Interactions of melatonin with membrane models: portioning of melatonin in AOT and lecithin reversed micelles

The interaction of melatonin with water containing either sodium bis (2-ethylhexyl) sulfosuccinate (AOT) or soybean phosphatidylcholine (lecithin) reversed micelles has been investigated by UV absorption spectroscopy, at a molar ratio of melatonin:surfactant 1:800 for AOT and 1:400 for lecithin reversed micelles, and by varying the water:surfactant molar ratio (R). Our results suggest that in the presence of domains from apolar organic solvent to surfactant and to water, melatonin positions itself in the micellar phase, with a preferential location in the surfactant polar head group domain, independent of the nature of the surfactant and the amount of water encapsulated into the micellar core. Effects are due to the hydrophilic and lipophilic moieties of melatonin. The effectiveness of melatonin as an electron donor and free radical scavenger has been recently recognized. While supporting the hypothesis that melatonin may provide antioxidant protection without the benefit of receptors, present findings may suggest that the molecule could easily scavenge aqueous as well as lipophilic radicals.     

Localization and interactions of melatonin in dry cholesterol/lecithin mixed reversed micelles used as cell membrane models

The state of melatonin confined in dry cholesterol/lecithin mixed reversed micelles dispersed in CCl4 was investigated using 1H-NMR and FT-IR spectroscopies as a function of the melatonin to lecithin molar ratio (R(MLT)) and of the cholesterol to lecithin molar ratio (R(CHL)). An analysis of experimental results leads to the hypothesis that, independent of R(MLT) and as a consequence of anisotropic melatonin/lecithin, melatonin/cholesterol and cholesterol/lecithin interactions, melatonin is totally solubilized in reversed micelles. Melatonin is mainly located in and oriented in the nanodomain constituted by the hydrophilic groups of cholesterol and lecithin. A competition of melatonin and cholesterol for the hydrophilic binding sites of the reversed micelles was observed by changing the R(CHL). Some possible biological implications of the specific interactions governing the solubilization process, the preferential location and the peculiar properties of melatonin confined in cholesterol/lecithin mixed reversed micelles are discussed.     

Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4

Chronic granulomatous disease (CGD) is characterized by overexuberant inflammation and autoimmunity that are attributed to deficient anti-inflammatory signaling. Although regulation of these processes is complex, phosphatidylserine (PS)-dependent recognition and removal of apoptotic cells (efferocytosis) by phagocytes are potently anti-inflammatory. Since macrophage phenotype also plays a beneficial role in resolution of inflammation, we hypothesized that impaired efferocytosis in CGD due to macrophage skewing contributes to enhanced inflammation. Here we demonstrate that efferocytosis by macrophages from CGD (gp91(phox)(-/-)) mice was suppressed ex vivo and in vivo. Alternative activation with interleukin 4 (IL-4) normalized CGD macrophage efferocytosis, whereas classical activation by lipopolysaccharide (LPS) plus interferon gamma (IFNgamma) had no effect. Importantly, neutralization of IL-4 in wild-type macrophages reduced macrophage efferocytosis, demonstrating a central role for IL-4. This effect was shown to involve 12/15 lipoxygenase and activation of peroxisome-proliferator activated receptor gamma (PPARgamma). Finally, injection of PS (whose exposure is lacking on CGD apoptotic neutrophils) in vivo restored IL-4-dependent macrophage reprogramming and efferocytosis via a similar mechanism. Taken together, these findings support the hypothesis that impaired PS exposure on dying cells results in defective macrophage programming, with consequent efferocytic impairment and has important implications in understanding the underlying cause of enhanced inflammation in CGD.     

