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S Milstien

Publications and source records attributed to S Milstien.

At least 55 records · Page 3Linked to original sources
PubMed1996

Sphingoid bases and phospholipase D activation.

There is increased interest in physiological functions and mechanisms of action of sphingolipids metabolites, ceramide, sphingosine, and sphingosine-1-phosphate (SPP), members of a new class of lipid second messengers. This review summarizes current knowledge regarding the role of these sphingolipids metabolites in the actions of growth factors and focuses on the second messenger roles of sphingosine and its metabolite, SPP, in the regulation of cell growth. We also discuss possible interactions with intermediates of the well known glycerophospholipid cycle. Sphingosine and SPP generally provide positive mitogenic signals whereas ceramide has been reported to induce apoptosis and cell arrest in several mammalian cell lines. Stimulation of phospholipase D leading to an increase in phosphatidic acid, a positive regulator of cell growth, by sphingosine and SPP, and its inhibition by ceramide, might be related to their opposite effects on cell growth. This also indicates that sphingolipid turnover could regulate the diacylglycerol cycle. Cross-talk between sphingolipid turnover pathways and the diacylglycerol cycle increases complexity of signaling pathways leading to cellular proliferation and adds additional sites of regulation.

Animals↗
PubMed1996

Effect of tetrahydrobiopterin on endothelial function in canine middle cerebral arteries.

Tetrahydrobiopterin is an essential cofactor required for activation of NO synthase. However, in intact arteries, the exact role of tetrahydrobiopterin in the regulation of NO synthase activity is not fully understood. The present study was designed to determine the effect of increasing intracellular tetrahydrobiopterin levels on endothelial function in isolated canine middle cerebral arteries. The arterial segments were incubated in MEM for 24 hours at 37 degrees C in the presence or absence of a tetrahydrobiopterin precursor, sepiapterin (10(-4) mol/L), and/or superoxide dismutase (150 U/mL). The rings were suspended for isometric tension recording. Tetrahydrobiopterin levels were assayed by high-performance liquid chromatography. Production of cGMP was measured by radioimmunoassay. Incubation with sepiapterin markedly increased intracellular tetrahydrobiopterin levels. In sepiapterin-treated arteries, endothelium-dependent relaxations to calcium ionophore A23187 and intracellular cGMP levels were significantly reduced. Superoxide dismutase alone did not affect either relaxation to A23187 or production of cGMP. However, when arteries were incubated with superoxide dismutase plus sepiapterin, endothelium-dependent relaxations to A23187, as well as cGMP production, were significantly augmented. The augmentation of cGMP was observed in rings with (but not without) endothelium. Incubation of arteries in calcium-free medium almost abolished the synergistic effect of tetrahydrobiopterin and superoxide dismutase on cGMP production. These results demonstrate that increased availability of tetrahydrobiopterin may activate endothelial NO synthase. This effect appears to be critically dependent on the presence of superoxide dismutase.

Animals↗
PubMed1995

The role of calcium influx in cellular proliferation induced by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin.

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. It was previously shown that the B subunit had no effect on cAMP, protein kinase C or phosphoinositide turnover, but did cause an increase in the influx of calcium from extracellular sources (Spiegel, S. and Panagiotopoulos, C. (1988) Exp. Cell Res. 177, 414-427). In contrast to the action of known growth factors, the B subunit induced significant DNA synthesis after only a 1-3 h treatment. We utilized this unique property to determine whether the increase in calcium influx plays a role in B subunit-induced mitogenicity. Cells were briefly treated with the B subunit in the presence of calcium channel blockers, followed by removal of the blockers and further incubation in B subunit-free medium for the remaining time required to measure DNA synthesis. When 1 mM cobalt was only present during the first 3 h incubation. DNA synthesis induced by either the B subunit or fetal bovine serum was completely abolished. However, both nickel (1 mM) adn the L-type voltage-gated calcium channel inhibitor nicardipin (10 microM) inhibited B subunit-induced cell proliferation without abrogating the response to fetal bovine serum. Using a gel retardation assay, we found that the B subunit markedly stimulated specific DNA-binding activity of the transcription factor, activator protein-1 (AP-1), which functions as a major convergence point coupling early events induced by a variety of mitogens to long term growth responses. Presence of c-Fos protein in the AP-1 complex was demonstrated as a supershift band in the gel mobility assay using c-Fos polyclonal antibody. Cobalt, which markedly inhibited B subunit-induced DNA synthesis, also completely abolished AP-1 DNA-binding activity stimulated by the B subunit. In sharp contrast, cobalt had no effect on DNA-binding activity of AP-1 induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate. Our results suggest that calcium influx is a key element for both DNA-binding activity of AP-1 and cell proliferation induced by binding of the B subunit of cholera toxin to cell surface ganglioside GM1.

