Effect of (R)-2-Amino-6-(1R,2S)-1,2-Dihydroxypropyl)-5,6,7,8-Tetrahydropterin-4(3H)-One and Its Structural Analogues on the Temperature Stability of Tryptophan Hydroxylase 2 with the P447R Mutation.

The enzyme tryptophan hydroxylase 2 (TPH2) catalyzes the hydroxylation of L-tryptophan to L-5-hydroxytryptophan (5-HTP), the first and the key step in 5-HT synthesis in the mammalian brain. Mutations in the human Tph2 gene reducing enzyme activity increase the risk of psychopathology. Pharmacological chaperones are small molecules that can specifically bind to mutant protein molecules, restore their disturbed 3D structure to the native state, and increase their stability and functional activity. The chaperone activity of (R)-2-amino-6-(1R,2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is expressed by increasing the in vitro thermal stability of mutant tyrosine hydroxylase and phenylalanine hydroxylase molecules which are similar to TPH2 in their structure and characteristics. The P447R substitution in the mouse TPH2 molecule results in a 2-fold decrease in enzyme activity in their brains. We studied the effect of this mutation on the TPH2 thermal stability, as well as on the ability of BH4 and its 8 structural analogues to increase the thermal stability of the mutant TPH2 from midbrain extracts of BALB/C mice. Temperature stability was studied by the decrease in enzyme activity during its heating for 2 min at increasing temperatures and was evaluated by the T50 value that is the temperature at which the enzyme activity decreased by half. For the mutant TPH2, the T50 value was decreased compared to the wild type enzyme. BH4 and its closest structural analogue, 6-methyl-5,6,7,8-tetrahydropterin, increased the T50 value, i.e., exhibited chaperone activity. Other close BH4 analogs, 6,7-dimethyl-5,6,7,8-tetrahydropterin and folic acid, were not effective. It can be assumed that BH4 can be effective in the treatment of mental disorders caused by mutations in the Tph2 gene.

Crystal structure of ricin toxin A chain complexed with a highly potent pterin-based small-molecular inhibitor.

Ricin toxin A chain (RTA), from Ricinus communis, is a deadly protein that inactivates ribosomes by degrading an adenine residue at position 4324 in 28S rRNA. Recently, we have demonstrated that pterin-7-carboxamides with peptide pendants were potent RTA inhibitors. Among these, N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY) is the most potent RTA inhibitor as a small organic molecule. However, despite this fascinating inhibitory activity, the mode of interaction of 7PCGY with RTA remains elusive. This study aimed to elucidate the factors responsible for the high RTA inhibitory activity of 7PCGY based on X-ray crystallographic analysis. Herein, we report the successfully resolved X-ray crystal structure of 7PCGY/RTA complexes, revealing that the interaction between the phenolic hydroxy group in 7PCGY and Asn78 of RTA through a hydrogen bonding and the conformational change of Tyr80 and Asn122 are responsible for the high RTA inhibitory activity of 7PCGY.

Diverse Aromatic Metabolites in the Solitary Tunicate Cnemidocarpa irene.

Fourteen aromatic metabolites (6-19) were isolated from an aqueous extract of the solitary tunicate Cnemidocarpa irene collected in Hokkaido, Japan. The structures of the metabolites were determined based on the spectroscopic interpretations, including one- and two-dimensional NMR, mass spectra, UV, and circular dichroism data. The biopterin analogue 10 modulated the behavior of mice after intracerebroventricular injection and showed a weak affinity to ionotropic glutamate receptor subtypes. Analyses of fluorescent coelomic fluid of the tunicate revealed that pterin 12 was responsible for the fluorescence of the blood cells, while ß-carbolines 1 and 3 were fluorescent compounds in the serum. The metabolic profiles in adults, juveniles, larvae, and eggs of the animal differed substantially, suggesting that the metabolism of the animal, especially biosynthesis of aromatic secondary metabolites, changes over different life stages.

