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Актуальність. Гіперфенілаланінемія (HPA), або підвищення концентрації фенілаланіну (Phe) в крові, є наслідком порушення перетворення фенілаланіну в тирозин. Яскравим прикладом цього клінічного стану є класична фенілкетонурія (PKU). У 1970-х роках стало зрозуміло, що в частини хворих із PKU, незважаючи на ефективну дієтотерапію, розвивалися тяжкі неврологічні порушення. Ці випадки верифікували як атипові, або злоякісні, варіанти PKU, що, як було досліджено, спричинялось мутаціями в генах, які регулюють синтез та регенерацію тетрагідробіоптерину (ВН4). Порушення метаболізму тетрагідробіоптерину, або ВН4-дефіцитні синдроми, — гетерогенна група спадкових захворювань ЦНС, спричинених дефіцитом моноамінів-нейротрансмітерів, які в низці випадків супроводжуються гіперфенілаланінемією. ВН4-дефіцитні синдроми, які є рідкісними вродженими порушеннями метаболізму, спричиненими недостатністю нейротрансмітерів, за відсутності своєчасного лікування призводять до прогресуючої, нерідко летальної, енцефалопатії. Упровадження в медичну практику ранньої диференціальної клінічної та лабораторної діагностики гіперфенілаланінемій дозволить запобігти розвитку летальних енцефалопатій та суттєво підвищити ефективність їх лікування. Матеріали та методи. Були обстежені 59 пацієнтів із діагнозом PKU віком від 2 до 16 років. Гіперфенілаланінемія була загальним симптомом і встановлена в усіх обстежених пацієнтів. Усім хворим було проведено молекулярно-генетичне обстеження на панель мутацій у гені РАН (R408W, Y414C (12-й екзон), R158Q (5-й екзон), Ivs10nt546 (5-й інтрон), Ivs12nt1 (12-й інтрон), R252W, R261W, R261Q, R261X, G272X, P281L, S273F (7-й екзон). Усі хворі були обстежені на спектр птеринів (біоптерин та неоптерин) в крові та сечі. У 4 пацієнтів із вираженими неврологічними симптомами додатково визначали активність дигідроптеринредуктази в сухих краплях крові. Двом пацієнтам проводилося вимірювання концентрації гомованілової кислоти та 5-гідроксііндолоцтової кислоти в спинномозковій рідині методом високоефективної рідинної хроматографії. Усі пацієнти лікувались за допомогою дієтотерапії з використанням продуктів спеціалізованого лікувального харчування. Результати. 59 пацієнтів із гіперфенілаланінемією були обстежені на птериновий профіль. У 55 пацієнтів був установлений діагноз PKU, у 4 пацієнтів із прогресуючим ураженням нервової системи та порушенням птеринового профілю було встановлено діагноз ВН4-дефіцитних синдромів: 3 пацієнти — із діагнозом «PTPS-синдром», 1 пацієнт — «DHPR-синдром». Основні клінічні прояви ВН4-дефіцитних синдромів у виявлених пацієнтів були такими: затримка розумового розвитку, судоми, прогресування екстрапірамідних симптомів, порушення тонусу м’язів і пози, ковтання, гіперсалівація. Перші симптоми виникали частіше протягом перших місяців життя дитини. Головним біохімічним маркером на початку захворювання була гіперфенілаланінемія. При застосуванні дієтотерапії рівень Phe швидко досягав рекомендованих безпечних для нервової системи значень, але хвороба прогресувала. Висновки. Гіперфенілаланінемія — основний прояв гетерогенної групи рідкісних нейрометаболічних захворювань, які вимагають подальшої діагностичної верифікації. У новонароджених, у яких під час проведення масового неонатального скринінгу на фенілкетонурію виявлений підвищений рівень фенілаланіну, необхідно застосовувати додатковий діагностичний протокол (визначення птеринового профілю в крові та сечі, обов’язкове дослідження активності дигідроптеринредуктази у сухій краплі крові). Дослідження птеринів необхідне в пацієнтів із раніше встановленим діагнозом «фенілкетонурія» за неефективності дієтотерапії, прогресування когнітивних і неврологічних порушень, зниження тонусу м’язів, виникнення судомного синдрому, наявності екстрапірамідних симптомів. Рання діагностика ВН4-дефіцитних синдромів та своєчасно розпочате лікування дозволяють суттєво покращити стан хворих. Для деяких ВН4-дефіцитних синдромів гіперфенілаланінемія не характерна, що унеможливлює раннє виявлення таких пацієнтів під час неонатального скринінгу. Тому виявлення прогресуючих неврологічних симптомів у пацієнтів віком до 1 року й у ранньому дитинстві, які супроводжуються втратою когнітивних функцій на тлі м’язової гіпотонії, дистонічних порушень і судом, зумовлює необхідність дослідження птеринового профілю в крові й сечі.

The aetiology of dystonia disorders is complex, and next-generation sequencing has become a useful tool in elucidating the variable genetic background of these diseases. Here we report a deleterious heterozygous truncating variant in the inosine monophosphate dehydrogenase gene (IMPDH2) by whole-exome sequencing, co-segregating with a dominantly inherited dystonia-tremor disease in a large Finnish family. We show that the defect results in degradation of the gene product, causing IMPDH2 deficiency in patient cells. IMPDH2 is the first and rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides, a dopamine synthetic pathway previously linked to childhood or adolescence-onset dystonia disorders. We report IMPDH2 as a new gene to the dystonia disease entity. The evidence underlines the important link between guanine metabolism, dopamine biosynthesis and dystonia.

Background and aims The apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. Methods Low-density lipoprotein (LDL) receptor null (LDLr−/−) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. Results Unexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. Conclusions Hypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

Aims Recent clinical trials indicate that sodium-glucose cotransporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We explored the direct effects of canagliflozin, an SGLT2 inhibitor with mild SGLT1 inhibitory effects, on myocardial redox signalling in humans. Methods and results Study 1 included 364 patients undergoing cardiac surgery. Right atrial appendage biopsies were harvested to quantify superoxide (O2.−) sources and the expression of inflammation, fibrosis, and myocardial stretch genes. In Study 2, atrial tissue from 51 patients was used ex vivo to study the direct effects of canagliflozin on NADPH oxidase activity and nitric oxide synthase (NOS) uncoupling. Differentiated H9C2 and primary human cardiomyocytes (hCM) were used to further characterize the underlying mechanisms (Study 3). SGLT1 was abundantly expressed in human atrial tissue and hCM, contrary to SGLT2. Myocardial SGLT1 expression was positively associated with O2.− production and pro-fibrotic, pro-inflammatory, and wall stretch gene expression. Canagliflozin reduced NADPH oxidase activity via AMP kinase (AMPK)/Rac1signalling and improved NOS coupling via increased tetrahydrobiopterin bioavailability ex vivo and in vitro. These were attenuated by knocking down SGLT1 in hCM. Canagliflozin had striking ex vivo transcriptomic effects on myocardial redox signalling, suppressing apoptotic and inflammatory pathways in hCM. Conclusions We demonstrate for the first time that canagliflozin suppresses myocardial NADPH oxidase activity and improves NOS coupling via SGLT1/AMPK/Rac1 signalling, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings reveal a novel mechanism contributing to the beneficial cardiac effects of canagliflozin., Graphical Abstract Proposed mechanism of canagliflozin-induced improvement of myocardial redox state. Canagliflozin increases intracellular AMP/ATP ratio through inhibition of SGLT1, which can activate AMPK/NOS signalling and increase NO that suppresses pro-inflammatory signalling. AMPK activation also inhibits activation of Rac1 and membrane translocation of Rac1 and p47phox, which decrease NADPH oxidase activity and superoxide (O2.−) production, attenuates inflammatory and apoptotic pathways and increasing the bioavailability of tetrahydrobiopterin (BH4), a key factor for NOS coupling.

Tetrahydrobiopterin (BH4) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH4 metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH4 deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine.

