bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Histological Assessment of Xenobiotic-Induced Liver Enzyme Induction and Pituitary-Thyroid Axis Stimulation in Rats Using Whole-Slide Automated Image Analysis
Authors: Garrido Rosario , Zabka Tanja S. , Tao Jianhua , Fielden Mark , Fretland Adrian , Albassam Mudher
Published: March 2013

Preclinical evaluation of a new compound, RO2910, identified a hypertrophic response in liver, thyroid gland, and pituitary gland (pars distalis). We aimed to develop and validate automated image analysis methods to quantify and refine the interpretation of semi-quantitative histology. Wistar-Han rats were administered RO2910 for 14 days. Liver, thyroid, and pituitary gland tissues were processed for routine histology and immunolabeled with anti–thyroid stimulating hormone (TSH) antibody (pituitary) and anti–topoisomerase II antibody (thyroid). Glass slides were scanned, image analysis methods were developed and applied to whole-slide images, and numerical results were compared with histopathology, circulating hormone levels, and liver enzyme mRNA expression for validation. Quantitative analysis of slides had strong individual correlation with semi-quantitative histological evaluation of all tissues studied. Hepatocellular hypertrophy quantification also correlated strongly with liver enzyme mRNA expression. In the pars distalis, measurement of TSH weak-staining areas correlated with both hypertrophy scores and circulating TSH levels. Whole-slide image analysis enabled automated quantification of semi-quantitative histopathology findings and a more refined interpretation of these data. The analysis also enabled a direct correlation with non-histological parameters using straightforward statistical analysis to provide a more refined dose- and sex-response relationship and integration among affected parameters. These findings demonstrate the utility of our image analysis to support preclinical safety evaluations.
Density and distribution of connexin 43 in corpus cavernosum tissue from diabetic and hypogonadal patients with erectile dysfunction
Authors: Traish Abdulmaged M. , Stottrup Casey , van Renterghem Koenraad , Achten Ruth , Roy Sayon
Published: March 2013

Aim: Altered expression of connexin 43 (Cx43) has been postulated to be involved in the development and progression of various diseases including erectile dysfunction (ED). The aim of this study was to determine whether distribution and density of the gap junction protein Cx43 are altered in human corpus cavernosum (HCC) tissue samples derived from diabetic or hypogonadal patients with ED compared to those from normal subjects.

Methods: HCC tissue sections derived from normal, diabetic and hypogonadal subjects were fixed in 4% formaldehyde, embedded in paraffin and immunostained with a monoclonal mouse anti-rat Cx43 antibody. Cx43 density was expressed as the cumulative number of gap junction plaques per unit area of tissue corrected for number of 4′,6-diamidino-2-phenylindole, dihydrochloride-labeled smooth muscle cells (dots per unit area corrected for number of cells).

Results: The distribution of Cx43 plaques in smooth muscle was not affected in tissues derived from diabetic or hypogonadal subjects with ED compared with those from normal subjects. However, the number of Cx43 plaques was significantly reduced in HCC tissues derived from diabetic or hypogonadal subjects (73±8% and 68±11% of normal, respectively), indicating reduced Cx43 gap junctions in diabetic and hypogonadal subjects with ED.

Conclusions: Cx43 density in the HCC was diminished in tissue samples derived from diabetic or hypogonadal patients with ED compared to tissue samples from normal non-diabetic subjects. This marked decrease in Cx43 gap junction channels may contribute to attenuated gap junction function and to diminished erectile physiology.
Correlation of Serum Interferon Gamma and Neopterin Concentrations in Africans with Various Diseases
Authors: Reibnegger Gilbert , Fuchs Dietmar , Hausen Arno , Schmutzhard Erich , Werner Ernst R. , Werner-Felmayer Gabriele , Dierich Manfred P. , Wachter Helmut
Published: March 2013

Concentrations of interferon gamma in serum and of neopterin in serum and urine were determined in 55 Tanzanians attending a local hospital for various diseases or complaints. For comparison, serum concentrations of interferon gamma and of neopterin were also measured in 76 clinically healthy Austrian blood donors. Both interferon gamma and neopterin concentrations were significantly higher in patients than in blood donors. Both analytes were significantly correlated with each other, and both exhibited similar behaviour in dependence on clinical diagnosis. The results support use of neopterin determination for indirect measurement of endogenous production of interferon gamma.
Immune Markers in Pregnancy A Study on Neopterin, β2-Microglobulin, Deoxythymidine Kinase and C-Reactive Protein
Authors: Norman Margareta , Bremme Katarina , Eneroth Peter
Published: March 2013

Neopterin and β2-microglobulin but neither C-reactive protein nor deoxythymidine kinase increased in maternal serum from pregnancy week 20 to 40. Only maternal C-reactive protein concentrations changed during vaginal delivery and after 4 days post partum. Retroplacental plasma levels of neopterin, deoxythymidine kinase and β2-microglobulin were significantly higher than in maternal peripheral serum which was interpreted as an indication of increased activity of the immune system as influenced by the fetoplacental unit. The concentrations of neopterin, deoxythymidine kinase and β2-microglobulin were significantly higher in mixed artero-venous umbilical plasma than in the retroplacental plasma, possibly reflecting activation signals to immunocompetent cells in the neonates. The possibility of a transfer of these compounds from fetal to maternal circulation was also pointed out.
Study on the Pteridines Metabolism in Children Affected by Hyperphenylalaninaemia and Phenylketonuria
Authors: Valsasina R. , Riva E. , Biasucci G. , Longhi R. , Giovannini M.
Published: March 2013

