H(2)O(2) increases de novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via GTP cyclohydrolase I and its feedback regulatory protein in vitiligo.

Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex.

Molecular evidence that halo in Sutton's naevus is not vitiligo.

Both halo naevus and vitiligo are acquired leucodermas of unknown aetiology. To date a significant contribution of oxidative stress through accumulation of hydrogen peroxide (H2O2) has been documented in the pathomechanism of vitiligo but not in halo naevus. Both epidermal pterin-4a-carbinolamine dehydratase (PCD) and catalase are sensitive markers to follow H2O2 concentration-dependent deactivation of these proteins. In situ protein expression of PCD and catalase was examined in full-skin biopsies from skin phototype-matched controls (n=5), untreated and treated vitiligo patients (n=5) and patients with untreated halo naevus in association with vitiligo (n=3). Vitiligo was treated with pseudocatalase (PC-KUS) only. Catalase levels were determined in epidermal suction blister extracts using fast protein liquid chromatography (FPLC). In addition, epidermal H2O2 levels were followed in vivo by Fourier-transform Raman spectroscopy. The results of this study ruled out a contribution of H2O2 in the millimolar range in the depigmentation process of halo naevus as previously documented in vitiligo. Therefore, it can be concluded that both leucodermas exercise distinct concentration-dependent H2O2 signalling in their pathomechanisms.

Serial transplants of DMBA-induced mammary tumors in Fischer rats as a model system for human breast cancer. VI. The role of different forms of tumor-associated stress for the regulation of pineal melatonin secretion.

Previous studies on human breast cancer patients showed a decline in circulating melatonin levels corresponding to primary tumor growth and an increase when relapse occurred. The aim of the current investigation was to study in an experimental model possible mechanisms involved. Inbred female F344 Fischer rats were used for serial passages derived from a chemically induced mammary adenocarcinoma. Animals with slow-growing carcinosarcomas at passage 2 showed a significant elevation of nocturnal urinary melatonin (23. 00-07.00 h; +50%, p < 0.05) and a nominal increase in plasma melatonin (+41%; 02.00-03.00 h). By contrast, these parameters were significantly depressed in animals with fast-growing sarcomas (urinary melatonin: -22%, p < 0.025; plasma melatonin: -56%, p < 0. 01). At passage 2 nocturnal pineal N-acetylserotonin (02.00-03.00 h) was significantly enhanced (+62%, p < 0.05) probably due to an increased activity of serotonin-N-acetyltransferase (SNAT, +45%), the rate-limiting step of pineal melatonin biosynthesis converting serotonin to N-acetylserotonin. The activation of SNAT may be due to a stimulation of the sympathetic nervous system (urinary noradrenaline; NA: +243%, p < 0.005) when the cellular immune system responded towards tumor growth (urinary biopterin, +214%, p < 0.005). At passage 12 SNAT and N-acetylserotonin were unaffected but a depletion of plasma tryptophan (-34%, p < 0.0001), the precursor amino acid of melatonin, was found. The marginal decline in pineal serotonin (-18%, p < 0.05) disputes that the drastic depletion in circulating melatonin (-56%, p < 0.01) can be exclusively explained by a reduced availability of tryptophan. Therefore, the involvement of an additional mechanism has to be postulated, such as a degradation of melatonin via indoleamine 2,3-dioxygenase, an extrahepatic enzyme which has been detected in tumor tissue and is related to tryptophan 2,3-dioxygenase (TDO). TDO occurs only in the liver, is highly specific for L-tryptophan and is induced by glucocorticoids which would account for the observed depletion of plasma tryptophan resulting from a tumor-associated activation of the hypothalamo-pituitary-adrenal axis (urinary corticosterone +208%, p < 0.01). These findings present first explanations for the previously observed modulation of melatonin levels in cancer patients but also illustrate the high degree of complexity of mechanisms involved in the interactions between tumor growth and the immunoneuroendocrine system.

Correlations between endotoxin, interferon-gamma, biopterin and serum phospholipase A2-activities during lethal gram negative sepsis in rats.

