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Biomedical subjects

H Wachter

Publications and source records attributed to H Wachter.

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

High-performance liquid chromatographic methods for the quantification of tetrahydrobiopterin biosynthetic enzymes.

Tetrahydrobiopterin is a cofactor in hydroxylation reactions, including phenylalanine 4-monooxygenase, tyrosine 3-monooxygenase, tryptophan 5-monooxygenase, alkyl glycol ether monooxygenase and nitric oxide synthase. Determination of its biosynthesis is carried out to diagnose inherited diseases leading to partial defects in tetrahydrobiopterin synthesis. In addition, tetrahydrobiopterin synthesis is induced by proinflammatory cytokines, and intracellular levels of tetrahydro-biopterin in many cases limit the activity of tetrahydrobiopterin-dependent reactions, such as nitric oxide synthase in intact cells. Biosynthesis of tetrahydrobiopterin from guanosine 5'-triphosphate (GTP) requires the action of three enzymes, GTP-cyclohydrolase I (E.C. 3.5.4.16), 6-pyruvoyl tetrahydropterin synthase (EC, 4.6.1.10) and sepiapterin reductase (E.C. 1.1.1.153). Methods for quantification of biopterin and related pteridines in biological matrices by HPLC and application of these for determining the activity of the three tetrahydrobiopterin biosynthetic enzymes are reviewed in this article.

Alcohol Oxidoreductases↗
PubMed1996

Interleukin 1 beta and cAMP trigger the expression of GTP cyclohydrolase I in rat renal mesangial cells.

Endogenous synthesis of tetrahydrobiopterin (BH4) is an important requirement for cytokine-stimulated nitric oxide (NO) production in mesangial cells. We have shown that inducible NO synthase is expressed in mesangial cells in response to two principal classes of activating signals, inflammatory cytokines such as interleukin 1 beta (IL-1 beta) and agents that elevate cellular levels of cAMP [Kunz, Mühl, Walker and Pfeilschifter (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5387-5391]. In the present paper we demonstrate that IL-1 beta and cAMP similarly increase the steady-state mRNA levels of GTP cyclohydrolase I (EC 3,5,4,16), the rate-limiting enzyme in BH4 biosynthesis, as measured by a sensitive and quantitative nuclease protection assay. Stimulation of cells with a combination of IL-1 beta plus cAMP revealed an additive induction profile of GTP cyclohydrolase I mRNA. Message stability studies established that GTP cyclohydrolase I mRNA induced by cAMP has a longer half-life than the IL-1 beta-induced message. Moreover, cAMP exposure markedly prolonged the half-life of GTP cyclohydrolase I mRNA, from 1.5 to 3.4 h. In a next step we generated a rabbit polyclonal antibody against rat GTP cyclohydrolase I expressed in Escherichia coli and demonstrated that IL-1 beta and cAMP elevated GTP cyclohydrolase I protein levels in mesangial cells. Furthermore, IL-1 beta and cAMP led to a marked increase in GTP cyclohydrolase I activity and to increased accumulation of biopterin in mesangial cells. Combinations of IL-1 beta and cAMP resulted in a synergistic stimulation of GTP cyclohydrolase I activity. This may suggest that, in addition to transcriptional and post-transcriptional regulation, there is a prominent post-translational modulation of enzyme activity.

Animals↗
PubMed1996

Neopterin activates transcription factor nuclear factor-kappa B in vascular smooth muscle cells.

We have previously shown that the pteridine compound neopterin stimulates inducible nitric oxide synthase (iNOS) gene expression in vascular smooth muscle cells in vitro. The mechanisms whereby neopterin exhibits these effects remained unclear. The present study demonstrates that neopterin induces the translocation of the transcription factor nuclear factor-kappa B (NF-kappa B) to the nucleus. Pretreatment of cells with the antioxidant pyrrolidine dithiocarbamate completely suppressed the effects of neopterin on NF-kappa B activation, iNOS gene expression, and nitric oxide release. From these data we conclude that neopterin activates the translocation of NF-kappa B subunits to the nucleus by modulating the intracellular redox state. This is one possible explanation for the impact of neopterin on iNOS gene expression.

Animals↗
PubMed1996

Self-organizing neural networks--an alternative way of cluster analysis in clinical chemistry.

Supervised learning schemes have been employed by several workers for training neural networks designed to solve clinical problems. We demonstrate that unsupervised techniques can also produce interesting and meaningful results. Using a data set on the chemical composition of milk from 22 different mammals, we demonstrate that self-organizing feature maps (Kohonen networks) as well as a modified version of error backpropagation technique yield results mimicking conventional cluster analysis. Both techniques are able to project a potentially multi-dimensional input vector onto a two-dimensional space whereby neighborhood relationships remain conserved. Thus, these techniques can be used for reducing dimensionality of complicated data sets and for enhancing comprehensibility of features hidden in the data matrix.

