Foam cell formation but not oxLDL cytotoxicity is inhibited by CD36 down regulation by the macrophage antioxidant 7,8-dihydroneopterin.

BACKGROUND AND AIMS: Cluster of differentiation 36 (CD36) is a key scavenger receptor in the control of macrophage uptake of oxidised low-density lipoproteins (oxLDL). CD36 expression levels are not down regulated by intracellular cholesterol but are upregulated by oxidised low density lipoprotein (oxLDL) leading to the formation of lipid loaded foam cells, a major constituent of atherosclerotic plaques. We have previous shown that CD36 is down regulated by 7,8-dihydroneopterin, an antioxidant generated by γ-interferon activated macrophages. How CD36 down regulation affects oxLDL induced cytotoxicity, CD36 oxLDL upregulation and foam cell formation is examined using human monocyte like U937 cell line as a model system of human macrophages. METHODS: Low density lipoprotein (LDL) was prepared by ultracentrifugation from human plasma and oxidised in copper chloride. CD36 levels in U937 cells were measured by western blot analysis. and lipid accumulation was measured by oil red-O staining and 7-ketocholesterol accumulation by high performance liquid chromatography. Cell viability was measured by flow cytometry analysis after propidium iodide staining. RESULTS: 7,8-dihydroneopterin concentrations above 100 µM caused a concentration and time dependent decrease in cellular CD36 levels to 20 % of the untreated cells after 24 h. Upregulation of CD36 by oxLDL was inhibited by 7,8-dihydroneopterin treatment. The CD36 down regulation was associated with decrease in foam cell formation but not a reduction on oxLDL cytotoxicity. CONCLUSIONS: 7,8-dihydroneopterin down regulated CD36 in U937 cells, inhibiting foam cell formation but not oxLDL mediated cell death. 7,8-dihydroneopterin may modulate foam cell formation in atherosclerotic plaques.

Early pregnancy metabolites predict gestational diabetes mellitus: implications for fetal programming.

BACKGROUND: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders. OBJECTIVE: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy. STUDY DESIGN: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus. RESULTS: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection. CONCLUSION: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity.

Nucleoside transporters are critical to the uptake and antioxidant activity of 7,8-dihydroneopterin in monocytic cells.

7,8-Dihydroneopterin protects cells intracellularly from oxidative stress-induced death, but its mode of transport across the cell membrane is unknown. Nucleosides, such as guanosine, are transported via nucleoside transporters of the equilibrative and concentrative forms. Therefore, the objective of this study was to identify which membrane transporters are responsible for 7,8-dihydroneopterin transport in cells and whether this is necessary for protection against oxidative stress. Monocytic cell lines U937, THP-1 and human monocytes were incubated with varying concentrations of 7,8-dihydroneopterin with or without nucleoside transporter inhibitors nitrobenzylthioinosine (NBMPR; ENT1), dipyridamole (DP; ENT1 and ENT2) or indomethacin (INDO; CNT). Only DP inhibited 7,8-dihydroneopterin uptake in U937 cells, while NBMPR and DP inhibited 7,8-dihydroneopterin uptake in THP-1 cells. All three inhibitors limited 7,8-dihydroneopterin uptake in human monocytes at short time points only. When the cells were incubated with 10 mM of the peroxyl radical generator 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) a 50-80% loss of cell viability was measured. 7,8-dihydroneopterin protected all cell lines against AAPH-induced cell death, which was prevented with DP in U937 cells, NBMPR in THP-1 cells and a combination of all three nucleoside inhibitors in human monocytes. These data indicate 7,8-dihydroneopterin is transported across the cell membrane of monocytic cells via equilibrative and concentrative nucleoside transporters in a cell lineage-dependent manner. The data also indicate protection from peroxyl radical-generated cell death with 7,8-dihydroneopterin is intracellular and facilitated through nucleoside transporters in monocytic cells.

Neopterin formation through radical scavenging of superoxide by the macrophage synthesised antioxidant 7,8-dihydroneopterin.