Atypical phenylketonuria (PKU) is caused by tetrahydrobiopterin (BH4) deficiency. In Italy a systematic screening service for BH4 deficiency is not currently performed, therefore its real frequency is not known yet. We determined urinary excretion of biopterin (B) and neopterin (N) by HPLC in 74 phenylketonuric and hyperphenylalaninemic subjects, including all newborns with positive Guthrie's test for PKU since 1984. We found two patients with N and B urinary values above the classical PKU range. In these subjects we also performed a BH4 loading test that restored a normal plasma phenylalanine level in one case, while it had no effect in the other one. In both of them, the enzymatic activity of dihydropteridine reductase on dried blood spots was absent. We conclude that the determination of urinary pteridines together with the BH4 loading test are useful and informative tools in the screening of BH4 deficiencies, but that a conclusive diagnosis can only be achieved by the enzymatic assays.
Synthesis and Dihydropteridine Reductase [EC 1.6.99.10] (Human) Activity of Reduced 2,7-Dimethylpteridin-4(3H)-one, 2-Methyl-2-desaminoneopterin [6-(1' S,2' R-1' ,2' ,3' -trihydroxypropyl)-2-methylpteridin-4(3H)one] and 2-Methyl-2-desaminobiopterin [6-(1' R,2' S-1',2'-dihydroxypropyl)- 2-methylpteridin-4(3 H)-one]
Authors: Matsuura Sadao , Traub Hermann , Armarego Wilfred L. F.
Published: March 2013

The syntheses of the title compounds have been achieved starting from 4,5-diamino-2-methylpyrimidin-6( 1 H)one. Sufficient amounts of the quinonoid 7,8-dihydropteridin-4(6H)-ones (12-14) were formed upon oxidation of the corresponding 5,6,7 ,8-tetrahydro derivatives (15 -17) to show that they are substrates of dihydroptcridine reductase (human). The apparent Km and Kcal values of (12-14) were determined. These provided evidence that the tautomeric form of the natural cofactor 7,8-dihydro(6H)biopterin has the endocyclic C2- N3 double bond structure (29).
Separation and Determination of Tetrahydrofolylpolyglutamates and their 5-Methyl Derivatives by Reversed-phase High-performance Liquid Chromatography with Electrochemical Detection
Authors: Kohashi Masahiro , Takahashi Asako , Iwai Kazuo
Published: March 2013

A highly sensitive method for the separation and quantitation of tetrahydrofolylpolyglutamates (H4PteGlun ) and their 5-methyl derivatives (CH3H4PteGlun) of different chain lengths by a reversed phase high-performance liquid chromatography with electrochemical detection is described. These compounds have been separated using a cosmosil 5-Ph column eluted with 50 mM potassium dihydrogen phosphate, pH 2.2, containing 0.05 M disodium EDTA and 1 % methanol at a tlow-rate of 1.5 ml/min, and an electrochemical detector (at a potential of + 350 mV versus the Ag/AgCI electrode). Good separation has been obtained on both H4PteGlun and CH3H4PteGIun derivatives containing up to 6 glutamyl residues. The detection limits at a signal-to-noise ratio of 3 for H4PteGlun and CH3H4PteGIun series (n = 1 - 6) were 1 -10 and 10 -15 pmol, respectivel y.
High Protein Diet and Phenylalanine Hydroxylase Activities in Rats
Authors: Carty Michael P. , Beirne Edel , Donlon John
Published: March 2013

The effects of a diet of 85% casein on the activities of the phenylalanine hydroxylases of rat liver and kidney have been compared. Whereas only the tetrahydrobiopterin-dependent activity of rat hepatic phenylalanine hydroxylase is significantly stimulated, both the tetrahydrobiopterin-dependent and the dimethyltetrahydropterin- dependent activities of the renal enzyme are significantly decreased, after five days of feeding a casein diet. The animals fed a high protein diet for seven days have an increased rate of phenylalanine catabolism in vivo, which is also reflected in increased flux of label from phenylalanine into glucose. The regulation of phenylalanine metabolism, under these conditions, is discussed.
Urinary Neopterin as Marker for Disease Activity in Children and Adolescents with Crohn’s Disease
Authors: Granditsch Gerhard , Fuchs Dietmar , Hausen Arno , Reibnegger Gilbert , Werner Ernst R. , Werner-Felmayer Gabriele , Wachter Helmut
Published: March 2013

Elevated production and excretion of neopterin indicate activation of cell-mediated immunity, since neopterin is produced in large quantities by human macrophages after stimulation with interferon gamma. In 21 patients, aged 9 to 19 years, with Crohn’s disease, urinary neopterin was measured on a total of 135 consecutive followup visits. Additionally, a large panel of anamnestic, clinical and laboratory data were obtained at every visit. Both the first visit data set (21 records) and the complete data set (135 records) were analyzed separately. As was previously demonstrated in adults with Crohn’s disease, neopterin excretion proved to be strongly correlated with overall rating of disease activity, and also with separate anamnestic, clinical and laboratory activity scores. Multiple regression analysis demonstrated that neopterin provides statistically independent information. Thus, measurement of neopterin concentrations as a marker for activation of cellular immune phenomena could aid in the monitoring of pediatric and juvenile Crohn’s disease.
Synthesis and Physico-Chemical Properties of 6- and 7-Monosubstituted Pyrazino|2,3-c̱|-1,2,6-thiadiazine 2,2-Dioxides
Authors: Alkorta Ibon , Goya Pilar , Paez Juan Antonio , Pfleiderer Wolfgang
Published: March 2013

Selective procedures for the synthesis of 6- and of 7-monosubstituted derivatives of pyrazinoI2,3-c̱|-1 ,2,6- thiadiazine 2,2-dioxide and their reactions with N-bromosuccinimide are reported. The structures of the compounds are discussed on the basis of 1 H -, 13C_ NMR and UV spectroscopic data. The pKa values of the compounds have also been determined.
Pteridines, XCVI. Synthesis and Reactions of 6-(1,2,3-Trihydroxypropyl)pteridines
Authors: Soyka Rainer , Pfleiderer Wolfgang
Published: March 2013