Animals↗
PubMed1996

Increase of tryptophan in serum and in cerebrospinal fluid of patients with HIV infection during zidovudine therapy.

A high percentage of patients with human immunodeficiency virus infection presents with decreased tryptophan concentrations in serum and cerebrospinal fluid. In parallel degradation products of tryptophan like kynurenine and quinolinic acid are increased. We investigated the behavior of tryptophan concentrations in 14 patients with HIV infection before and during treatment with zidovudine, and we found a significant increase of tryptophan in serum and cerebrospinal fluid after 4-14 months of therapy. In parallel, neopterin concentrations decreased significantly. Moreover, an association existed in cerebrospinal fluid between the degree of tryptophan increase and neopterin decrease. Thus, treatment with zidovudine contributes to a gradual normalization of tryptophan metabolism in patients with HIV-1 infection. The data imply that zidovudine therapy is associated not only with a reduction of virus replication but also immune activation is reduced.

Anti-HIV Agents↗
PubMed1996

Activated cellular immunity and decreased serum tryptophan in healthy pregnancy.

We analyzed plasma samples from 45 randomly selected healthy pregnant women and from 20 healthy nonpregnant female controls of corresponding age. Tryptophan and kynurenine concentrations were measured by reverse-phase high performance liquid chromatography. The kynurenine per tryptophan ratio was calculated to allow a more accurate estimate of tryptophan degradation. In addition, neopterin was measured by radioimmunoassay. There were significantly lower tryptophan concentrations in pregnant women compared to nonpregnant controls. Kynurenine per tryptophan ratios were increased in pregnant women in the 3rd trimester. Significant correlations existed between neopterin increase and tryptophan decrease as well as kynurenine increase and tryptophan decrease. Also significant correlations between week of pregnancy and lower tryptophan and higher kynurenine per tryptophan ratio were found (p < 0.01). From our data, cellular immune activation is likely to be the cause of enhanced tryptophan degradation during pregnancy.

Biopterins↗
PubMed1996

Decreased plasma tryptophan in pregnancy.

OBJECTIVE: To examine levels of serum tryptophan and its degradation product kynurenine in uncomplicated pregnancy, according to the week of pregnancy and the concentrations of neopterin. METHODS: Plasma was analyzed from 45 healthy pregnant women (15 in each trimester), 15 healthy puerperas, and 20 nonpregnant controls. Tryptophan and kynurenine were measured by reverse-phase, high-performance liquid chromatography, and neopterin by radioimmunoassay. RESULTS: In healthy pregnant women, tryptophan values decreased (median first trimester: 72 mumol/L; second trimester: 51 mumol/L; third trimester: 46 mumol/L; P < .001) in a manner correlated with the duration of pregnancy (Spearman rank correlation coefficient ra = -0.771, P < .001) and normalized in the puerperium (median 60 mumol/L). No change in kynurenine, a tryptophan degradation product, was observed, but the ratio of kynurenine to tryptophan increased during pregnancy and correlated positively with gestational age (ra = 0.714, P < .001). In addition, an inverse correlation existed between neopterin and tryptophan concentrations (ra = -0.566, P < .001), as well as a positive one between neopterin and the kynurenine to tryptophan ratio (ra = 0.660, P < .001). CONCLUSION: Tryptophan levels decrease during normal pregnancy and the decrease may be related to immune activation phenomena.

Biopterins↗
PubMed1996

T-cell activation, expression of adhesion molecules and response to ethanol in alcoholic cirrhosis.

Abnormal immune function is a well-recognized feature in patients with alcoholic cirrhosis. It may contribute to the pathogenesis of the disease and to the clinical consequences. Nevertheless, a potential role of ethanol to elicit immune disturbances in patients is still unclear. To further examine the immune mechanisms which potentially are involved in alcoholic cirrhosis and the relationship to ethanol, we have determined the expression of surface antigens CD4, CD8, and of adhesion molecules CD25, LFA-1, ICAM-1 and LFA-3 in patients and in response to stimulation with OKT-3, IL-2 and with ethanol in vitro. In addition, we quantified the production of IL-2, TNF-alpha and IFN-gamma by lymphocytes of alcoholic cirrhosis patients compared to controls. Lymphocytes from patients showed increased basal and stimulated expression of CD4, CD25, LFA-1, ICAM-1 and LFA-3 molecules and increased TNF-alpha production in comparison to controls. When lymphocytes from patients were co-cultured with ethanol, the overexpression of activation markers and TNF-alpha production was similar to that obtained with mitogens. In contrast, a predominant suppressive effect of ethanol was observed in lymphocytes from controls. Our study underlines the importance of a chronic state of immune activation in alcoholic cirrhosis. The data further suggest a role of ethanol to stimulate immune response and to be directly involved in the development of disease.

Cell Adhesion Molecules↗
PubMed1996

Attenuated SIV imparts immunity to challenge with pathogenic spleen-derived SIV but cannot prevent repair of the nef deletion.

