition of amino acids as potent inhibitors of intracellular proteolysis. During glucagon administration, bile canalicular area and pericanalicular distribution of secondary lysosomes show a rapid increase, which persists during the entire infusion period and thus does not explain the transient biliary release of lysosomal enzymes. We therefore postulate that regulation of this process must be located beyond the lysosomal compartment, either involving transport processes or intracellular kinetics of lysosome formation or altered fusion kinetics at the bile canalicular membrane compartment. Metabolic and biliary effects of glucagon seem to occur independent of each other and to underly different regulatory mechanisms.
Amino Acids↗
Epidermal keratinocyte expression of inducible nitric oxide synthase in skin lesions of psoriasis vulgaris.
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Adult↗
Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9.
Nitric oxide, generated from S-nitrosocysteine or applied as gas mediates metal ion release from the Zn2+/Cd(2+)-complexing protein metallothionein via oxidation of SH-groups. Time-dependent S-nitrosylation and subsequent disulfide formation of metallothionein are demonstrated. Furthermore, nitric oxide inhibits DNA binding activity of the yeast transcription factor LAC9 containing a zinc finger like DNA binding domain. These results show that nitric oxide interacts with and destroys zinc-sulfur clusters in proteins.
Animals↗
Inflammatory islet damage in patients bearing HLA-DR 3 and/or DR 4 haplotypes does not lead to islet autoimmunity.
The hypothesis was tested that islet autoimmunity is induced by ongoing islet cell destruction in subjects with susceptibility genes HLA-DR 3 and/or DR 4. Sixty-one patients with confirmed chronic pancreatitis were analysed, 30 of whom expressed HLA-DR 3 and/or DR 4. Electron microscopy studies in 10 patients showed that the inflammatory process also affected islets, as recognisable from islet cell lysis, intrainsular fibrosis and immune cell infiltrates. None of the sera tested contained any of three markers of islet autoimmunity, ICA, IAA or GAD antibodies. A correlation was seen between the loss of exocrine function, as determined by the ALTAB-test, and of beta-cell function, as determined by the C-peptide response to i.v. glucagon. However, there was no preferential loss of beta-cell function in patients with HLA-DR 3 and/or DR 4. We conclude that islet cell destruction occurs during chronic pancreatitis, but does not trigger islet autoimmunity, even in the presence of HLA-DR 3 and/or DR 4.
Adult↗
In situ nick-translation detects focal apoptosis in thymuses of glucocorticoid- and lipopolysaccharide-treated mice.
In this study we used in situ nick-translation to analyze apoptotic events in the thymus and in cultured thymocytes at the level of individual cell nuclei. In vitro nuclear DNA strand breaks were observed 3 hr after exposure of thymocytes to dexamethasone (Dex) in 30% of cells and increased to 78% after 15 hr. In sections of 10-day-old mouse thymus, single cells with DNA strand breaks were dispersed throughout the cortex and to a lesser degree in the medulla. In contrast, a large number of clusters of apoptotic cells were seen in the thymic cortex 3-18 hr after injection of Dex or lipopolysaccharide (LPS). After 48 hr apoptotic cells were no longer detectable. Positive signals correlated with the detection of DNA ladders of multimers of about 180 BP size on agarose gels. Electron microscopy confirmed the presence of apoptotic cell clusters and showed that apoptotic foci were located around capillaries in LPS-injected animals. We conclude that in situ nick translation is a suitable method to detect apoptotic nuclei in cultured cells and on cryostat sections. With this method we could demonstrate that in vivo spontaneous apoptosis occurs in single dispersed thymocytes, also including the medulla, whereas experimentally induced apoptosis affects cell clusters, possibly due to high local concentrations of apoptosis inducers.
Animals↗
Linear loss of insulin secretory capacity during the last six months preceding IDDM. No effect of antiedematous therapy with ketotifen.