SIGNIFICANCE: Tetrahydrobiopterin (BH4) is most well known as a required cofactor for enzymes regulating cellular redox homeostasis, aromatic amino acid metabolism, and neurotransmitter synthesis. Less well known are the effects dependent on the cofactor's availability, factors governing its synthesis and recycling, redox implications of the cofactor itself, and protein–protein interactions that underlie cell death. This review provides an understanding of the recent advances implicating BH4 in the mechanisms of cell death and suggestions of possible therapeutic interventions. RECENT ADVANCES: The levels of BH4 often reflect the sum of synthetic and recycling enzyme activities. Enhanced expression of GTP cyclohydrolase, the rate-limiting enzyme in biosynthesis, increases BH4, leading to improved cell function and survival. Pharmacologically increasing BH4 levels has similar beneficial effects, leading to enhanced production of neurotransmitters and nitric oxide or reducing oxidant levels. The GTP cyclohydrolase-BH4 pairing has been implicated in a type of cell death, ferroptosis. At the cellular level, BH4 counteracts anticancer therapies directed to enhance ferroptosis via glutathione peroxidase 4 (GPX4) activity inhibition. CRITICAL ISSUES: Because of the multitude of intertwined mechanisms, a clear relationship between BH4 and cell death is not well understood yet. The possibility that the cofactor directly influences cell viability has not been excluded in previous studies when modulating BH4-producing enzymes. FUTURE DIRECTIONS: The importance of cellular BH4 variations and BH4 biosynthetic enzymes to cell function and viability makes it essential to better characterize temporal changes, cofactor activity, and the influence on redox status, which in turn would help develop novel therapies. Antioxid. Redox Signal. 37, 171–183.

Growing evidence links high aldosterone levels with atrial fibrillation and other heart diseases. Here, we have investigated the functional consequences of culturing adult rat atrial myocytes with aldosterone, at the level of cell size, homeostasis of Ca2+, reactive oxygen species (ROS), and nitrogen oxide (NO). The protein levels of NO synthase (NOS), aldehyde dehydrogenase 2 (ALDH2), NADPH oxidase (NOX), and Na+-Ca2+ exchanger (NCX) were also studied. Aldosterone did not alter the expression of these proteins, except for the NCX, which was enhanced by nearly 100%. Additionally, the hormone inhibited and stimulated, respectively, the production of NO and ROS (the effect on ROS appeared after 24 h of treatment and reached a maximum by 4-6 days, with an EC50 of 1.2 nM). These changes in reactive species generation were blunted by tetrahydrobiopterin (BH4, a NOS cofactor), suggesting the involvement of an uncoupled NOS. An activator (Alda-1) and an inhibitor (daidzin) of ALDH2 were used, to determine if this enzyme activity is related to aldosterone effects, through possible modulation of ROS. Aldosterone produced a ∼10% increase in cell size and, remarkably, this hypertrophic effect, along with the corresponding changes in ROS and NO, were all mimicked by daidzin and prevented by Alda-1. Something different happened with SR Ca2+ release. Aldosterone increased both the magnitude of Ca2+ transients and the incidence of spontaneous Ca2+ oscillations, but these actions were not reproduced by daidzin. Moreover, rather than being prevented, they were further promoted by Alda-1, which also increased the rate of SR Ca2+ reuptake. These results suggest that NOS and ALDH2 may prevent some adverse consequences of aldosteronism (in the case of ALDH2, at the expense of exacerbating SR Ca2+ release). Our data also suggest a hierarchical model in which aldosterone promotes: SR Ca2+ release, then ROS production, and finally hypertrophy.

Aims We have previously reported that nano-selenium quantum dots (SeQDs) prevented endothelial dysfunction in atherosclerosis. This study is to investigate whether amorphous SeQDs (A-SeQDs) increase endogenous tetrahydrobiopterin biosynthesis to alleviate pulmonary arterial hypertension. Results Both A-SeQDs and C-SeQDs were stable under physiological conditions, while the size of A-SeQDs was smaller than C-SeQDs by high resolution-transmission electron microscopy scanning. In monocrotaline-injected mice, oral administration of A-SeQDs was more effective to decrease pulmonary arterial pressure, compared to C-SeQDs and organic selenium. Further, A-SeQDs increased both nitric oxide productions and intracellular BH4 levels, upregulated dihydrofolate reductase activity in lungs, and improved pulmonary arterial remodeling. Gene deletion of dihydrofolate reductase abolished these effects produced by A-SeQDs in mice. Finally, the blood levels of tetrahydrobiopterin and selenium were decreased in patients with pulmonary arterial hypertension. Conclusion A-SeQDs increase intracellular tetrahydrobiopterin to prevent pulmonary arterial hypertension through recoupling endothelial nitric oxide synthase. Methods Two polymorphs of SeQDs and A-SeQDs, and a crystalline form of SeQDs (C-SeQDs) were prepared through self-redox decomposition of selenosulfate precursor. Mice were injected with monocrotaline to induce pulmonary arterial hypertension in vivo. Pulmonary arterial pressure was measured.

Personalized medicine means the selection of therapy for patients, taking into account the assessment of genetic risk factors for side effects. A number of studies show that folate metabolism disorders, including single nucleotide polymorphisms (SNPs) in the genes of folate-metabolizing enzymes, are more frequently detected in schizophrenic patients than in the general population. The role of SNPs of the key folate cycle enzymes in developing the extrapyramidal side effects of antipsychotics has not yet been studied, although there is evidence of their association with other movement disorders.Objective: to analyze the association between the carriage of SNP alleles of MTHFR 677C>T, MTR 2756A>G, and MTRR 66A>G and the severity of extrapyramidal side effects of antipsychotics in patients with schizophrenia.Patients and methods. The investigation included 61 patients with schizophrenia (according to the criteria for ICD-10 Code F20). All the patients took antipsychotics for at least 7 hospital days were examined using real-time polymerase chain reaction (PCR) with allele-specific primers, followed by detection for the carriage of SNP alleles of MTHFR 677C>T, MTR 2756A>G, and MTRR 66A>G. The standardized Simpson–Angus scale (SAS) was used to evaluate the severity of extrapyramidal symptoms; the PCR test results were unknown during their examination.Results and discussion. In the patients carrying a low-functional 677 T allele in the gene of the key folate cycle enzyme MTHFR, the severity of extrapyramidal side effects of antipsychotics was statistically significantly higher than in the carriers of the wild-type genotype: 13.27±5.10 versus 9.84±6.03 SAS scores, respectively (t=-2.40; p=0.020). In addition, the carriage of the wild allele A of SNP in the MTRR 66A>G gene (F=3.83; p=0.0283; pcorr.=0.043) is associated with the severity of extrapyramidal symptoms. There was a direct moderate correlation of the number of risk alleles at two loci with the total SAS score (r=0.51; p=0.00017).Conclusion. The polymorphic allele of MTHFR 677T and the wild allele of MTRR 66A can be regarded as risk alleles for the development of extrapyramidal side effects of antipsychotics.

Background Hypertension is the the primary cause of diastolic heart failure. Oxidative stress plays an important role in cardiac diastolic dysfunction caused by hypertension. The occurrence of oxidative stress is related to the level of nitric oxide (NO) in the body. Tetrahydrobiopterin (BH4) is an essential cofactor for NO synthesis. Nebivolol can reduce myocardial oxidative stress and increase NO activity. Therefore, we investigated the effects of monotherapy or combination therapy of different doses of BH4 and nebivolol on cardiac diastolic function in spontaneously hypertensive rats, and preliminarily expounded the related mechanisms. Methods Left ventricular function was evaluated by non-invasive echocardiographic assessment and invasive right carotid artery catheterization methods. ELISA was used to measure myocardial 3-nitrotyrosine content, NO production, and cyclic guanosine monophosphate (cGMP) concentration in the myocardium; quantitative real-time PCR (qRT-PCR) was used to determine endothelial nitric oxide synthase (eNOS), phospholamban and sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) mRNA expression levels; Western blot was used to detect the protein expression levels of eNOS and eNOS dimers in myocardial tissue, and immunohistochemical detection of cGMP expression in the myocardium was performed. Results Studies have shown that compared with those in the control group, NO generation and the expression level of myocardial eNOS mRNA, eNOS expression of dimers, phospholamban, SERCA2a and cGMP increased significantly after the combined intervention of BH4 and nebivolol, while the expression of 3-nitrotyrosine was significantly decreased. Conclusions The combined treatment group had a synergistic effect on reducing myocardial oxidative stress, increasing eNOS content, and increasing NO production, and had a more obvious protective effect on diastolic dysfunction through the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway.