To date, some success has been achieved with several experimental vaccines against AIDS in the available animal models. In the simian immunodeficiency virus (SIV) macaque model protection against superinfection was obtained by preinfection with a virus attenuated by a deletion in nef. To investigate the efficacy of SIVmac32H(pC8), a nef deletion mutant of SIVmac251, as a live-attenuated vaccine, rhesus monkeys were infected intravenously (i.v.) with this virus. All monkeys became productively infected by the pC8 virus. The animals had low cell-associated viral loads but developed a strong cellular and humoral antiviral immune response. Two out of eight preinfected monkeys developed signs of immunodeficiency and were excluded from the challenge. Sequence analysis of reisolates from one of them revealed a complete repair of the nef deletion. The remaining six monkeys, two preinfected for 42 weeks and four for 22 weeks, were challenged i.v. with a pathogenic SIV derived ex vivo from the spleen of a SIV infected macaque. Four of the monkeys challenged resisted the second infection whereas in two monkeys preinfected for 22 weeks full length nef was detectable. All monkeys maintained a virus-specific CD4-cell proliferative response after challenge. Thus, even after short preinfection periods with an attenuated SIV sterilising immunity against a challenge with a pathogenic SIV can be obtained. However, such a vaccine is unsafe since the attenuated virus frequently reverts to a more virulent form.

AIDS Vaccines↗
PubMed1996

Effects of neopterin-derivatives on H2O2-induced luminol chemiluminescence: mechanistic aspects.

Neopterin, 6-D-erythro-1',2',3'-trihydroxypropyl-pterin, and its dihydroform, 7,8-dihydro-neopterin, are synthesized by human monocytes/macrophages upon stimulation by interferon-gamma. In the presence of iron chelator complexes neopterin enhances hydrogen peroxide-induced luminol chemiluminescence at neutral or slightly alkaline pH (7.5). In contrast, 7,8-dihydroneopterin scavenges chemiluminescence independently from the pH value and iron. In this study, we explored in more detail the mechanism possibly involved: analysis of the reaction products shows that 7,8-dihydroneopterin is oxidized and degraded to 7,8-dihydroxanthopterin and xanthopterin, whereas the neopterin molecule is not chemically altered during the chemiluminescence reaction. Investigations of the neopterin-induced effect show that mannitol, a scavenger of hydroxyl radicals, does not alter the enhancing effect of neopterin. L-histidine, which scavenges singlet oxygen almost as effective as hydroxyl radicals, reduces the enhancing effect of neopterin. However, singlet oxygen was not detectable during the reaction by measuring monomol light emission (1270 nm). When replacing hydrogen peroxide by 3-morpholinosydnonimine, a generator of hydroxyl radicals, or naphthalene-endoperoxide, a generator of singlet oxygen, in the luminol chemiluminescence assay, neopterin shows no enhancing effect irrespective of the presence of iron-(III)-EDTA. The data suggest that neopterin enhances hydrogen peroxide-induced luminol chemiluminescence in the presence of iron-(III)-EDTA by formation of a catalytic complex that seems to favor the formation of oxygen intermediates which derive from hydrogen peroxide and react with luminol.

Biopterins↗
PubMed1996

Streptococcal erythrogenic toxins induce neopterin formation in human peripheral blood mononuclear cells but not in the human myelomonocytoma cell line THP-1.

We tested whether the exposure of human monocytic cells to streptococcal erythrogenic toxins A, B, C and a streptococcal-derived Mitogert BX is associated with synthesis of neopterin in vitro. Neopterin production was not induced when the human myelomono-cytoma cell line THP-1 was stimulated with these toxins, and there was only a slight co-stimulatory effect of streptococcal erythrogenic toxin A together with interferon-gamma stimulation. However, these toxins induced interferon-gamma and further neopterin production in peripheral blood mononuclear cells of three healthy individuals. This neopterin formation could be blocked by anti-human interferon-gamma. From our investigations we conclude that there is no direct effect of streptococcal erythrogenic toxins on neopterin production by monocytic cells. However, the data obtained in peripheral blood mononuclear cell culture imply that these toxins are able to stimulate neopterin production in humans via the induction of huge amounts of interferon-gamma.

Biopterins↗
PubMed1996

7,8-Dihydroneopterin upregulates interferon-gamma promoter in T cells.

Activated cell-mediated immunity is accompanied by elevated concentrations of interferon-gamma leading to the secretion of neopterin-derivatives which are known as sensitive immune activation markers in clinical laboratory diagnosis. Recent data imply a potential role of neopterin-derivatives in oxygen-free radical-mediated processes and a direct impact of 7,8-dihydroneopterin on tumor necrosis factor alpha-mediated programmed cell death. We report here that 7,8-dihydroneopterin and hydrogen peroxide upregulate the production of interferon-gamma, thereby establishing an autocrine feed-back loop. Data put emphasis on the role of neopterin-derivates within the cytokine network.

Antioxidants↗