Clinical measurement of neopterin has been extensively used as a marker of inflammation but the in vivo mechanism generating neopterin is poorly understood. Neopterin is described as the oxidation product of 7,8-dihydroneopterin, a potent antioxidant generated by monocyte/macrophages in response to interferon-γ. While peroxyl and hydroxyl scavenging generates dihydroxanthopterin, hypochlorite efficiently oxidises 7,8-dihydroneopterin into neopterin, but this reaction alone does not explain the high levels of neopterin seen in clinical data. Here, we examine whether superoxide scavenging by 7,8-dihydroneopterin generates neopterin. U937 cells incubated with oxLDL showed a time dependent increase superoxide and 7,8-dihydroneopterin oxidation to neopterin. Neopterin generation in oxLDL or phorbol ester treated U937 cells or human monocytes was inhibited by apocynin and PEG-SOD. Addition of the myeloperoxidase inhibitor 4-aminobenzoic acid hydrazide (ABAH) had no effect of the superoxide generation or neopterin formation. 7,8-Dihydroneopterin reacted with superoxide/hydroxy radical mixtures generated by X-ray radiolysis to give neopterin. Formation of neopterin by superoxide derived from the xanthine/xanthine oxidase system was inhibited by superoxide dismutase. Neopterin formation was inhibited by apocynin in phorbol ester treated human carotid plaque rings in tissue culture. These results indicate that 7,8-dihydroneopterin scavenges superoxide and is subsequently oxidised into neopterin in cellular and cell-free experimental systems.

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target.

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53 µM; vmax = 180 nmol mg-1 min-1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Š(Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Knee replacement surgery significantly elevates the urinary inflammatory biomarkers neopterin and 7,8-dihydroneopterin.

CONTEXT: Knee arthroplasty surgery is significant trauma, leading to an activated immune system causing inflammation and oxidative stress. Many current biomarkers are invasive, costly, and often slow to analyse, limiting their use for rapid inflammatory measurements. OBJECTIVES: We have examined the use of urinary neopterin and total neopterin in knee arthroplasty patients to non-invasively measure oxidative stress and inflammation from immune system activation. We aim to validate the use of these biomarkers for quick, cost effective and predictive measurements of post-surgical inflammation assessment. METHODOLOGY: 19 Knee arthroplasty patients were analysed pre-operatively and for a defined post-operative period to determine the urinary levels of neopterin and total neopterin (neopterin +7,8-dihydroneopterin) using high performance liquid chromatography with fluorescence detection. These results were then compared to a control group of 20 participants with normal knee function. RESULTS: 7,8-Dihydroneopterin was stable in urine over 12 h when refrigerated. Knee arthritis was associated with an increase in pre-operative neopterin (oxidative stress) and total neopterin (inflammation). The subsequent arthroplasty surgery generated a significant increase neopterin and total neopterin. Both biomarkers were reduced immediately post-operatively, before becoming elevated on the following days. There was no clear evidence of an association between initial neopterin and total neopterin levels and a patient's level of inflammation during in-hospital recovery. CONCLUSIONS: The stability of 7,8-dihydroneopterin in urine allows for its use as an inflammatory marker. Urinary neopterin and total neopterin provided a fast, non-invasive, and simple measure of oxidative stress and inflammation after knee arthroplasty.

Neopterin/7,8-dihydroneopterin is elevated in Duchenne muscular dystrophy patients and protects mdx skeletal muscle function.

NEW FINDINGS: What is the central question of this study? We examined whether the macrophage-synthesized antioxidant 7,8-dihydroneopterin was elevated in Duchenne muscular dystrophy (DMD) patients. We then examined whether 7,8-dihydroneopterin could protect dystrophic skeletal mouse muscle from eccentric contraction-induced force loss and improve recovery. What is the main finding and its importance? Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients. 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss of dystrophic skeletal mouse muscle and accelerated recovery of force. These results suggest that eccentric contraction-induced force loss is mediated, in part, by an oxidative component and provides a potential protective role for 7,8-dihydroneopterin in DMD. ABSTRACT: Macrophage infiltration is a hallmark of dystrophin-deficient muscle. We tested the hypothesis that Duchenne muscular dystrophy (DMD) patients would have elevated levels of the macrophage-synthesized pterins, neopterin and 7,8-dihydroneopterin, compared with unaffected age-matched control subjects. Urinary neopterin/creatinine and 7,8-dihydroneopterin/creatinine were elevated in DMD patients, and 7,8-dihydroneopterin/creatinine was associated with patient age and ambulation. Urinary 7,8-dihydroneopterin corrected for specific gravity was also elevated in DMD patients. Given that 7,8-dihydroneopterin is an antioxidant, we then identified a potential role for 7,8-dihydroneopterin in disease pathology. We assessed whether 7,8-dihydroneopterin could: (i) protect against isometric force loss in wild-type skeletal muscle exposed to various pro-oxidants; and (ii) protect wild-type and mdx muscle from eccentric contraction-induced force loss, which has an oxidative component. Force loss was elicited in isolated extensor digitorum longus (EDL) muscles by 10 eccentric contractions, and recovery of force after the contractions was measured in the presence of exogenous 7,8-dihydroneopterin. 7,8-Dihydroneopterin attenuated isometric force loss by wild-type EDL muscles when challenged by H2 O2 and HOCl, but exacerbated force loss when challenged by SIN-1 (NO• , O2• , ONOO- ). 7,8-Dihydroneopterin attenuated eccentric contraction-induced force loss in mdx muscle. Isometric force production by EDL muscles of mdx mice also recovered to a greater degree after eccentric contractions in the presence of 7,8-dihydroneopterin. The results corroborate macrophage activation in DMD patients, provide a potential protective role for 7,8-dihydroneopterin in the susceptibility of dystrophic muscle to eccentric contractions and indicate that oxidative stress contributes to eccentric contraction-induced force loss in mdx skeletal muscle.