Several risk factors are known to be involved in the initiation and development of gastric cancer. Among them, H. pylori is one of the most prominent with multiple virulence factors contributing to its pathogenicity. In this study, we have discussed an interesting immunological cycle exploring the interplay between H. pylori, aryl hydrocarbon receptor (AHR), tryptophan, arginine, and the metabolites of these two amino acids in the development of gastric cancer. AHR is a ligand-activated transcription factor which acts as a regulator for a diverse set of genes and has various types of exogenous and endogenous ligands. The tryptophan metabolite, kynurenine, is one of these ligands that can interact with AHR, leading to immune suppression and subsequently, susceptibility to gastric cancer. On the other hand, H. pylori downregulates the expression of AHR and AHR repressor (AHRR), leading to increased inflammatory cytokine production. A metabolite of the kynurenine pathway, xanthurenic acid, is a potent inhibitor of a terminal enzyme in the synthetic pathway of tetrahydrobiopterin (BH4). BH4, itself, is a cofactor in the process of nitric oxide (NO) production from arginine that has been shown to have immune-enhancing properties. Arginine has also been evidenced to have anti-tumoral function through inducing apoptosis in gastric cell lines; however, controversy exists regarding the anti-tumor role of arginine and BH4, since they are also associated with increased NO production, subsequently promoting tumor angiogenesis. Hence, although several synergistic connections result in immunity improvement, these correlations can also act as a double-edged sword, promoting tumor development. This emphasizes on the need for further investigations to better understand this complex interplay.

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5’-serially deleted eNOS promoters revealed that Tax-responsive element site, located at −962 to −873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominant-negative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser133 level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser1981 and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser473, p-CREB-Ser133, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser1981, p-Akt-Ser473, p-CREB-Ser133, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

PURPOSE: The present study assesses the change in tetrahydrobiopterin (BH4), one of the most important products in the guanosine triphosphate (GTP) pathway and in other parameters that might affect nitric oxide (NO) production, in gestational diabetes mellitus (GDM). METHODS: The study included 100 healthy pregnant women and 100 women diagnosed with GDM. Serum levels of neopterin, BH4 and NO were measured. The levels of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GCHI/GTPCH) gene expression were determined. RESULTS: It was found that diabetes led to an increase in neopterin and NO levels, and a decrease in BH4 levels. A stimulation was observed in eNOS gene expression in the GDM group when compared to the control group, while GCHI levels were found to decrease when compared to the control group. iNOS gene expression was detected in neither the healthy controls nor the patient group. CONCLUSIONS: Decreased NO bioavailability plays an important role in the progression of such macrovascular diseases as diabetes. BH4 levels decrease in diabetes patients, while the increased gene expression of GCHI reverses the diabetes-related BH4 deficiency and allows the endothelial cells to regain their ability to produce NO. Since GCHI is the rate-limiting enzyme in the biosynthesis of BH4, changes in GCHI levels directly affect the BH4 levels and the NO metabolism, leading to an increased risk of macrovascular complications. The significant increase in neopterin levels suggest that this is a potential biomarker for the early diagnosis of GDM.

GTP cyclohydrolase I enzyme (GTPCH-I) is a rate limiting enzyme in the biosynthesis pathway of tetrahydrobiopterin (BH4) and tetrahydrofolate (THF) compounds; latter being are an essential compounds involved in many biological functions. This enzyme has been evaluated structurally and functionally in many organisms to understand its putative role in cell processes, kinetics, regulations, drug targeting in infectious diseases, pain sensitivity in humans, and so on. In Mycobacterium tuberculosis (a human pathogen causing tuberculosis), this GTPCH-I activity has been predicted to be present in Rv3609c gene (folE) of H37 Rv strain, which till date has not been studied in detail. In order to understand in depth, the structure and function of folE protein in M. tuberculosis H37 Rv, in silico study was designed by using many different bioinformatics tools. Comparative and structural analysis predicts that Rv3609c gene is similar to folE protein ortholog of Listeria monocytogenes (cause food born disease), and uses zinc ion as a cofactor for its catalysis. Result shows that mutation of folE protein at 52th residue from tyrosine to glycine or variation in pH and temperature can lead to high destability in protein structure. Studies here have also predicted about the functional regions and interacting partners involved with folE protein. This study has provided clues to carry out experimentally the analysis of folE protein in mycobacteria and if found suitable will be used for drug targeting.

Phenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]–1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066−11G>A (IVS10−11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066−11G>A];[1066−11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome. Fil: Hillert, Alicia. No especifíca; Fil: Anikster, Yair. No especifíca; Fil: Belanger Quintana, Amaya. No especifíca; Fil: Burlina, Alberto. No especifíca; Fil: Burton, Barbara K.. No especifíca; Fil: Carducci, Carla. No especifíca; Fil: Chiesa, Ana Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina Fil: Christodoulou, John. No especifíca; Fil: Dordevic, Maja. No especifíca; Fil: Desviat, Lourdes R.. No especifíca; Fil: Eliyahu, Aviva. No especifíca; Fil: Evers, Roeland A.F.. No especifíca; Fil: Fajkusova, Lena. No especifíca; Fil: Feillet, Francois. No especifíca; Fil: Bonfim Freitas, Pedro E.. No especifíca; Fil: Gizewska, María. No especifíca; Fil: Gundorova, Polina. No especifíca; Fil: Karall, Daniela. No especifíca; Fil: Kneller, Katya. No especifíca; Fil: Kutsev, Sergey I.. No especifíca; Fil: Leuzzi, Vincenzo. No especifíca; Fil: Levy, Harvey L.. No especifíca; Fil: Lichter Koneck, Uta. No especifíca; Fil: Muntau, Ania C.. No especifíca; Fil: Namour, Fares. No especifíca; Fil: Oltarzewsk, Mariusz. No especifíca; Fil: Paras, Andrea. No especifíca; Fil: Perez, Belén. No especifíca; Fil: Polak, Emil. No especifíca; Fil: Polyakov, Alexander V.. No especifíca; Fil: Porta, Francesco. No especifíca; Fil: Rohrbach, Marianne. No especifíca; Fil: Scholl Bürgi, Sabine. No especifíca; Fil: Spécola, Norma. No especifíca; Fil: Stojiljkovic, Maja. No especifíca; Fil: Shen, Nan. No especifíca; Fil: Santana da Silva, Luiz C.. No especifíca; Fil: Skouma, Anastasia. No especifíca; Fil: van Spronsen, Francjan. No especifíca; Fil: Stoppioni, Vera. No especifíca; Fil: Thöny, Beat. No especifíca; Fil: Trefz, Friedrich K.. No especifíca; Fil: Vockley, Jerry. No especifíca; Fil: Yu, Youngguo. No especifíca; Fil: Zschocke, Johannes. No especifíca; Fil: Hoffmann, Georg F.. No especifíca; Fil: Garbade, Sven F.. No especifíca; Fil: Blau, Nenad. No especifíca

BACKGROUND The number of erectile dysfunction (ED) patients is increasing annually. How to improve the effectiveness of ED treatment is an important issue for the field of andrology. OBJECTIVES To investigate whether low androgen status impairs the erectile function of rats by regulated endothelial nitric oxide synthase (eNOS) uncoupling. MATERIALS AND METHODS Thirty-six 8-week-old male Sprague Dawley (SD) rats were randomly divided into six groups as follows: 4-week sham-operated group (4w-sham), 4-week castration group (4w-cast), 4-week castration + testosterone (T) group (4w-cast + T), 8-week sham-operated group (8w-sham), 8-week castration group (8w-cast), and 8-week castration + T group (8w-cast + T). Three mg/kg of T was subcutaneously injected every other day in castration + T groups. The ratio of the maximum intracavernous pressure/the mean arterial pressure (ICPmax/MAP), the level of serum T, dihydrobiopterin(BH2 ), tetrahydrobiopterin (BH4 ), nitric oxygen(NO), 3-nitrotyrosine(3NT), dihydrofolate reductase (DHFR), guanosine triphosphate cyclohydrolase 1 (GTPCH1), nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), and eNOS monomers/dimers in the corpus cavernosum were detected. RESULTS The ratio of ICPmax/MAP and BH4 /BH2 , the level of serum T, NO, and GTPCH1 decreased significantly in castration groups compared with sham-operated groups and castration + T groups (P

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH(4)) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH(4) triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators. SIGNIFICANCE STATEMENT: The heat shock protein 70 (Hsp70):c-terminus of Hsp70-interacting protein (CHIP) complex facilitates the ubiquitination and subsequent degradation of several hundred-client proteins, and activation of Hsp70 has been suggested as a therapeutic strategy to enhance the degradation of disease-causing proteins. The current study shows that the pharmacological activation of Hsp70 enhances the ubiquitination of dysfunctional but not native nNOS, and it suggests that this therapeutic strategy will likely be highly selective.