3T3 Cells↗
PubMed1995

Quinolinic acid in tumors, hemorrhage and bacterial infections of the central nervous system in children.

A potential mechanism that may contribute to neurological deficits following central nervous system infection in children was investigated. Quinolinic acid (QUIN) is a neurotoxic metabolite of the kynurenine pathway that accumulates within the central nervous system following immune activation. The present study determined whether the levels of QUIN are increased in the cerebrospinal fluid of children with infections of the CNS, hydrocephalus, tumors or hemorrhage. Extremely high QUIN concentrations were found in patients with bacterial infections or the CNS, despite treatment with antimicrobial agents. CSF QUIN levels were also elevated to a lesser degree in patients with hydrocephalus or tumors. CSF L-kynurenine levels increased in parallel to the accumulations in QUIN, which is consistent with increased activity of the first enzyme of the kynurenine pathway, indoleamine-2,3-dioxygenase. The CSF levels of neopterin, a marker of immune and macrophage activation, were also increase in patients with infections. The cytokines tumor necrosis factor-alpha and interleukin-6 were also detected in some patients' samples, and were highest in patients with infection. These results suggest that QUIN is a sensitive marker of the presence of immune activation within the CNS. Further studies of QUIN as a potential contributor to neurologic dysfunction and neurodegeneration in children with CNS inflammation are warranted.

Adolescent↗
PubMed1995

Monoaminergic effects of folinic acid, L-DOPA, and 5-hydroxytryptophan in dihydropteridine reductase deficiency.

Plasma and CSF concentrations of endogenous L-DOPA, catecholamines, and metabolites of monoamines were assayed in a patient with atypical phenylketonuria due to absent dihydropteridine reductase (DHPR), before and during treatment with folinic acid, Sinemet, and 5-hydroxytryptophan. The patient had low but detectable levels of L-DOPA, 3,4-dihydroxyphenylacetic acid (DOPAC), and 3,4-dihydroxyphenylglycol (DHPG) in plasma and low but detectable levels of these compounds and of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in CSF, with approximately normal plasma and CSF levels of norepinephrine [noradrenaline (NA)]. Folinic acid treatment approximately doubled plasma levels of L-DOPA, NA, DOPAC, and DHPG, compared with values during dietary phenylalanine restriction alone. Detection of L-DOPA, catecholamines, and monoamine metabolites in this patient indicates that monoamine synthesis in humans does not absolutely require DHPR. The results are consistent with the existence of an alternative biochemical pathway, with folinic acid treatment augmenting activity along this pathway. Low plasma levels of L-DOPA, DOPAC, and DHPG may reflect decreased catecholamine synthesis and turnover in sympathetic nerves, with compensatory increases in exocytotic release normalizing plasma NA levels.

5-Hydroxytryptophan↗
PubMed1995

Parallel induction of nitric oxide and tetrahydrobiopterin synthesis by cytokines in rat glial cells.

Activation of monocyte-derived macrophages with cytokines leads to the induction of nitric oxide synthase. Much less is known about the effects of cytokines on microglia, resident brain macrophages, or on astrocytes. In this study, we compared the induction by lipopolysaccharide, interferon-gamma, and tumor necrosis factor-alpha of nitric oxide production and synthesis of tetrahydrobiopterin, the required cofactor for nitric oxide synthase, in microglia and peritoneal macrophages. Activation of microglia induced parallel increases in nitric oxide and intracellular tetrahydrobiopterin levels, although induction of the latter appears to be somewhat more sensitive to diverse stimulators. As with macrophages, inducible nitric oxide production in microglia was blocked by inhibitors of tetrahydrobiopterin biosynthesis. Interleukin-2, an important component of the neuroimmunomodulatory system, was only a weak activator of microglia by itself but potently synergized with interferon-gamma to stimulate production of both nitric oxide and tetrahydrobiopterin. Astrocytes were also activated by lipopolysaccharide and combinations of cytokines but showed a somewhat different pattern of responses than microglia. Biopterin synthesis was increased to higher levels in astrocytes than in microglia, but maximal induction of nitric oxide production required higher concentrations of cytokines than microglia and the response was much lower. These results suggest that tetrahydrobiopterin synthesis in glial cells is a potential target for therapeutic intervention in acute CNS infections whose pathology may be mediated by overproduction of nitric oxide.