Various hydropyrano [3.2-g] pteridines (7, 10, 12, 21 , 33) were oxidized to the corresponding 6-(1,2,3- trihydroxypropyl)pteridine derivatives (8,9, 11, 13,32, 34), which are valuable intermediates for the synthesis of new 6-substituted pteridines (15 - 46). Periodate oxidation led to pteridine-6-carboxaldehydes (15, 16, 17, 38), which easily formed oximes (19 - 22, 39, 40). The aldehydes were further oxidized to 6-carboxylic acids (25,26,42) or reduced to 6-hydroxymethyl derivatives (27, 28, 43). Reductive condensations of the aldehydes (15, 16, 38) with p-aminobenzoylglutamic acid afforded folic acid analogues (29, 30, 44). Dehydration of the oximes (19, 20, 39) resulted in the formation of 6-carbonitriles (23, 24, 41, 46). The newly synthesized compounds were characterized by elemental analysis and by UV and NMR spectra.
Activity of 5, 10-Methenyltetrahydrofolate Synthetase in Rat Tissues and in Tumor Cell Lines
Authors: Li Mingxia , Collier Christine , Gritsman Helena , Bertino Joseph R.
Published: March 2013

The enzyme 5.1O-methenyltetrahydrofolate synthetase catalyzes the key enzymatic reaction in the pathway by which 5-formyl tetrahydrofolate (leucovorin) enters the folate coenzyme pool. The distribution of this enzyme in different organs and tumor cell lines was studied. This enzyme has a wide distribution among organs and tumor cell lines with highest levels of activity in liver and kidney. Tumor cell lines also contain significant levels of this enzyme activity. Thus, leucovorin can be converted to active folate coenzyme forms in organs and tumor cells, and does not require conversion in liver alone. The implications of these findings for leucovorin usage as "rescue" after methotrexate treatment or to augment fluoropyrimidine cytotoxicity are discussed.
Distinct Neopterin Excretion Patterns after Vaccination
Authors: Fuchs Dietmar , Hausen Arno , Reibnegger Gilbert , Werner Ernst R. , Werner-Felmayer Gabriele , Wachter Helmut
Published: March 2013

To compare the involvement of cellular immunity in response to vaccination we have investigated urinary neopterin levels in daily follow-ups of children after vaccination with live measles/mumps vaccine and of adults after boosting with the soluble antigen tetanus toxoid. Neopterin levels distinctly peaked 8 - 11 days after vaccination with measles/mumps vaccine. In contrast, after boosting with soluble antigen tetanus toxoid neopterin levels remained unaffected. Large amounts of neopterin are produced by human monocytes/ macro phages on stimulation with gamma interferon. In patients neopterin concentrations reflect activation of cell mediated immunity. The data imply that distinct pathways of T cell activation are triggered in humans after immunization with live vaccine and with soluble antigen .
Urinary Neopterin, Antigenaemia and CD4/CD8 Ratio in HIV Positive Children under AZT Therapy
Authors: Zuccotti Gian Vincenzo , Biasucci Giacomo , Brioschi Manuela , Brioschi Manuela , Antonucci Angela , Riva Enrica , Giovannini Marcello
Published: March 2013

We evaluated urinary neopterin in seven HIV positive children, aged 4 months to 4 years, 11 months, under Zidovudine (AZT) treatment, 300 mg/mq/12 hourly per os.

The results were compared with HIV antigenaemia (HIV Ag) and CD4/CD8 ratios.

Neopterin (μmol/mol creatinine) was determined by HPLC; HIV Ag assessed by enzyme-immunoassay and T-cell subsets by flow-citometry.

Blood and urine samples were obtained before and at 3 and 6 months after starting therapy.

There was no significant trend in CD4/CD8 ratios during AZT treatment. Neopterin and HIV Ag showed a statistically significant decrease (median values 3239, 1679, 2062 μmol/mol creatinine; 2.7, 1.3, 1.2 sample optic density/cut off optic density respectively; p < 0.05 by Friedman's test) and were also significantly correlated (p = 0.01 by Spearman's ranks correlation test).

We conclude that urinary neopterin is useful in monitoring AZT therapy in HIV positive children.
Localization of Sepiapterin Deaminase and Pteridines In the Granules In Epidermal Cells of the Silkworm, Bombyx mori
Authors: Tsusué M. , Kuroda S. , Sawada H.
Published: March 2013

A new method for assay of sepiapterin deaminase (3.5.4.24) activity by use of high performance liquid chromatography (HPLC) was developed. By this sensitive method the enzyme activity in the cell organelle was assayed. After cell fractionation, the enzyme was extracted with deoxycholate from pteridine granules of epidermal cells of the lemon mutant silkworm. On stepwise sucrose gradient centrifugation, most of the enzyme activity localized in the aggregated pteridine granules fraction, while the soluble fraction contained only one fourth of the total enzyme activity. The enzyme in the granule fraction had the same properties as the previously reported sepiapterin deaminase. These data show that the enzyme is localized in pteridine granules in the living cells. The attachment of the enzyme to the granule membrane is rather loose and previous papers studied the enzyme released from the granules. Cell fractionation and morphological observation showed that sepiapterin, sepialumazine and other pteridines were also localized in the granules together with uric acid.
Effect of Hormones and Protein Level on Induction of Enzymes Involved in Histidine Degradation and Folate Metabolism
Authors: Nair C.P.P. , Stokstad E. L. Robert
Published: March 2013