OBJECTIVE: To establish a standardised reproducible animal model of intraperitoneal sepsis, and to investigate early immunoserological responses to find a mediator-based system for evaluation and grading of diffuse peritonitis in patients DESIGN: Prospective experimental study SETTING: 4 Teaching hospitals, Germany and Austria MATERIAL: 42 LEW. 1W rats, 12 of which acted as controls INTERVENTIONS: Gram negative sepsis was induced by intraperitoneal injection of 6 ml of a mixture of Escherichia coli (K1:H+) 10(10) organisms/ml, autogenous haemoglobin 2.9 ml (haemoglobin concentration 3%), 0.9% sodium chloride 3 ml, and suspension 0.1 ml. Control rats were given physiological saline 6 ml alone. MAIN OUTCOME MEASURES: Concentrations of endotoxin, interferon gamma (IFN-gamma), and biopterin, and serum phospholipase A2 (PLA2) activity. RESULTS: There were significant differences between the septic and control rats in concentrations of endotoxin (EU/ml) (median (interquartile range) 21.85 (2.02-159.5) compared with 0, p < 0.0001; IFN-gamma (pg/ml) 1263.0 (271.0-7575.0) compared with 101.0 (89.0-141.0), p < 0.0001; biopterin (nmol/L) 111.0 (66.4-156.3) compared with 53.7 (38.3-67.6), p < 0.001; and PLA2 (U/L) 163.0 (125.8-209.0) compared with 112.5 (88.5-126.5) p < 0.01. CONCLUSIONS: Measurements of concentrations of endotoxin, IFN-gamma, pteridines, and PLA2 activity may well be adequate markers for early recognition of sepsis, and perhaps for grading it during the first 6 hours after induction. The allow a clear distinction to be made between septic and non-septic disorders in 87% of cases.

Serial transplants of DMBA-induced mammary tumors in Fischer rats as model system for human breast cancer. IV. Parallel changes of biopterin and melatonin indicate interactions between the pineal gland and cellular immunity in malignancy.

Nocturnal (23.00-07.00 h) urinary melatonin and total biopterin (tBI; after acidic oxidation of reduced biopterins) were analyzed during the growth of two passages of a mammary tumor line in female F344 Fischer rats. In addition, nocturnal (02.00-03.00 h) peak concentrations of pineal melatonin in plasma were analyzed when tumors had reached comparable average tumor volumes of 25-30 cm3. Since tetrahydrobiopterin (BH4) is produced by murine macrophages in response to interferon-gamma released by activated T lymphocytes, measurements of tBI can serve to estimate the state of cellular immunity. At passage 2, a slow-growing localized carcinosarcoma, tBI showed a progressing increase during tumor growth reaching more than 200% (p < 0.05-0.005) of controls by the end of the experiment. Urinary and plasma melatonin were elevated by 30-50% (p < 0.05) and 42% respectively. At passage 12, a fast-growing metastasizing sarcoma, a depression of about 20-30% was found for tBI (p < 0.05) and urinary melatonin (p < 0.025); plasma melatonin was depleted by 70% (p < 0.005). Parallel changes of both parameters at each tumor passage indicate a close link between the pineal hormone melatonin and cellular immunity. The opposite trends observed at the two passages indicate a clear stimulation of the immune system and the pineal gland at early but inhibition at advanced stages of cancer.

3,5-Di-iodo-L-thyronine suppresses TSH in rats in vivo and in rat pituitary fragments in vitro.

3,5-Di-iodo-L-thyronine (T2) is a naturally occurring metabolite of thyroxine (T4). Contrary to earlier findings, T2 has recently been shown to have rapid effects in rat liver and in mononuclear blood cells. In the experiments described here, T2 was tested to determine whether it has a TSH suppressive effect in rats in vivo and in rat pituitary fragments in vitro. In experiments over 2 weeks in rats in vivo, low doses of T2 (20-200 micrograms/100 g body weight per day) had no significant influence on body and organ weights, but significantly decreased TSh and T4 serum concentrations. At 200 micrograms/100 g per day, T2 suppressed TSH to 43% and T4 to 29% of control levels. At 1-15 micrograms/100 g per day, 3,5,3'-tri-iodo-L-thyronine (T3), used as a comparison to T2, had significant effects on TSH and T4 levels, and also on body weight. Fifteen micrograms T3/100 g per day decreased TSH to 44%, T4 to 25%, and body weight to 59% of control levels. In experiments over 3 months in rats in vivo, a low dose (25 micrograms/100 g per day) of T2 suppressed TSH to 60% and T4 to 57% of control levels and had no significant influence on other parameters. Conversely, 0.1 microgram/100 g per day T3 had significant effects on body and organ weights as well as pellet intake, but a less pronounced TSH suppressive effect: TSH concentrations were unchanged and T4 concentrations were down to 80% of control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum biopterin--a novel marker for immune activation during pre-diabetes in the BB rat.

