Physio-pathological effects of m6A modification and its potential contribution to melanoma.

Methylation of N6-adenosine (m6A) is the most prevalent internal RNA modification and is especially common among the messenger RNAs. These m6A modifications regulate splicing, translocation, stability and translation of RNA through dynamic and reversible interactions with m6A-binding proteins, namely the writers, erasers and readers. RNA methyltransferases catalyze the m6A modifications, while demethylases reverse this methylation. Deregulation of the m6A modification process has been implicated in human carcinogenesis, including melanoma-which carries one of the highest mutant rates. In this review, we provide an up-to-date summary of m6A regulation and its biological impacts on normal and cancer cells, with emphasis on the deregulation of m6A modification and m6A regulators in melanoma. In addition, we highlight the prospective potential of exploiting m6A modification in the treatment of melanoma and non-cancer diseases.

Inferring lanosterol functions in the female rabbit reproductive tract based on the immunolocalization of lanosterol 14-demethylase and farnesoid beta-receptor.

Female reproductive organs have de novo synthesis of cholesterol. Some sterol molecules, intermediaries in the cholesterol synthesis, have important paracrine/autocrine actions. Lanosterol binds to the farnesoid beta-receptor (FXRß), a molecule widely expressed in the ovaries, suggesting that it may play a role in reproduction. Up to date, we know little about lanosterol functions across female reproductive organs. We described immunolocalized lanosterol 14-demethylase (LDM or CYP51A1), responsible for catalyzing the conversion of lanosterol in cholesterol, and FXRß in the ovary, oviduct, uterus, and vagina of virgin and pregnant rabbits. In virgin rats, we found CYP51A1 and FXRß immunoreactivity was found in all ovarian follicles, epithelial cells, stroma, and Graafian follicles. Also, the epithelium and stroma, as well as the smooth muscle of the oviduct, vagina, and uterus showed CYP51A1 and FXRß immunoreactivity. In pregnant dams, we observed the presence of CYP51A1 and FXRß immunoreactivity in the corpora lutea, giant uterine cells, and trophoblastic cells. The presence of CYP51A1 and FXRß support that lanosterol participates in diverse reproductive processes, including follicular maturation, transport of gametes and zygote, implantation of blastocyst, lubrication, and contraction of the vagina, secretion of female prostate, and control of delivery mediated by pelvic muscles contraction.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP.

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.

Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways.

The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl(3). Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K(m) for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.

CYP2B6 variants and plasma efavirenz concentrations during antiretroviral therapy in Port-au-Prince, Haiti.

BACKGROUND: Polymorphisms in CYP2B6 are known to predict increased steady-state plasma concentrations of efavirenz. We characterized relationships between genetic polymorphisms and plasma efavirenz concentrations among 45 Haitians who initiated antiretroviral therapy in Port-au-Prince. METHODS: An observational study characterized relationships between clinical factors, pharmacokinetics, and treatment response among antiretroviral-naive patients initiating once-daily treatment with efavirenz plus twice-daily treatment with zidovudine and lamivudine. Plasma drug concentrations were determined at weeks 2 and 4. Drug doses were directly observed by field workers or designated family members. We retrospectively characterized relationships between efavirenz concentrations and 50 single-nucleotide polymorphisms in CYP2B6 and several polymorphisms in CYP2A6, CYP3A4, CYP3A5, and ABCB1. RESULTS: Plasma specimens for efavirenz analysis were obtained from study participants a mean (+/- standard deviation) of 13.9 +/- 1.6 h after they received the dose. As expected, CYP2B6 516G-->T was associated with increased plasma efavirenz concentrations (Spearman rho = 0.71; P < .001), as were 10 polymorphisms in linkage disequilibrium with 516G-->T. Distinct CYP2B6 polymorphisms were associated with decreased plasma efavirenz concentrations (greatest absolute rho = 0.48; P = .001). Associations were replicated by results from a recent pharmacokinetic study involving 34 healthy, human immunodeficiency virus-negative African Americans. CONCLUSIONS: Relatively frequent CYP2B6 polymorphisms may predict decreased plasma efavirenz exposure in patients of African descent. If replicated in other cohorts, the implications of these novel associations for treatment response warrant further study.

Cytochrome P450 2E1 and 3A activities do not differ between Mexicans and European Americans.

