Biomedical subjects

Jean-Luc Boucher

Publications and source records attributed to Jean-Luc Boucher.

At least 19 recordsLinked to original sources

PubMed2006

Analogies and surprising differences between recombinant nitric oxide synthase-like proteins from Staphylococcus aureus and Bacillus anthracis in their interactions with l-arginine analogs and iron ligands.

Genome sequencing has recently shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles of these proteins remain unclear. The interactions of a series of l-arginine (l-arg) analogs and iron ligands with two recombinant NOS-like proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) have been studied by UV-visible spectroscopy. SaNOS and baNOS in their ferric native state, as well as their complexes with l-arg analogs and with various ligands, exhibit spectral characteristics highly similar to the corresponding complexes of heme-thiolate proteins such as cytochromes P450 and NOSs. However, saNOS greatly differs from baNOS at the level of three main properties: (i) native saNOS mainly exists under an hexacoordinated low-spin ferric state whereas native baNOS is mainly high-spin, (ii) the addition of tetrahydrobiopterin (H4B) or H4B analogs leads to an increase of the affinity of l-arg for saNOS but not for baNOS, and (iii) saNOS Fe(II), contrary to baNOS, binds relatively bulky ligands such as nitrosoalkanes and tert-butylisocyanide. Thus, saNOS exhibits properties very similar to those of the oxygenase domain of inducible NOS (iNOS(oxy)) not containing H4B, as expected for a NOSoxy-like protein that does not contain H4B. By contrast, the properties of baNOS which look like those of H4B-containing iNOS(oxy) are unexpected for a NOS-like protein not containing H4B. The origin of these surprising properties of baNOS remains to be determined.

Arginine↗

PubMed2006

Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites.

Although primary and memory responses against bacteria and viruses have been studied extensively, T helper type 2 (T(H)2) effector mechanisms leading to host protection against helminthic parasites remain elusive. Examination of the intestinal epithelial submucosa of mice after primary and secondary infections by a natural gastrointestinal parasite revealed a distinct immune-cell infiltrate after challenge, featuring interleukin-4-expressing memory CD4(+) T cells that induced IL-4 receptor(hi) (IL-4R(hi)) CD206(+) alternatively activated macrophages. In turn, these alternatively activated macrophages (AAMacs) functioned as important effector cells of the protective memory response contributing to parasite elimination, demonstrating a previously unknown mechanism for host protection against intestinal helminths.

Adoptive Transfer↗

PubMed2006

Differential effects of alkyl- and arylguanidines on the stability and reactivity of inducible NOS heme-dioxygen complexes.

NO-Synthases are heme proteins that catalyze the oxidation of L-arginine into NO and L-citrulline. Some non-amino acid alkylguanidines may serve as substrates of inducible NOS (iNOS), while no NO* production is obtained from arylguanidines. All studied guanidines induce uncoupling between electrons transferred from the reductase domain and those required for NO formation. This uncoupling becomes critical with arylguanidines, leading to the exclusive formation of superoxide anion O2*- as well as hydrogen peroxide H2O2. To understand these different behaviors, we have conducted rapid scanning stopped-flow experiments with dihydrobiopterin (BH2) and tetrahydrobiopterin (BH4) to study, respectively, the (i) autoxidation and (ii) activation processes of heme ferrous-O2 complexes (Fe(II)O2) in the presence of eight alkyl- and arylguanidines. The Fe(II)O2 complex is more easily autooxidized by alkylguanidines (10-fold) and arylguanidines (100-fold) compared to L-arginine. In the presence of alkylguanidines and BH4, the oxygen-activation kinetics are very similar to those observed with L-arginine. Conversely, in the presence of arylguanidines, no Fe(II)O2 intermediate is detected. To understand such variations in reactivity and stability of Fe(II)O2 complex, we have characterized the effects of alkyl- and arylguanidines on Fe(II)O2 structure using the Fe(II)CO complex as a mimic. Resonance Raman and FTIR spectroscopies show that the two classes of guanidine derivatives induce different polar effects on Fe(II)CO environment. Our data suggest that the structure of the substituted guanidine can modulate the stability and the reactivity of heme-dioxygen complexes. We thus propose differential mechanisms for the electron- and proton-transfer steps in the NOS-dependent, oxygen-activation process, contingent upon whether alkyl- or arylguanidines are bound.

Animals↗

PubMed2006

Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates.