The effects of the hormones thyroxine, glucagon and hydrocortisone, and of protein level on hepatic histidase, urocanase, and certain folate enzymes were studied in rats. Hypothyroidism, induced either by feeding thiouracil or by thyroidectomy, increased histidase, urocanase, and the folate dependent enzyme formiminotransferase, and increased histidine metabolism as measured by oxidation of the 2-ring carbon of histidine to carbon dioxide. Hyperthyroidism, produced by feeding thyroid powder, decreased histidine oxidation and liver levels of this enzyme. Glucagon and hydrocortisone increased histidine oxidation and increased levels of histidase, urocanase and formiminotransferase. Elevated levels of casein and soy protein produced four to eight fold increases in histidase and urocanase, and 50% increases in formiminotransferase. Thus, increases in histidase and urocanase involved in the formation of formiminoglutamic acid (FIGlu) were accompanied by increases in the FIGlu metabolizing enzyme formiminotransferase.
6-Acetonylisoxanthopterin: A Potent Inhibitor of Sepiapterin Reductase
Authors: Nakanishi A. , Goto M. , Katoh S. , Sueoka T.
Published: March 2013

The inhibitory effect of 6-/7-substituted (2'-oxo) pteridines and ichthyopterin was examined for sepiapterin reductase. 6-Acetonylisoxanthopterin and 6-acetonyl-2,4-diamino-7-hydroxypteridine showed pronounced competitive inhibitory effects to sepiapterin reductase and Ki values were 0.38 μM and 90 μM, respectively. Ichthyopterin and 6-acetonyl-7-hydroxylumazine showed weak noncompetitive inhibitory effects and Ki values were 2.4 mM and 1.1 mM, respectively. 7-Acetonylxanthopterin exhibited no inhibition to sepiapterin reductase.
An in vitro System for Studying Pteridine Biosynthesis In Drosophila melanogaster
Authors: Escriche B. , Silva F. J.
Published: March 2013

In vitro organ culture is a system especially useful for the study of metabolic pathways and their regulation. This report applies such a system to the study of the biosynthesis of pteridines in Drosophila melanogaster, determining in vitro optimal conditions for head cultures, in relation to this metabolic pathway. The validity of the system was tested by applying it to the study of a well-known enzyme, xanthine dehydrogenase, which catalyzes the transformation of pterin into isoxanthopterin. Supplementation experiments with pterin were carried out, determining that, as expected, only those strains having xanthine dehydrogenase activity were able to transform this compound into isoxanthopterin. These results confirm the usefulness of this system for future studies on pteridine metabolism.
Measurement of Neopterin and Biopterin in Urine from Phenylketonuria Heterozygotes and Normal Controls
Authors: Ruiz-Vázquez P. , Bel Y. , Garcia A. M. , Cabello M. L. , Dalmau J. , Alós T. , Catalá J. L. , Ferré J.
Published: March 2013

The effect of an oral load of phenylalanine (100 mg/kg body weight) on the levels of neopterin and biopterin in urine has been determined in 8 heterozygotes for classical phenylketonuria and 25 supposed normal controls. In basal conditions, neopterin and biopterin levels were significantly different between males and females. A significant increase in urinary biopterin was found two hours after the phenylalanine load, both in heterozygotes and in normal homozygotes. This increase was maintained at least until the fourth hour. Neopterin levels did not suffer any change during that period. Comparison of urinary pteridines from normal controls and phenylketonuria carriers showed that there were no significant differences between both groups neither before nor after the phenylalanine load. From these data we concluded that measurement of biopterin and neopterin in urine cannot help in the identification of heterozygotes for phenylketonuria.
The Influence of 2,4-Diamino-6-Hydroxypyrimidine and Food Restriction on Tetrahydrobiopterin Metabolism, Neurotransmitters and Behaviour in the Rat
Authors: Cox J. , Armstrong R. A. , Blair J. A.
Published: March 2013

Administration of 2,4-diamino-6-hydroxypyrimidine (DAHP) in the diet of rats reduced total biopterins in plasma, brain and liver. The levels of dihydropteridine reductase (DHPR) and the rate of biopterin biosynthesis were unaffected. Animals on the DAHP diet consistently lost weight and ate less. Hence, the effects of DAHP and food restriction on biopterin metabolism, neurotransmitters and behaviour were investigated. Food restriction had similar effects to DAHP in that brain, liver and plasma biopterins were reduced. Neither DAHP nor food restriction significantly influenced the levels of neurotransmitters in the brain. However, DAHP reduced general activity and rearing of the animals in an open field arena while food restriction increased these parameters. These results suggest that the behavioural changes are either due to neurotransmitter changes not revealed by gross brain neurochemical analyses or to a more direct effect of tetrahydrobiopterin. In addition, the effects of DAHP may be confounded to some degree with these of reduced dietary intake and this has implications for the use of DAHP as an animal model of tetrahydrobiopterin (BH4) deficiency.
Ab Initio Quantum Chemical Calculations on the Stability of Different Tautomers of 6- and 7-Phenacetyl Pterin
Authors: Reibnegger Gilbert , Denny Brian J. , Wachter Helmut
Published: March 2013

In order to investigate the stabilities of different tautomeric forms of pteridine derivatives with a phenacetyl side chain in the 6- or 7-position, we have performed ab initio quantum chemical geometry optimizations of both the keto form and the vinylogous amide form of 6- and 7-phenacetyl pterin. The results are in accordance with experimental expectations: the keto form is slightly more stable for the 6-substituted derivative (1 .8 kcal per mol). while the vinylogous amide form is substantially more stable for the 7-substituted compound (8 .7 kcal per mol). The vinylogous amide forms are planar molecules, and this geometry enables the interaction between the hydrogen atom at N-5 (or N-8) and the carbonyl function of the side chain. Inspection of the calculated electron densities (population analysis) underlines the suggestion that the vinylogous amide form of 7-phenacetyl pterin is stabilized by a shift of electron density from the nitrogen bonded to C-2 to the carbonyl oxygen atom of the side chain (resonance stabilization via an additional highly conjugated vinylogous amide structure).
Investigation of Spectra and Ionization Constants of 8-Alkyl Pterins by Fluorimetry
Authors: Jeong Soon-Seog , Wormell Paul , Gready Jill E.
Published: March 2013

