The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele.

Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.

Identification and age-dependence of pteridines in bed bugs (Cimex lectularius) and bat bugs (C. pipistrelli) using liquid chromatography-tandem mass spectrometry.

Determining the age of free-living insects, particularly of blood-sucking species, is important for human health because such knowledge critically influences the estimates of biting frequency and vectoring ability. Genetic age determination is currently not available. Pteridines gradually accumulate in the eyes of insects and their concentrations is the prevailing method. Despite of their stability, published extractions differ considerably, including for standards, for mixtures of pteridines and even for light conditions. This methodological inconsistency among studies is likely to influence age estimates severely and to hamper their comparability. Therefore we reviewed methodological steps across 106 studies to identify methodological denominators and results across studies. Second, we experimentally test how different pteridines vary in their age calibration curves in, common bed (Cimex lectularius) and bat bugs (C. pipistrelli). Here we show that the accumulation of particular pteridines varied between a) different populations and b) rearing temperatures but not c) with the impact of light conditions during extraction or d) the type of blood consumed by the bugs. To optimize the extraction of pteridines and measuring concentrations, we recommend the simultaneous measurement of more than one standard and subsequently to select those that show consistent changes over time to differentiate among age cohorts.

Age-dependent changes in cuticular color and pteridine levels in a clonal ant.

Social insects are emerging models for studying aging and the longevity/fecundity trade-off. Research on the demography of colonies and populations are hampered by the lack of reliable age markers. Here we investigate the suitability of cuticular pigmentation and pteridine fluorescence for age grading individuals of the clonal ant Platythyrea punctata. We found that both traits varied with age. Cuticular color darkened with individual's age until 25-30 days after hatching. For pteridine fluorescence, we found that P. punctata workers show a decrease in head pteridine levels over time until 70-80 days of age. Together with other markers, such as age-based behavior, cuticular coloration and pteridine fluorescence may help to estimate the age structure of colonies.

Characterisation of white and yellow eye colour mutant strains of house cricket, Acheta domesticus.

Two eye-colour mutant strains, white (W) and yellow (Y) of house cricket Acheta domesticus were established in our laboratory. We phenotyped and genotyped the mutants, performed genetic crossings and studied the eye structure and pigment composition using light and electron microscopy and biochemical analysis. We show that W and Y phenotypes are controlled by a single autosomal recessive allele, as both traits are metabolically independent. The analysis of the mutants`eye structure showed a reduced number of dark pigment granules while simultaneously, and an increased amount of light vacuoles in white eye mutants was observed. Significant differences in eye pigment composition between strains were also found. The Y mutant had a lower number of ommochromes, while the W mutant had a lower number of ommochromes and pteridines. This indicates that mutated genes are involved in two different, independent metabolic pathways regulating tryptophan metabolism enzymes, pigment transporter granules or pigment granule formation.

New approach: Chemical and fluorescence profiling of NZ honeys.

New Zealand manuka (Leptospermum scoparium) and kanuka (Kunzea ericoides) honeys contain a unique array of chemical markers useful for chemical fingerprinting. We investigated the presence of 13 potential marker compounds in nectars of the major honey crop species. We confirmed that leptosperin, lepteridine, 2'-methoxyacetophenone, and 2-methoxybenzoic acid are exclusive to manuka nectar whereas lumichrome is unique to kanuka nectar. 3-Phenyllactic acid and 4-hydroxyphenyllactic acid are present in manuka and kanuka nectars. Leptosperin, lepteridine, 3-phenyllactic acid, and 4-hydroxyphenyllactic acid are chemically stable over prolonged storage, but not 2-methoxybenzoic acid and 2'-methoxyacetophenone. Accordingly, leptosperin and lepteridine are definitive chemical markers for authentication of manuka honey. An optimal concentration cut-off was established for the floral source-specific markers: leptosperin (94mg/kg), lepteridine (2.1mg/kg), 2'-methoxyacetophenone (2.0mg/kg) for manuka honey, and lumichrome (4.5mg/kg) for kanuka honey. The use of leptosperin and lepteridine as fluorescence markers for manuka honey authentication is reinforced.

Kinetics of drosopterin release as indicator pigment for heat-induced color changes of brown shrimp (Crangon crangon).

