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Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host-pathogen interactions.

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.

Tetrahydrobiopterin (BH4) and 4-amino-tetrahydrobiopterin (ABH4) prevent acute rejection after solid organ transplantation. Moreover, BH4 also attenuates ischemia-reperfusion injury (IRI). The mechanisms underlying these protective effects are poorly defined. Activation of intracellular signaling proteins, including the mitogen-activated protein kinases (MAPKs) ERK, p38, and JNK, and the excessive production of mitochondrial reactive oxygen species (ROS) are observed mainly during early reperfusion. While the role of ROS in the initiation and progression of IRI is well understood, the contribution of individual signaling pathways is less clear. Here, we tested the potential effects of BH4 and ABH4 on MAPK activity and mitochondrial ROS levels. During hypoxia and reoxygenation (H/R), all three MAPKs were activated during early reoxygenation in cardiomyocytes and endothelial cells. p38 and JNK activation were further augmented by BH4 and ABH4, whereas ERK activation was not affected. Pretreatment with BH4 and ABH4 reduced the basal mitochondrial ROS levels as well as the H/R-induced increase in ROS. Prolonged incubation with ABH4, however, showed pro-apoptotic effects in cardiomyocytes. These data suggest that a protective effect of BH4 and ABH4 pretreatment may be attributed mainly to their antioxidant capacity. The effects on intracellular signaling are complex and warrant further investigations.

Alkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli. Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation.

Ischemia-reperfusion injury is a primarily non-allospecific event leading to the depletion of the essential nitric oxide synthase cofactor and potent antioxidant tetrahydrobiopterin. Suboptimal concentrations of tetrahydrobiopterin result in a reduced biosynthesis of nitric oxide leading to vascular endothelial dysfunction. Tetrahydrobiopterin supplementation has been shown to protect from this pathological state in a plethora of cardiovascular diseases including transplant-related ischemia-reperfusion injury. Even though still controversially discussed, there is increasing evidence emerging from both human as well as animal studies that tetrahydrobiopterin-mediated actions rely on its nitric oxide synthase cofactor activity rather than on its antioxidative properties. Herein, we review the current literature regarding the role of tetrahydrobiopterin in ischemia-reperfusion injury including our experience acquired in a murine pancreas transplantation model.

Alkylglycerol monooxygenase is a tetrahydrobiopterin-dependent enzyme that cleaves the O-alkyl-bond of alkylglycerols. It is an exceptionally unstable, hydrophobic membrane protein which has never been purified in active form. Recently, we were able to identify the sequence of alkylglycerol monooxygenase. TMEM195, the gene coding for alkylglycerol monooxygenase, belongs to the fatty acid hydroxylases, a family of integral membrane enzymes which have an 8-histidine motif crucial for catalysis. Mutation of each of these residues resulted in a complete loss of activity. We now extended the mutational analysis to another 25 residues and identified three further residues conserved throughout all members of the fatty acid hydroxylases which are essential for alkylglycerol monooxygenase activity. Furthermore, mutation of a specific glutamate resulted in an 18-fold decreased affinity of the protein to tetrahydrobiopterin, strongly indicating a potential important role in cofactor interaction. A glutamate residue in a comparable amino acid surrounding had already been shown to be responsible for tetrahydrobiopterin binding in the aromatic amino acid hydroxylases. Ab initio modelling of the enzyme yielded a structural model for the central part of alkylglycerol monooxygenase where all essential residues identified by mutational analysis are in close spatial vicinity, thereby defining the potential catalytic site of this enzyme.

The tetrahydrobiopterin-dependent degradation of ether lipids by alkylglycerol monooxygenase (AGMO) produces fatty aldehydes, which are toxic to cells. Therefore, it is of great physiological importance that these harmful compounds are converted into their corresponding, less toxic fatty acids by fatty aldehyde dehydrogenase (FALDH). Dysfunction of this enzyme causes Sjögren-Larsson syndrome. This severe inherited disorder is accompanied by symptoms such as ichthyosis, mental retardation and spasticity. Surprisingly, fatty alcohols and not fatty aldehydes were found to accumulate in fibroblasts of Sjögren-Larsson syndrome patients, suggesting that there can be wide-ranging alterations in the lipid composition of patient cells. In particular, this has to be considered when searching for possible treatment options for patients suffering from Sjögren-Larsson syndrome. For example, inhibition of fatty aldehyde producing ether lipid degradation would have multiple implications on ether lipid- and fatty alcohol-mediated signalling pathways.

We investigated a possible involvement of nitric oxide formed by inducible nitric oxide synthase (iNOS) in the signaling cascade leading to growth inhibition and differentiation in the human neuroblastoma cell line SK-NSII. Treatment of SK-N-SH with interferon-γ (IFN-γ) plus interleukin-lß (IL-lß) led to induction of iNOS, growth inhibition and an altered cell shape. However two inhibitors of iNOS were not able to prevent cytokine induced changes. In addition, IFN-γ alone led to growth inhibition in absence of iNOS induction. Inhibition of the induced indoleamine 2,3-dioxygenase (IDO) activity also did not prevent growth inhibition. Our findings show that mechanisms other than NO and IDO can control interferon-y-induced growth inhibition of SK-N-SH cells.