Spectrofluorimetric studies of the properties of 8-alkyl-pterins arc reported, together with preliminary results on the use of fluorimetry in studying the binding of these new mechanism-based substrates to dihydrofolate reductase (DHFR). The compounds fluoresce strongly in the 460-490 nm range with emission maxima for the cations being blue-shifted by ~5-20 nm. Quantum yields for the cations of ~0.14-0.23 have been measured. pKa values for the basic ionization of ~5.3-5.7 have been determined from fits to both the emission and excitation spectra using sample concentrations in the 1-2 µM range. Although the UV /vis spectra between pH 0 and 4 show little change. there were significant sigmoidal changes in fluorescence intensity which could he fitted to apparent ionization constant values of ~1.4-1.0 with fluorescence intensity decaying to almost zero at pH 0. Studies of the fluorescence for the cations and neutral forms in organic acids and polar organic solvents respectively indicate little solvent dependence. Spectral modelling using the CNDO/S-CI method confirms that the spectra are due to π→π* transitions. and that the long wavelength absorption hand of ~400 nm is due to transitions involving mostly the pyrazine ring and N1. Significant differences in emission wavelength and intensity between 6-methyl- and 7 -methyl-8-alkyl-pterins have been characterised with the latter exhibiting fluorescence stronger by a factor of ~2 and blue-shifted by ~20 nm which arc readily detectable visually: aqua-blue and green fluorescence respectively for the 7-methyl and 6-methyl isomers. From this understanding of the fluorescence properties of 8-alkyl-pterins and their dependence on charge state and pyrazine-ring substituents, and taking into account the fluorescence characteristics of DHFR and cofactor NADP. we have assessed the potential of fluorimetric experiments for studying both pterin dissociation constants and the form of enzyme-bound pterin
Mutagenesis at Five Pteridine Pathway Loci In Drosophila melanogaster: Genetic and Biochemical Characterization
Authors: Millán J.M. , Nájera C.
Published: March 2013

In order to carry out a genetic and biochemical analysis of pteridin pathway eye colour loci, sixteen strains of five eye colour mutants of Drosophila melanogaster (2 dke, 7 sf. 3 se, 3 Hnr and 1 bw) from natural populations were used. Four EMS mutagenesis experiments were carried out to produce induced mutants of the same loci. 54 mutants (mosaics and completes) were obtained but only 7 (4 sf, 1 bw, 1 Hnr and I dke) could be isolated. The 40.48% of the mutations were mosaics. The percentage of mutants appeared during the four first days (85.19%) was significatively higher to the percentage of mutants appeared during the four following (14.18%). Viabilities of EMS-induced mutants were similar to that of the natural ones. For the induced mutants viability at 25°C was higher than at 16°C and heterozygotes had a higher Viability than mutant homozygotes. The low mutagenesis frequencies and the lack of some metabolites for Hnr and se mutants suggest an important role of these mutants in the pteridine pathway.
Activated Macrophages in Preeclampsia
Authors: Schröcksnadel H. , Fuchs D. , Herold M. , Wachter H. , Dapunt O.
Published: March 2013

Neopterin (serum. urine) and other markers of macrophage activation (lL-1 IL-6, TNF alpha) were compared in preeclamptic patients and healthy pregnant controls. The prepartal urine neopterin median in preeclampsia (337 μmol/mol creat.) was 33% higher than that of healthy pregnant controls (253 μmol/mol creat.). There was no statistically significant difference between the groups (p = 0.08). In plasma there were no statistically significant differences in cytokine concentrations between healthy pregnant and nonpregnant controls with the exception of neopterin. that showed higher values in pregnant women (p = O.OO4). Elevated levels of IL-6. TNF-alpha and neopterin were observed in hypertensive women. Differences to healthy pregnant controls were statistically significant for IL-6 (p = 0.008). TNF-alpha (p = 0.009) and neopterin (p=0.04) and were more pronounced in severe forms of the disease. These three parameters of monocytic origin showed positive significant correlations amongst each other. A participation of macrophages in the pathomechanism of hypertensive disorders of pregnancy can thus be assumed.
Cyclosporin A Treatment in Psoriasis: Monitoring by Neopterin Concentrations In Serum and Urine
Authors: Sepp N. , Benedikter S. , Kofler H. , Fritsch P. , Wachter H. , Fuchs D.
Published: March 2013

Psoriasis is a hyperproliferative skin disorder in which immune mechanisms are thought to play a role. Recently, cyclosporin A has been found effective to treat psoriasis. However, mechanisms of action of cyclosporin A in patients are not well understood. We investigated seven patients (6 males. 1 female, aged 29-58 years) with severe psoriasis, three of them associated with arthropathy. The patients were treated with cyclosporin A given twice daily in gelatine capsules. Four patients were given 5 mg/kg, the others received 2.5 mg/kg initially. Two of them were put on 5 mg/kg (after 4 and 12 weeks of treatment). Clinical evaluation before and during therapy was carried out using the Psoriasis Area and Severity Index (PASI). Patients were followed up for 6 and 15 months. Their cyclosporin A blood levels were adjusted by a monoclonal radioimmunoassay. In parallel, serum and urine neopterin levels were quantified by HPLC. Mean neopterin concentrations in patients prior to therapy were found only slightly higher (serum 6.5± 0.8 mmol/l) than in healthy controls. There was no correlation of neopterin values and the degree of PASIscore. Onset of cyclosporin A was paralleled by decreasing neopterin concentrations and improvement of the skin status. Decrease of cyclosporin A doses was accompanied by an increase of PASI and significantly decreasing neopterin levels. In patients with severe relapses of psoriasis after withdrawal of cyclosporin A, neopterin rose higher than pretreatment levels. In 6/7 patients PASI and neopterin levels were closely correlated.
Immune Stimulation of Interferon-Gamma- and Biopterin Synthesis with Endotoxin in Animal-Experimental Peritonitis
Authors: Kullich W. , Weissenhofer W. , Hunsicker A. , Rokos H.
Published: March 2013