Heat-induced color changes of crustaceans are commonly described as the release of astaxanthin. In this study on Crangon crangon, it was found that astaxanthin plays a minor role in the (dis)coloration. By LC-HRMS, two polar, process dependent pigments were found. One pigment was identified as riboflavin and one as drosopterin (level-2 certainty). Thermal treatments had highest effect on drosopterin concentration changes and were chosen as indicator for a kinetic study of heat-induced color changes. The kinetic data fitted a consecutive step model (r2 = 0.971), including a first step in which drosopterin was released (kd,85°C = 0.95 ±â€¯0.09 min-1; Ead = 105 ±â€¯4 kJ/mol) and a second step where drosopterin is degraded (kb,85°C = 0.02 ±â€¯0.002 min-1; Eab = 190 ±â€¯15 kJ/mol). The kinetic model shows that shrimp should be heated at lower temperatures (<80 °C) than the heating temperatures used by fishermen (86-101 °C), creating opportunities for quality optimization. Therefore, this study delivers essential information needed in a comprehensive quality optimization study of the cooked brown shrimp.

Simultaneous determination of twelve polar pteridines including dihydro- and tetrahydropteridine in human urine by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

Pteridines and their derivatives are important cofactors in the process of cell metabolism, and the level of urinary excretion of these compounds is considered as an important clinical criterion. In this work, a new separation method involving hydrophilic interaction chromatography (HILIC) with tandem mass spectrometric detection has been developed for the simultaneous analysis of 12 pteridines including oxidized, di- and tetrahydroforms, namely neopterin, 7,8-dihydroneopterin, biopterin, 7,8-dihydrobiopterin, 5,6,7,8-tetrahydrobiopterin, dimethylpterin, dimethyltetrahydropterin, pterin, isoxanthopterin, xanthopterin, sepiapterin and pterin-6-carboxylic acid, in human urine without oxidative pretreatments. The stabilizing agent (dithiothreitol) at various concentrations and the stability of oxidized, di- and tetrahydroforms during the sample's short-term storage and processing and of the extracts were tested. In the developed method, 12 pteridines were chromatographically separated on an ZIC-HILIC column by gradient elution, and the run time was 20 min. Matrix effect was evaluated and several dilutions of urine were tested in order to study the evolution of signal suppression. Spiked recovery studies demonstrated that the technique was both accurate (83.1-116.7%) and precise (RSD 1.4-15.6%). Finally, several clinical urine specimens without oxidative pretreatments were examined with the new technique and compared with previous reports.

Quantitative pteridine fluorescence analysis: A possible age-grading technique for the adult stages of the blow fly Calliphora vicina (Diptera: Calliphoridae).

Age estimation of adult flies could extend the possible window of time for calculating the minimal postmortem interval (PMImin) by means of entomological methods. Currently, this is done by estimating the time required by necrophagous Diptera to reach certain juvenile developmental landmarks, and the method only works until the end of metamorphosis and emergence of the adult fly. Particularly at indoor crime scenes, being able to estimate the age of trapped adult flies would be an important tool with which to extend the calculable PMI beyond the developmental period. Recently, several promising age-dependent morphological and physiological characteristics of adult insects have been investigated in medical and forensic entomology, but the results are still preliminary and restricted to a few species. We examined adults of the forensically relevant blow fly species Calliphora vicina and investigated the fluorescence levels of pteridine, a group of metabolites that accumulates in the eyes during aging. From Day 1 to Day 25 post-emergence, flies were kept at three different temperature regimes (20°C, 25°C, and fluctuating temperatures in the context of a field study) and 12:12 L:D. From Day 1 until Day 7, the fluorescence of pteridine was determined on a daily basis, and thereafter, every three days. The achieved fly age was multiplied with the relevant temperature and converted into accumulated degree-days (ADD). The fluorescence level of pteridine increased linear with increasing ADD (females: R2=0.777; males: R2=0.802). The difference between sexes was significant (p<0.001). Neither head weight nor temperature had an effect on pteridine fluorescence. Because the variation in pteridine fluorescence increased with increasing ADD, it seems favorable to combine several aging methods for more precise results. In context, we emphasize that different body parts of the same specimen can be used to analyze cuticular hydrocarbons (legs), pteridine fluorescence (head/eyes), and gonotrophic stage (female abdomen).

Folic acid and its photoproducts, 6-formylpterin and pterin-6-carboxylic acid, as generators of reactive oxygen species in skin cells during UVA exposure.

