In vivo evaluation in cynomolgus monkey brain and metabolism of [¹⁸F]fluorodeprenyl: a new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO-B) radioligand, [¹⁸F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [¹⁸F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [¹¹C]deprenyl, an established MAO-B radioligand. The highest uptake was observed in the striatum, one of the MAO-B-rich regions, with a peak at approximately 2-3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [¹⁸F]fluorodeprenyl was largely inhibited by preadministration of the MAO-B inhibitor, L-deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO-B. [¹⁸F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [¹¹C]deprenyl. These results suggest that [¹⁸F]fluorodeprenyl is a potential PET radioligand for visualization of MAO-B activity.

Cerebral formation in situ of S-carboxymethylcysteine after ifosfamide administration to mice: a further clue to the mechanism of ifosfamide encephalopathy

The clinical use of the alkylating oxazaphosphorine ifosfamide is hampered by a potentially severe encephalopathy. S-carboxymethylcysteine (SCMC), a metabolite of ifosfamide (IF), activates the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor, causes neuronal acidification, and could thus be responsible for the encephalopathy. Since the presence of SCMC in brain has not been documented following administration of IF, SCMC was measured in the brain of mice following both the individual i.p. administration of IF and SCMC. SCMC was found in a concentration of 108.2 +/- 29.7 nmol/g following IF, but was detectable at much lower levels following the administration of SCMC (21.1 +/- 21.2 nmol/g). Together with the observation that the concentration of SCMC was 10-fold higher in liver than in brain 1h after administration of SCMC, these findings suggest that the SCMC found after IF was formed in the brain in situ. The concentration of glutamic acid was similar in IF and SCMC treated animals. Methylene blue, which is used clinically to treat and to prevent IF encephalopathy, did not decrease the formation of SCMC in brain. By inhibiting monoamine oxidase activity it did, however, markedly increase the concentration of serotonin in brain which could modulate the effects of SCMC on AMPA/kainate receptors. Thus, SCMC is present in brain following the administration of IF and could contribute to the IF-associated encephalopathy by activation of AMPA/kainate receptors.

Comparative aromatic hydroxylation and N-demethylation of MPTP neurotoxin and its analogs, N-methylated beta-carboline and isoquinoline alkaloids, by human cytochrome P450 2D6

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin is a chemical inducer of Parkinson's disease (PD) whereas N-methylated beta-carbolines and isoquinolines are naturally occurring analogues of MPTP involved in PD. This research has studied the oxidation of MPTP by human CYP2D6 (CYP2D6*1 and CYP2D6*10 allelic variants) as well as by a mixture of cytochrome P450s-resembling HLM, and the products generated compared with those afforded by human monoamine oxidase (MAO-B). MPTP was efficiently oxidized by CYP2D6 to two main products: MPTP-OH (p-hydroxylation) and PTP (N-demethylation), with turnover numbers of 10.09 min-1 and Km of 79.36+/-3 microM (formation of MPTP-OH) and 18.95 min-1 and Km 69.6+/-2.2 microM (PTP). Small amounts of dehydrogenated toxins MPDP+ and MPP+ were also detected. CYP2D6 competed with MAO-B for the oxidation of MPTP. MPTP oxidation by MAO-B to MPDP+ and MPP+ toxins (bioactivation) was up to 3-fold higher than CYP2D6 detoxification to PTP and MPTP-OH. Several N-methylated beta-carbolines and isoquinolines were screened for N-demethylation (detoxification) that was not significantly catalyzed by CYP2D6 or the P450s mixture. In contrast, various beta-carbolines were efficiently hydroxylated to hydroxy-beta-carbolines by CYP2D6. Thus, N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline (a close MPTP analog) was highly hydroxylated to 6-hydroxy-N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline and a corresponding 7-hydroxy-derivative. Thus, CYP2D6 could participate in the bioactivation and/or detoxification of these neuroactive compounds by an active hydroxylation pathway. The CYP2D6*1 enzymatic variant exhibited much higher metabolism of both MPTP and N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline than the CYP2D6*10 variant, highlighting the importance of CYP2D6 polymorphism in the oxidation of these toxins. Altogether, these results suggest that CYP2D6 can play an important role in the metabolic outcome of both MPTP and beta-carbolines.