Animals↗
PubMed1994

Cerebrospinal fluid nitrite/nitrate levels in neurologic diseases.

Nitric oxide has been proposed to mediate cytotoxic effects in inflammatory diseases. To investigate the possibility that overproduction of nitric oxide might play a role in the neuropathology of inflammatory and noninflammatory neurological diseases, we compared levels of the markers of nitric oxide, nitrite plus nitrate, in the CSF of controls with those in patients with various neurologic diseases, including Huntington's and Alzheimer's disease, amyotrophic lateral sclerosis, and HIV infection. We found that there were no significant increases in the CSF levels of these nitric oxide metabolites, even in patients infected with HIV or in monkeys infected with poliovirus, both of which have significantly elevated levels of the neurotoxin quinolinic acid and the marker of macrophage activation, neopterin. However, CSF quinolinic acid, neopterin, and nitrite/nitrate levels were significantly increased in a small group of patients with bacterial and viral meningitis.

Alzheimer Disease↗
PubMed1994

Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity.

We have examined cytokine regulation of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). 24-h treatment with IFN-gamma (200 U/ml) plus TNF (200 U/ml) or IL-1 beta (5 U/ml) increased NOS activity in HUVEC lysates, measured as conversion of [14C]L-arginine to [14C]L-citrulline. Essentially, all NOS activity in these cells was calcium dependent and membrane associated. Histamine-induced nitric oxide release, measured by chemiluminescence, was greater in cytokine-treated cells than in control cells. Paradoxically, steady-state mRNA levels of endothelial NOS fell by 94 +/- 2.0% after cytokine treatment. Supplementation of HUVEC lysates with exogenous tetrahydrobiopterin (3 microM) greatly increased total NOS activity, and under these assay conditions, cytokine treatment decreased maximal NOS activity. IFN-gamma plus TNF or IL-1 beta increased endogenous tetrahydrobiopterin levels and GTP cyclohydrolase I activity, the rate-limiting enzyme of tetrahydrobiopterin synthesis. Intracellular tetrahydrobiopterin levels were higher in freshly isolated HUVEC than in cultured cells, but were still limiting. We conclude that inflammatory cytokines increase NOS activity in cultured human endothelial cells by increasing tetrahydrobiopterin levels in the face of falling total enzyme; similar regulation appears possible in vivo.

Amino Acid Oxidoreductases↗
PubMed1994

An eosinophilia-myalgia syndrome related disorder associated with exposure to L-5-hydroxytryptophan.

OBJECTIVE: To determine whether L-5-hydroxytryptophan (L-5-HTP) associated with eosinophiliamyalgia syndrome (EMS) like illness contains impurities in a fashion similar to that described in L-tryptophan associated with EMS. METHODS: Members of a family who became ill after exposure to L-5-HTP were evaluated at the National Institutes of Health. Data from patients with extended exposure to L-5-HTP were also examined. Samples of L-5-HTP were examined using high performance liquid chromatography. RESULTS: One member of the family had EMS, and 2 others had eosinophilia. No patient in the other group reviewed developed the syndrome, although 2 patients developed eosinophilia. The L-5-HTP used by the family contained an impurity not present in samples from the other patient group. After replacement with L-5-HTP not containing this impurity, eosinophilia in 2 family members resolved. CONCLUSION: Some L-5-HTP contains impurities that may be related to L-5-HTP associated EMS.

5-Hydroxytryptophan↗
PubMed1993

Induction of pterin synthesis is not required for cytokine-stimulated tryptophan metabolism.

