Quantitative measurement of genome-wide DNA methylation by a reliable and cost-efficient enzyme-linked immunosorbent assay technique.

DNA methylation, the conversion of cytosine to 5-methylcytosine, is an important epigenetic modification involved in gene regulation. DNA methylation is essential for normal development whereas abnormal methylation has been implicated in pathological conditions including cancer. To evaluate the extent and variation of genome-wide DNA methylation and its changes during cellular differentiation and tumorgenesis as well as the interplay with histone modifications, accurate and reproducible quantification of the genomic DNA methylation level is required. These measurements have so far been achieved only by sophisticated and costly techniques. Here we report the generation of an enzyme-linked immunosorbent assay (methDNA-ELISA) for the accurate quantification of global DNA methylation levels. The linear region of this methDNA-ELISA ranges from 1 to 10%, making it highly suitable for the typical ranges from 2 to 6% in mammalian genomes. This method requires 10 ng of isolated DNA per sample, thus permitting investigation with minimal amounts of DNA previously not applicable for global DNA methylation analysis, e.g., clinical biopsies or cells collected by microdissection.

A proinflammatory activity of interleukin 8 in human skin: expression of the inducible nitric oxide synthase in psoriatic lesions and cultured keratinocytes.

Psoriasis is a common chronic skin disease mediated by cellular immune mechanisms and characterized by an intense neutrophil cell infiltrate and proliferative activation of epidermal keratinocytes. We have previously described the expression of the inducible nitric oxide synthase (iNOS) in epidermal keratinocytes of psoriatic skin lesions. In this study, the role of iNOS in psoriatic inflammation was explored ex vivo in psoriatic skin biopsies and in vitro in primary cultures of human keratinocytes. Messenger RNA for the iNOS enzyme (iNOS mRNA) was detected by reverse transcriptase polymerase chain reaction in skin biopsies from patients with psoriasis, but not in skin specimens from patients with atopic eczema or from healthy volunteers. As demonstrated by in situ hybridization and immunohistochemistry, expression of iNOS mRNA and its gene product was localized to the epidermal keratinocytes of psoriatic skin lesions. In situ hybridization further revealed a complete colocalization of mRNA expression for iNOS with interleukin (IL) 8 receptor-specific mRNA either in the basal germinative cell layer or at focal sites of ongoing neutrophil inflammation in suprabasal cell layers. Because psoriatic keratinocytes have previously been shown to express mRNA transcripts for IL-8, it seemed reasonable to hypothesize that iNOS expression could be induced in an autocrine loop by IL-8. This hypothesis was substantiated by our in vitro experiments showing that a combination of IL-8 and interferon gamma induces the expression of iNOS-specific mRNA and of the functional enzyme in cultured human keratinocytes. These results suggest an important role for iNOS in concert with IL-8 and its receptor early during the formation of psoriatic lesions.

Uptake of toxic silica particles by isolated rat liver macrophages (Kupffer cells) is receptor mediated and can be blocked by competition.

Silica particles (quartz dust) are toxic to macrophages after their uptake into these cells. These experiments describe the opsonization mechanism(s) and macrophage receptor(s) involved in silica uptake. Freshly isolated rat liver macrophages (Kupffer cells) were incubated at 37 degrees C with silica particles in the presence or absence of autologous or heterologous plasma or purified plasma fibronectin and cell viability was assessed at various times. Within 60 min of coincubation, > 80% of macrophages were lysed in the presence of plasma or purified fibronectin but not in their absence (viability > 90%). Lysis was slower with defibronectinized plasma (28% in 60 min). Macrophages could be protected from lysis by addition of the monosaccharide N-acetyl-D-galactosamine but not by N-acetyl-D-glucosamine. Galactosylated serum albumin but not mannosylated albumin or native albumin exerted full protection from lysis. The pentapeptide GRGDS also prevented macrophage lysis in synergy with N-acetyl-galactosamine. Enzymatic deglycosylation of fibronectin reduced lysis significantly. These findings indicate an important opsonizing activity for fibronectin and dual recognition via the lectin-like galactose-specific binding activity of membrane-associated C-reactive protein and by integrin receptor(s). Binding experiments (at 4 degrees C) revealed initial binding as primarily galactose-inhibitable, suggesting integrin-mediated binding as a later event necessary for effective uptake.

Hepatic receptor for asialo-glycoproteins. Ultrastructural demonstration of ligand-induced microaggregation of receptors.