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the ß-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

Effect of 7,8-dihydroneopterin mediated CD36 down regulation and oxidant scavenging on oxidised low-density lipoprotein induced cell death in human macrophages.

The role of CD36 in oxidised low-density lipoprotein (oxLDL) mediated cell death was examined by down regulating the receptor level with the macrophage generated antioxidant 7,8-dihydroneopterin. Down regulation of CD36 protein levels in human monocyte derived macrophages by 7,8-dihydroneopterin corresponded to a decrease in CD36-mRNA. The oxidation products of 7,8-dihydroneopterin, dihydroxanthopterin and neopterin did not significantly down regulate CD36. The CD36 down regulation resulted in a decrease in oxLDL uptake measured as 7-ketocholesterol accumulation. Though less oxLDL was taken up by the macrophages as a result of the 7,8-dihydroneopterin induced down regulation in CD36 levels, the cytotoxicity of the oxLDL was not decreased. Addition of 7,8-dihydroneopterin to oxLDL treated macrophages decreased the concentration of intracellular oxidants. In the presence of oxLDL, 7,8-dihydroneopterin was oxidised to neopterin showing that the 7,8-dihydroneopterin was scavenging intracellular oxidants generated in response to the oxLDL. The results show CD36 down regulation does not protect human macrophages form oxLDL cytotoxicity but 7,8-dihydroneopterin intracellular oxidant scavenging is protective.

Deletion of nudB Causes Increased Susceptibility to Antifolates in Escherichia coli and Salmonella enterica.

Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in increased susceptibility to both SMX and TMP in Salmonella enterica Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.

Repetitive cryotherapy attenuates the in vitro and in vivo mononuclear cell activation response.

What is the central question of this study? Acute and repetitive cryotherapy are routinely used to accelerate postexercise recovery, although the effect on resident immune cells and repetitive exposure has largely been unexplored and neglected. What is the main finding and its importance? Using blood-derived mononuclear cells and semi-professional mixed martial artists, we show that acute and repetitive cryotherapy reduces the in vitro and in vivo T-cell and monocyte activation response whilst remaining independent of the physical performance of elite athletes. We investigated the effect of repetitive cryotherapy on the in vitro (cold exposure) and in vivo (cold water immersion) activation of blood-derived mononuclear cells following high-intensity exercise. Single and repeated cold exposure (5°C) of a mixed cell culture (T cells and monocytes) was investigated using in vitro tissue culture experimentation for total neopterin production (neopterin plus 7,8-dihydroneopterin). Fourteen elite mixed martial art fighters were also randomly assigned to either a cold water immersion (15 min at 10°C) or passive recovery protocol, which they completed three times per week during a 6 week training camp. Urine was collected and analysed for neopterin and total neopterin three times per week, and perceived soreness, fatigue, physical performance (broad jump, push-ups and pull-ups) and training performance were also assessed. Single and repetitive cold exposure significantly (P < 0.001) reduced total neopterin production from the mixed cell culture, whereas cold water immersion significantly (P < 0.05) attenuated urinary neopterin and total neopterin during the training camp without having any effect on physical performance parameters. Soreness and fatigue showed little variation between the groups, whereas training session performance was significantly (P < 0.05) elevated in the cold water immersion group. The data suggest that acute and repetitive cryotherapy attenuates in vitro T-cell and monocyte activation. This may explain the disparity in in vivo neopterin and total neopterin between cold water immersion and passive recovery following repetitive exposure during a high-intensity physical impact sport that remains independent of physical performance.