As a result to a microhial stimulus different defence systems are activated. Bacterial lipopolysaccharids (endotoxins) can lead to activation of the immune system in which phagocytic cells like macrophages. secrete cytokines and inflammatory mediators. Owing to a hyperactivation of immune mechanisms and also to generalized inflammatory processes organic lesions or even multiple failure of organs can evolve due to influences by cytokines and anaphylatoxines. On a standardized peritonitis model with wistar-pragrats. release of endotoxin. interferon-gamma synthesis and release of hiopterin into plasma has heen measured 6 hours after intraperi to neal administration of E.-coli-haemoglobin suspension. In the initial phase of the immune response interferon-gamma - being endogenously induced by endotoxin - stimulates synthesis of pteridines. Due to antagonistic effects to the cytokines (which stimulate the inflammatory processes) interferon-gamma regulatorily intervenes with the inflammatory mechanisms. The production of biopterin serves as a measure of activation of the monocyte/macrophage system. In comparison to a control group. treated with sodium chloride. statistically significant different have been found in the measuring parameters endotoxin. biopterin and interferon-gamma. The significant correlation of all parameters confirm the quick activation of immune mechanisms as response of the immune system to endotoxinaemia in diffuse purulent peritonitis.
Association between Urinary Zinc and Neopterin Excretion: A Manifestation of the Acute Phase Response in the Kidney?
Authors: Melichar Bohuslm
Published: March 2013

An elevation in urinary neopterin excretion is established to be a sensitive index of immune system activation in various disorders. In many of these conditions, a simultaneous increase in urine zinc output is observed, but this phenomenon remains largely unexplained. In previous studies in patients with different diseases, a significant correlation between urine zinc and neopterin was demonstrated. This suggests that the immune system may be involved in inducing an augmentation in urine zinc loss. As both zinc and neopterin are now known to influence oxidative processes, and lipid peroxidation has been demonstrated 10 play an important role in renal pathology, a protective role of the increase in urine zinc and neopterin, which might represent a manifestation of the acute phase reaction in the kidney, is discussed.
Pteridines CI Synthesis and Properties of Lumazine-6- and -7-carboxylic Acids
Authors: Eisele Roland , Aritomo Keiichi , Pfleiderer Wolfgang
Published: March 2013

Lumazine-6-(8) and 7-carboxylic acid (23) and their N-methyl derivatives (12, 13, 21, 26, 28, 29) have been synthesized and characterized by the determination of their pK values and the UV spectra of the neutral, mono and dianion species. It was noticed that the acidities of the two N-H protons of the pyrimidine moiety are almost the same indicating that the dianions of 8 and 23, respectively, are I: 1 mixtures of the corresponding N-l and N-3 deprotonated forms. The lumazine-7-carboxylic acids (23, 26, 28, 29, 31, 33) are in comparison to their 6-carboxyJic acid counterparts (8, 12, 13, 21, 39) stronger acids by about I pK unit due to the resonance effect of the 4-oxo function on C-7 but not C-6. Alkaline KMn04 oxidation of 6.7-dimethyl-Iumazine (30) forms expectedly 6-methyl-Iumazine-7-carboxylic acid (31) in high yield according to the activation of the 7-methyl group by the electron-attracting 4-carbonyl residue.
Stereoelectronic Effects in the 02-mediated Autoxidation of Reduced Folate Derivatives
Authors: Fitzhugh Anthony L.
Published: March 2013

For over forty years pteridinologists have studied the O2-mediated autoxidation of reduced folate derivatives. This effort has resulted in the identification of several transient intermediates and many products. Until now. however, no clear explanation has emerged of why certain reduced folate derivatives arc less susceptible to autoxidation than others. Presented below is a detailed theoretical model which readily explains the chemical behavior of reduced folate derivatives towards O2. Of note are the steric and electronic forces at work during the formation of the 4a-hydroperoxy intermediate and the stereoelectronic factors that predominate during its breakdown.
Effect of 5-Fluorouracil Administered at Low Doses on Cytotoxic Properties of Methotrexate in the in vivo Model of Mice L1210 Leukemia; According to the Schedule of Treatment
Authors: Graczyk Julita
Published: March 2013

The study compared antineoplastic effects of combined methotrexate and 5-fluorouracil therapy on LI210 lymphoid leukemia in mice according to the schedule of treatment. Methotrexate was administered at doses of: LOw.. 0.5 LOw. 5-Fluorouracil was administered at doses of: 0.2 LOIO, 0.1 LOw, 0.02 LOro. The parameters characterizing the efficacy of the therapy were survival time of mice inoculated with LI2l0 leukemic cells .. as well as 1151 excretory capacity of mice inoculated with 1251-iododeoxyuridine-Iabelled leukemic cells. It has been observed that the longest survival time of mice with LI210 leukemia is obtained when methotrexate is administered prior to 5-fluorouracil. Reversing the schedule, or simultaneous administration of the drugs results in shorter survival time. despite the administration of 5-fluorouracil at low doses, ineffective on LI210 leukemia in monotherapy. Cytotoxic effects on LI210 leukemic cells labelled with 1151UDR were present only when methotrexate was administered prior to 5-fluorouracil.
Serum Neopterin and Angiotensin-converting Enzyme In Cancer Patients
Authors: Melichar Bohuslav , Jandik Pavel , Vavrova Jaroslava , Mergancová Jindřiška , Maliřová Eva , Vobořil Zbyněk
Published: March 2013