Nicotine induces endothelial dysfunction and promotes atherosclerosis via GTPCH1.

Smoking is a major preventable risk factor for atherosclerosis. However, the causative link between cigarette smoke and atherosclerosis remains to be established. The objective of this study is to characterize the role of GTP cyclohydrolase 1 (GTPCH1), the rate-limiting enzyme for de novo tetrahydrobiopterin (BH4) synthesis, in the smoking-accelerated atherosclerosis and the mechanism involved. In vitro, human umbilical vein endothelial cells were treated with nicotine, a major component of cigarette smoke, which reduced the mRNA and protein levels of GTPCH1 and led to endothelial dysfunction. GTPCH1 overexpression or sepiapterin could attenuate nicotine-reduced nitric oxide and -increased reactive oxygen species levels. Mechanistically, human antigen R (HuR) bound with the adenylateuridylate-rich elements of the GTPCH1 3' untranslated region and increased its stability; nicotine inhibited HuR translocation from the nucleus to cytosol, which downregulated GTPCH1. In vivo, nicotine induced endothelial dysfunction and promoted atherosclerosis in ApoE-/- mice, which were attenuated by GTPCH1 overexpression or BH4 supplement. Our findings may provide a novel and promising approach to atherosclerosis treatment.

Sepiapterin alleviates impaired gastric nNOS function in spontaneous diabetic female rodents through NRF2 mRNA turnover and miRNA biogenesis pathway.

An impaired nitrergic system and altered redox signaling contribute to gastric dysmotility in diabetics. Our earlier studies show that NF-E2-related factor 2 (NRF2) and phase II antioxidant enzymes play a vital role in gastric neuronal nitric oxide synthase (nNOS) function. This study aims to investigate whether supplementation of sepiapterin (SEP), a precursor for tetrahydrobiopterin (BH4) (a cofactor of NOS) via the salvage pathway, restores altered nitrergic systems and redox balance in spontaneous diabetic (DB) female rats. Twelve-week spontaneous DB and age-matched, non-DB rats, with and without dietary SEP (daily 20 mg/kg body wt for 10 days) treatment, were used in this study. Gastric antrum muscular tissues were excised to investigate the effects of SEP in nitrergic relaxation and the nNOS-nitric oxide (NO)-NRF2 pathway(s). Dietary SEP supplementation significantly ( P < 0.05) reverted diabetes-induced changes in nNOS dimerization and function; nitric oxide (NO) downstream signaling molecules; HSP-90, a key regulator of nNOSα activity and dimerization; miRNA-28 that targets NRF2 messenger RNA (mRNA), and levels of microRNA (miRNA) biogenesis pathway components, such as DGCR8 (DiGeorge Syndrome Critical Region Gene 8) and TRBP (HIV1-1 transactivating response RNA-binding protein). These findings emphasize the importance of the BH4 pathway in regulating gastric motility functions in DB animals by modulating nNOSα dimerization in association with changes in enteric NRF2 and NO downstream signaling. Our results also identify a new pathway, wherein SEP regulates NRF2 mRNA turnover by suppressing elevated miRNA-28, which could be related to alterations in miRNA biogenesis pathway components. NEW & NOTEWORTHY This study is the first to show a causal link between NF-E2-related factor 2 (NRF2) and neuronal nitric oxide synthase (nNOS) in gastric motility function. Our data demonstrate that critical regulators of the miRNA biosynthetic pathway are upregulated in the diabetic (DB) setting; these regulators were rescued by sepiapterin (SEP) treatment. Finally, we show that low dihydrofolate reductase expression may lead to impaired nNOS dimerization/function-reduced nitric oxide downstream signaling and elevate oxidative stress by suppressing the NRF2/phase II pathway through miRNA; SEP treatment restored all of the above in DB gastric muscular tissue. We suggest that tetrahydrobiopterin supplementation may be a useful therapy for patients with diabetes, as well as women with idiopathic gastroparesis.