The rabbit pyrogen assay identifies pyrogenic contaminations in drugs i.l1tend~d for parenteral use. In order to replace this test because of ethical and economical reasons, various in vitro methods have been developed in recent years. Here, we summarize our results on optimizing a test based on using interferony- treated monocytoid cell lines from man (THP-l) and mouse (RAW264.7). The read-out of the test is neopterin or nitrite, respectively, which is released into the supernatant in response to bacterial compounds. These are costimuli of the enzymes involved in neopterin and nitrite formation, i.e. GTP cyclohydrolase I and inducible nitric oxide synthase. The test reproducibly detects cell wall components from Gram-negative as well as from Gram-positive bacteria and mycobacteria. Furthermore, DNA and cell-free supernatants containing a number of bioactive but not further characterized proteins, both from Staphylococcus aureus, can be detected. Thus, this test is superior over the only in vitro alternative accepted in certain cases, namely the limulus amebocyte lysate assay. Results obtained by measuring neopterin or nitrite correlate well with formation of the endogenous pyrogen tumor necrosis factor-a, a read-out used by some other cell-based in vitro alternatives to the rabbit pyrogen test. We therefore think that the assay presented here has the potential to reduce or even replace the animal test.

Urine concentrations of neopterin, a marker for the activation of cellular immunity, were determined concomitantly with a large panel of hematological and immunological variables on 24 women with gynaecological cancers (12 uterine cervix, 5 endometrium, 7 ovary). There were 14 women investigated at first diagnosis of carcinoma, and 10 women were studied during follow-up (evidence of disease, only palliative treatment during the preceding three months). A statistically significant correlation was found between neopterin concentrations and number of circulating non-B lymphocytes expressing HLA-DR antigen. Neopterin concentrations, numbers of HLA-DR positive cells, and numbers of circulating leukocytes were significantly higher in women studied during follow-up, when compared with women freshly diagnosed. In contrast. numbers of CD4 +, CD3 +, CD19 + lymphocytes and of total lymphocytes in peripheral blood were significantly lower in women studied during follow-up. The results suggest that high neopterin levels in tumor patients indicate activation of early steps of cellular immune mechanisms.

Elevated production and excretion of neopterin indicate activation of cell-mediated immunity, since neopterin is produced in large quantities by human macrophages after stimulation with interferon gamma. In 21 patients, aged 9 to 19 years, with Crohn’s disease, urinary neopterin was measured on a total of 135 consecutive followup visits. Additionally, a large panel of anamnestic, clinical and laboratory data were obtained at every visit. Both the first visit data set (21 records) and the complete data set (135 records) were analyzed separately. As was previously demonstrated in adults with Crohn’s disease, neopterin excretion proved to be strongly correlated with overall rating of disease activity, and also with separate anamnestic, clinical and laboratory activity scores. Multiple regression analysis demonstrated that neopterin provides statistically independent information. Thus, measurement of neopterin concentrations as a marker for activation of cellular immune phenomena could aid in the monitoring of pediatric and juvenile Crohn’s disease.

Nitric oxide (NO) synthase has been found to be induced in Physarum polycephalum sporulation. P. polycephalum expresses a calcium-independent NO synthase like the inducible NO synthase (NOS) isoenzyme in mammals. It has been cloned and sequnced and is the first nonanimal NOS identified. Less than 39% of its amino acids are identical to those of known NOSs. The second messenger used for NO siglnaling in sporulation is cGMP. It links the signaling pathway to expression of lig1.

The effective collection and management of personal data of rapidly migrating populations is important for ensuring adequate healthcare and monitoring of a displaced peoples' health status. With developments in ICT data sharing capabilities, electronic personal health records (ePHRs) are increasingly replacing less transportable paper records. ePHRs offer further advantages of improving accuracy and completeness of information and seem tailored for rapidly displaced and mobile populations. Various emerging initiatives in Europe are seeking to develop migrant-centric ePHR responses. This paper highlights their importance and benefits, but also identifies a number of significant ethical, legal and social issues (ELSI) and challenges to their design and implementation, regarding (1) the kind of information that should be stored, (2) who should have access to information, and (3) potential misuse of information. These challenges need to be urgently addressed to make possible the beneficial use of ePHRs for vulnerable migrants in Europe. [ABSTRACT FROM AUTHOR]

To compare the involvement of cellular immunity in response to vaccination we have investigated urinary neopterin levels in daily follow-ups of children after vaccination with live measles/mumps vaccine and of adults after boosting with the soluble antigen tetanus toxoid. Neopterin levels distinctly peaked 8 - 11 days after vaccination with measles/mumps vaccine. In contrast, after boosting with soluble antigen tetanus toxoid neopterin levels remained unaffected. Large amounts of neopterin are produced by human monocytes/ macro phages on stimulation with gamma interferon. In patients neopterin concentrations reflect activation of cell mediated immunity. The data imply that distinct pathways of T cell activation are triggered in humans after immunization with live vaccine and with soluble antigen .