Myrsinoic acid B inhibits the production of hydrogen sulfide by periodontal pathogens in vitro

Recently, we reported that myrsinoic acid B purified from Myrsine seguinii inhibited methyl mercaptan (CH(3)SH) production by Fusobacterium nucleatum JCM8532. Since hydrogen sulfide (H(2)S) is the main component of physiological halitosis, while CH(3)SH is involved in pathological oral halitosis, the objective of this study is to determine whether myrsinoic acid B inhibits H(2)S production by oral microorganisms. F. nucleatum, Porphyromonas gingivalis and Treponema denticola were incubated with myrsinoic acid B and a substrate such as l-cysteine or l-methionine. H(2)S or CH(3)SH concentration in the headspace air, was determined using a gas chromatograph. The concentration of myrsinoic acid B inhibiting 50% (IC(50)) of H(2)S production by F. nucleatum was 0.142 μg ml(-1), and the IC(50) of P. gingivalis and T. denticola were 2.71 μg ml(-1) and 28.9 μg ml(-1), respectively. The presence of pyruvate, a by-product of H(2)S production, was determined. The IC(50) values of myrsinoic acid B for pyruvate production were 22.9 μg ml(-1) for F. nucleatum, 87.7 μg ml(-1) for P. gingivalis and 165 μg ml(-1) for T. denticola. We concluded that myrsinoic acid B inhibited the production of both H(2)S and pyruvate by periodontal pathogens.     

Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

The iron and acid-labile sulfide contents and the electron paramagnetic resonance (EPR) properties of hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum (strain W5) have been determined on the basis of quantitative amino acid analyses. The iron and acid-labile sulfide values are approximately 20 and 18 atoms per molecule of hydrogenase I and 14 and 11 atoms per molecule of hydrogenase II, respectively. These amounts are substantially greater than previously reported values, which relied on protein concentration determined by colorimetric assay. The oxidized hydrogenases exhibit unusual EPR signals that originate from a novel type of iron-sulfur center, termed the hydrogenase or H cluster, which covalently binds the inhibitor CO. This EPR signal represents approximately one unpaired electron per molecule in each enzyme with and without bound CO, which is consistent with the presence of one oxidized H cluster (S = 1/2) per enzyme molecule. The two enzymes also contain ferredoxin-type four-iron centers or F clusters. The EPR signals from the F clusters observed in the reduced forms of hydrogenase I and hydrogenase II account for approximately four and one unpaired electron per molecule, respectively. We conclude from the iron determinations and the EPR results, together with a reevaluation of previous spectroscopic data, that in both hydrogenases the H cluster probably comprises six iron atoms. Mechanistic models of the two hydrogenases are presented that account for their cluster compositions and the dramatic differences in their catalytic activities.     

Reproducibility of orocecal transit time and hydrogen production measured by breath tests]

The hydrogen breath test after a lactulose oral load in the fasting period is currently used to measure mouth to cecum transit time (MCTT). However, the reproducibility of this test is poor, and normal values are very scattered. The aim of the study was to determine the reproducibility of hydrogen breath test for MCTT measurement and hydrogen production after administration of 2 disaccharides: lactulose and lactitol ingested in the fasting state and postprandial period. Twelve healthy volunteers (6 men and 6 women; mean age = 34.6 +/- 9.6 years) were studied eight times in a random order, each disaccharide being studied twice in the fasting state and twice in the postprandial period. In the later, lactulose or lactitol was ingested 30 min after a liquid meal completely absorbed (400 kcal; glucide: 55 p. 100, lipid: 30 p. 100, protein: 15 p. 100; 400 ml of Inkopeptide). The MCTT was significantly increased with both disaccharides in the postprandial period as compared with the fasting state (P less than 0.0001). There was nos significant correlation between the 2 measurements of the MCTT in the fasting state, in contrast, the 2 measurements of the MCTT were closely related in the fed state (r = 0.62, P less than 0.05, et r = 0.79, P less than 0.003 for lactulose and lactitol respectively). During both periods no significant difference was found in the MCTT between lactulose and lactitol. As well, hydrogen production did not differ between the 2 disaccharides, but was significantly increased in the postprandial period, and in non methane producers.(ABSTRACT TRUNCATED AT 250 WORDS)     