An elevation of serum angiotensin-converting enzyme (ACE) has been reported in some disorders associated with immune activation, e.g. sarcoidosis, HIV infection or ulcerative colitis. Both endothelial cells and macrophages are thought to be the possible source of increased ACE levels. We have investigated serum ACE and neopterin, an indicator of macrophage activation, in 23 patients with solid tumors as well as in 8 controls. Similar ACE levels have been observed in the cancer patients and the controls (2.6 ± 2.1 vs 3.4 ± 1.1 μkat/1, while neopterin has been significantly higher in the cancer patients (15.4 ± 12.6 vs 7.2 ± 2.2 nmol/I, P<O.02). No correlation has been observed between serum ACE and neopterin. We conclude that an elevation of ACE is not present in cancer patients. Macrophage activation in cancer is not accompanied by increased ACE release.
Pteridines CV
Authors: Hanaya Tadashi , Torigoe Kiyoshi , Soranaka Kazuyuki , Yamamoto Horoshi , Qizhengt Yao , Pfleiderer Wolfgang
Published: March 2013

Treatment of L-biopterin (I) with N,N-dimethyformamide dimethyl- (or diethyl)acetal and then with acetic anhydride in pyridine gave 1',2'-di-O-acetyl-N'-(N,N-dimethylaminomethylene)-L-biopterin (4), which was converted by the Mitsunobu reaction into 3-methyl (5) and 3-p-nitrophenetyl derivatives (7). The protective groups on the side chain diols and N2 of these compounds were selectively cleaved to furnish products 6, 8-10, among which 9 is naturally occurring 3-methyl-L-biopterin and 8 is N',N(3)-protected biopterin, a versatile intermediate for various reactions on the side-chain diol. In contrast, the same Mitsunobu reactions of tri-N2:I',2'-0-acetyl-L-biopterin (II) afforded 04-methyl (12) and 04-NPE derivatives (13), both of which yielded 0 4-methyl-L-biopterin (14) and subsequently led to 4-amino-L-biopterin (18).
Neopterin-induced Suppression of Erythropoietin Production In Vitro
Authors: Schobersberger W. , Jelkmann W. , Fandrey J. , Frede S. , Wachter H. , Fuchs D.
Published: March 2013

The production of neopterin increases in several diseases with activation of the ceIlular immune response. As previously shown serum concentrations of neopterin are inversely correlated with blood hemoglobin concentrations in the anemia of hematological and malignant disorders. Besides the role of chronic immune activation on the disturbed iron metabolism, an inhibitory influence of pteridines on cellular erythropoietin production could not be excluded. To test the possibility that pteridines are able to suppress the hypoxia-induced production of erythropoietin, the effects of neopterin and 7,8-dihydroneopterin on the human ceIl line HepG2 (hepatoceIlular carcinoma) were investigated. 24 h incubation with neopterin induced a dose-dependent reduction of erythropoietin production. The erythropoietin concentration significantly decreased by - 57.6% with 300 11M and by - 34.9% with 100 11M neopterin, respectively. 7,8 dihydroneopterin did not influence erythropoietin production. The inhibitory effect of neopterin on erythropoietin production was a consequence of reduced erythropoietin-mRNA levels. The results of this study show a neopterin-induced suppression of hypoxia-induced erythropoietin formation in HepG2 cultures in a dose dependent manner. We speculate that under in vivo conditions high concentrations of neopterin can aggravate the anemia of chronic disease.
An Improved Synthesis of t-Butyl N-(4-propargylaminobenzoyl)-γ-oligo (L-glutamate )s
Authors: Kempny Michal , Rzeszotarska Barbara , Pawelczak Krzysztof
Published: March 2013

A simple synthesis in solution, of the series of six consecutive t-butyl N-(4-propargylaminobenzoyl)-yoligo( L-glutamate)s (8-13) containing from two to seven L-glutamic acid residues (Scheme 1) is described. a-t-Butyl N-(4-propargylaminobenzoyl)-L-glutamate (1), the versatile synthon is coupled with one of the t-butyl y-oligo(L-glutamate)s (2-7) by means of 2-chloro-l-methylpyridinium tosylate to give the title compounds in 91-95% yield subsequent to edulcoration of a post-reaction mixture and crystallization. These compounds were characterized by melting points, elemental analyses and HPLC and are 96.7-100.0% pure according to the last method (Table I).
Expression and Characterization of Recombinant Drosophila 6-pyruvoyl tetrahydropterin Synthase
Authors: Shik Park Young , Kim Nacksung , Kim Hajeong , Park Dongkook , Yim Jeongbin
Published: March 2013

6-Pyruvoyl tetrahydropterin synthase is involved in the synthesis of pteridine eye pigments in Drosophila. The purple gene which was known to be one of the target loci of the suppressor mutation su(sj2 has been identified to encode the enzyme, and its cDNA has been cloned recently. The cDNA encoding the 19.3 kDa subunit of the 6-pyruvoyl tetrahydropterin synthase was expressed as fusion proteins in E. coli. The recombinant protein was shown to be active and purified from the E. coli crude extract by metal-chelation chromatography. The fused metal-chelating oilgopeptide was removed by thrombin for further characterization. Apparent Km for the substrate dihydroneopterin triphosphate was determined to be 590 IlM, which was slightly higher than the value of the native enzyme. The isoelectric point of 6.4 was also different from the known value of 4.3 determined by the native enzyme. Heat stability and the stimulatory effect of reducing agents were similar to the native enzyme. The modification of cysteine residues in the recombinant enzyme, one of which is known to be conserved in human and rat enzymes, by iodoacetamide inhibited its activity by up to 80%.
Functional Studies on the Promoter Region of the Human GTP Cyclohydrolase I Gene
Authors: Gütlich Markus , Witter Klaus , Katzenmeier Gerd , Rödl Wolfgang , Wernert Thomas , Ziegler Irmgard , Bacher Adelbert
Published: March 2013