Endocytosis of asialo-glycoproteins in hepatocytes is mediated by a lectin-like receptor with specificity for D-galactose. Early events of receptor-ligand interactions have been studied by ultrastructural analysis. Hepatocytes were isolated from the rat liver by collagenase perfusion and incubated with a galactosylated electron dense marker (gold-Gal-BSA, glactosylated bovine serum albumin adsorbed onto colloidal gold particles). Initial binding of gold-Gal-BSA particles occurs to receptors diffusely distributed at hepatic microvilli of the former space of Disé. No lectin activity was found in membrane areas that had formed in situ the region of hepatic cell contact or bile canaliculi. Microaggregation of receptor-ligand complexes is seen as an early consequence of particle binding. Microaggregates contain 2-5 particles and are located outside coated pits. After prolonged incubation larger clusters are formed, these are found associated with coated membrane areas. It is concluded that at least three steps precede the uptake of galactosylated proteins by hepatocytes. These are: (i) binding of ligand at diffusely distributed binding sites; (ii) local microaggregation of receptor-ligand complexes; (iii) formation of larger clusters and association with coated pits.

Galactose-specific receptors on liver cells. I. Hepatocyte and liver macrophage receptors differ in their membrane anchorage.

Colloidal gold particles coated with asialoglycoproteins are bound by hepatocytes as well as by liver macrophages. Binding by both cell types is inhibited by N-acetylgalactosamine and related saccharides and is dependent on the presence of Ca2+. We have now performed an electron microscopic study on receptor anchorage in the plasma membranes. Cells with prebound ligand were treated with 20 mM EDTA at 4 degrees C, washed free of chelator and tested for residual galactose-specific receptor activity. Whereas hepatocytes preserve binding activity (73% of untreated control), liver macrophages lose galactose-specific receptor activity (12% of untreated control). Liver macrophages regain binding activity after a 2 min incubation at 37 degrees C allowing for receptor recycling. If the macrophages were fixed with low glutaraldehyde concentration prior to EDTA treatment they fully retained their receptor activity (74% of control). Ligands were also removed from both cell types by incubation with 80 mM N-acetylgalactosamine. After washing the cells free of the competing monosaccharide, both the hepatocytes as well as the macrophages show full binding activity (120% and 85% of untreated controls). Therefore, membrane anchorage sites of the macrophage receptors are not identical to ligand-binding sites. These results suggest a Ca2+-Mg2+-dependent receptor anchorage on the macrophage plasma membrane. As shown in the accompanying paper (Roos, P.H., Hartmann, H.J., Schlepper-Schäfer, J., Kolb, H. and Kolb-Bachofen, V. (1985) Biochim. Biophys. Acta 847, 115-121), EDTA-induced dissociation from the membrane can be used for isolation of the galactose-specific receptors of liver macrophages.

Galactose-particle receptor on liver macrophages. Quantitation of particle uptake.

Liver macrophages have been shown previously to bind and ingest gold particles coated with asialoglycoproteins via a N-acetyl-D-galactosamine / D-galactose-specific lectin (Kolb-Bachofen, V., Schlepper-Schäfer, J., Vogell, W. and Kolb, H. (1982) Cell 29, 859-866). We present here a quantitative analysis of lectin-dependent particle endocytosis. We used a conjugate of asialofetuin with colloidal gold as ligand, the cellular uptake of which could be followed by spectrophotometry. Freshly isolated Kupffer cells from the rat liver ingest asialofetuin at a rate of approx. 4200 particles/cell per min. Uptake is inhibited by saccharides related in structure to D-galactose and depends on the presence of Ca2+. The rate of endocytosis is zero below 10 degrees C, shows a modest increase until 20 degrees C and a steep increase between 20 and 37 degrees C. Uptake is energy-dependent and strongly inhibited by cytochalasin B but only slightly by colchicine.

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.

Galactose-specific receptors on liver cells. II. Characterization of the purified receptor from macrophages reveals no structural relationship to the hepatocyte receptor.

We have isolated a galactose-specific receptor protein from rat liver macrophages by three techniques, all using EDTA extraction and subsequent affinity chromatography. The purified receptor has an apparent molecular mass of 30 kDa and exhibits hemagglutinating activity. Monospecific receptor-antisera produce one precipitation line with the macrophage receptor in Ouchterlony double diffusion but show no cross-reaction with the hepatocyte receptor. Sinusoidal cells, but not hepatocytes, are stained with monoclonal antibodies to the macrophage receptor, whereas anti-hepatocyte receptor antibodies stain hepatocyte surfaces but not sinusoidal cells. We conclude that the galactose-specific receptor from liver macrophages is structurally different from the hepatocyte receptor, although the two lectins share a similar binding specificity.