Activation of the immune system which occurs in inflammatory disease leads to parallel increases in pterin synthesis and increased production of neuroactive L-tryptophan metabolites. Several model systems were studied to determine whether pterins, which are cofactors for hydroxylation reactions, could be required in the oxidative kynurenine pathway of L-tryptophan degradation. Treatment of mice with interferon-gamma increased L-tryptophan metabolism without any corresponding change in tissue biopterin concentrations. Cytokine-treated human fibroblasts, macrophages and glioblastoma cells all showed increases in kynurenine production, which were completely independent of pterin synthesis. When pterin synthesis de novo was blocked, either by an inhibitor of GTP cyclohydrolase or because of a genetic deficiency of one of the enzymes of the pathway of pterin biosynthesis, cytokine-stimulated increases in tryptophan metabolism were unaffected. Furthermore, increasing intracellular tetrahydrobiopterin concentrations by treating cells with sepia-pterin also had no effect on markers of tryptophan metabolism. Therefore, both normal and cytokine-stimulated L-tryptophan metabolism appears to be completely independent of pterin biosynthesis.

Animals↗
PubMed1993

Availability of tetrahydrobiopterin is not a factor in the inability to detect nitric oxide production by human macrophages.

Human macrophages, in contrast to murine macrophages, do not produce nitric oxide after stimulation with cytokines. This failure has been attributed to the known lack of production by human macrophages of tetrahydrobiopterin, an essential cofactor for nitric oxide synthase. Increasing intracellular levels of tetrahydrobiopterin in cytokine-stimulated murine cells results in an increase in nitrite production. However, this treatment does not result in any detectable accumulation of nitrite by stimulated human monocyte-derived macrophages. Thus, the inability of these cells to produce nitric oxide appears to be unrelated to a lack of tetrahydrobiopterin and suggests that proper in vitro conditions may not yet have been discovered that permit nitric oxide synthesis by activated human macrophages.

Biological Availability↗
PubMed1993

Tetrahydrobiopterin biosynthesis defects examined in cytokine-stimulated fibroblasts.

Incubation of primary skin fibroblast cultures with the cytokines interferon-gamma and tumour necrosis factor-alpha stimulates the de novo pathway of tetrahydrobiopterin biosynthesis. Fibroblasts from patients with the two most common types of genetic defects of tetrahydrobiopterin metabolism that cause hyperphenylalaninaemia show characteristic pterin responses predicted by the nature of the defect. Cells from a child with 6-pyruvoyl-tetrahydropterin synthase deficiency produce higher than normal levels of neopterin and no biopterin. Fibroblasts from dihydropteridine reductase-deficient children produce normal levels of tetrahydrobiopterin, which gradually becomes partially oxidized, and higher than normal levels of neopterin. As a model for cells with the rarest form of tetrahydrobiopterin deficiency, lack of GTP cyclohydrolase activity, normal fibroblasts were treated with 2,4-diamino-6-hydroxypyrimidine to inhibit GTP cyclohydrolase activity by > 90%, the level expected in patients with a GTP cyclohydrolase deficiency. Neopterin and biopterin synthesis rates of < 10% of normal levels were readily detectable. Therefore, analysis of the patterns of the pterins synthesized by fibroblasts can aid in the diagnosis of the hyperphenylalaninaemias caused by disorders of tetrahydrobiopterin metabolism.

Adult↗
PubMed1993

A mechanism of quinolinic acid formation by brain in inflammatory neurological disease. Attenuation of synthesis from L-tryptophan by 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilate.