Background. Shear stress is an effective modulator of endothelial progenitor cells (EPCs) and has been suggested to play an important role in angiogenesis. The phosphatase and tensin homolog (PTEN)/Akt and guanosine triphosphate cyclohydrolase (GTPCH)/tetrahydrobiopterin (BH4) pathways regulate the function of early EPCs. However, the role of these pathways in the shear stress-induced angiogenesis of late EPCs remains poorly understood. Therefore, we aim to investigate whether shear stress could upregulate the angiogenesis capacity of late EPCs and to further explore the possible underlying mechanisms. Methods. Late EPCs were subjected to laminar shear stress (LSS), and their in vitro migration, proliferation, and tube formation capacity were determined. In addition, the in vivo angiogenesis capacity was explored, along with the expression of molecules involved in the PTEN/Akt and GTPCH/BH4 pathways. Results. LSS elevated the in vitro activities of late EPCs, which were accompanied by downregulated PTEN expression, accelerated Akt phosphorylation, and GTPCH/BH4 pathway activation (all P<0.05). Following Akt inhibition, LSS-induced upregulated GTPCH expression, BH4, and NO level of EPCs were suppressed. LSS significantly improved the migration, proliferation, and tube formation ability (15 dyn/cm2 LSS vs. stationary: 72.2±5.5 vs. 47.3±7.3, 0.517±0.05 vs. 0.367±0.038, and 1.664±0.315 vs. 1±0, respectively; all P<0.05) along with the in vivo angiogenesis capacity of late EPCs, contributing to the recovery of limb ischemia. These effects were also blocked by Akt inhibition or GTPCH knockdown (P<0.05, respectively). Conclusions. This study provides the first evidence that shear stress triggers angiogenesis in late EPCs via the PTEN/Akt/GTPCH/BH4 pathway, providing a potential nonpharmacologic therapeutic strategy for promoting angiogenesis in ischemia-related diseases.

The fraction of nitric oxide in exhaled gas (FENO) is decreased after exposure to hyperoxia in vivo, although the mechanisms for this decrease is not clear. A key co-factor for nitric oxide synthase (NOS), tetrahydrobiopterin (BH4), has been shown to be oxidized in vitro when exposed to hyperoxia. We hypothesized that the decrease of FENO is due to decreased enzymatic generation of NO due to oxidation of BH4. The present study was performed to investigate the relationship between levels of FENO and plasma BH4 following hyperoxic exposure in humans. Two groups of healthy subjects were exposed to 100% oxygen for 90 minutes. FENO was measured before and 10 minutes (n = 13) or 60 minutes (n = 14) after the exposure. Blood samples were collected at the same time points for quantification of biopterin levels (BH4, BH2 and B) using LC-MS/MS. Each subject was his or her own control, breathing air for 90 minutes on a separate day. Hyperoxia resulted in a 28.6 % decrease in FENO 10 minutes after exposure (p < 0.001), confirming previous findings. Moreover, hyperoxia also caused a 14.2% decrease in plasma BH4 (p = 0.012). No significant differences were observed in the group measured 60 minutes after exposure. No significant correlation was found between the changes in FENO and BH4 after the hyperoxic exposure (r = 0.052, p = 0.795), this might be due to the recovery of BH4 being faster than the recovery of FENO.

Tetrahydrobiopterin-dependent hydroxylation of aromatic amino acids is the first step in the biosynthesis of many neuroactive compounds in humans. A fundamental challenge in building these pathways in Escherichia coli is the provision of the non-native hydroxylase cofactor, tetrahydrobiopterin. To solve this, we designed a genetic selection that relies on the tyrosine synthesis activity of phenylalanine hydroxylase. Using adaptive laboratory evolution, we demonstrate the use of this selection to discover: (1) a minimum set of heterologous enzymes and a host folE (T198I) mutation for achieving this type of hydroxylation chemistry in whole cells, (2) functional complementation of tetrahydrobiopterin by indigenous cofactors, and (3) a tryptophan hydroxylase mutation for improving protein abundance. Thus, the goal of having functional aromatic-amino-acid hydroxylation in E. coli was achieved through directed metabolic pathway evolution.

Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Tetrahydrobiopterin (BH4) is an important cofactor in regulating the balance between NO (eNOS coupling) and superoxide production (eNOS uncoupling). However, whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling remains completely unknown. Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH-1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with MitoTEMPO treatment replenished BH4 levels by elevating levels of GCH-1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.

Nitric oxide is a signalling molecule with an extensive range of functions in both health and disease. Discovered in the 1980s through work that earned the Nobel prize, nitric oxide is an essential factor in regulating cardiovascular, immune, neurological and haematological function in normal homeostasis and in response to infection. Early work implicated exaggerated nitric oxide synthesis as a potentially important driver of septic shock; however, attempts to modulate production through global inhibition of nitric oxide synthase were associated with increased mortality. Subsequent work has shown that regulation of nitric oxide production is determined by numerous factors including substrate and co-factor availability and expression of endogenous regulators. In sepsis, nitric oxide synthesis is dysregulated with exaggerated production leading to cardiovascular dysfunction, bioenergetic failure and cellular toxicity whilst at the same time impaired microvascular function may be driven in part by reduced nitric oxide synthesis by the endothelium. This bench to bedside review summarises our current understanding of the ways in which nitric oxide production is regulated on a tissue and cellular level before discussing progress in translating these observations into novel therapeutic strategies for patients with sepsis.

Dopa-responsive dystonia (DRD) is a rare movement disorder associated with defective dopamine synthesis. This impairment may be due to the fact of a deficiency in GTP cyclohydrolase I (GTPCHI, GCH1 gene), sepiapterin reductase (SR), tyrosine hydroxylase (TH), or 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) enzyme functions. Mutations in GCH1 are most frequent, whereas fewer cases have been reported for individual SR-, PTP synthase-, and TH deficiencies. Although termed DRD, a subset of patients responds poorly to L-DOPA. As this is regularly observed in severe cases of TH deficiency (THD), there is an urgent demand for more adequate or personalized treatment options. TH is a key enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis, and THD patients often present with complex and variable phenotypes, which results in frequent misdiagnosis and lack of appropriate treatment. In this expert opinion review, we focus on THD pathophysiology and ongoing efforts to develop novel therapeutics for this rare disorder. We also describe how different modeling approaches can be used to improve genotype to phenotype predictions and to develop in silico testing of treatment strategies. We further discuss the current status of mathematical modeling of catecholamine synthesis and how such models can be used together with biochemical data to improve treatment of DRD patients.

ispartof: PARKINSONISM & RELATED DISORDERS vol:82 pages:10-12 ispartof: location:England status: published

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP into dihydroneopterin triphosphate (DHNP). DHNP is the first intermediate of the folate de novo biosynthesis pathway in prokaryotic and lower eukaryotic microorganisms and the tetrahydrobiopterin (BH4) biosynthesis pathway in higher eukaryotes. The de novo folate biosynthesis provides essential cofactors for DNA replication, cell division, and synthesis of key amino acids in rapidly replicating pathogen cells, such as Plasmodium falciparum(P. falciparum), a causative agent of malaria. In eukaryotes, the product of the BH4 biosynthesis pathway is essential for the production of nitric oxide and several neurotransmitter precursors. An increased copy number of the malaria parasite P. falciparum GCH1 gene has been reported to influence antimalarial antifolate drug resistance evolution, whereas mutations in the human GCH1 are associated with neuropathic and inflammatory pain disorders. Thus, GCH1 stands as an important and attractive drug target for developing therapeutics. The GCH1 intrinsic dynamics that modulate its activity remains unclear, and key sites that exert allosteric effects across the structure are yet to be elucidated. This study employed the anisotropic network model to analyze the intrinsic motions of the GCH1 structure alone and in complex with its regulatory partner protein. We showed that the GCH1 tunnel-gating mechanism is regulated by a global shear motion and an outward expansion of the central five-helix bundle. We further identified hotspot residues within sites of structural significance for the GCH1 intrinsic allosteric modulation. The obtained results can provide a solid starting point to design novel antineuropathic treatments for humans and novel antimalarial drugs against the malaria parasite P. falciparum GCH1 enzyme.