Sepiapterin Enhances Tumor Radio- and Chemosensitivities by Promoting Vascular Normalization.

Previously, we demonstrated that nitric oxide (NO) synthase (NOS) is uncoupled in a wide range of solid tumors and that restoring NOS coupling with the tetrahydrobiopterin precursor sepiapterin (SP) inhibits tumor progression. Endothelial dysfunction characterizes the poorly functional vasculature of solid tumors, and since NO is critical for regulation of endothelial function we asked whether SP, by recoupling NOS, improves tumor vasculature structure and function-enhancing chemotherapeutic delivery and response to radiotherapy. MMTV-neu mice with spontaneous breast tumors were treated with SP by oral gavage and evaluated by multispectral optoacoustic tomographic analysis of tumor HbO2 and by tissue staining for markers of hypoxia, blood perfusion, and markers of endothelial and smooth muscle proteins. Recoupling tumor NOS activity results in vascular normalization observed as reduced tumor hypoxia, improved tumor percentage of HbO2 and perfusion, as well as increased pericyte coverage of tumor blood vessels. The normalized vasculature and improved tumor oxygenation led to a greater than 2-fold increase in radiation-induced apoptosis compared with radiation or SP alone. High-performance liquid chromatography analysis of tumor doxorubicin levels showed a greater than 50% increase in doxorubicin uptake and a synergistic effect on tumor cell apoptosis. This study highlights for the first time the importance of NOS uncoupling and endothelial dysfunction in the development of tumor vasculature and presents a new approach for improving the tumoricidal efficacies of chemotherapy and radiotherapy.

Lipophilic Decyl Chain-Pterin Conjugates with Sensitizer Properties.

A new series of decyl chain [-(CH2)9CH3] pterin conjugates have been investigated by photochemical and photophysical methods, and with theoretical solubility calculations. To synthesize the pterins, a nucleophilic substitution (SN2) reaction was used for the regioselective coupling of the alkyl chain to the O site over the N3 site. However, the O-alkylated pterin converts to N3-alkylated pterin under basic conditions, pointing to a kinetic product in the former and a thermodynamic product in the latter. Two additional adducts were also obtained from an N-amine condensation of DMF solvent molecule as byproducts. In comparison to the natural product pterin, the alkyl chain pterins possess reduced fluorescence quantum yields (ΦF) and increased singlet oxygen quantum yields (ΦΔ). It is shown that the DMF-condensed pterins were more photostable compared to the N3- and O-alkylated pterins bearing a free amine group. The alkyl chain pterins efficiently intercalate in large unilamellar vesicles, which is a good indicator of their potential use as photosensitizers in biomembranes. Our study serves as a starting point where the synthesis can be expanded to produce a wider series of lipophilic, photooxidatively active pterins.

Determining the Relative Binding Affinity of Ricin Toxin A Inhibitors by Using Molecular Docking and Nonequilibrium Work.

Ricin is a ribosome-inactivating protein (RIP type 2) consisting of two subunits, ricin toxin A (RTA) and ricin toxin B (RTB). Because of its cytotoxicity, ricin has worried world authorities for its potential use as a chemical weapon; therefore, its inhibition is of great biotechnological interest. RTA is the target for inhibitor synthesis, and pterin derivatives are promising candidates to inhibit it. In this study, we used a combination of the molecular docking approach and fast steered molecular dynamics (SMD) to assess the correlation between nonequilibrium work, ⟨ W⟩, and the IC50 for six RTA inhibitors. The results showed that molecular docking is a powerful tool to predict good bioactive poses of RTA inhibitors, and ⟨ W⟩ presented a strong correlation with IC50 ( R2 = 0.961). Such a profile ranked the RTA inhibitors better than the molecular docking approach. Therefore, the combination of docking and fast SMD simulation was shown to be a promising tool to distinguish RTA-active inhibitors from inactive ones and could be used as postdocking filtering approach.