Pancreatic islet cells are highly susceptible towards the cytotoxic effects of chemically generated nitric oxide.

To compare the sensitivity of different mammalian cell types towards the cytotoxic action of nitric oxide, freshly isolated rat pancreatic islet cells, hepatocytes, resident and activated macrophages, cultured aortic endothelial cells and two murine tumor cell lines were tested for susceptibility towards exogenous nitric oxide. As sources for nitric oxide nitroprusside, S-nitroso-N-acetyl-penicillamine and the sydnonimine-derivative SIN-1 were used. These generate nitric oxide by different mechanisms and kinetics. Among the cell types tested we found large differences in their susceptibility towards the three nitric oxide donors. Islet cells were by far the most sensitive of the investigated cells and were completely lysed by all three nitric oxide donors. Hepatocytes and endothelial cells were sensitive towards nitroprusside but relatively resistant towards toxicity of SIN-1 and S-nitroso-N-acetyl-penicillamine. Activated and resident macrophages were lysed by SIN-1, whereas high concentrations of nitroprusside and S-nitroso-N-acetyl-penicillamine led to partial cell lysis only. The tumor cell lines were both lysed by SIN-1 but showed differences in their sensitivity towards S-nitroso-N-acetyl-penicillamine. Nitric oxide, which is produced in large amounts during infection and inflammation, may play an important role in the destruction of islet cells during insulitis leading to insulin-dependent diabetes mellitus.

Even after UVA-exposure will nitric oxide protect cells from reactive oxygen intermediate-mediated apoptosis and necrosis.

Reactive oxygen species (ROS) play a pivotal role in UVA-induced cell damage. As expression of the inducible nitric oxide synthase (iNOS) is a normal response of human skin to UV radiation we examined the role of nitric oxide (NO) as a protective agent during or even after UVA1- or ROS-exposure against apoptosis or necrosis of rat endothelial cells. When added during or up to 2 h subsequent to UVA1 or ROS exposure the NO-donor S-nitroso-cysteine (SNOC) at concentrations from 100-1000 microM significantly protects from both apoptosis as well as necrosis. The NO-mediated protection strongly correlates with complete inhibition of lipid peroxidation (sixfold increase of malonedialdehyde formation in untreated versus 1.2-fold with 1 mM SNOC). NO-mediated protection of membrane function was also shown by the inhibition of cytochrome c leakage in UVA1 treated cells, a process not accompanied by alterations in Bax and Bcl-2 protein levels. Thus, the experiments presented demonstrate that NO exposure during or even after a ROS-mediated toxic insult fully protects from apoptosis or necrosis by maintaining membrane integrity and function.

Low-dose streptozocin-induced diabetes in mice. Electron microscopy reveals single-cell insulitis before diabetes onset.

We investigated the morphology of mouse islets 5 days after completion of low-dose streptozocin treatment of C57BL/6 mice by electron microscopy. At this stage, mice were still normoglycemic and light microscopy did not reveal massive islet infiltration. The electron-microscopic investigation revealed two characteristics indicative of ongoing islet cell destruction. In all islets investigated, lysed islet beta-cells were recognized by disrupted plasma membranes and concomitantly decreased plasma contrast. Many of the lysed islet beta-cells still contained numerous insulin granules. We also found immunocytes scattered throughout the islets, most of which could be identified as macrophages. Some were found engaged in phagocytosis of islet beta-cell debris. This early stage of islet lesion termed single-cell insulitis is followed by the well-known later stage of massive infiltration easily recognized in light microscopy. Administration of silica particles to mice treated with low-dose streptozocin inhibited macrophage infiltration of islets as shown by immunocytochemistry with macrophage-specific monoclonal antibody F4/80. In parallel, the development of hyperglycemia was suppressed. The observations favor a pathogenic role of macrophages in islet destruction.

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.

Quantification and localization of galactose-specific binding sites in rat liver during postnatal development.