We have isolated genomic clones that contained the gene for human GTP cyclohydrolase I. One clone containing the 5'-regulatory region of this gene was further analysed. It encompassed the first exon, parts of the first intron and about 2.6kb of the promoter region. The transcription start site was localised by rapid amplification of cDNA ends (5'-RACE). The 2.6 kb region upstream of the transcription start site showed promoter activity when ligated upstream of a reporter gene. 5'-truncations of the promoter region increased its activity as long as the CAAT and TATA boxes remained unchanged. Several putative Spl responsive elements were located within a GC-rich region close to the transcription start site.
Effect of 7,8 -Dihydroneopterin, Biopterin and Isoxanthopterin on Cholesterol and Phospholipid Content and Phospholipid Biosynthesis in Vitro
Authors: Rudzite Vera , Jurika Edite , Baier-Bitterlich Gabriele , Wachterl Helmut , Fuchs Dietmar
Published: March 2013

Incorporation of fatty acids into phospholipids has been investigated using samples of rat liver tissue homogenate (Krebs-Ringer-phosphate buffer, pH=7.4, containing 0.3% albumin, fatty acid mixture and glycerol). The addition of 7,8-dihydroneopterin and biopterin (5 and 30 pmol/g wet weight) to incubation medium induced a decrease of palmitic (7,8-dihydroneopterin) and stearic acid (7,8-dihydroneopterin, biopterin) and an increase of linolenic (7,8-dihydroneopterin) and arachidonic acid (7,8 - dihydroneopterin, biopterin) incorporation into phospholipids. Cholesterol content decreased but phospholipid content did not change in samples containing 7,8-dihydroneo-pterin or biopterin. No changes of fatty acid incorporation into phospholipids as well as of the content of cholesterol and phospholipids were observed in samples after addition of isoxanthopterin (5 and 30 pmoljg wet weight) to incubation medium for phospholipid biosynthesis in vitro. Despite the fact that 7,8-dihydroneopterin and biopterin have different oxidation states and some similar effects on phospholipid biosynthesis and cholesterol content have been observed in our study, we suggest that the oxidation state of pteridines is more important than the pterin-( 6 )-side chain in the deviations of fatty acid incorporation into phopholipids as well as in the regulation of cholesterol content in samples
Chronopharmacokinetics of Methotrexate in Mice: Modulation by Administration of 5-Fluorouracil in Combination Chemotherapy
Authors: Graczyk Julita
Published: March 2013

The basic pharmacokinetic parameters of MTX administered as monotherapy as well as in the combined therapy with simultaneous and prior administation of FU were investigated. The parameters were determined in the two-compartment open system on the basis of measurement of MTX levels in mice after i.p. administration of a single dose 20 mg/ kg of the drug. The FU dose was 20 mg/kg, too. The effect of the circadian rhythm on the kinetics of MTX have been taken into consideration. In one series of experiments the drugs were administered in the morning and in the other during midday hours . The statistical analysis of the obtained results showed the essential effect of the administration time on the pharmacokinetic parameters of MTX in mice. The statistical significal differences could be found: C max, AUC, CL, T0,5 . The differences in T0,5 , parameter resulting from coadministration of FU with the same circadian rhythm of MTX administration have been observed.
Increased Urinary Neopterin Excretion in Vaccinated Adults
Authors: Giovannoni Gavin , Bell Joan , Feldmann Marc , Thompson Edward J.
Published: March 2013

Serial urinary neopterin :creatinine ratios of 58 healthy adult sub jects were measured from 2 days prior to and up to 12 days post vaccination. An increase in the urinary neopterin of at least twice the baseline was detected in 24/ 46 subjects who received either a Mantoux skin test or a live viral vaccine (cell mediated immune group, median average increase above baseline = 206%(interquartile range 150-338% ), and 'in 5/12 who received non-live vaccines known to stimulate mainly humoral immunity (humoral immune group, median average increase above baseline = 165%((interquartile range 142-412%) ). The peaks in the urinary neopterin in the cell mediated group occurred between days 5 and 10 post-vaccination, with the mode on day 5. In contrast the 5 subjects in the humoral immune group all developed their peaks on day 7 or 8 . Three of these subjects received an influenza vaccine, one a typhoid vaccine and one a tetanus toxoid. No association was found between local or systemic side-effects of vaccination and raised urinary neopterin . In conclusion, the monitoring of urinary neopterin excretion in adults is sensitive enough to detect an immunological response to vaccination with immunogens that induce either cell mediated or humoral immune responses.
Pteridines CVIII Reactions of 6, 7-Dichloro-I, 3-dimethyllumazine with Sulfur-Nucleophiles
Authors: Abou-Hadeed Khaled , Pfleiderer Wolfgang
Published: March 2013

6,7-Dichloro-1,3-dimethyllumazine (1) and various 6-chloro-7-O-, S- and N-subst.-1,3-dimethyllumazines (2-7) react under relatively mild reaction condiditions with S-nucleophiles to the corresponding 6-mercapto derivatives 8-16. Treatment of 1 with sodium benzylmercaptide at room temperature led to di-( 6- chloro-1,3-dimethyllumazine-7-yl)sulfide (17) whereas thiourea caused transformation to the corresponding 7,7-disulfide 18. 7-Mercapto-1,3-dimethyl-6(5H)lumazinethione (12) can be modified widely by alkylations to open-chain (21-23, 27-29) or cyclic 6,7-bis-alkylthio derivatives (24-26) and acylations with phosgene and thiophosgene, respectivly, gave the derivatives 36 and 37 of the new 1 ,3-dithiolo[ 4,5-g]pteridine ring system. Ribosylation of 12 afforded a 6,7-bis-S-riboside (40) in high yield.