Single turnover reactions of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) in the presence of several non alpha-amino acid N-hydroxyguanidines and guanidines were studied by stopped-flow visible spectroscopy, and compared with reactions using the native substrates L-arginine (L-arg) or N(omega)-hydroxy-L-arginine (NOHA). In experiments containing dihydrobiopterin, a catalytically incompetent pterin, and each of the studied substrates, L-arg, butylguanidine (BuGua), para-fluorophenylguanidine (FPhGua), NOHA, N-butyl- and N-(para-fluorophenyl)-N'-hydroxyguanidines (BuNOHG and FPhNOHG), the formation of a iron(II) heme-dioxygen intermediate (Fe(II)O2) was always observed. The Fe(II)O2 species then decayed to iron(III) iNOSoxy at rates that were dependent on the nature of the substrate. Identical reactions containing the catalytically competent cofactor tetrahydrobiopterin (BH4), iNOSoxy and the three N-hydroxyguanidines, all exhibited an initial formation of an Fe(II)O2 species that was successively converted to an Fe(III)NO complex and eventually to high-spin iron(III) iNOSoxy. The formation and decay kinetics of the Fe(III)NO complex did not vary greatly as a function of the N-hydroxyguanidine structure, but the formation of Fe(III)NO was substoichiometric in the cases of BuNOHG and FPhNOHG. Reactions between BH4-containing iNOSoxy and BuGua exhibited kinetics similar to those of the corresponding reaction with L-arginine, with formation of an Fe(II)O2 intermediate that was directly converted to high-spin iron(III) iNOSoxy. In contrast, no Fe(II)O2 intermediate was observed in the reaction of BH4-containing iNOSoxy and FPhGua. Multi-turnover reaction of iNOS with FPhGua did not lead to formation of NO or to hydroxylation of the substrate, contrary to reactions with BuGua or L-arg. Our results reveal how different structural and chemical properties of NOS substrate analogues can impact on the kinetics and reactivity of the Fe(II)O2 intermediate, and support an important role for substrate pKa during NOS oxygen activation.

Animals↗

PubMed2005

Dual oxidase-2 has an intrinsic Ca2+-dependent H2O2-generating activity.

Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.

Animals↗

PubMed2005

Nomega-hydroxy-L-arginine homologues and hydroxylamine as nitric oxide-dependent vasorelaxant agents.

Endothelium-independent relaxant activities of N(omega)-hydroxy-L-arginine (L-NOHA) homologues and hydroxylamine, a possible intermediate in nitric oxide (NO) formation, were examined in rat aortic rings. Addition of one -CH(2)- group to the -(CH(2))(x)- chain between the alpha-amino acid and the hydroxyguanidine group (x=4) almost abolished-while deletion of one or two -CH(2)- (x=1 or 2) enhanced-the relaxant activity of L-NOHA homologues. N(omega)-hydroxy-nor-L-arginine- (x=2) and hydroxylamine-induced relaxations were blunted by a NO scavenger and by inhibitors of the guanylyl cyclase pathway, but not by NO synthase or cytochrome P(450) inhibitors (except 7-ethoxyresorufin). However, aortic NO formation was detected (using electron paramagnetic resonance) in the presence of concentrations of these compounds higher than those producing relaxation. These findings support the view that endothelium-independent vasorelaxations induced by both L-NOHA homologues with a required chain length x</=3 and hydroxylamine are mediated by NO-dependent activation of guanylyl cyclase, through a 7-ethoxyresorufin-inhibited mechanism.

Animals↗

PubMed2005

Putative role of nitric oxide synthase isoforms in the changes of nitric oxide concentration in rat brain cortex and cerebellum following sevoflurane and isoflurane anaesthesia.

We have previously observed an increase in nitric oxide (NO) content in rat brain cortex following halothane, sevoflurane or isoflurane anaesthesia. This study was undertaken in order to determine whether isoform-specific nitric oxide synthase (NOS) inhibitors and inducers could modify these increases in NO contents. Rats were subjected to isoflurane and sevoflurane anaesthesia with concomitant administration of neuronal nitric oxide synthase (nNOS) inhibitor 7-Nitro-indazole (7-NI), inducible nitric oxide synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) or lipopolysaccharide. NO concentration in different organs was measured by electron paramagnetic resonance (EPR) spectroscopy. 7-NI significantly decreased NO concentration in cerebellum but not in brain cortex, whereas AMT decreased NO in all the organs studied. Anaesthesia significantly increased NO concentration in brain cortex and decreased that in cerebellum. AMT abolished the NO increase in brain cortex. Anaesthesia enhanced the drastic increase in NO concentration in brain cortex after intraventricular lipopolysaccharide administration. Isoflurane was found to inhibit recombinant nNOS and iNOS activities at high concentrations (EC50=20 mM). Our data suggest a putative role for iNOS in the increase in NO levels produced by isoflurane and sevoflurane, whereas nNOS activity is probably inhibited during anaesthesia.