Effects of opioids on human serotonin transporters.

The serotonin (5-hydroxtryptamine, 5-HT) system plays a role in analgesia and emesis. The aim of this study was to test whether opioids or ketamine inhibit the human 5-HT transporter and whether this increases free plasma 5-HT concentrations. HEK293 cells, stably transfected with the human 5-HT transporter cDNA, were incubated with morphine, hydromorphone, fentanyl, alfentanil, pethidine (meperidine), tramadol, ketamine, and the reference substance citalopram (specific 5-HT transporter inhibitor). The uptake of [(3)H]5-HT was measured by liquid scintillation counting. In a second series of experiments, study drugs were incubated with plasma of ten healthy blood donors and change of 5-HT plasma-concentrations were measured (ELISA). The end point was the inhibition of the 5-HT transporter by different analgesics either in HEK293 cells or in human platelets ex vivo. Tramadol, pethidine, and ketamine suppressed [(3)H]5-HT uptake dose-dependently with an IC50 of 1, 20.9, and 230 μM, respectively. These drugs also prevented 5-HT uptake in platelets with an increase in free plasma 5-HT. Free 5-HT concentrations in human plasma were increased by citalopram 1 μM, tramadol 20 μM, pethidine 30 μM, and ketamine 100 μM to 280 [248/312]%, 269 [188/349]%, and 149 [122/174]%, respectively, compared to controls without any co-incubation (means [95 % CI]; all p < 0.005). No change in both experimental settings was observed for the other opioids. Tramadol and pethidine inhibited the 5-HT transporter in HEK293 cells and platelets. This inhibition may contribute to serotonergic effects when these opioids are given in combination, e.g., with monoamine oxidase inhibitors or selective serotonin reuptake inhibitors.

Lysyl oxidase expression is an independent marker of prognosis and a predictor of lymph node metastasis in oral and oropharyngeal squamous cell carcinoma (OSCC)

Proteins of the lysyl oxidase (LOX) family are important modulators of the extracellular matrix. However, they have an important role in the tumour development as well as in tumour progression. To evaluate the diagnostic and prognostic value of the LOX protein in oral and oropharyngeal squamous cell carcinoma (OSCC) we performed QRT-PCR and immunohistochemical analysis on two tissue microarrays (622 tissue samples in total). Significantly higher LOX expression was detected in high grade dysplastic oral mucosa as well as in OSCC when compared to normal oral mucosa (P < 0.001). High LOX expression was correlated with clinical TNM stage (P = 0.020), lymph node metastases for the entire cohort (P < 0.001), as well as in the subgroup of small primary tumours (T1/T2, P < 0.001). Moreover, high LOX expression was correlated with poor overall survival (P = 0.004) and disease specific survival (P = 0.037). In a multivariate analysis, high LOX expression was an independent prognostic factor, predicting unfavourable overall survival. In summary, LOX expression is an independent prognostic biomarker and a predictor of lymph node metastasis in OSCC. Moreover, LOX overexpression may be an early phenomenon in the pathogenesis of OSCC and thus an attractive novel target for chemopreventive and therapeutic strategies.

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Identification of the bovine Arachnomelia mutation by massively parallel sequencing implicates sulfite oxidase (SUOX) in bone development

Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a approximately 7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development.