肝星状细胞合成气体信号分子H2S的测定及其意义

目的:测定活化肝星状细胞(hepatic stellate cells,HSC)H2S的生成率及对细胞增殖的影响.方法:采用活化大鼠HSC-T6,分4组:对照组、炔丙基甘氨酸(D-Propargylglycine,DL-PPG)组、N-硝基-L-精氨酸甲酯(Nitro-L-arginine methyl ester,L-NAME)组、氯化高铁血红素(hemin)组,每组重复6个皿.RT-PCR检测HSC-T6胱硫醚-γ-裂解酶(cystathionine-γ-lyase,CSE)mRNA水平,亚甲基蓝分光光度法测定H2S生成率,MTT法观察不同浓度外源性H2S供体-NaSH(0,50,100,500,1000 μmol/L)对HSC-T6增殖的影响.结果:HSC-T6能自分泌H2S,DL-PPG可明显减少HSC-T6 H2S生成率(4.55 nmol/min±1.06 nmol/min vs 6.79 nmol/min±1.27 nmol/min,P<0.05).RT-PCR显示:HSC-T6 CSEmRNA阳性,DL-PPG、hemin均降低HSC-T6CSEmRNA水平(P<0.05);L-NAME对HSC-T6CSE mRNA无明显影响.50 μmol/L NaSH可明显促进HSC-T6增殖(细胞存活率为116%),500-1000 μmol/L NaSH对细胞增殖无明显影响.结论:活化HSC自分泌H2S,并且影响其增殖.H2S在肝纤维化及肝硬化门脉高压症的发生机制可能有重要作用.     

Neuroprotective Sirtuin ratio reversed by ApoE4

The canonical pathogenesis of Alzheimer’s disease links the expression of apolipoprotein E ε4 allele (ApoE) to amyloid precursor protein (APP) processing and Aβ peptide accumulation by a set of mechanisms that is incompletely defined. The development of a simple system that focuses not on a single variable but on multiple factors and pathways would be valuable both for dissecting the underlying mechanisms and for identifying candidate therapeutics. Here we show that, although both ApoE3 and ApoE4 associate with APP with nanomolar affinities, only ApoE4 significantly (i) reduces the ratio of soluble amyloid precursor protein alpha (sAPPα) to Aβ; (ii) reduces Sirtuin T1 (SirT1) expression, resulting in markedly differing ratios of neuroprotective SirT1 to neurotoxic SirT2; (iii) triggers Tau phosphorylation and APP phosphorylation; and (iv) induces programmed cell death. We describe a subset of drug candidates that interferes with the APP–ApoE interaction and returns the parameters noted above to normal. Our data support the hypothesis that neuronal connectivity, as reflected in the ratios of critical mediators such as sAPPα:Aβ, SirT1:SirT2, APP:phosphorylated (p)-APP, and Tau:p-Tau, is programmatically altered by ApoE4 and offer a simple system for the identification of program mediators and therapeutic candidates.Alzheimer’s disease (AD) poses a health problem of pandemic proportions. It is estimated that by the year 2030 there will be greater than 60 million people worldwide with AD, and $375 billion will be spent annually in the United States to care for affected individuals. The tedious and expensive process of drug discovery in AD is complicated by the fact that the causes and underlying mechanisms of the disease are still incompletely defined, and presymptomatic diagnosis is not yet in routine clinical use (1).The apolipoprotein E ε4 allele (ApoE; chromosomal locus 19q13) is the single most important genetic risk factor associated with AD. This allele confers increased risk for sporadic and familial AD (2). Individuals with two copies of the ApoE ε4 allele have an approximately eightfold increased risk of AD and have a significantly lower age of onset compared with AD patients not carrying this allele (2). Recent data indicate that the greater risk of AD associated with the ApoE4 isoform might relate to ApoE’s susceptibility to proteolysis and neurotoxicity or through its role in inhibiting Aβ clearance and/or stimulating Aβ deposition, leading to plaque formation (3, 4). Contrary to the previously reported work, one recent study provides evidence that ApoE and soluble Aβ have very minimal direct interaction, thus ApoE may influence