Anesthetics, Inhalation↗

PubMed2005

Synthesis and evaluation of new omega-borono-alpha-amino acids as rat liver arginase inhibitors.

Recent studies have demonstrated that arginase plays important roles in pathologies such as asthma or erectile dysfunctions. We have synthesized new omega-borono-alpha-amino acids that are analogues of the previously known arginase inhibitors S-(2-boronoethyl)-l-cysteine (BEC) and 2-amino-6-boronohexanoic acid (ABH) and evaluated them as inhibitors of purified rat liver arginase (RLA). In addition to the distance between the B(OH)(2) and the alpha-amino acid functions, the position of the sulfur atom in the side chain also appears as a key determinant for the interaction with the active site of RLA. Furthermore, substitution of the alkyl side chain of BEC by methyl groups and conformational restriction of ABH by incorporation of its side chain in a phenyl ring led to inactive compounds. These results suggest that subtle interactions govern the affinity of inhibitors for the active site of RLA.

Amino Acids↗

PubMed2005

New 2-bromomethyl-8-substituted-benzo[c]chromen-6-ones. Synthesis and biological properties.

New 2-bromomethyl-8-substituted-benzo[c]chromen-6-ones have been synthesized and their bioactive properties have been evaluated on different enzymatic models: serine proteases (trypsin and alpha-chymotrypsin), HIV aspartyl protease, nitric oxide synthase and a panel of protein kinases. These new derivatives can provide upon chemical or enzymatic attack, very reactive quinonimine methide intermediates, which could be utilized for the design of enzyme inhibitors. We found that some of these new derivatives exhibit modest inhibitory activities on the studied enzyme models, but it could be improved after structure optimization.

Aspartic Acid Endopeptidases↗

PubMed2005

Spectroscopic investigation of isonitrile complexes of ferric and ferrous microperoxidase 8.

Microperoxidase 8 (MP8) is able to react with alkyl- and aryl-isonitriles (RNC) both in its reduced and oxidized states, to form MP8Fe(II)- and MP8Fe(III)-CNR complexes. The coordination and spin states of these complexes have been fully characterized by UV-visible and resonance Raman spectroscopies. Both MP8Fe(II)- and MP8Fe(III)-CNR complexes are hexacoordinate low-spin complexes, which bear a single RNC ligand on the distal face of the heme and keep the His 18 ligand on its proximal face, trans to the RNC ligand. A comparison of these characteristics with those of the Fe-CNR complexes of other hemoproteins suggests that both MP8Fe(II)- and MP8Fe(III)-CNR complexes present a Fe-C-N linear arrangement. This may be due to the lack of any interactions of the RNC ligand with the octapeptide of MP8 that is mainly located over the opposite face of the heme. Finally the formation of hexacoordinate low-spin MP8Fe(II)- and MP8Fe(III)-CNR complexes constitutes a new example of the reactivity of MP8 with a new class of weak sigma-donating and strong pi-accepting ligands, which adds to its already very rich coordination chemistry.

Animals↗

PubMed2005

Relationship between the structure of guanidines and N-hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS-catalysed oxidation to NO.