OBJECTIVES: Population differences in the activity of various cytochrome P450 (CYP) enzymes have been demonstrated on the basis of either genetic or environmental determinants. Hispanics are a large demographic group both worldwide and within the United States; hence the possibility of differences in metabolism between one such group-Mexicans-and a European-derived population was determined with respect to CYP2E1 and CYP3A. METHODS: Young healthy Mexican immigrants living in Los Angeles, Calif, who had maintained a traditional diet were compared with previously and identically studied groups of age-, sex-, and weight-matched European Americans who resided in middle Tennessee and ate a "western" diet (15 men and 15 women). In one study carried out in 15 women, the disposition of chlorzoxazone after an oral dose (250 mg) was compared. In the other investigation, all of the 15 subjects were men and received intravenous [(15)N(3)]-labeled midazolam (1 mg) and oral midazolam (2 mg) simultaneously to characterize the disposition of benzodiazepine in the two populations. RESULTS: Plasma concentration-time profiles of chlorzoxazone and its 6-hydroxy metabolite and the 0- to 24-hour urinary recovery of the latter were not different between Mexicans and European Americans. This indicates that CYP2E1 activity is similar in the two populations. Similarly, no significant intergroup differences were noted in the plasma concentration-time profiles of midazolam after either intravenous or oral administration. Accordingly, CYP3A does not appear to be different between Mexicans and European Americans. CONCLUSIONS: Similarity in the metabolism of chlorzoxazone between Mexicans and European Americans suggests that the risk associated with CYP2E1-mediated activation of procarcinogens is not different between these two populations. Likewise, the absence of any difference in the disposition of midazolam indicates that, from a pharmacokinetic standpoint, dosages of drugs metabolized by CYP3A need not be different between Mexicans and European Americans.

Cytochrome P450 2B (CYP2B)-mediated activation of methyl-parathion in rat brain extracts.

The role of cytochrome P450 (CYP) and the CYP isoform involved in the activation of the widely used pesticide methyl-parathion (MePA) were investigated in rat brain extracts by measuring the effect of different CYP inhibitors on acetylcholinesterase (AChE) inhibition by MePA. Brain extracts provide a useful tool to study the activation mechanisms of organophosphorus compounds (OP) since they contain both the activating enzyme(s) and the molecular target for OP toxicity. As expected, in incubations of rat brain extract supplemented with NADPH, AChE activity was non-competitively inhibited by the presence of MePA, indicating that MePA was activated to its reactive metabolite methyl-paraoxon (MePO). Indeed, Vmax(app) decreased from 13.4 to 8.7 micromol thionitrobenzoic acid (TNB)/min per mg protein. MePA activation by rat brain extracts, as measured by the AChE inhibition produced by the presence of the pesticide in the incubation, was fully prevented by previously bubbling the incubation mix with CO, by the presence of monoclonal anti-rat CYP2B1/2B2 antibodies and by the addition of phenobarbital (PB), a CYP2B substrate. Interestingly, MePA showed a greater affinity for CYP2B than PB. CYP1A1 antibodies showed no effect on MePA activation. The presence of cytochrome P450 2B (CYP2B) in the rat brain extracts was confirmed by immunoblotting. These results demonstrate indisputably the responsibility of CYP2B in MePA activation in the rat brain in vitro, suggesting that metabolic activation of OP compounds in situ might be crucial for their organ specific toxicity to the central nervous system also in vivo.

Inhibitory effect of naringin on the micronuclei induced by ifosfamide in mouse, and evaluation of its modulatory effect on the Cyp3a subfamily.

Naringin (Nar) is a flavonone found in high amount in grapefruit. In in vitro studies to determine its antimutagenicity results have been both positive and negative. On the other hand, an increase in the bioavailability of some medicaments have been observed when these are ingested together with grapefruit. It has been suggested that the effect may be related to the inhibition of the human enzyme Cytochrome P450 (CYP) 3A4 by Nar, an enzyme with a high aminoacid sequence homology with the Cyp3a in mouse. The present study was designed for three main purposes: (1) to determine whether Nar has a genotoxic effect in mouse in vivo. This was evaluated by measuring the rate of micronucleated polychromatic erythrocytes (MNPE); (2) to determine its antigenotoxic and its anticytotoxic potential on the damage produced by ifosfamide (Ifos). The first study was done by scoring the rate of MNPE, and the second one by establishing the index polychromatic erythrocytes/normochromatic erythrocytes (PE/NE); and (3) to explore whether its antigenotoxic mechanism of action is related to an inhibitory effect of Nar on the expression of the Cyp3a enzyme, an effect which could avoid the biotransformation of Ifos. A single oral administration was used for all groups in the experiment: three groups were given different doses of Nar (50, 250, and 500 mg/kg), other groups received the same doses of Nar plus an administration of Ifos (60 mg/kg), another group treated with distilled water and another with Ifos (60 mg/kg) were used as negative and positive controls, respectively. The micronuclei and the cell scoring were made in blood samples taken from the tail of the animals at 0, 24, 48, 72, and 96 h. The results showed that Nar was neither genotoxic nor cytotoxic with the doses tested, but Ifos produced an increase in the rate of MNPE at 24 and 48 h. The highest value was 24+/-1.57 MNPE per thousand cells at 48 h. The index PE/NE was significantly reduced by Ifos at 24 and 48 h. Concerning the antigenotoxic capacity of Nar, a significant decrease was observed in the MNPE produced by Ifos at the three tested doses. This effect was dose-dependent, showing the highest reduction in MNPE frequency (54.2%) at 48 h with 500 mg/kg of Nar. However, no protection on the cytotoxicity produced by Ifos was observed. Immunoblot analysis was used to assess the Cyp3a expression in liver and intestinal microsomes from mouse exposed orally to Nar. An induction in the Cyp3a protein was observed in both intestinal and hepatic microsomes from treated mice. This induction correlated with an increase in erythromycin N-demethylase activity. These data suggest that other mechanism(s) are involved in the antigenotoxic action of naringin.