soluble Aβ metabolism through its interactions with other receptors or transporters (5). Thus, despite knowing for over a decade that the ApoE ε4 allele is somehow contributory to the disease process, the precise molecular mechanisms underlying ApoE and APP interactions, direct or indirect, resulting in ApoE4-mediated toxicity, remain unclear.The amyloid precursor protein, APP, has been shown to function as a molecular switch: cleavage at the β, γ, and caspase sites results in the production of four pro-AD-peptides—soluble amyloid precursor protein beta (sAPPβ) (from which N-APP is derived), Aβ, Jcasp, and C31—that mediate neurite retraction, synaptic reorganization, and ultimately programmed cell death. In contrast, cleavage at the α site produces the trophic peptide sAPPα and the inhibitor of APP γ-site cleavage, alpha-COOH-terminal fragment of APP (αCTF) (1, 6). The decision between these two proteolytic pathways is governed at least in part by ligand binding: interaction with the axon guidance and trophic factor netrin-1 increases α-site cleavage, whereas interaction with the antitrophin Aβ inhibits α-site cleavage and increases net production of the four neurite-retractive peptides (1, 6). According to a recent study, the level of cerebrospinal fluid (CSF) sAPPα is significantly lower in AD patients possessing one or two ApoE4 alleles than in those not possessing the ApoE4 allele (7). Therefore, it was of interest to determine whether ApoE isoforms impact this trophic–antitrophic peptide balance differentially, and, if so, by what mechanism.Our data reveal differential effects of ApoE4 vs. ApoE3 on APP interaction, signaling, and processing and are compatible with the notion that the Alzheimer’s phenotype represents an imbalance between the trophic and antitrophic signaling of APP, reflected by the ratio of the four APP-derived neurite-retractive peptides to the two APP-derived trophic peptides. Our current results show that ApoE4 interacts with high affinity with APP, shifting the processing balance in the antitrophic direction, decreasing sAPPα secretion, and reducing sAPPα/Aβ and sAPPα/sAPPβ ratios in comparison with ApoE3. In addition, the presence of ApoE4 results in increased APP-Thr668 phosphorylation (p-APP) and Tau phosphorylation (p-Tau). Some of these ApoE4-mediated events were blocked by proteasomal inhibitors, and in a small pilot study, the proteasomal inhibitor disulfiram and a CDK inhibitor were shown to be effective in reversing some of the ApoE4-mediated effects. Furthermore, we identified initial therapeutic candidates—F03 and F05—that normalize some of the ApoE4 effects.In addition to these effects on APP processing and signaling, ApoE4 expression was associated with a marked reduction in the ratio of SirT1 to SirT2, both in cultured neural cells and in the brains of patients with AD. Because SirT1 has been implicated in neuroprotection and SirT2 in neurodegeneration (8), this effect of ApoE4 may be important from both mechanistic and therapeutic development standpoints.Our data support the view that ApoE4 modulates the connectivity balance, as reflected in the ratios of sAPPα:sAPPβ, sAPPα:Aβ, SirT1:SirT2, APP:p-APP, and Tau:p-Tau. The network of proteins that mediates this balance thus represents a set of candidate targets for the prevention and treatment of ApoE4-associated processes such as mild cognitive impairment and AD.     