Tetrahydrobiopterin (BH4) is a pteridine product which is released by rodent macrophages on activation by cytokines. We have used serial pancreatic biopsy, and measurement of serum biopterin at 30, 60, 90 and 120 days in the BB/S rat to relate histological change to macrophage activation during the course of pre-diabetes. Using immunohistochemistry, and an arbitrary scoring system read blind and standardised against day 30, we found that pancreatic MHC class I, MHC class II and infiltrating macrophage staining were up-regulated in the BB/S diabetes-prone rats (n = 17) at day 60, markedly so at day 90, and less so at day 120. Staining for resident pancreatic macrophages remained unchanged throughout in diabetes prone, diabetes resistant and Wistar (n = 28) control animals. Serum biopterin fell progressively and identically with age in BB diabetes resistant rats (n = 11) and Wistar controls. No change in weight gain or biopterin levels was observed in the biopsied animals. Mean serum biopterin levels in diabetes prone rats (of which 13 of 17 became diabetic at median 85 days) were the same as in diabetes resistant and Wistar rats at days 30, 60 and 120, but showed a striking and highly significant elevation (p < 0.001) at day 90. Although macrophages infiltrate the islet early in pre-diabetes, the timing of their activation is unknown. The rise in biopterin we observed is a potentially important immunological event which occurred late in the progression of pre-diabetes. This acute terminal event has not been reported previously, and may modify current concepts concerning the tempo of cell destruction during pre-diabetes.

Structural requirements of iodothyronines for the rapid inactivation and internalization of type II iodothyronine 5'-deiodinase in glial cells.

3,3'5,5'-Tetraiodothyronine (T4), but not 3,3'5-triiodothyronine (T3), acutely regulates the activity of the plasma membrane-bound enzyme, type II iodothyronine 5'-deiodinase (5'D-II), by inducing internalization of the enzyme through an extranuclear, energy-dependent mechanism that requires an intact actin cytoskeleton. The affinity label, N-bromoacetyl-L-T4, binds to 5'D-II and irreversibly inhibits the enzyme but does not initiate internalization. To determine the structural elements of T4 which are required for enzyme internalization, T4 analogs were modified in the alanine side chain and were then evaluated for their ability to induce enzyme internalization, to inhibit enzyme activity, and to promote actin polymerization in hypothyroid cells. The analogs studied showed marked variability in their ability to inactivate 5'D-II. The rank order of potency for enzyme inactivation was T4 > COOH-blocked analogs > NH3 and COOH blocked analogs >> NH3 blocked analogs (EC50 values range from 1 to > 1000 nM). In contrast, all T4 analogs tested and T4 were excellent competitive inhibitors of 5'D-II with respect to substrate (Ki values ranged from 4 to 27 nM). The differential capability of iodothyronines to inactivate the enzyme was not related to their ability to enter the cell, since Ki values measured in intact glial cells were equivalent to those measured in cell sonicates. The power of the T4 analogs to inactivate 5'D-II was paralleled by their ability to polymerize actin in hypothyroid cells and to induce 5'D-II binding to F-actin. The data show that modification of the alanine side chain markedly alters the ability of T4 analogs to induce 5'D-II inactivation and actin polymerization. A net negative charge on the alanine side chain of T4 is detrimental for the hormone-dependent inactivation of 5'D-II and polymerization of actin, whereas uncharged or positively charged molecules retain significant activity.

Affinity labeling of rat liver and kidney type I 5'-deiodinase. Identification of the 27-kDa substrate binding subunit.

Extrathyroidal production of 3,3',5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5'-deiodinases. Type I 5'-deiodinase (5'D-I) produces greater than 75% of the T3 found in the circulation and in thyroid hormone-responsive tissues and is most abundant in rat liver and kidney. In this study, we used the bromoacetyl derivatives of T4 (N-bromoacetyl-[125I]L-thyroxine, BrAcT4) and T3 (N-bromoacetyl-[125I]3,3',5-triiodothyronine, BrAcT3) as alkylating affinity labels to identify 5'D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5'D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity labeled 5'D-I preparations showed that approximately 80% of the affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5'D-I substrates and inhibitors specifically blocked affinity labeling of p27 with a rank order of potency (BrAcT4 greater than BrAcT3 greater than 3,5,3'-triiodothyronine (rT3) approximately flavone EMD 21388 greater than iodoacetate greater than N-acetyl-T4 (NAcT4) greater than N-acetyl-T3 (NAcT3] identical to that determined for inhibition of 5'-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5'D-I activity, respectively, were matched by comparable changes in the quantity of affinity labeled p27. BrAcT3 was a less effective affinity label for p27 and minor labeling of a new band with 53 kDa was observed. Molecular sieve chromatography of detergent-solubilized 5'D-I showed coincident peaks of p27 and 5'-deiodinating activity with an apparent Mr approximately 51,000. Two-dimensional gel electrophoresis showed that p27 was a single polypeptide with a pI of 6.1. Approximately 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5'D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5'-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3.