OBJECTIVE: To investigate the effect of an antiedematous therapy with the histamine antagonist ketotifen on beta-cell function in late prediabetes. RESEARCH DESIGN AND METHODS: In a randomized double-blind placebo-controlled study, ketotifen was administered for 3 months to 9 islet cell antibody positive (ICA+) prediabetic patients with a first-phase insulin response (FPIR) below the 2.5th percentile to preserve residual beta-cell function. Patients were followed by intravenous glucose tolerance tests (IVGTTs) every 4-6 weeks for determination of FPIR, HbA1, ICAs, and insulin autoantibodies. In 5 patients, the immune activation state was followed by determination of serum levels of tumor necrosis factor-alpha (TNF-alpha), beta 2-microglobulin, and C-reactive protein (CRP). RESULTS: Seven of nine patients developed diabetes within one year of follow-up. Irrespective of treatment with ketotifen, a slow and linear decline (P < 0.05) of 1 + 3-min insulin values was observed in sequential IVGTTs in those 7 patients who developed insulin-dependent diabetes mellitus (IDDM) during follow-up. The 2 other patients showed wide fluctuations of the insulin response with a threefold increase of initial insulin levels. HbA1 did not correlate with FPIR. Fasting blood glucose increased significantly during the study (P < 0.05). Individual levels of serum TNF-alpha, CRP, and beta 2-microglobulin did not change during the study. CONCLUSIONS: The study could not demonstrate preservation of beta-cell function by ketotifen in the late stage before manifestation of clinical diabetes. Manifestation is preceded in the last 6 months by a steady loss of the FPIR without rapid deterioration immediately before diagnosis and without signs of increased immune activity.
Adolescent↗
Primary cultures of rat islet capillary endothelial cells. Constitutive and cytokine-inducible macrophagelike nitric oxide synthases are expressed and activities regulated by glucose concentration.
We have succeeded in obtaining cultures of pure rat islet capillary endothelial cells. These multiply in vitro and exhibit the same antigenic phenotype as expressed in situ: von Willebrand factorhigh, Ox43 (rat endothelial marker)weak, and Ox2 (thymocyte and brain endothelium marker)high. This phenotype differs from both exocrine endothelium stained in situ and rat aorta endothelial cells cultured in vitro under identical conditions. Islet and aorta endothelial cells were cultured in the presence of various glucose concentrations. Nitrite and citrulline concentrations in culture supernatants were measured as an indirect quantification of nitric oxide formation. In islet endothelia, both nitrite and citrulline levels were found to be strongly glucose-dependent, with high levels at high glucose concentrations and vice versa, in contrast to aorta endothelial cells, where no glucose effect was found. Shifting islet endothelial cultures from high to low glucose levels or the reverse led to a slow decrease or increase in nitrite and citrulline formation with several cell generations needed to reach steady levels. Adding a combination of the cytokines interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma to both endothelial cell cultures led to a dramatic increase of nitric oxide formation. Again with islet but not with aorta endothelial cells a modulating effect by glucose concentrations was found. Reverse-transcription-polymerase chain reaction with specific primers demonstrated the presence of constitutively expressed nitric oxide synthase-RNA in the islet capillary endothelial cells and confirmed the glucose effect. In addition, we found that cytokines indeed induce the expression of inducible synthase messenger RNA in both endothelial cells, which was not found in the absence of cytokines. Electron paramagnetic resonance spectroscopy of islet endothelial cells confirmed intracellular synthesis of nitric oxide in the presence of cytokines. In conclusion, we here for the first time provide evidence that constitutive nitric oxide synthase is also expressed in capillary endothelium and that cytokine challenge leads to the expression of the inducible isoform in these cells.
Amino Acid Oxidoreductases↗
Coating particles with a block co-polymer (poloxamine-908) suppresses opsonization but permits the activity of dysopsonins in the serum.