Folic acid (FA) is the synthetic form of folate (vitamin B9), present in supplements and fortified foods. During ultraviolet (UV) radiation FA is degraded to 6-formylpterin (FPT) and pterin-6-carboxylic acid (PCA) which generate reactive oxygen species (ROS) and may be phototoxic. The aim of the present study was to investigate the production of ROS and phototoxicity of FA, FPT and PCA in skin cells during UVA exposure. The production of ROS and phototoxicity of FA, FPT and PCA were studied in the immortal human keratinocytes (HaCaT) and malignant skin cells (A431 and WM115) during UVA exposure. Increased ROS production and the photoinactivation of cells in vitro were observed during UVA exposure in the presence of FA, FPT and PCA. HPLC analysis revealed that 10 µM FA photodegradation was around 2.1 and 5.8-fold faster than that of 5 µM and 1 µM FA. Photodegradation of FA is concentration dependent, and even non-phototoxic doses of FA and its photoproducts, FPT and PCA, generate high levels of ROS in vitro. FA, FPT and PCA are phototoxic in vitro. The photodegradation of topical or unmetabolized FA during UV exposure via sunlight, sunbeds or phototherapy may lead to ROS production, to the cutaneous folate deficiency, skin photocarcinogenesis and other deleterious skin effects. Further studies are needed to confirm whether UV exposure can decrease cutaneous and serum folate levels in humans taking FA supplements or using cosmetic creams with FA.

A new lumazine peptide penilumamide E from the fungus Aspergillus terreus.

A new rare lumazine peptide, penilumamide E (1), together with 13 known compounds (2-14) were isolated from the fungus Aspergillus terreus. Their structures were identified by spectroscopic techniques. The relative configuration of 1 was confirmed by single-crystal X-ray diffraction analysis. Compound 10 exhibited antimalarial activity against Plasmodium falciparum with IC50 values of 2.83 µg/mL. Compounds 4 and 6 showed weak cytotoxicity against cholangiocarcinoma (CCA) cell lines. In addition, 4 and 11 exhibited weak cytotoxicity against human hepatoma cell line.

High-throughput intracellular pteridinic profiling by liquid chromatography-quadrupole time-of-flight mass spectrometry.

Pteridines are a diverse family of endogenous metabolites that may serve as useful diagnostic biomarkers for disease. While many preparative and analytical techniques have been described for analysis of selected pteridines in biological fluids, broad intracellular pteridine detection remains a significant analytical challenge. In this study, a novel, specific and sensitive extraction and high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) method was developed to simultaneously quantify seven intracellular pteridines and monitor 18 additional, naturally-occurring intracellular pteridines. The newly developed method was validated through evaluation of spiked recoveries (84.5-109.4%), reproducibility (2.1-5.4% RSD), method detection limits (0.1-3.0 µg L(-1)) and limits of quantitation (0.1-1 µg L(-1)), and finally application to non-small cell lung cancer A549 cells. Twenty-three pteridine derivatives were successfully detected from cell lysates with an average RSD of 12% among culture replicates. Quantified intracellular pteridine levels ranged from 1 to 1000 nM in good agreement with previous studies. Finally, this technique may be applied to cellular studies to generate new biological hypotheses concerning pteridine physiological and pathological functions as well as to discovery new pteridine-based biomarkers.

Determination of pteridines in biological samples with an emphasis on their stability.

Pteridines are a group of endogenous heterocyclic compounds whose concentrations in biological fluids may be increased in some disorders, such as infections, autoimmune disorders and cancer. In particular, pteridine concentrations in urine may represent promising noninvasive markers. However, their specificity requires further investigation. Pteridines can occur in three oxidation states with different stability. In order to enable the analysis of the unstable di- and tetra-hydroforms either an oxidation (mainly with iodine) or stabilization by reducing agents is applied. Due to the high polarity of pteridines, many analytical procedures employed ion-pair, ion-exchange or hydrophilic interaction liquid chromatography using mostly fluorescence detection. In the last decade, MS was found to be applicable. The objective of this Review is to show possibilities and different approaches in pteridine analysis in biological samples.

Hydrophilic interaction liquid chromatography with tandem mass spectrometric detection applied for analysis of pteridines in two Graphosoma species (Insecta: Heteroptera).