The binding of several alkyl- and aryl-guanidines and N-hydroxyguanidines to the oxygenase domain of inducible NO-synthase (iNOS(oxy)) was studied by UV/Vis difference spectroscopy. In a very general manner, monosubstituted guanidines exhibited affinities for iNOS(oxy) that were very close to those of the corresponding N-hydroxyguanidines. The highest affinities were observed for the natural substrates, L-arginine and N(omega)-hydroxy-L-arginine (K(d) at the microm level). The deletion of either the CO2H or the NH2 function of their amino acid moiety led to dramatic decreases in the affinity. However, alkylguanidines with a relatively small alkyl chain exhibited interesting affinities, the best being observed for a butyl chain (K(d) =20 microM). Arylguanidines also bound to iNOS(oxy), however, with lower affinities (K(d) > 250 microm). Many N-alkyl- and N-aryl-N'-hydroxyguanidines are oxidized by iNOS with formation of NO, whereas only few alkylguanidines led to significant production of NO under identical conditions, and all the arylguanidines tested to date were unable to lead to the production of NO. The k(cat) values of NO production from the oxidation by iNOS of the studied N-hydroxyguanidines were found to vary independently of their affinity for the protein. The k(cat) values determined for the two-step oxidation of alkylguanidines to NO were not clearly related to the K(d) of these substrates toward iNOS(oxy). However, there is a qualitative relationship between these k(cat) values and the apparent rate constants of dissociation of the complex between iNOS(oxy) and the corresponding N-alkyl-N'-hydroxyguanidine (k(off) (app)) that were determined by stopped-flow UV/Vis spectroscopy. These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation. This explains why 4,4,4-trifluorobutylguanidine is so far the best non-alpha-amino acid guanidine substrate of iNOS with formation of NO, because the k(off) (app) of the corresponding N-hydroxyguanidine is particularly low. This suggests that the rational design of guanidines as new NO donors upon in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine but also with the corresponding N-hydroxyguanidine.

Binding Sites↗

PubMed2004

Alternative nitric oxide-producing substrates for NO synthases.

Nitric oxide (NO) is a key inter- and intracellular molecule involved in the maintenance of vascular tone, neuronal signaling, and host response to infection. The biosynthesis of NO in mammals involves a two-step oxidation of L-arginine (L-Arg) to citrulline and NO catalyzed by a particular class of heme-thiolate proteins, called NO-synthases (NOSs). The NOSs successively catalyze the Nomega-hydroxylation of the guanidine group of L-Arg with formation of Nomega-hydroxy-L-arginine (NOHA) and the oxidative cleavage of the CN(OH) bond of NOHA with formation of citrulline and NO. During the last decade, a great number of compounds bearing a CNH or CNOH function have been synthesized and studied as possible NO-producing substrates of recombinant NOSs. This includes derivatives of L-Arg and NOHA, N-alkyl (or aryl) guanidines, N,N'- or N,N-disubstituted guanidines, N-alkyl (or aryl) N'-hydroxyguanidines, N- (or O-) disubstituted N'-hydroxyguanidines, as well as amidoximes, ketoximes, and aldoximes. However, only those involving the NHC(NH2)=NH (or NOH) moiety have led to a significant formation of NO. All the N-monosubstituted N'-hydroxyguanidines that are well recognized by the NOS active site lead to NO with catalytic efficiences (kcat/Km) up to 50% of that of NOHA. This is the case of many N-aryl and N-alkyl N'-hydroxyguanidines, provided that the aryl or alkyl substituent is small enough to be accommodated by a NOS hydrophobic site located in close proximity of the NOS "guanidine binding site." As far as N-substituted guanidines are concerned, few compounds bearing a small alkyl group have been found to act as NO-producing substrates. The kcat value found for the best compound may reach 55% of the kcat of L-Arg oxidation. However, the best catalytic efficiency (kcat/Km) that was obtained with N-(4,4,4-trifluorobutyl) guanidine is only 100-fold lower than that of L-Arg. In a general manner, NOS II is a better catalyst that NOS I and III for the oxidation of exogenous guanidines and N-hydroxyguanidines to NO. This is particularly true for guanidines as the ones acting as substrates for NOS II have been found to be almost inactive for NOS I and NOS III. Thus, a good NO-producing guanidine substrate for the two latter isozymes remains to be found.

Alkylation↗

PubMed2004

Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Animals↗

PubMed2004

Inhibitor coordination interactions in the binuclear manganese cluster of arginase.

Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. The structure and stability of the binuclear manganese cluster are critical for catalytic activity as it activates the catalytic nucleophile, metal-bridging hydroxide ion, and stabilizes the tetrahedral intermediate and its flanking states. Here, we report X-ray structures of a series of inhibitors bound to the active site of arginase, and each inhibitor exploits a different mode of coordination with the Mn(2+)(2) cluster. Specifically, we have studied the binding of fluoride ion (F(-); an uncompetitive inhibitor) and L-arginine, L-valine, dinor-N(omega)-hydroxy-L-arginine, descarboxy-nor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid. Some inhibitors, such as fluoride ion, dinor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid, cause the net addition of one ligand to the Mn(2+)(2) cluster. Other inhibitors, such as descarboxy-nor-N(omega)-hydroxy-L-arginine, simply displace the metal-bridging hydroxide ion of the native enzyme and do not cause any net change in the metal coordination polyhedra. The highest affinity inhibitors displace the metal-bridging hydroxide ion (and sometimes occupy a Mn(2+)(A) site found vacant in the native enzyme) and maintain a conserved array of hydrogen bonds with their alpha-amino and -carboxylate groups.