Oxidation of tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase (NOS), by reactive oxidative species (ROS), leads to NOS uncoupling and superoxide production instead of NO. Further, oxidative stress plays a major role in ethanol-evoked cardiac dysfunction in proestrus female rats, and acute ethanol administration reduces brain BH4 level. Therefore, we discerned the unknown role of BH4 in ethanol-evoked cardiac dysfunction by pharmacologically increasing BH4 levels or inhibiting its effect in proestrus female rats. Acute ethanol (1.5 g/kg, i.v, 30 min) caused myocardial dysfunction (lowered dP/dtmax and LVDP) and hypotension, along with increases in myocardial: (i) levels of NO, ROS and malondialdehyde (MDA), (ii) activities of catalase, ALDH2 and NADPH oxidase (Nox), and (iii) phosphorylation of eNOS, nNOS. Further, ethanol suppressed myocardial arginase and superoxide dismutase (SOD) activities and enhanced eNOS uncoupling. While ethanol had no effect on cardiac BH4 levels, BH4 (19 mg/kg, i.v) supplementation paradoxically caused cardiac oxidative stress, but mitigated the cardiac dysfunction/hypotension and most of the adverse molecular responses caused by ethanol. Equally important, the BH4 inhibitor DAHP (1 g/kg, i.p) exacerbated the adverse molecular and cardiovascular effects caused by ethanol. Our pharmacological studies support a protective role for the NOS co-factor BH4 against ethanol-evoked cardiac dysfunction and hypotension in female rats.

BackgroundDuring the initial 26-week SPARK (Safety Paediatric efficAcy phaRmacokinetic with Kuvan®) study, addition of sapropterin dihydrochloride (Kuvan®; a synthetic formulation of the natural cofactor for phenylalanine hydroxylase, tetrahydrobiopterin; BH4), to a phenylalanine (Phe)-restricted diet, led to a significant improvement in Phe tolerance versus a Phe-restricted diet alone in patients aged 0–4 years with BH4-responsive phenylketonuria (PKU) or mild hyperphenylalaninaemia (HPA). Based on these results, the approved indication for sapropterin in Europe was expanded to include patients ResultsAll 51 patients who completed the 26-week SPARK study period entered the extension period. Patients who were previously treated with a Phe-restricted diet only (‘sapropterin extension’ group; n = 26), were initiated on sapropterin at 10 mg/kg/day, which could be increased up to 20 mg/kg/day. Patients previously treated with sapropterin plus Phe-restricted diet, remained on this regimen in the extension period (‘sapropterin continuous’ group; n = 25). Dietary Phe tolerance increased significantly at the end of the study versus baseline (week 0), by 38.7 mg/kg/day in the ‘sapropterin continuous’ group (95% CI 28.9, 48.6;p p = 0.1929). Patients in both groups had normal neuromotor development and growth parameters.ConclusionsLong-term treatment with sapropterin plus a Phe-restricted diet in patients who initiated sapropterin at 4-responsive PKU or mild HPA maintained improvements in dietary Phe tolerance over 3.5 years. These results continue to support the favourable risk/benefit profile for sapropterin in paediatric patients (4-responsive PKU. Frequent monitoring of blood Phe levels and careful titration of dietary Phe intake to ensure adequate levels of protein intake is necessary to optimise the benefits of sapropterin treatment.Trial registrationClinicalTrials.gov, NCT01376908. Registered 17 June 2011,https://clinicaltrials.gov/ct2/show/NCT01376908.

Nitric oxide synthase (NOS) catalyzes the transformation of l -arginine, molecular oxygen (O2), and NADPH-derived electrons to nitric oxide (NO) and l -citrulline. Under some conditions, however, NOS catalyzes the reduction of O2 to superoxide (O2−) instead, a phenomenon that is generally referred to as uncoupling. In principle, both the heme in the oxygenase domain and the flavins in the reductase domain could catalyze O2− formation. In the former case the oxyferrous (Fe(II)O2) complex that is formed as an intermediate during catalysis would dissociate to heme and O2−; in the latter case the reduced flavins would reduce O2 to O2−. The NOS cofactor tetrahydrobiopterin (BH4) is indispensable for coupled catalysis. In the case of uncoupling at the heme this is explained by the essential role of BH4 as an electron donor to the oxyferrous complex; in the case of uncoupling at the flavins it is assumed that the absence of BH4 results in NOS monomerization, with the monomers incapable to sustain NO synthesis but still able to support uncoupled catalysis. In spite of little supporting evidence, uncoupling at the reductase after NOS monomerization appears to be the predominant hypothesis at present. To set the record straight we extended prior studies by determining under which conditions uncoupling of the neuronal and endothelial isoforms (nNOS and eNOS) occurred and if a correlation exists between uncoupling and the monomer/dimer equilibrium. We determined the rates of coupled/uncoupled catalysis by measuring NADPH oxidation spectrophotometrically at 340 nm and citrulline synthesis as the formation of [3H]-citrulline from [3H]-Arg. The monomer/dimer equilibrium was determined by FPLC and, for comparison, by low-temperature polyacrylamide gel electrophoresis. Uncoupling occurred in the absence of Arg and/or BH4, but not in the absence of Ca2+ or calmodulin (CaM). Since omission of Ca2+/CaM will completely block heme reduction while still allowing substantial FMN reduction, this argues against uncoupling by the reductase domain. In the presence of heme-directed NOS inhibitors uncoupling occurred to the extent that these compound allowed heme reduction, again arguing in favor of uncoupling at the heme. The monomer/dimer equilibrium showed no correlation with uncoupling. We conclude that uncoupling by BH4 deficiency takes place exclusively at the heme, with virtually no contribution from the flavins and no role for NOS monomerization.

Nitric oxide synthases (NOSs) are heme-based monooxygenases that convert l-Arg to l-citrulline and nitric oxide (NO), a key signaling molecule and cytotoxic agent in mammals. Bacteria also contain NOS proteins, but the role of NO production within these organisms, where understood, differs considerably from that of mammals. For example, a NOS protein in the marine cyanobacterium Synechococcus sp. PCC 7335 (syNOS) has recently been proposed to function in nitrogen assimilation from l-Arg. syNOS retains the oxygenase (NOSox) and reductase (NOSred) domains present in mammalian NOS enzymes (mNOSs), but also contains an N-terminal globin domain (NOSg) homologous to bacterial flavohemoglobin proteins. Herein, we show that syNOS functions as a dimer and produces NO from l-Arg and NADPH in a tetrahydrobiopterin (H4B)-dependent manner at levels similar to those produced by other NOSs but does not require Ca2+-calmodulin, which regulates NOSred-mediated NOSox reduction in mNOSs. Unlike other bacterial NOSs, syNOS cannot function with tetrahydrofolate and requires high Ca2+ levels (>200 μm) for its activation. NOSg converts NO to NO3− in the presence of O2 and NADPH; however, NOSg did not protect Escherichia coli strains against nitrosative stress, even in a mutant devoid of NO-protective flavohemoglobin. We also found that syNOS does not have NOS activity in E. coli (which lacks H4B) and that the recombinant protein does not confer growth advantages on l-Arg as a nitrogen source. Our findings indicate that syNOS has both NOS and NO oxygenase activities, requires H4B, and may play a role in Ca2+-mediated signaling.