MR1 presents microbial vitamin B metabolites to MAIT cells.

Antigen-presenting molecules, encoded by the major histocompatibility complex (MHC) and CD1 family, bind peptide- and lipid-based antigens, respectively, for recognition by T cells. Mucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T cells in humans that are activated by an antigen(s) bound to the MHC class I-like molecule MR1. Although the identity of MR1-restricted antigen(s) is unknown, it is present in numerous bacteria and yeast. Here we show that the structure and chemistry within the antigen-binding cleft of MR1 is distinct from the MHC and CD1 families. MR1 is ideally suited to bind ligands originating from vitamin metabolites. The structure of MR1 in complex with 6-formyl pterin, a folic acid (vitamin B9) metabolite, shows the pterin ring sequestered within MR1. Furthermore, we characterize related MR1-restricted vitamin derivatives, originating from the bacterial riboflavin (vitamin B2) biosynthetic pathway, which specifically and potently activate MAIT cells. Accordingly, we show that metabolites of vitamin B represent a class of antigen that are presented by MR1 for MAIT-cell immunosurveillance. As many vitamin biosynthetic pathways are unique to bacteria and yeast, our data suggest that MAIT cells use these metabolites to detect microbial infection.

Evaluation of Pterin, a Promising Drug Candidate from Cyanide Degrading Bacteria.

Pterin is a member of the compounds known as pteridines. They have the same nucleus of 2-amino-4-hydroxypteridine (pterin); however, the side-chain is different at the position 6, and the state of oxidation of the ring may exist in different form viz. tetrahydro, dihydro, or a fully oxidized form. In the present study, the microorganisms able to utilize cyanide, and heavy metals have been tested for the efficient production of pterin compound. The soil samples contaminated with cyanide and heavy metals were collected from Salem steel industries, Tamil Nadu, India. Out of 77 isolated strains, 40 isolates were found to utilize sodium cyanate as nitrogen source at different concentrations. However, only 13 isolates were able to tolerate maximum concentration (60 mM) of sodium cyanate and were screened for pterin production. Among the 13 isolates, only 1 organism showed maximum production of pterin, and the same was identified as Bacillus pumilus SVD06. The compound was extracted and purified by preparative high-performance liquid chromatography and analyzed by UV/visible, FTIR, and fluorescent spectrum. The antioxidant property of the purified pterin compound was determined by cyclic voltammetry. In addition, antimicrobial activity of pterin was also studied which was substantiated by antagonistic activity against Escherichia coli, and Pseudomonas aeruginosa. Besides that the pterin compound was proved to inhibit the formation of biofilm. The extracted pterin compounds could be proposed further not only for antioxidant and antimicrobial but also for its potency to aid as anticancer and psychotic drugs in future.

Tetrahydrobiopterin Role in human umbilical vein endothelial dysfunction in maternal supraphysiological hypercholesterolemia.

Maternal physiological hypercholesterolemia (MPH) allows a proper foetal development; however, maternal supraphysiological hypercholesterolemia (MSPH) associates with foetal endothelial dysfunction and early development of atherosclerosis. MSPH courses with reduced endothelium-dependent dilation of the human umbilical vein due to reduced endothelial nitric oxide synthase activity compared with MPH. Whether MSPH modifies the availability of the nitric oxide synthase cofactor tetrahydrobiopterin is unknown. We investigated whether MSPH-associated lower umbilical vein vascular reactivity results from reduced bioavailability of tetrahydrobiopterin. Total cholesterol <7.2mmol/L was considered as maternal physiological hypercholesterolemia (n=72 women) and ≥7.2mmol/L as MSPH (n=35 women). Umbilical veins rings were used for vascular reactivity assays (wire myography), and primary cultures of human umbilical vein endothelial cells (HUVECs) to measure nitric oxide synthase, GTP cyclohydrolase 1, and dihydrofolate reductase expression and activity, as well as tetrahydrobiopterin content. MSPH reduced the umbilical vein rings relaxation caused by calcitonine gene-related peptide, a phenomenon partially improved by incubation with sepiapterin. HUVECs from MSPH showed lower nitric oxide synthase activity (l-citrulline synthesis from l-arginine) without changes in its protein abundance, as well as reduced tetrahydrobiopterin level compared with MPH, a phenomenon reversed by incubation with sepiapterin. Expression and activity of GTP cyclohydrolase 1 was lower in MSPH, without changes in dihydrofolate reductase expression. MSPH is a pathophysiological condition reducing human umbilical vein reactivity due to lower bioavailability of tetrahydrobiopterin leading to lower NOS activity in the human umbilical vein endothelium.