A new separation method involving hydrophilic interaction chromatography with tandem mass spectrometric detection has been developed for the analysis of pteridines, namely biopterin, isoxanthopterin, leucopterin, neopterin, xanthopterin and erythropterin in the cuticle of heteropteran insect species. Two columns, Atlantis HILIC Silica and ZIC(®)-HILIC were tested for the separation of these pteridines. The effect of organic modifier content, buffer type, concentration and pH in mobile phase on retention and separation behavior of the selected pteridines was studied and the separation mechanism was also investigated. The optimized conditions for the separation of pteridines consisted of ZIC(®)-HILIC column, mobile phase composed of acetonitrile/5mM ammonium acetate, pH 6.80, 85/15 (v/v), flow rate 0.5mL/min and column temperature 30°C. Detection was performed by tandem mass spectrometry operating in electrospray ionization with Agilent Jet Stream technology using the selected reaction monitoring mode. The optimized method provided a linearity range from 0.3 to 5000ng/mL (r>0.9975) and repeatability with relative standard deviation<8.09% for all the studied pteridines. The method was applied to the analysis of pteridines in the cuticle of larvae and three adult color forms of Graphosoma lineatum and one form of Graphosoma semipunctatum (Insecta: Hemiptera: Heteroptera: Pentatomidae). The analysis shows that different forms of Graphosoma species can be characterized by different distribution of individual pteridines, which affects the coloration of various forms. Only isoxanthopterin was found in all the five forms tested.

HPLC analysis of tetrahydrobiopterin and its pteridine derivatives using sequential electrochemical and fluorimetric detection: application to tetrahydrobiopterin autoxidation and chemical oxidation.

Tetrahydrobiopterin (BH(4)) is an essential cofactor of endothelial nitric oxide (NO) synthase and when depleted, endothelial dysfunction results with decreased production of NO. BH(4) is also an anti-oxidant being a good "scavenger" of oxidative species. NADPH oxidase, xanthine oxidase, and mitochondrial enzymes producing reactive oxygen species (ROS) can induce elevated oxidant stress and cause BH(4) oxidation and subsequent decrease in NO production and bioavailability. In order to define the process of ROS-mediated BH(4) degradation, a sensitive method for monitoring pteridine redox-state changes is required. Considering that the conventional fluorescence method is an indirect method requiring conversion of all pteridines to oxidized forms, it would be beneficial to use a rapid quantitative assay for the individual detection of BH(4) and its related pteridine metabolites. To study, in detail, the BH(4) oxidative pathways, a rapid direct sensitive HPLC assay of BH(4) and its pteridine derivatives was adapted using sequential electrochemical and fluorimetric detection. We examined BH(4) autoxidation, hydrogen peroxide- and superoxide-driven oxidation, and Fenton reaction hydroxyl radical-driven BH(4) transformation. We demonstrate that the formation of the primary two-electron oxidation product, dihydrobiopterin (BH(2)), predominates with oxygen-induced BH(4) autoxidation and superoxide-catalyzed oxidation, while the irreversible metabolites, pterin and dihydroxanthopterin (XH(2)), are largely produced during hydroxyl radical-driven BH(4) oxidation.

Metabolic profiling of pteridines for determination of potential biomarkers in cancer diseases.

Cancer disease is the second leading cause of death in the world. Epidemiology data indicate that early diagnosis of a tumour increases a patient's chance of recovery. Biomarkers are effective instruments which can potentially lead to precancer screening or precancer diagnosis and may provide useful information on the cancer type and the disease's stage of progression. The analysis of new biomarkers for cancer is currently a popular area of study in clinical and cancer research. Pteridines are a class of potential cancer biomarkers. The monitoring levels of pteridines may have prospective value for controlling the course of the malignant process. This review describes the functional employment of pteridines, as biomarkers, in cancer diagnosis. It also contains the description of application of analytical techniques such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) used for pteridine analysis.

Evaluation of hybrid hydrophilic interaction chromatography stationary phases for ultra-HPLC in analysis of polar pteridines.

Retention characteristics of ultra-HPLC (UHPLC) hybrid stationary phases (bridged ethyl hybrid (BEH) and BEH Amide) were studied in hydrophilic interaction chromatography system in the group of polar basic pteridines (neopterin, biopterin, dihydroneopterin and dihydrobiopterin). The effect of mobile phase composition, buffer type, pH and concentration on retention of pteridines were examined in detail under UHPLC-fluorescence detection and UHPLC-MS conditions. The selectivity, retention properties and column performance were examined. BEH HILIC did not provide sufficient retention and selectivity for the separation of four pteridines under any tested conditions. BEH Amide provided strong retention for all pteridines especially at high pH values such as 9.8. However, at pH 9.8 the selectivity of separation for the pairs neopterin-dihydroneopterin and biopterin-dihydrobiopterin substantially decreased and resulted in very long analysis time. The best separation of four pteridine derivatives was obtained in the pH range 4.8-7.8 within a reasonable analysis time up to 8 min for UHPLC-fluorescence detection using higher concentrations of ammonium acetate buffer and up to 4 min for UHPLC-MS using lower concentrations of ammonium acetate.

Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus.

The gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2(1), with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 A, beta = 104.9 degrees; the crystal diffracted to a resolution of 1.9 A. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 A, and diffracted to 1.75 A resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2(1) and R32, respectively.

Undiagnosed coeliac disease and nutritional deficiencies in adults screened in primary health care.

OBJECTIVE: To compare the nutritional parameters of individuals with a previous diagnosis of coeliac disease (CD) with those of screen-detected patients in a large cohort of adults in primary care. MATERIAL AND METHODS: A cohort of 1900 adults (aged 18-64 years) was screened for tissue transglutaminase antibodies (TG2A) in primary care in the capital region of Finland. IgA-endomysium antibodies (EmA), HLADQ2/8 associated with CD were determined in positive individuals. Folate, iron, and transferrin receptor in sera were assessed in patients reporting a previous diagnosis of CD and patients positive for the above tests. RESULTS: Twenty-two out of 1900 (1%) patients reported a previous diagnosis of CD (biopsy-based 16/22 cases; 6/22 cases diagnostic criteria unknown). Among the screen-detected cases with TG2A> or = the cut-off value, 14/32 cases were considered to have CD based on high levels of both TG2A and EmA, DQ2/8 genotype and/or biopsy results. The prevalence of CD was as high as 1:53 in the total study population (36/1900), and in women even 1:46 (2.2%). Nutritional deficiencies were rare among CD patients diagnosed earlier (low iron = 1; low folate n=1) but common among those who had an undiagnosed CD (low folate n=6; p<0.005; concomitant iron deficiency n=2). One-third of the screen-detected CD patients were obese. Screen-detected patients did not present more abdominal symptoms than those with no CD. CONCLUSIONS: CD is common, the proportion among women possibly being as high as 2.2%. Although the great majority of screen-detected patients do not present any gastrointestinal symptoms at primary care, nutritional deficiencies such as low folate levels and iron deficiency are common.

[Time course of the content of folates and free amino acids in leaves of Pisum sativum L. after irradiation with ultraviolet C].

The effect of irradiation with UV-C on the time course of the content of total folates and free amino acids in leaves of pea (Pisum sativum L.) cultivar Neistoshchimyi was studied. It was shown that photolysis of folates is a rapid response to exposure to ultraviolet, as a result of which the plant produces a stable compound, pterin-6-carboxylic acid, with a relative fluorescence quantum yield approximately 2.0 at 20 degrees C (total value, 0.58). Presumably, this compound may be involved in the pterin-mediated photosensitization of singlet oxygen production. The kinetics of changes in the composition of free amino acids after exposure to UV-C has been studied. Exposure to UV-C for 0.5 and 1min induced utilization of free amino acids, suggesting activation of the synthesis of hormones and alkaloids that may facilitate resistance to the stressor. Greater doses as a result of exposure to radiation for 10 and 40 min decreased the content of free hydrophobic amino acids. This phenomenon could be due to the formation of covalent cross-links in membranes, which decrease the accessibility of hydrophobic amino acids. It is concluded that the changes in the qualitative and quantitative composition of free amino acids in leaves of irradiated plants were due to glycolysis.

Multifrequency cw-EPR investigation of the catalytic molybdenum cofactor of polysulfide reductase from Wolinella succinogenes.

Electron paramagnetic resonance (EPR) spectra of the molybdenum centre in polysulfide reductase (Psr) from Wolinella succinogenes with unusually high G-tensor values have been observed for the first time. Three different Mo(V) states have been generated (by the addition of the substrate polysulfide and different redox agents) and analysed by their G- and hyperfine tensors using multifrequency (S-, X- and Q-band) cw-EPR spectroscopy. The unusually high G-tensor values are attributed to a large number of sulfur ligands. Four sulfur ligands are assumed to arise from two pterin cofactors; one additional sulfur ligand was identified from mutagenesis studies to be a cysteine residue of the protein backbone. One further sulfur ligand is proposed for two of the Mo(V) states, based on the experimentally observed shift of the g(av) value. This sixth sulfur ligand is postulated to belong to the polysulfide substrate consumed within the catalytic reaction cycle of the enzyme. The influence of the co-protein sulfur transferase on the Mo(V) G-tensor supports this assignment.