6 pags., 3 figs., Phenylalanine hydroxylase (PAH) is a key enzyme in the catabo-lism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH. BH forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic Fe. Upon BH binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH. Importantly, BH binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations., The work was supported by grants from the MICINN Spanish Ministry of Research, Innovation & Universities BFU2017-90030-P (to J.A.H.) and BFU2017-87316 (to R.F.-L.); programs Forny (248889/O30) and FRIMEDBIO (261826) from the Research Council of Norway (to A.M.); the Western Norway Regional Health Authorities (Helse Vest Projects 911959 to M.I.F. and 912246 to A.M.); and the K.G. Jebsen foundation (to M.I.F. and A.M.), the staff from ALBA synchrotron facility (Barcelona) and the European Synchrotron Radiation Facility (ESRF)

The nature of phenylalanine hydroxylase (PAH) variants determines residual enzyme activity, which modifies the clinical phenotype in phenylketonuria (PKU). We exploited the statistical power of a large genotype database to determine the relationship between genotype and phenotype in PKU. A total of 9336 PKU patients with 2589 different genotypes, carrying 588 variants, were investigated using an allelic phenotype value (APV) algorithm. We identified 251 0-variants encoding inactive PAH, and assigned APVs (0 = classic PKU; 5 = mild PKU; 10 = mild hyperphenylalaninaemia) to 88 variants in PAH-functional hemizygous patients. The genotypic phenotype values (GPVs) were set equal to the higher-APV allele, which was assumed to be dominant over the lower-APV allele and to determine the metabolic phenotype. GPVs for 8872 patients resulted in cut-off ranges of 0.0–2.7 for classic PKU, 2.8–6.6 for mild PKU and 6.7–10.0 for mild hyperphenylalaninaemia. Genotype-based phenotype prediction was 99.2% for classic PKU, 46.2% for mild PKU and 89.5% for mild hyperphenylalaninaemia. The relationships between known pretreatment blood phenylalanine levels and GPVs (n = 4217), as well as tetrahydrobiopterin responsiveness and GPVs (n = 3488), were significant (both P

AIMS: Reduced nitric oxide (NO) availability may adversely affect renal perfusion and glomerular filtration. The aim of the present study was to characterize in detail the pharmacological effects of VAS203, an inhibitor of NO synthase, on renal haemodynamics in humans. METHODS: This double‐blind, randomized, placebo‐controlled, cross‐over phase‐I‐study comprised 18 healthy men. Renal haemodynamics were assessed with constant‐infusion input‐clearance technique with p‐aminohippurate and inulin for renal plasma flow (RPF) and glomerular filtration rate (GFR), respectively. After baseline measurement, a constant infusion of the tetrahydrobiopterin analogue ronopterin (VAS203, total 10 mg/kg body weight) or placebo was administered at random order for 6 hours additionally. After a wash‐out phase of 28 days, the second course was applied. In parallel, markers of early kidney injury and renal function were assessed repeatedly up to 48 hours after starting VAS203/placebo‐infusion. RESULTS: VAS203‐infusion resulted in a significant decrease of RPF (P < .0001) and GFR (P < .001) compared to placebo, but magnitude was within the physiological range. RPF and GFR recovered partly 2 hours after end of VAS203‐infusion and was normal at beginning of the second infusion period. Compared to placebo, preglomerular resistance (P < .0001), and to lesser extent postglomerular resistance (P < .0001) increased, resulting in a decrease of intraglomerular pressure (P < .01). No treatment related effect on markers of early kidney injury, and on renal function (P for all >.20) have been observed. CONCLUSIONS: Our phase‐I‐study in healthy humans indicates that VAS203 (10 mg/kg body weight) reduces renal perfusion and glomerular function within the physiological range mainly due to vasoconstriction at the preglomerular site.

Aims Exogenous tetrahydrobiopterin (BH4), an indispensable cofactor of endothelial nitric oxide synthase (eNOS), supplementation has been proved to be of advantage to improve cardiovascular function. Nevertheless, due to its highly redox-sensitive and easy to be oxidized, there is an urgent need to develop an appropriate BH4 formulation for clinical therapy. Gum Arabic (GA) has been considered as an alternative biopolymer for the stabilization and coating of drugs. The effects of GA on protecting BH4 from being oxidized were investigated in a rat model of myocardial ischemia-reperfusion (I/R). Main methods Rats were subjected to 60-min of in vivo left coronary artery occlusion and varying periods of reperfusion with or without pre-ischemic GA-coated BH4 supplementation (10 mg/kg, oral). Myocardial infarction, fibrotic area and left ventricle ejection fraction were correlated with cardiac BH4 content, eNOS protein, NOS enzyme activity, and ROS/NO generation. Key findings Pretreatment of rats with GA-coated 6R-BH4, 24 h before myocardial ischemia, resulted in smaller myocardial infarction, improved left ventricular function and inhibited fibrosis, correlated with maintained high levels of cardiac BH4 content, preserved eNOS activation and dimerization, and decreased ROS generation. However in uncoated group, 6R-BH4 treatment did not reduce acute and chronic myocardial I/R injury compared with control I/R rats, which was closely related with the marked loss of myocardial BH4 levels during I/R. Significance These findings provide evidence that in vivo pre-ischemic oral GA-coated BH4 administration preserves eNOS function secondary to maintaining cardiac BH4 content, and confers cardioprotection after I/R.

Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild-type PAH of 20 selected PKU-linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild-type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH4 ), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single-site missense variants using the full-length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease-linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.

6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions. Human PTS has two cysteines: Cys-43 at the catalytic site and Cys-10 at the N terminus. PTS can be oxidized and consequently inactivated by H2O2 treatment, oxidized GSH, or S-nitrosoglutathione, and determining the oxidized modifications of PTS induced by each oxidant by MALDI–TOF MS, we show that PTS is S-glutathionylated in the presence of GSH and H2O2. S-Glutathionylation at Cys-43 protected PTS from H2O2-induced irreversible sulfinylation and sulfonylation. We also found that PTS expressed in HeLa and THP-1 cells is reversibly modified under oxidative stress conditions. Our findings suggest that PTS activity and S-glutathionylation is regulated by the cellular redox environment and that reversible S-glutathionylation protects PTS against oxidative stress.

Acute liver failure (ALF) is associated with deregulated nitric oxide (NO) signaling in the brain, which is one of the key molecular abnormalities leading to the neuropsychiatric disorder called hepatic encephalopathy (HE). This study focuses on the effect of ALF on the relatively unexplored endothelial NOS isoform (eNOS). The cerebral prefrontal cortices of rats with thioacetamide (TAA)-induced ALF showed decreased eNOS expression, which resulted in an overall reduction of NOS activity. ALF also decreased the content of the NOS cofactor, tetrahydro-L-biopterin (BH4), and evoked eNOS uncoupling (reduction of the eNOS dimer/monomer ratio). The addition of the NO precursor L-arginine in the absence of BH4 potentiated ROS accumulation, whereas nonspecific NOS inhibitor L-NAME or EDTA attenuated ROS increase. The ALF-induced decrease of eNOS content and its uncoupling concurred with, and was likely causally related to, both increased brain content of reactive oxidative species (ROS) and decreased cerebral cortical blood flow (CBF) in the same model.