Extracellular ATP-induced production of hydrogen peroxide in porcine thyroid cells

Hydrogen peroxide (H2O2) is an essential substrate for the peroxidase reaction in thyroid hormone biosynthesis. We demonstrated the production of H2O2 from porcine thyroid cells stimulated with extracellular ATP, using a scopoletin-horseradish peroxidase (HRP) system. Incubation of isolated cells for 1 day in the presence of 10% (v/v) newborn calf serum was necessary for the detection of induction by ATP of H2O2 production. The rate of H2O2 production induced by the addition of ATP increased in a dose-dependent manner, and the concentration of ATP required for half-maximum stimulation was about 10 mumol/l. ADP and GTP were also effective, but only at higher concentrations than ATP. In the absence of extracellular Ca2+, the production rate was very low. Production of H2O2 from thyroid cells was also measured by a method which discriminated between H2O2 and superoxide anion (O2-); in this, diacetyldeuteroheme-substituted HRP was employed as the trapping agent for both O2 metabolites. The thyroid cells produced H2O2, but not O2-, when the cells were stimulated by extracellular ATP.     

Molecular mechanism for metal-independent production of hydroxyl radicals by hydrogen peroxide and halogenated quinones

We have shown previously that hydroxyl radicals (HO*) can be produced by H2O2 and halogenated quinones, independent of transition metal ions; however, the underlying molecular mechanism is still unclear. In the present study, using the electron spin resonance secondary radical spin-trapping method, we found that tetrachloro-1,4-benzoquinone (TCBQ), but not its corresponding semiquinone anion radical, the tetrachlorosemiquinone anion radical (TCSQ*-), is essential for HO* production. The major reaction product between TCBQ and H2O2 was identified by electrospray ionization quadrupole time-of-flight mass spectrometry to be the ionic form of trichlorohydroxy-1,4-benzoquinone (TrCBQ-OH), and H2O2 was found to be the source and origin of the oxygen atom inserted into the reaction product TrCBQ-OH. On the basis of these data, we propose that HO* production by H2O2 and TCBQ is not through a semiquinone-dependent organic Fenton reaction but rather through the following mechanism: a nucleophilic attack of H2O2 to TCBQ, forming a trichlorohydroperoxyl-1,4-benzoquinone (TrCBQ-OOH) intermediate, which decomposes homolytically to produce HO*. This represents a mechanism of HO* production that does not require redox-active transition metal ions.     

Photosensitized Inactivation of Plasmodium falciparum- and Babesia divergens-Infected Erythrocytes in Whole Blood by Lipophilic Pheophorbide Derivatives

Background and objectives: Blood transfusions can transmit parasitic infections, such as those caused by Plasmodium (malaria), Trypanosoma cruzi (Chagas' disease), and Babesia (babesiosis). A higher degree of blood transfusion safety would be reached if methods were available for inactivating such parasites. Materials and methods: We evaluated the effectiveness of photosensitization using lipophilic pheophorbide and red light illumination to eradicate red blood cells infected with Plasmodium falciparum, and with Babesia divergens, in whole blood. Fluorescence microscopy and conventional fluorometry showed the specific accumulation of pheophorbide derivatives in the RBC infected with either parasite, compared with uninfected RBC. The effectiveness of different derivatives in eradicating infected RBC was first estimated in parasite cultures. Results: The best photosensitizer was the N-(4-butanol) phrophorbide derivative (Ph4-OH) at 0.2 μM concentration and 5-min illumination. In whole blood, the eradication of RBC infected with B. divergens and P. falciparum was obtained with 2 μM Ph4-OH and 10 and 20 min illumination, respectively. Under these conditions of photosensitization, low levels of RBC hemolysis were noted even after 2 weeks of storage at 4 °C and a subsequent 48-hour incubation at 37 °C. No reduction of negative charges on treated RBC was noted and no increase in methemoglobin content. Conclusion: In plasma, Ph4-OH is mainly transported by high-density lipoproteins (HDL). This high affinity for HDL may explain the selective accumulation of lipophilic pheophorbide derivatives in the intracellular parasites. Photosensitization with pheophorbide derivatives may be a promising approach to inactivation of transfusion-transmissible parasites and viruses in blood bank units.     

Effect of beta-lapachone on hydrogen peroxide production in Trypanosoma cruzi.

beta-Lapachone, an antimicrobial agent, markedly increase the generation of H2O2 in intact Trypanosoma cruzi epimastigotes (Y strain). Increase in H2O2 was determined by the horseradish peroxidase-H2O2 Compound II formation as well as by a cytochemical technique.     

Hypotension produced by platelet-activating factor is reversed by thyrotropin-releasing hormone

Platelet-activating factor (PAF), a vasoactive phospholipid implicated in anaphylactic reactions, causes severe hypotension in experimental animals that is highly resistant to pharmacological therapy. In the present studies, we showed that PAF (1 nmol/600 g body weight, IV) produced profound hypotension in unanesthetized guinea pigs that was promptly and completely reversed by thyrotropin-releasing hormone (TRH) (2 mg/kg, IV) or by the synthetic TRH analog MK771 (2 mg/kg, IV). TRH also reversed this hypotension when administered intracerebroventricularly (ICV) at a dose (0.02 mg/kg) that was systemically ineffective. The opiate receptor antagonist naloxone (5 mg/kg) was less effective than TRH in reversing the cardiovascular consequences of PAF administration. These data suggest that TRH reverses PAF-induced shock through central receptor-mediated mechanisms. This therapeutic action of TRH may partially account for the beneficial cardiovascular effects of this peptide in anaphylactic shock.