Rapid stimulation of hepatic oxygen consumption by 3,5-di-iodo-L-thyronine.

Tri-iodothyronine (T3) and thyroxine (T4) as well as 3,5-di-iodothyronine (T2) stimulated O2 consumption by isolated perfused livers from hypothyroid rats at a concentration as low as 1 pM by about 30% within 90 min. Application of T2 resulted in a faster stimulation than with application of T3 or T4. Inhibition of iodothyronine monodeiodinase by propylthiouracil, thereby blocking the degradation of T4 to T3 and of T3 to T2, demonstrated that only T2 is the active hormone for the rapid stimulation of hepatic O2 consumption: T3 and T4 lost all of their stimulative activity, whereas T2 was as potent as in the absence of propylthiouracil. Perfusion experiments with thyroid-hormone analogues confirmed the specificity of the T2 effect. The nucleus is unlikely to contribute to the rapid T2 effect, as can be deduced from perfusion experiments with cycloheximide and lack of induction of malic enzyme by T2. In conclusion, a new scheme of regulation of mitochondrial activity is proposed: T2 acts rapidly and directly via a mitochondrial pathway, whereas T3 exerts its long-term action indirectly by induction of specific enzymes.

[Results of a phase II study on the treatment of hairy cell leukemias with various doses of alpha-2-recombinant interferon]. / Ergebnisse einer Phase-II-Studie zur Behandlung von Haarzell-Leukämien mit unterschiedlichen Dosen von alpha-2-rekombinantem Interferon.

Hairy-cell leukemia has been shown to be extraordinary sensitive to treatment with alpha-interferon. In order to define clinically effective interferon doses associated with minimal toxicity two different dose regimens were applied in this clinical trial: firstly, a conventional dose schedule, and secondly, a biologically defined dose regimen. For dose finding in the latter group, neopterin, a GTP degradation product produced by macrophages under control of interferon, was chosen. Six patients (Group A) received conventional doses of recombinant interferon--alpha-2 (rIFN-alpha-2) 3 X 10(6) U/sqm/daily by the subcutaneous route. Five patients (Group B) were treated with the minimal dose of rIFN-alpha-2 which had previously been shown to induce maximum neopterin levels in urine. Already interferon doses in the range of 3 to 5 X 10(5) U/sqm2/daily administered subcutaneously proved to be sufficient for triggering maximum neopterin excretion in the urine. After six months of interferon treatment all patients were evaluable. At this time both doses regimens proved to be effective in terms of their anti-leukemic activity, but differed significantly in toxicity, which was only seen in Group A patients.

Molecular structure and biochemical activity of 3,5,3'-triiodothyronamine.

The thyroid hormone decarboxylation product, 3,5,3'-triiodothyronamine (T3AM), has been shown to inhibit the cAMP production stimulated by isoproterenol in turkey erythrocytes. This adrenergic receptor binding inhibition was not shown by the thyroid hormones nor by tyramine, but was observed for 3,5-diiodotyramine, 3,5-diiodothyronamine, and thyronamine. T3AM also inhibits prolactin secretion in cultured pituitary cells as well as domperidon binding in rat corpora striata membranes. T3AM has no thyromimetic activity at the nuclear level. The molecular structure of T3AM, determined as a borosalicylate salt by X-ray diffraction techniques, is the first report of a decarboxylated thyroid hormone analogue.

Altered urinary excretion of pteridines in neoplastic disease. Determination of biopterin, neopterin, xanthopterin, and pterin.

Urinary pteridine concentrations in healthy control subjects and patients with cancer and non-malignant diseases were determined by HPLC and TLC after partial purification by ion exchange and Sephadex chromatography. Elevated concentrations of neopterin were found in 70% of the 50 cancer patients investigated. In patients with non-Hodgkin's lymphoma (seven cases) or with liver metastases (12 cases) neopterin concentrations were significantly higher than in control subjects (p < 0.01). Biopterin was less frequently increased (22%). Xanthopterin was generally raised when neopterin and/or biopterin excretion was high. Neopterin/biopterin ratios were higher in some patients with cancer or with severe renal insufficiency than in controls. These findings suggest that alterations in pteridine metabolism are common in malignant disease. The pathogenic, diagnostic and therapeutic significance of these changes remains to be established.