We investigated the number and distribution of galactose-specific binding sites in developing livers from suckling rats of various ages using Lac-BSA-Au5 (lactosylated bovine serum albumin adsorbed onto colloidal gold particles 5 nm in diameter) as electron-dense ligand, and performing transmission electron microscopy of the specimen. It has been reported that the number of galactose-specific binding sites increases rapidly during organ development post partum (p.p.) and this was ascribed to hepatocyte receptor increase only. We now have investigated in in situ and in vitro experiments whether the binding sites of identical sugar specificity but located on sinusoidal cells show the same increase in expression or are independently regulated. We therefore quantified the number of particles bound by isolated hepatocytes and liver macrophages and found a gradual increase of both binding activities with age, the binding levels of adult liver cells being reached at day 15 p.p. This was confirmed with experiments using in situ prefixed organs thus proving the validity of this finding also for the intact organ. In both sets of experiments--in vitro as well as in vivo--ligand was found binding statistically distributed as single particles on hepatocytes of all ages, whereas on liver macrophages the binding pattern changed during development. On liver macrophages from rats 15 days of age ligand binding occurs in the preclustered pattern described for macrophages from adult rat livers whereas liver macrophages of newborn rats express a different binding pattern: they bind the ligands mostly as single particles with only few and small microaggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide in human skin: current status and future prospects.

The gaseous free radical nitric oxide is an important biologic mediator with physiologic and pathophysiologic roles in nearly every organ system. Because of its unique biologic activity, unusual chemical structure, and unprecedented mechanisms of action, nitric oxide, arguably more than any other natural product, has opened new avenues to investigate cellular processes. Nitric oxide is generated in biologic tissues by specific nitric oxide synthases that metabolize arginine and molecular oxygen to citrulline and nitric oxide. Besides its function as a diffusible messenger in the vasculature and in neurons, nitric oxide also plays a key role in innate immunity and inflammation. Recent progress has allowed the identification of the nitric oxide pathway in several cell types that reside in the skin, including keratinocytes, melanocytes, Langerhans cells, fibroblasts, and endothelial cells. Convincing evidence suggests that nitric oxide synthesis in these cells can be modulated by calcium-mobilizing agonists as well as diverse inflammatory and immune stimuli, and thereby contributes to the pathogenesis of several human skin diseases. Characterization of these intrinsic and extrinsic regulatory stimuli of nitric oxide synthesis has afforded substantial insights into the role of nitric oxide in inflammatory, hyperproliferative, and autoimmune skin diseases, as well as skin cancer, and may ultimately form the basis for future therapeutic intervention. The demonstrable and potential roles of nitric oxide in skin disease pathogenesis and treatment are the subjects of this review.

Aberrant timing in epidermal expression of inducible nitric oxide synthase after UV irradiation in cutaneous lupus erythematosus.

Photosensitivity is a main criterion for the diagnosis of systemic lupus erythematosus (LE), and ultraviolet (UV) irradiation plays a key role in the pathogenesis of cutaneous LE. Patients with a tentative diagnosis of LE are routinely tested for skin lesion development after experimental UV irradiation, providing an ideal opportunity to evaluate early, preclinical events involved in the pathogenesis of LE. Several reports have shown expression of the cytokine-inducible nitric oxide synthase (iNOS) in autoimmune diseases. Therefore, we investigated the role of iNOS expression at mRNA and protein level in the pathogenesis of LE lesions. Skin biopsies from patients with different subtypes of LE were examined, and iNOS expression was found in six of 18 biopsies from cutaneous LE patients and two of three biopsies from systemic LE patients. In biopsies taken 4-20 d after UV irradiation, epidermal iNOS expression was seen in all patients (n = 10) after UVB and in four of 10 patients provoked by UVA. In healthy controls (n = 8) epidermal iNOS expression was detected 24 h after UV irradiation, persisting for another day before subsiding on day 3. In LE patients (n = 8) the exact reverse situation was seen: an iNOS-specific signal was undetectable in keratinocytes for 2 d after UV irradiation, but became positive on day 3 and persisted for up to 25 d in the evolving skin lesions. Our findings demonstrate a time-restricted, UV-induced iNOS expression in human skin; moreover, the results indicate that both the kinetics of iNOS induction as well as the time span of local iNOS expression may be critical to the development of cutaneous LE lesions.

Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts.

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.

Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.