Animals↗

PubMed2004

Endothelium- and nitric oxide-dependent vasorelaxing activities of gamma-butyrobetaine esters: possible link to the antiischemic activities of mildronate.

Mildronate [3-(2,2,2-trimethylhydrazine) propionate (THP)] is an antiischemic drug acting mainly via inhibition of fatty acid beta-oxidation. Some effects of the drug cannot be explained by the latter mechanism. We tested the eventual nitric oxide (NO) dependence of the mildronate action. Mildronate, gamma-butyrobetaine (GBB) and GBB methyl ester induced transient increases in nitric oxide (NO) concentrations in rat blood and myocardium. In vitro, these compounds neither modified the activities of purified neuronal and endothelial recombinant nitric oxide synthases (NOSs) nor were able to interact with their active site. GBB induced vasodilatation at high concentrations only (EC50 = 5 x 10(-5) M) while mildronate alone displayed no vasodilating effect although it enhanced the GBB vasodilating activity. GBB methyl and ethyl esters were found more potent vasodilators (EC50 = 2.5 x 10(-6) M). Pretreatment of aortic rings with NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) abolished vasodilating effects of the compounds. A hypothesis explaining NO and endothelium-dependent effects of mildronate and its analogues is proposed.

Animals↗

PubMed2004

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin.

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.

Antioxidants↗

PubMed2004

Mouse strain susceptibility to trypanosome infection: an arginase-dependent effect.

We previously reported that macrophage arginase inhibits NO-dependent trypanosome killing in vitro and in vivo. BALB/c and C57BL/6 mice are known to be susceptible and resistant to trypanosome infection, respectively. Hence, we assessed the expression and the role of inducible NO synthase (iNOS) and arginase in these two mouse strains infected with Trypanosoma brucei brucei. Arginase I and arginase II mRNA expression was higher in macrophages from infected BALB/c compared with those from C57BL/6 mice, whereas iNOS mRNA was up-regulated at the same level in both phenotypes. Similarly, arginase activity was more important in macrophages from infected BALB/c vs infected C57BL/6 mice. Moreover, increase of arginase I and arginase II mRNA levels and of macrophage arginase activity was directly induced by trypanosomes, with a higher level in BALB/c compared with C57BL/6 mice. Neither iNOS expression nor NO production was stimulated by trypanosomes in vitro. The high level of arginase activity in T. brucei brucei-infected BALB/c macrophages strongly inhibited macrophage NO production, which in turn resulted in less trypanosome killing compared with C57BL/6 macrophages. NO generation and parasite killing were restored to the same level in BALB/c and C57BL/6 macrophages when arginase was specifically inhibited with N(omega)-hydroxy-nor-L-arginine. In conclusion, host arginase represents a marker of resistance/susceptibility to trypanosome infections.

Animals↗

PubMed2004

Aromatic N-hydroxyguanidines as new reduction cosubstrates for dopamine beta-hydroxylase.

Conversion of neurotransmitter dopamine into norepinephrine is catalyzed by dopamine beta-hydroxylase (DbH). The reaction requires the presence of both molecular oxygen and a reducing cosubstrate, the assumed physiological cosubstrate being ascorbic acid. We have investigated the ability of a new family of molecules, N-aryl-N'-hydroxyguanidines, to serve as cosubstrates for DbH. N-(4-Methoxyphenyl)-N'-hydroxyguanidine proved to be an efficient reducing agent for DbH. The complete N-hydroxyguanidine moiety was required for activity, as any modification of this function resulted in non-cosubstrate compounds. Moreover, analysis of the products formed from N-(4-methoxyphenyl)-N'-hydroxyguanidine showed that the main oxidation product was a nitrosoimine. Modification of the aromatic para-substituent evidenced an influence of its electronic properties on the catalytic activity whereas steric factors seemed less important. In addition, changing the methoxy-substituent from the para- to the ortho-position led to an inactive compound. Our results demonstrate that N-aryl-N'-hydroxyguanidines are new efficient reducing cosubstrates for DbH and prove that specific interactions with the reducing cosubstrate do take place at the active site of the enzyme.

Copper↗