Impact-induced muscle damage and urinary pterins in professional rugby: 7,8-dihydroneopterin oxidation by myoglobin.

Muscle damage caused through impacts in rugby union is known to increase oxidative stress and inflammation. Pterins have been used clinically as markers of oxidative stress, inflammation, and neurotransmitter synthesis. This study investigates the release of myoglobin from muscle tissue due to force-related impacts and how it is related to the subsequent oxidation of 7,8-dihydroneopterin to specific pterins. Effects of iron and myoglobin on 7,8-dihydroneopterin oxidation were examined in vitro via strong cation-exchange high-performance liquid chromatography (SCX-HPLC) analysis of neopterin, xanthopterin, and 7,8-dihydroxanthopterin. Urine samples were collected from 25 professional rugby players pre and post four games and analyzed for myoglobin by enzyme-linked immunosorbent assay, and 7,8-dihydroneopterin oxidation products by HPLC. Iron and myoglobin oxidized 7,8-dihydroneopterin to neopterin, xanthopterin, and 7,8-dihydroxanthopterin at concentrations at or above 10 µM and 50 µg/mL, respectively. All four games showed significant increases in myoglobin, neopterin, total neopterin, biopterin, and total biopterin, which correlated between each variable (P < 0.05). Myoglobin and iron facilitate 7,8-dihydroneopterin oxidation to neopterin and xanthopterin. In vivo delocalization of myoglobin due to muscle damage may contribute to oxidative stress and inflammation after rugby. Increased concentrations of biopterin and total biopterin may indicate production of nitric oxide and monoamine neurotransmitters in response to the physical stress.

Oxidised low density lipoprotein causes human macrophage cell death through oxidant generation and inhibition of key catabolic enzymes.

Oxidised low density lipoprotein (oxLDL) is thought to be a significant contributor to the death of macrophage cells observed in advanced atherosclerotic plaques. Using human-derived U937 cells we have examined the effect of cytotoxic oxLDL on oxidative stress and cellular catabolism. Within 3h of the addition of oxLDL, there was a rapid, concentration dependent rise in cellular reactive oxygen species followed by the loss of cellular GSH, and the enzyme activity of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aconitase. The loss of these catabolic enzymes was accompanied by the loss of cellular ATP and lower lactate generation. Addition of the macrophage antioxidant 7,8-dihydroneopterin inhibited the ROS generation, glutathione loss and catabolic inactivation. NOX was shown to be activated by oxLDL addition while apocynin inhibited the loss of GSH and cell viability. The data suggests that oxLDL triggers an excess of ROS production through NOX activation, and catabolic failure through thiol oxidation resulting in cell death.

Assessing the Effectiveness of Selected Biomarkers in the Acute and Cumulative Physiological Stress Response in Professional Rugby Union through Non-invasive Assessment.

Rugby union is a sport involving high force and frequency impacts making the likelihood of injury a significant risk. The aim of this study was to measure and report the individual and group acute and cumulative physiological stress response during 3 professional rugby games through non-invasive sampling. 24 professional rugby players volunteered for the study. Urine and saliva samples were collected pre and post 3 matches. Myoglobin, salivary immunoglobulin A, cortisol, neopterin and total neopterin (neopterin+7,8-dihydroneopterin) were analysed by high performance liquid chromatography or enzyme linked immunosorbent assay. Significant increases in cortisol, myoglobin, neopterin and total neopterin when urine volume was corrected with specific gravity were observed (p<0.05). Significant decreases in salivary immunoglobulin A concentration were observed for games 1 and 2 while secretion rate decreased after games 2 and 3. Significant decreases were seen with the percent of 7,8-dihydroneopterin being converted to neopterin following games 2 and 3. The intensity of 3 professional rugby games was sufficient to elicit significant changes in the physiological markers selected for our study. Furthermore, results suggest the selected markers not only provide a means for analysing the stress encountered during a single game of rugby but also highlight the unique pattern of response for each individual player.

Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.

A prospective study of the immune system activation biomarker neopterin and colorectal cancer risk.