Quinolinic acid (QUIN), kynurenic acid (KYNA) and L-kynurenine (L-KYN) are neuroactive kynurenine pathway metabolites that accumulate in inflammatory neurological diseases. These increases were attributed to the induction of indoleamine-2,3-dioxygenase (IDO), the enzyme that converts L-tryptophan into L-KYN. Direct conversion of L-tryptophan into QUIN by brain tissue occurs in conditions of CNS inflammation, but not by normal brain tissue. To investigate whether increased activity of enzymes distal to IDO may determine L-KYN conversion to QUIN, rhesus macaques were inoculated with poliovirus directly into the spinal cord, as a model of focal inflammatory neurological disease (FASEB J. 6, 2977-2989, 1992). Induction of spinal cord IDO (35.9-fold) accompanied smaller, but proportional increases in kynurenine-3-hydroxylase (2.4-fold) and kynureninase (2.3-fold) activities, which were correlated to CSF and tissue QUIN levels, as well as to measures of inflammatory lesions. 3-Hydroxyanthranilate-3,4-dioxygenase activity was unchanged. Cerebrospinal fluid KYNA levels increased in proportion to both IDO activity and L-KYN accumulation, though kynurenine aminotransferase activity was unaffected. Cerebrospinal fluid neopterin, a marker of macrophage and immune activation, accumulated in proportion to the responsive enzymes and metabolites. The cell types involved in producing QUIN were investigated in vitro. Human foetal brain cultures consisting of astrocytes and neurons converted large quantities of [13C6]L-tryptophan into L-KYN when stimulated by gamma-interferon, but very little [13C6]QUIN was formed unless macrophages (THP-1 cells) were first added to the cultures (to model a key component of brain inflammation). [13C6]L-Tryptophan was converted into [13C6]QUIN by either gamma-interferon stimulated macrophages, or following intracisternal administration into poliovirus-infected macaques. Inhibitors of the kynurenine pathway, 6-chlorotryptophan and 4-chloro-3-hydroxyanthranilic acid, attenuated [13C6]QUIN formation by macrophages, and when co-infused with [13C6]L-tryptophan into poliovirus-infected macaques. These results suggest roles for increased activities of IDO, kynurenine-3-hydroxylase and kynureninase in accelerating the synthesis of QUIN, L-KYN and KYNA in conditions of brain inflammation. Macrophage infiltrates, and perhaps microglia, are important sources of QUIN, whereas constitutive brain cells and macrophages are sources of L-KYN. Drugs that inhibit kynurenine pathway enzymes attenuate QUIN formation in the CNS, and provide tools to examine the consequences of reduced QUIN accumulation.

3-Hydroxyanthranilic Acid↗
PubMed1993

Mutation in the 4a-carbinolamine dehydratase gene leads to mild hyperphenylalaninemia with defective cofactor metabolism.

Hyperphenylalaninemias represent a major class of inherited metabolic disorders. They are most often caused by mutations in the phenylalanine hydroxylase gene and, less frequently but with usually more serious consequences, in genes necessary for the synthesis and regeneration of the cofactor, tetrahydrobiopterin. This cofactor is absolutely required for all aromatic amino acid hydroxylations, and, recently, nitric oxide production from L-arginine has also been found to be dependent on tetrahydrobiopterin. Phenylalanine hydroxylase catalyzes a coupled reaction in which phenylalanine is converted to tyrosine and in which tetrahydrobiopterin is converted to the unstable carbinolamine, 4a-hydroxytetrahydrobiopterin. The enzyme, carbinolamine dehydratase, catalyzes the dehydration of the carbinolamine to quinonoid dihydropterin. A decreased rate of dehydration of this compound has been hypothesized to be responsible for the production of 7-biopterin found in certain mildly hyperphenylalaninemic individuals. We have now identified nonsense and missense mutations in the 4a-carbinolamine dehydratase gene in a hyperphenylalaninemic child who excretes large amounts of 7-biopterin. This finding is consistent with the role of the carbinolamine dehydratase in the phenylalanine hydroxylation reaction. Together with previously identified inherited disorders in phenylalanine hydroxylase and dihydropteridine reductase, there are now identified mutations in the three enzymes involved in the phenylalanine hydroxylation system. In addition, the genetics of this system may have broader implications, since the product of the dehydratase gene has previously been shown to play an additional role (as dimerization cofactor for hepatocyte nuclear factor-1 alpha) in the regulation of transcription, through interaction with hepatocyte nuclear factor-1 alpha.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins.

The principal pathway for the metabolism of phenylalanine in mammals is via conversion to tyrosine in a tetrahydrobiopterin-dependent hydroxylation reaction occurring predominantly in the liver. Recently, the proposal that certain hyperphenylalaninemic children may have a deficiency of carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, has widened the interest in this area of metabolism. Upon cloning and sequencing the dehydratase, we discovered that this protein is identical to DCoH, the cofactor which regulates the dimerization of hepatic nuclear factor 1 alpha, a homeodomain transcription factor. The identity of the nuclear and cytoplasmic proteins is demonstrated by size, immunoblotting, stimulation of phenylalanine hydroxylase, and dehydratase activity. The evolution of the dual functions of regulation of phenylalanine hydroxylation activity and transcription activation in a single polypeptide is unprecedented.

Amino Acid Sequence↗