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.

Pterin-7-carboxamides as a new class of aldose reductase inhibitors.

Aldose reductase is related to the onset and progression of diabetic complications, such as neuropathy, retinopathy, angiopathy, and so on: therefore molecules that are capable of inhibiting the enzyme are potential drugs for treatment of diabetic complications. Epalrestat is the sole aldose reductase inhibitor that is clinically used, but still has some drawbacks. Thus, the development of new aldose reductase inhibitors is still desired. We have synthesized a series of new pterin-7-carboxamides, and evaluated their in vitro inhibitory activities against human aldose reductase. All newly synthesized compounds exhibited the inhibitory activity. Among them, 1a having a glycine side chain exhibits the highest activity comparable to that of sorbinil, a highly active aldose reductase inhibitor. Molecular docking of 1a on the active site of the enzyme indicated this compound interacts with amino acid residues that are specific to the enzyme and related to suppressing side effects. Based on these results, we proved perin-7-carboxamides to be a new class of aldose reductase inhibitors, and particularly compound 1a was found to be a good candidate for further biological investigations as a drug for treatment of diabetic complications with fewer side effects.

Netrin-1 abrogates ischemia/reperfusion-induced cardiac mitochondrial dysfunction via nitric oxide-dependent attenuation of NOX4 activation and recoupling of NOS.

Despite an established role of mitochondrial dysfunction in cardiac ischemia/reperfusion (I/R) injury, the upstream activators have remained incompletely defined. We have recently identified an innovative role of exogenously applied netrin-1 in cardioprotection, which is mediated by increased nitric oxide (NO) bioavailability. Here, we tested the hypothesis that this "pharmacological" treatment of netrin-1 preserves mitochondrial function via novel mechanisms that are NO dependent. Freshly isolated C57BL6 mouse hearts were perfused using a Langendorff system, and subjected to a 20min global ischemia/60min reperfusion, in the presence or absence of netrin-1. I/R induced marked increases in infarct size, total superoxide and hydrogen peroxide production, activity and protein abundance of NADPH oxidase (NOX) isoform 4 (NOX4), as well as impaired mitochondrial integrity and function, all of which were attenuated by netrin-1. This protective effect of netrin-1 is attributed to cGMP, a downstream effector of NO. The protein levels of NOX1 and NOX2 were however unaffected, and infarct size from NOX1 and NOX2 knockouts was not different from wild type animals. Scavenging of NO with PTIO reversed inhibitory effects of netrin-1 on NOX4, while NO donor attenuated NOX4 protein abundance. In vivo NOX4 RNAi, or sepiapterin perfusion, resulted in recoupling of NOS, decreased infarct size, and blockade of dysfunctional mitochondrial swelling and mitochondrial superoxide production. These data demonstrate that netrin-1 induces cardioprotection through inhibition of NOX4 activity, which leads to recoupling of NOS, augmented NO bioavailability, reduction in oxidative stress, and ultimately preservation of mitochondrial function. The NO-dependent NOX4 inhibition connects with our previously established pathway of DCC/ERK1/2/eNOS/NO/DCC feed-forward mechanism, to maintain NOS in the coupling state to attenuate oxidative stress to preserve mitochondrial function. These findings may promote development of novel therapeutics for cardiac I/R injury. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Sepiapterin prevents left ventricular hypertrophy and dilatory remodeling induced by pressure overload in rats.