BACKGROUND: Neopterin may be relevant for colorectal cancer (CRC) development, as a biomarker of cellular immune activity exerting pleiotropic effects on cellular ageing, oxidative stress, and inflammation. So far, the association between prediagnostic neopterin and colon and rectal cancer risk has not been evaluated in human populations. METHODS: A nested case-control study was conducted within the European Prospective Investigation into Cancer and Nutrition cohort using data on plasma concentrations of total neopterin (T-N, sum of neopterin and 7,8-dihydroneopterin) in 830 incident CRC case patients (561 colon and 269 rectal) matched within risk sets to 830 control participants. A subsequent replication study used data from the Hordaland Health Study, where 173 CRC case patients have been diagnosed among 6594 healthy participants over 12 years of follow-up. RESULTS: After multivariable adjustment for a priori chosen CRC risk factors, a "U-shaped" association of T-N with CRC was revealed. Compared with the second quintile of the T-N distribution, the relative risks for the first, third, fourth, and fifth quintiles were 2.37 (95% CI = 1.66 to 3.39), 1.24 (95% CI = 0.87 to 1.77), 1.55 (95% CI = 1.08 to 2.22), and 2.31 (95% CI = 1.63 to 3.27), respectively. Replication of these associations within the Hordaland Health Study yielded similar results. No differences have been observed when the associations were explored by colon and rectal cancer site (two-sided P difference = .87) and after excluding case patients diagnosed within the first four follow-up years. CONCLUSIONS: These novel findings provide evidence of the role of both suppressed and activated cell-mediated immunity as reflected by prediagnostic T-N concentrations in the development of CRC.

Simultaneous determination of all forms of biopterin and neopterin in cerebrospinal fluid.

In humans, genetic defects of the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor in hydroxylation reactions, are associated with severe neurological disorders. The diagnosis of these conditions relies on the determination of BH4, dihydrobiopterin (BH2), and dihydroneopterin (NH2) in cerebrospinal fluid (CSF). As MS/MS is less sensitive than fluorescence detection (FD) for this purpose, the most widely used method since 1980 involves two HPLC runs including two differential off-line chemical oxidation procedures aiming to transform the reduced pterins into their fully oxidized fluorescent counterparts, biopterin (B) and neopterin (N). However, this tedious and time-consuming two-step indirect method underestimates BH4, BH2, and NH2 concentrations. Direct quantification of BH4 is essential for studying its metabolism and for monitoring the efficacy of BH4 supplementation in patients with genetic defects. Here we describe a single step method to simultaneously measure BH4, BH2, B, NH2, and N in CSF by HPLC coupled to FD after postcolumn coulometric oxidation. All target pterins were quantified in CSF with a small volume (100 µL), and a single filtration step for sample preparation and analysis. As compared to the most widely used method in more than 100 CSF samples, this new assay is the easiest route for accurately determining in a single run BH4, BH2, and NH2 in CSF in deficit situations as well as for monitoring the efficacy of the treatment.

Comparative genomics guided discovery of two missing archaeal enzyme families involved in the biosynthesis of the pterin moiety of tetrahydromethanopterin and tetrahydrofolate.

C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H(4)-MPT) or tetrahydrofolate (H(4)-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2',3'-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586-37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658-6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.

Intracellular glutathione protects human monocyte-derived macrophages from hypochlorite damage.

AIMS: Macrophages must function in an inflammatory environment of high oxidative stress due to the production of various oxidants. Hypochlorous acid (HOCl) is a potent cytotoxic agent generated by neutrophils and macrophages within inflammatory sites. This study determines whether glutathione is the key factors governing macrophage resistance to HOCl. MAIN METHODS: Human monocyte derived macrophages (HMDM) were differentiated from human monocytes prepared from human blood. The HMDM cells were exposed to micromolar concentrations of HOCl and the timing of the cell viability loss was measured. Cellular oxidative damage was measured by loss of glutathione, cellular ATP, tyrosine oxidation, and inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). KEY FINDINGS: HOCl causes a rapid loss in HMDM cell viability above threshold concentrations. The cell death occurred within 10 min of treatment with the morphological characteristics of necrosis. The HOCl caused the extensive cellular protein oxidation with the loss of tyrosine residue and inactivation of GAPDH, which was accompanied with the loss of cellular ATP. This cellular damage was only observed after the loss of intracellular GSH from the cell. Removal of intracellular GSH with diethyl maleate (DEM) increased the cells' sensitivity to HOCl damage while protecting the intracellular GSH pool with the antioxidant 7,8-dihydroneopterin prevented the HOCl mediated viability loss. Variations in the HOCl LD(50) for inducing cell death were strongly correlated with initial intracellular GSH levels. SIGNIFICANCE: In HMDM cells scavenging of HOCl by intracellular glutathione is sufficient to protect against oxidative loss of key metabolic functions within the cells.