Background Hyperphenylalaninemia (HPA) is the most common amino acid metabolic disease involving phenylalanine hydroxylase (PAH, OMIM*612,349) deficiency or coenzyme tetrahydrobiopterin (BH4) deficiency. Patients with severe HPA often have a difficult life. Early diagnosis of HPA before the development of symptoms is possible via neonatal screening, facilitating appropriate treatment and reducing mortality and disability rates. This study revealed the prevalence, mutational and phenotypic spectrum, and prognosis of HPA by neonatal screening from January 2001 to September 2020 in Nanjing, Jiangsu Province, China. Methods Through a retrospective analysis of the information available in the neonatal screening database, the clinical presentations, laboratory data, molecular characteristics and treatment follow-up data of HPA patients detected by neonatal screening were evaluated. Results We diagnosed 181 patients with HPA from 1 to 957 newborns, giving an incidence of 1:6873. Among these patients, 177 were identified as PAH deficient and four patients were BH4 deficient. The average current age of the patients was 6.38 years old. The most common mutations of PAH were c.728 C > A/ p.Arg243Gln (13.83 %), c.158G > A/ p.Arg53His (9.57 %), c.611 A > G/ p.Tyr204Cys (7.44 %), and c.721 C > T/ p.Arg241Cys (6.38 %). Conclusions This study revealed the prevalence, phenotype-genotype, and prognosis of HPA in China and contributes to the updating of PAHD data for China and worldwide. Our study not only expanded the spectrum of phenotypes and genotype but also provided a valuable tool for improved genetic counseling and management of future cases.

Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30–100 µM and 5–20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.

After radiation exposure, endothelium-dependent vasorelaxation is impaired due to impaired nitric oxide production. Endothelial dysfunction is characterized by uncoupled endothelial nitric oxide synthase activity, oxidation of the reduced cofactor tetrahydrobiopterin to dihydrobiopterin as one well recognized mechanism. Oral treatment with sepiapterin, a tetrahydrobiopterin precursor, decreased infiltrating inflammatory cells and cytokine levels in mice with colitis. We therefore tested whether a synthetic sepiapterin, PTC923, might mitigate radiation-induced cardiac and pulmonary injuries. C57L/J wild-type 6-8-week-old mice of both sexes received 5 Gy total-body irradiation (TBI), followed by a top-up dose of 6.5 Gy to the thorax (total thoracic dose of 11.5 Gy). Starting from 24 h postirradiation, mice were treated once daily with 1 mg/kg PTC923 for six days by oral gavage. Assessment of lung injury by breathing rate was measured every other week and echocardiography to assess heart function was performed at different time points (8, 30, 60, 90 and 180 days). Plasma proteins (fibrinogen, neutrophil elastase, C-reactive protein, and IL-6) were assessed as well. TBI induced a reduction in cardiac contractile reserve and an impairment in diastolic function restored by daily oral PTC923. Postirradiation lung injury was significantly delayed by PTC923. TBI mice treated with PTC923 experienced a longer survival compared to nonirradiated mice (71% vs. 40% of mice alive after 180 days). PTC923-treated mice showed a reduction in inflammatory mediators, especially IL-6 and IL-1b. In conclusion, these findings support the proposal that PTC923 is a potential mitigator of cardiac and lung injury caused by TBI.

Maternal physiological dyslipidemia (MPD) supports fetal development in human pregnancy. However, some women develop maternal supraphysiological dyslipidemia (MSPD: increased total cholesterol (TC) and triglycerides (TG) levels). MSPH is present in normal and also in gestational diabetes mellitus (GDM) pregnancies. MSPD and GDM associate with fetoplacental endothelial dysfunction, producing alterations in nitric oxide (NO)-L-arginine/arginase metabolism. Nevertheless, the effect of MSPD on GDM, and how this synergy alters fetoplacental endothelial function is unknown. Therefore, the aim of this study was to evaluate in human umbilical vein endothelial cells, the effects of MSPD in GDM and how these pathologies together affect the fetoplacental endothelial function. 123 women at term of pregnancy were classified as MPD (n = 40), MSPD (n = 35), GDM with normal lipids (GDM-MPD, n = 23) and with increased lipids (GDM-MSPD, n = 25). TC ≥291 mg/dL and TG ≥275 mg/dL were considered as MSPD. Endothelial NO synthase (eNOS), human cationic amino acid transporter 1 (hCat1), and arginase II protein abundance and activity, were assayed in umbilical vein endothelial cells. In MSPD and GDM-MSPD, TC and TG increased respect to MPD and GDM-MPD. eNOS activity was reduced in MSPD and GDM-MSPD, but increased in GDM-MPD compared with MPD. However, decreased tetrahydrobiopterin levels were observed in all groups compared with MPD. Increased hCat1 protein and L-arginine transport were observed in both GDM groups compared with MPD. However, the transport was higher in GDM-MSPD compared to GDM-MPD. Higher Arginase II protein and activity were observed in GDM-MSPD compared with MPD. Thus, MSPD in GDM pregnancies alters fetal endothelial function associated with NO metabolism.

There is a high incidence of radiation enteritis (RE) after abdominal radiotherapy. The occurrence of RE seriously affects the treatment and quality of life of patients; however, its pathogenesis is complex and there are no effective drugs for its prevention or treatment. Intestinal ischemia plays an important role in the occurrence of enteritis. Previous studies have shown that targeting GTP-cyclohydrolase 1 (Gch1) to improve intestinal ischemia could be a new strategy to prevent and treat RE. A high content of the naturally occurring phthalide derivative ligustilide (LIG) has been found in the plant drug Rhizoma Ligustici Chuanxiong for the treatment of cardiovascular diseases. The purpose of this study was to evaluate the protective effects of LIG on RE. Ionizing radiation (IR) rat and endothelial cell models were used to observe and record rat body weights and stool morphologies, measure intestinal blood perfusion by laser Doppler blood flow imaging, determine the diastolic functions of mesenteric arteries, detect the levels of Gch1/BH4/eNOS pathway-related proteins and regulatory molecules in the mesenteric arteries and endothelial cells, and predict affinity by molecular docking technology. The results showed that LIG significantly improved the body weights, loose stools, intestinal villi lengths, intestinal perfusion and vasodilatory functions of IR rats. LIG also significantly improved Gch1 protein and BH4 levels in the mesenteric arteries and endothelial cells after IR, increased the NO content, reduced superoxide accumulation, and improved p-eNOS (Ser1177) levels in endothelial cells. LIG has good affinity for Gch1, which significantly improves its activity. These results indicate that LIG is the preferred compound for the prevention and treatment of RE by improving intestinal ischemia through the Gch1/BH4/eNOS pathway. This study provides a theoretical basis and new research ideas for the development of new drugs for RE.

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies., Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH) and can lead to neurotoxicity. Here the authors describe a mouse model of PKU based on a mutation in phenylalanine hydroxylase (R261Q) which replicates traits of this disease and shows a proteostasis defect and oxidative stress, implying a gain-of-function contribution to the disease phenotype.

The core symptoms of many neurological disorders have traditionally been thought to be caused by genetic variants affecting brain development and function. However, the gut microbiome, another important source of variation, can also influence specific behaviors. Thus, it is critical to unravel the contributions of host genetic variation, the microbiome, and their interactions to complex behaviors. Unexpectedly, we discovered that different maladaptive behaviors are interdependently regulated by the microbiome and host genes in the Cntnap2-/- model for neurodevelopmental disorders. The hyperactivity phenotype of Cntnap2-/- mice is caused by host genetics, whereas the social-behavior phenotype is mediated by the gut microbiome. Interestingly, specific microbial intervention selectively rescued the social deficits in Cntnap2-/- mice through upregulation of metabolites in the tetrahydrobiopterin synthesis pathway. Our findings that behavioral abnormalities could have distinct origins (host genetic versus microbial) may change the way we think about neurological disorders and how to treat them.