Production of reactive oxygen species by microsomes enriched in specific human cytochrome P450 enzymes.

Few studies have evaluated the production of reactive oxygen intermediates by human microsomes, especially the influence of the specific form of cytochrome P450. Experiments were carried out to evaluate the ability of CYP1A1, 1A2, 2B6, and 3A4 to consume NADPH, reduce iron, and catalyze production of reactive oxygen species. Microsomes enriched in each of these CYPs were obtained from commercial +/- lymphoblast cells that had been transfected with cDNA encoding the specific human CYP. On a per nanomole cytochrome P450 basis, CYP3A4 was the most active P450 evaluated in catalyzing NADPH oxidation, production of superoxide anion radical, NADPH-dependent chemiluminescence, oxidation of dichlorofluorescein diacetate, and reduction of either ferric-EDTA or ferric-citrate. CYP1A1 was the next most reactive CYP, whereas CYP1A2 and 2B6 displayed a comparable, lower activity. Nitric oxide, which reacts with and inactivates hemoproteins, inhibited superoxide production by all the CYPs to a similar extent. Because CYP3A4 is present in high amounts in human liver microsomes and is active in catalyzing the formation of reactive oxygen species, this CYP may make an important contribution in the overall ability of human liver microsomes to generate active oxygen species.

The heme-binding region polymorphism of cytochrome P450IA1 (CypIA1), rather than the RsaI polymorphism of IIE1 (CypIIE1), is associated with lung cancer in Rio de Janeiro.

Ile-Val polymorphism in exon 7 of cytochrome P450IA1 (CypIA1) and RsaI polymorphism of cytochrome P450IIE1 (CypIIE1) were examined in a case-control study of lung cancer in Rio de Janeiro, Brazil. The Val-containing genotype in exon 7 of CypIA1 was found to be associated with lung cancer in this population (odds ratio, 2.26; 95% confidence interval, 1.14-4.47 for 99 cases versus 108 controls of 123 matched pairs), whereas RsaI polymorphism in CypIIE1 was not associated with lung cancer susceptibility. In squamous cell carcinoma, the degree of association of Val-containing genotype was greater in those with fewer pack-years of smoking. The RsaI polymorphism of CypIIE1 has a different distribution from the Japanese pattern and is not associated with lung cancer. When we analyzed the association of Ile-Val polymorphism to MspI polymorphism of CypIA1, the Val/Val homozygote was found only in the subpopulation with the MspI site-present homozygote. The apparent lack of association of CypIA1 MspI polymorphism with lung cancer in this area reported in our previous study and the results of the present study indicate that the "true" responsible site for lung cancer susceptibility should be the Ile-Val polymorphism in the catalytic site of CypIA1.

Sanguinarine: its potential as a liver toxic alkaloid present in the seeds of Argemone mexicana.

The alkaloid sanguinarine reported to be responsible for several outbreaks of epidemic dropsy in the tropics was examined for its hepatotoxic potential in rats. The studies showed that a single i.p. dose (10 mg/kg) of sanguinarine not only increased the activity of SGPT and SGOT substantially but also caused a significant loss of microsomal cytochrome P-450 and benzphetamine N-demethylase activity. Furthermore, the treated rats exhibited considerable loss of body and liver weight, peritoneal edema and slightly enlarged livers with fibrinous material. Microscopic examination of the liver tissue showed progressive cellular degeneration and necrosis further substantiating that sanguinarine is a potential hepatotoxic alkaloid.