The surfaces of polystyrene microspheres (60 nm in diameter) and colloidal gold particles (17 nm in diameter) were coated with a polyoxyethylene (POE)/polyoxypropylene (POP) block co-polymer; poloxamine-908. The polymer adsorb strongly to the microspheres via its relatively hydrophobic POP segments. This leaves the POE chains in a mobile state as they extend outward from the surface and thereby provide stability to the particle suspension by suppressing aggregation. The blood clearance and biodistribution of uncoated vs. poloxamine-908-coated 125I-labelled polystyrene microspheres were compared 1 h after intravenous administration into rats. Poloxamine coating dramatically reduced liver accumulation of microspheres and kept them within the systemic circulation. These observations were further confirmed by electron microscopy, demonstrating that Kupffer cells were loaded with uncoated latex but had ingested few if any of the poloxamine-908-coated particles. The interaction of uncoated and poloxamine-coated gold particles with freshly isolated rat liver sinusoidal cells was examined by electron microscopy. The accumulation in Kupffer cells of gold particles after opsonization with autologous plasma was in accordance with previous observations where the dominant opsonizing activity had been identified as fibronectin. In contrast, coating of gold particles with poloxamine-908 prior to plasma opsonization prevented the adsorption of fibronectin onto their surface. Simultaneously, Kupffer cells failed to recognize poloxamine-908-coated gold particles before and after opsonization. Unlike Kupffer cells, liver endothelial cells endocytosed poloxamine-908-coated gold particles prior to opsonization but failed to recognize them after the opsonization process. This was taken as an indication of the presence of dysopsonic activity in plasma. This dysopsonic activity was studied using polystyrene latex microspheres, where the uptake of such particles by phagocytes is known to be independent of opsonization. The coating of 125I-labelled polystyrene microspheres with poloxamine-908 dramatically reduced their interaction with liver sinusoidal cells. This interaction was further reduced in the presence of either autologous plasma or serum. A heat-stable (60 degrees C for 15 min) serum component of molecular mass > 100 kDa was found to mediate this suppressive effect. Thus, we demonstrate that organ-specific receptors, opsonin activities and plasma dysopsonins regulate the in vivo clearance of particulate materials from the circulation. Poloxamine-908 coating modulates particle clearance by effectively blocking opsonization but still allowing for dysopsonization.
Animals↗
Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma.
We investigated the effects of murine rTNF-alpha, human rIL-1 beta, and rat rIFN-gamma in various concentrations and/or combinations on inducible nitric oxide (NO) production in primary cultures of rat aortic endothelial cells. Northern blot analysis of total RNA from induced and control cultures using the cloned mouse macrophage gene of inducible NO synthase as probe as well as polymerase chain reaction using a specific primer sequence gave a positive signal for activated cells only. A RNA approximately 4.4 kb of length similar to the inducible form of NO synthase in macrophages was labeled. The concentration of nitrite as a stable reaction product of NO in culture supernatants was determined 24 h after incubation with the various cytokines. IL-1 beta alone (40 to 1000 U/ml) induced formation of increasing amounts of nitrite with increasing concentrations of IL-1 beta present. Neither TNF-alpha alone (10 to 2000 U/ml) nor IFN-gamma alone 25 to 500 U/ml) showed significant effects on nitrite production. Simultaneous incubation with low concentrations of TNF-alpha (< or = 100 U/ml) and IL-1 beta abrogated the induction effect of IL-1 beta. Conversely, addition of high concentrations of TNF-alpha (> or = 500 U/ml) led to near maximal levels of nitrite formation even at lowest IL-1 beta concentrations (40 U/ml). In addition, simultaneous incubation of endothelial cells with IFN-gamma plus IL-1 beta and/or TNF-alpha led to near maximal NO production of endothelial cells, even at lowest IFN-gamma concentrations (25 U/ml). We hypothesize that the regulating effect of TNF-alpha may in vivo help to prevent local inflammatory responses from spreading to intact sites.
Amino Acid Oxidoreductases↗