Mycophenolate acid inhibits endothelial NAD(P)H oxidase activity and superoxide formation by a Rac1-dependent mechanism

Endothelial dysfunction precedes hypertension and atherosclerosis and predicts cardiac allograft vasculopathy and death in heart transplant recipients. Endothelial overproduction of reactive oxygen species, such as superoxide anions produced by NAD(P)H oxidase, induces endothelial dysfunction. Because immunosuppressive drugs have been associated with increased reactive oxygen species production and endothelial dysfunction, we sought to elucidate the underlying mechanisms. Reactive oxygen species, release of superoxide anions, and NAD(P)H oxidase activity were studied in human umbilical vein endothelial cells and in polymorphonuclear neutrophils. Gp91ds-tat was used to specifically block NAD(P)H oxidase. Transcriptional activation of different subunits of NAD(P)H oxidase was assessed by real-time RT-PCR. Rac1 subunit translocation and activation were studied by membrane fractionation and pull-down assays. Calcineurin inhibitors significantly increased endothelial superoxide anions production because of NAD(P)H oxidase, whereas mycophenolate acid (MPA) blocked it. MPA also attenuated the respiratory burst induced by neutrophil NAD(P)H oxidase. Because transcriptional activation of NAD(P)H oxidase was not affected, but addition of guanosine restored endothelial superoxide anions formation after MPA treatment, we speculate that the inhibitory effect of MPA was mediated by depletion of cellular guanosine triphosphate content. This prevented activation of Rac1 and, thus, of endothelial NAD(P)H oxidase. Because all heart transplant recipients are at risk for cardiac allograft vasculopathy development, these differential effects of immunosuppressants on endothelial oxidative stress should be considered in the choice of immunosuppressive drugs.

Nitric oxide down-regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells

Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS-generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO-releasing compound DETA-NO in a concentration- and time-dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL-1beta on nox1 transcription is counteracted by an inhibitory effect of IL-1beta-evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA-NO attenuated platelet-derived growth factor (PDGF)-BB or serum stimulated production of superoxide as assessed by real-time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine-tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs. mycoides fail to generate neutralizing antibodies and succumb to disease on challenge

The membrane-associated enzyme L-α-glycerol-3-phosphate oxidase (GlpO) of Mycoplasma mycoides subs. mycoides (Mmm), the causal agent of contagious bovine pleuropneumonia (CBPP) has been identified as a virulence factor responsible for the release of toxic by-products such as H2O2 that mediate host cell injury. Since CBPP pathogenesis is based on host inflammatory reactions, we have determined the capacity of recombinant GlpO to generate in vivo protective responses against challenge in immunized cattle. We also investigated whether sera raised against recombinant GlpO in cattle and mice inhibit production of H2O2 by Mmm. Immunization of cattle with recombinant GlpO did not protect against challenge with a virulent strain of Mmm. Further, although both murine and bovine antisera raised against recombinant GlpO detected recombinant and native forms of GlpO in immunoblot assays with similar titres, only murine antibodies could neutralize GlpO enzymatic function. The data raise the possibility that Mmm has adapted to evade potential detrimental antibody responses in its definitive host.

NADPH oxidase-independent formation of extracellular DNA traps by basophils

Basophils are primarily associated with a proinflammatory and immunoregulatory role in allergic diseases and parasitic infections. Recent studies have shown that basophils can also bind various bacteria both in the presence and the absence of opsonizing Abs. In this report, we show that both human and mouse basophils are able to produce mitochondrial reactive oxygen species and to form extracellular DNA traps upon IL-3 priming and subsequent activation of the complement factor 5 a receptor or FcεRI. Such basophil extracellular traps (BETs) contain mitochondrial, but not nuclear DNA, as well as the granule proteins basogranulin and mouse mast cell protease 8. BET formation occurs despite the absence of any functional NADPH oxidase in basophils. BETs can be found in both human and mouse inflamed tissues, suggesting that they also play a role under in vivo inflammatory conditions. Taken together, these findings suggest that basophils exert direct innate immune effector functions in the extracellular space.

Mutation of a single residue in the ba 3 oxidase specifically impairs protonation of the pump site

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative "pump site" was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.