Skin exposure to ultraviolet radiation from sunlight causes erythema and edema formation as well as inflammatory responses. As some of these ultraviolet-induced effects are potentially mediated by nitric oxide synthases, we examined the role of cytokines and ultraviolet A1 radiation (340-400 nm) on the expression of the nitric oxide synthase-2 in endothelia of normal human skin biopsies during short-term organ culture as well as expression and activity of the nitric oxide synthase-2 in in vitro cell cultures of human dermal endothelial cells. Both, cytokine challenge (interleukin-1beta + tumor necrosis factor-alpha + interferon-gamma) but also ultraviolet A1 exposure (50 J per cm2) in the absence of cytokines led to the expression of nitric oxide synthase-2 in human skin organ cultures as shown by immunohistochemistry. Moreover, exposing human dermal endothelial cell cultures to proinflammatory cytokines but also to ultraviolet A1 radiation (6-24 J per cm2) in the absence of cytokines resulted in significant nitric oxide synthase-2 mRNA and protein expression as well as enzyme activity. Ultraviolet A1 irradiation of cytokine activated cells led to further increases in nitric oxide synthase-2 mRNA, protein expression, and enzyme activity. Moreover, a reporter gene assay using a human nitric oxide synthase-2 promoter construct provide evidence that ultraviolet A1, in the absence of cytokines, induces nitric oxide synthase-2 expression and activity, as previously shown for cytokines. Thus, the results presented here demonstrate for the first time that in dermal endothelia of human skin ultraviolet A1 radiation alone represents a proinflammatory stimulus sufficient to initiate nitric oxide synthase-2 expression as well as activity comparable with the respective response seen in the presence of proinflammatory cytokines.

Influence of nitric oxide on the intracellular reduced glutathione pool: different cellular capacities and strategies to encounter nitric oxide-mediated stress.

Different cell types exhibit huge differences towards the cytotoxic action of NO. In search for an explanation, we used subtoxic concentrations of the NO-donors S-nitrosocysteine (SNOC) for short-term challenge and of (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO) for longer periods of exposure, respectively, and subtoxic concentrations of the oxidant H2O2 to determine the impact on intracellular reduced glutathione (GSH) concentrations. We find that GSH concentrations are always decreased, but that different cell types show different responses. Incubation of the relatively NO-sensitive murine lymphocytes with both NO-donors, but not with H2O2, resulted in a nearly complete loss of intracellular GSH. Short-term NO-treatment of P815 mastocytoma cells, also sensitive to NO-mediated cell death, decreased GSH to a similar extent only if either glutathione reductase (GSHR) activity or y-glutamylcysteine synthetase (gammaGCS) activity were inhibited concomitantly by specific inhibitors. Long-term NO-treatment of P815 cells, however, resulted in a significant decrease of GSH that could be further enhanced by inhibiting gammaGCS activity. In contrast, neither short-term nor long-term NO-exposure nor H2O2-treatment affected intracellular GSH levels of L929 fibroblasts, which were previously shown to be extremely resistant towards NO, whereas concomitant gammaGCS inhibition, but not GSHR inhibition, completely decreased GSH concentrations. These results show that different cell types use different pathways trying to maintain glutathione concentrations to cope with nitrosative stress, and the overall capability to maintain a critical amount of GSH correlates with susceptibility to NO-induced cell death.

Two galactose-specific receptors in the liver with different function.

In the rat liver both hepatocytes and macrophages have been shown to express on the surface lectins with similar binding specificity for galactose residues. Functionally the two lectins differ in the uptake of ligands. Whereas the hepatocytes ingest molecules and small particles (less than 10 nm), the macrophages take up particles only. Antisera raised against hepatic galactose-specific receptor failed to react with the macrophage lectin but blocked ligand binding to the hepatocyte only, indicating either a different antigenic structure or membrane localization of the two lectins.

Transcription and translation of inducible nitric oxide synthase in the pancreas of prediabetic BB rats.

The inducible NO synthase (iNOS) was found to be expressed in pancreatic lesions of adult diabetes-prone BB rats. Pancreatic iNOS mRNA was detected by reverse transcriptase PCR in pancreatic RNA of adult diabetes-prone BB rats but not in normal Wistar rats, young diabetes-prone BB rats without insulitis or in diabetes-resistant BB rats. Immunohistochemistry of pancreatic sections using an iNOS-specific antiserum labeled the pancreas of adult diabetes-prone BB rats but not Wistar rats. Parallel staining for ED1-positive macrophages showed restriction of iNOS expression to areas of islet infiltration by macrophages. In conclusion, the data provide direct evidence for enhanced expression of inducible NO synthase in tissue lesions during the development of autoimmune diabetes.