Uncoupling of nitric oxide (NO) synthase (NOS) has been implicated in left ventricular (LV) hypertrophy (LVH) and dilatory remodeling induced by pressure overload. We investigated whether administration of sepiapterin, a substrate of the salvage pathway of tetrahydrobiopterin synthesis, prevents LVH and dilatory LV remodeling by inhibiting NOS uncoupling and increasing bioavailable NO. Pressure overload was induced in rats by transverse aortic constriction (TAC). Concentric LVH developed during 8 wk after TAC, and dilatory LV remodeling and dysfunction developed between 8 and 16 wk after TAC associated with a decrease in capillary density. Oral administration of sepiapterin or the superoxide/peroxynitrite scavenger N-(2-mercaptopropionyl)-glycine for 8 wk after TAC inhibited oxidative stress, but only sepiapterin increased bioavailable NO and inhibited cardiomyocyte hypertrophy associated with a further increase in capillary density. When sepiapterin was administered between 8 and 16 wk after TAC, cardiomyocyte hypertrophy was regressed and capillary density was restored. This was associated with the inhibition of interstitial fibrosis and dilatory LV remodeling. N-nitro-l-arginine methyl ester abrogated all the beneficial effects of sepiapterin in rats with TAC. These results suggest that sepiapterin prevents concentric LVH and dilatory remodeling after TAC primarily by increasing the bioavailability of NO.

Photodynamic inactivation induced by carboxypterin: a novel non-toxic bactericidal strategy against planktonic cells and biofilms of Staphylococcus aureus.

Microbial related contamination is of major concern and can cause substantial economic losses. Photodynamic inactivation (PDI) has emerged as a suitable approach to inhibit microorganism proliferation. In this work, PDI induced by 6-carboxypterin (Cap), a biocompatible photosensitizer (PS), was analyzed. The growth inhibition of Staphylococcus aureus exposed to artificial UV-A radiation and sunlight in the presence of Cap was investigated. After UV-A irradiation, 50 µM Cap was able to decrease by three orders (with respect to the initial value) the number of S. aureus cells in early biofilms. However, this concentration was 500 times higher than that needed for eradicating planktonic cells. Importantly, under solar exposure, 100 µM Cap was able to suppress sessile bacterial growth. Thus, this strategy is able to exert a bactericidal effect on sessile bacteria and to eradicate planktonic cells by exposing the Cap-containing sample to sunlight.

An acute rise in intraluminal pressure shifts the mediator of flow-mediated dilation from nitric oxide to hydrogen peroxide in human arterioles.

Endothelial nitric oxide (NO) is the primary mediator of flow-mediated dilation (FMD) in human adipose microvessels. Impaired NO-mediated vasodilation occurs after acute and chronic hypertension, possibly due to excess generation of reactive oxygen species (ROS). The direct role of pressure elevation in this impairment of human arteriolar dilation is not known. We tested the hypothesis that elevation in pressure is sufficient to impair FMD. Arterioles were isolated from human adipose tissue and cannulated, and vasodilation to graded flow gradients was measured before and after exposure to increased intraluminal pressure (IILP; 150 mmHg, 30 min). The mediator of FMD was determined using pharmacological agents to reduce NO [N(G)-nitro-l-arginine methyl ester (l-NAME), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)], or H2O2 [polyethylene glycol (PEG)-catalase], and mitochondrial (mt) ROS was quantified using fluorescence microscopy. Exposure to IILP decreased overall FMD (max %dilation: 82.7 ± 4.9 vs. 62 ± 5.6; P < 0.05). This dilation was abolished by treatment with l-NAME prepressure and PEG-catalase after IILP (max %dilation: l-NAME: 23.8 ± 6.1 vs. 74.8 ± 8.6; PEG-catalase: 71.8 ± 5.9 vs. 24.6 ± 10.6). To examine if this change was mediated by mtROS, FMD responses were measured in the presence of the complex I inhibitor rotenone or the mitochondrial antioxidant mitoTempol. Before IILP, FMD was unaffected by either compound; however, both inhibited dilation after IILP. The fluorescence intensity of mitochondria peroxy yellow 1 (MitoPY1), a mitochondria-specific fluorescent probe for H2O2, increased during flow after IILP (%change from static: 12.3 ± 14.5 vs. 127.9 ± 57.7). These results demonstrate a novel compensatory dilator mechanism in humans that is triggered by IILP, inducing a change in the mediator of FMD from NO to mitochondria-derived H2O2.