Aim Loss of dystrophin causes oxidative stress and affects nitric oxide synthase-mediated vascular function in striated muscle. Because tetrahydrobiopterin is an antioxidant and co-factor for nitric oxide synthase, we tested the hypothesis that tetrahydrobiopterin would be low in mdx mice and humans deficient for dystrophin. Methods Tetrahydrobiopterin and its metabolites were measured at rest and in response to exercise in Duchenne and Becker muscular dystrophy patients, age-matched male controls as well as wild-type, mdx and mdx mice transgenically overexpressing skeletal muscle-specific dystrophins. Mdx mice were also supplemented with tetrahydrobiopterin and pathophysiology was assessed. Results Duchenne muscular dystrophy patients had lower urinary dihydrobiopterin + tetrahydrobiopterin/specific gravity1.020 compared to unaffected age-matched males and Becker muscular dystrophy patients. Mdx mice had low urinary and skeletal muscle dihydrobiopterin + tetrahydrobiopterin compared to wild-type mice. Overexpression of dystrophins that localize neuronal nitric oxide synthase restored dihydrobiopterin + tetrahydrobiopterin in mdx mice to wild-type levels while utrophin overexpression did not. Mdx mice and Duchenne muscular dystrophy patients did not increase tetrahydrobiopterin during exercise and in mdx mice tetrahydrobiopterin deficiency was likely because of lower levels of sepiapterin reductase in skeletal muscle. Tetrahydrobiopterin supplementation improved skeletal muscle strength, resistance to fatiguing and injurious contractions in vivo, increased utrophin and capillary density of skeletal muscle and lowered cardiac muscle fibrosis and left ventricular wall thickness in mdx mice. Conclusion These data demonstrate that impaired tetrahydrobiopterin synthesis is associated with dystrophin loss and treatment with tetrahydrobiopterin improves striated muscle histopathology and skeletal muscle function in mdx mice.

5-Hydroxytryptophan (5-HTP) plays an important role in the regulation of emotion, behavior, sleep, pain, body temperature, and other physiological functions. It is used in the treatment of depression, insomnia, migraine, and other diseases. Due to a lack of effective biosynthesis methods, 5-HTP is mainly obtained by natural extraction, which has been unable to meet the needs of the market. Through the directed evolution of enzymes and the introduction of substrate supply pathways, 5-HTP biosynthesis and yield increase have been realized. This review provides examples that illustrate the production mode of 5-HTP and the latest progress in microbial synthesis.

A timely detection of patients with tetrahydrobiopterin (BH4) -deficient types of hyperphenylalaninemia (HPABH4) is important for assignment of correct therapy, allowing to avoid complications. Often HPABH4 patients receive the same therapy as phenylalanine hydroxylase (PAH) -deficiency (phenylketonuria) patients—dietary treatment—and do not receive substitutive BH4 therapy until the diagnosis is confirmed by molecular genetic means. In this study, we present a cohort of 30 Russian patients with HPABH4 with detected variants in genes causing different types of HPA. Family diagnostics and biochemical urinary pterin spectrum analyses were carried out. HPABH4A is shown to be the prevalent type, 83.3% of all HPABH4 cases. The mutation spectrum for the PTS gene was defined, the most common variants in Russia were p.Thr106Met—32%, p.Asn72Lys—20%, p.Arg9His—8%, p.Ser32Gly—6%. We also detected 7 novel PTS variants and 3 novel QDPR variants. HPABH4 prevalence was estimated to be 0.5–0.9% of all HPA cases in Russia, which is significantly lower than in European countries on average, China, and Saudi Arabia. The results of this research show the necessity of introducing differential diagnostics for HPABH4 into neonatal screening practice.

BACKGROUND: In patients with phenylketonuria, stability of blood phenylalanine and tyrosine concentrations might influence brain chemistry and therefore patient outcome. This study prospectively investigated the effects of tetrahydrobiopterin (BH4), as a chaperone of phenylalanine hydroxylase on diurnal and day-to-day variations of blood phenylalanine and tyrosine concentrations.METHODS: Blood phenylalanine and tyrosine were measured in dried blood spots (DBS) four times daily for 2 days (fasting, before lunch, before dinner, evening) and once daily (fasting) for 6 days in a randomized cross-over design with a period with BH4 and a period without BH4. The sequence was randomized. Eleven proven BH4 responsive PKU patients participated, 5 of them used protein substitutes during BH4 treatment. Natural protein intake and protein substitute dosing was adjusted during the period without BH4 in order to keep DBS phenylalanine levels within target range. Patients filled out a 3-day food diary during both study periods. Variations of DBS phenylalanine and Tyr were expressed in standard deviations (SD) and coefficient of variation (CV).RESULTS: BH4 treatment did not significantly influence day-to-day phenylalanine and tyrosine variations nor diurnal phenylalanine variations, but decreased diurnal tyrosine variations (median SD 17.6 μmol/l, median CV 21.3%, p = 0.01) compared to diet only (median SD 34.2 μmol/l, median CV 43.2%). Consequently, during BH4 treatment diurnal phenylalanine/tyrosine ratio variation was smaller, while fasting tyrosine levels tended to be higher.CONCLUSION: BH4 did not impact phenylalanine variation but decreased diurnal tyrosine and phenylalanine/tyrosine ratio variations, possibly explained by less use of protein substitute and increased tyrosine synthesis.

Preeclampsia (PE) is a multifactorial pregnancy disease, characterized by new-onset gestational hypertension with (or without) proteinuria or end-organ failure, exclusively observed in humans. It is a leading cause of maternal morbidity affecting 3–7% of pregnant women worldwide. PE pathophysiology could result from abnormal placentation due to a defective trophoblastic invasion and an impaired remodeling of uterine spiral arteries, leading to a poor adaptation of utero-placental circulation. This would be associated with hypoxia/reoxygenation phenomena, oxygen gradient fluctuations, altered antioxidant capacity, oxidative stress, and reduced nitric oxide (NO) bioavailability. This results in part from the reaction of NO with the radical anion superoxide (O2•−), which produces peroxynitrite ONOO-, a powerful pro-oxidant and inflammatory agent. Another mechanism is the progressive inhibition of the placental endothelial nitric oxide synthase (eNOS) by oxidative stress, which results in eNOS uncoupling via several events such as a depletion of the eNOS substrate L-arginine due to increased arginase activity, an oxidation of the eNOS cofactor tetrahydrobiopterin (BH4), or eNOS post-translational modifications (for instance by S-glutathionylation). The uncoupling of eNOS triggers a switch of its activity from a NO-producing enzyme to a NADPH oxidase-like system generating O2•−, thereby potentiating ROS production and oxidative stress. Moreover, in PE placentas, eNOS could be post-translationally modified by lipid peroxidation-derived aldehydes such as 4-oxononenal (ONE) a highly bioreactive agent, able to inhibit eNOS activity and NO production. This review summarizes the dysfunction of placental eNOS evoked by oxidative stress and lipid peroxidation products, and the potential consequences on PE pathogenesis., Graphical abstract Image 1, Highlights • Physiological ROS production is enhanced during pregnancy. • eNOS is one of the main target of oxidative stress in PE placenta. • eNOS is S-glutathionylated in PE placentas. • eNOS is modified by lipid oxidation products in PE placentas.

Objectives: The present study describes clinical, biochemical, molecular genetic data, current treatment strategies and follow-up in nine patients with tetrahydrobiopterin (BH4) deficiency due to various inherited genetic defects. Methods: We analyzed clinical, biochemical, and molecular data of nine patients with suspected BH4 deficiency. All patients were diagnosed at Ege University Faculty of Medicine in Izmir, Turkey and comprised data collected from 2006 to 2019. The diagnostic laboratory examinations included blood phenylalanine and urinary or plasma pterins, dihydropteridine reductase (DHPR) enzyme activity measurement in dried blood spots, folic acid and monoamine neurotransmitter metabolites in cerebrospinal fluid, as well as DNA sequencing. Results: Among the nine patients, we identified one with autosomal recessive GTP cyclohydrolase I (ar GTPCH) deficiency, two with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, three with sepiapterin reductase (SR) deficiency, and three with DHPR deficiency. Similar to previous observations, the most common clinical symptoms are developmental delay, intellectual disability, and movement disorders. All patients received treatment with l-dopa and 5-hydroxytryptophan, while only the ar GTPCH, the PTPS, and one DHPR deficient patients were supplemented in addition with BH4. The recommended dose range varies among patients and depends on the type of disease. The consequences of BH4 deficiencies are quite variable; however, early diagnosis and treatment will improve outcomes. Conclusions: As BH4 deficiencies are rare group of treatable neurometabolic disorders, it is essential to diagnose the underlying (genetic) defect in newborns with hyperphenylalaninemia. Irreversible brain damage and progressive neurological deterioration may occur in untreated or late diagnosed patients. Prognosis could be satisfying in the cases with early diagnose and treatment. © 2021 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.