Methotrexate analogues display enhanced inhibition of TNF-α production in whole blood from RA patients.

OBJECTIVES: Although methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis (RA), patients experience clinical resistance to MTX upon prolonged treatment. We explored whether new-generation antifolates elicit superior anti-inflammatory properties when compared to MTX, based on their capacity to inhibit tumour necrosis factor (TNF)-α production. METHOD: T cells in whole blood from 18 RA patients (including MTX-naïve, MTX- responsive, and MTX non-responsive patients) and seven healthy volunteers were stimulated with αCD3/αCD28 antibodies and incubated ex vivo for 72 h with MTX and eight novel antifolate drugs with potentially favourable biochemical and pharmacological properties. Drug concentrations exerting 50% inhibition (IC-50) of TNF-α production (by enzyme-linked immunosorbent assay, ELISA) were determined as an estimate for their anti-inflammatory capacity. In addition, induction of T-cell apoptosis was evaluated by flow cytometry. RESULTS: The new-generation antifolates PT523, PT644, raltitrexed, and GW1843 proved to be potent inhibitors of TNF-α production in activated T cells from all three groups of RA patients and from healthy volunteers. Based on IC-50 values, these antifolates were up to 10.3 times more potent than MTX. The anti-inflammatory effects were observed at drug concentrations that provoked suppression of T-cell activation and induction of apoptosis in 20-40% of activated T cells. CONCLUSION: In an ex-vivo setting, novel antifolates elicited marked inhibition of TNF-α production in activated T cells from RA patients. Further clinical evaluation is warranted to investigate whether a low dosage of these antifolates can elicit immunosuppressive effects equivalent to MTX, and whether they are superior to MTX in patients who fail to respond to MTX.

Photosensitized isomerization of resveratrol: Evaluation of energy and electron transfer pathways.

Resveratrol (3,5,4'-trihydroxystilbene, RSV) is a natural stilbene synthetized as trans-isomer in plants exposed to oxidative stress. In order to understand the mechanism involved during photosensitized degradation of trans-resveratrol, steady-state and time-resolved experiments were performed and compared with quantum-chemical calculations using density functional theory (DFT). Pterin (Ptr), a well-known photosensitizer, under UV-A radiation induces the oxidation of several biomolecules mainly through electron-transfer mechanisms. On the one hand, it was observed that trans-RSV participates in an energy-transfer pathway with Ptr triplet excited state (3Ptr*) forming 3trans-RSV*, which dissipates the energy by isomerization to cis-RSV. On the other hand, RSV neutral radical (trans-RSV(-H)•) was detected in laser flash photolysis experiments, evidencing an electron-transfer mechanism. The electron-transfer from 3Ptr* to trans-RSV is a barely feasible reaction, however, more favorable is the formation of trans-RSV(-H)• in a reaction between trans-RSV and Ptr radical cation (Ptr•+), which is produced during irradiation. The combination of experimental and theoretical approaches evidences the capability of trans-RSV to undergo energy-transfer (feasible by DFT calculations) and/or one-electron transfer pathways with 3Ptr*. These findings reveal the mechanisms involved in the interaction of trans-RSV and pterin excited states and provide information on the antioxidant action of resveratrol during photosensitized oxidation of biomolecules.