Molecular origin of cancer: Catechol estrogen-3,4-quinones as endogenous tumor initiators

Cancer is a disease that begins with mutation of critical genes: oncogenes and tumor suppressor genes. Our research on carcinogenic aromatic hydrocarbons indicates that depurinating hydrocarbon–DNA adducts generate oncogenic mutations found in mouse skin papillomas (Proc. Natl. Acad. Sci. USA 92:10422, 1995). These mutations arise by mis-replication of unrepaired apurinic sites derived from the loss of depurinating adducts. This relationship led us to postulate that oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE) of estrone (E1) and estradiol (E2) to catechol estrogen-3,4-quinones (CE-3, 4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts. The resultant apurinic sites in critical genes can generate mutations that may initiate various human cancers. The noncarcinogenic 2-hydroxy CE are oxidized to CE-2,3-Q and form only stable DNA adducts. As reported here, the CE-3,4-Q were bound to DNA in vitro to form the depurinating adduct 4-OHE1(E2)-1(α,β)-N7Gua at 59–213 μmol/mol DNA–phosphate whereas the level of stable adducts was 0.1 μmol/mol DNA–phosphate. In female Sprague–Dawley rats treated by intramammillary injection of E2-3,4-Q (200 nmol) at four mammary glands, the mammary tissue contained 2.3 μmol 4-OHE2-1(α,β)-N7Gua/molDNA–phosphate. When 4-OHE1(E2) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87–440 μmol of 4-OHE1(E2)-1(α, β)-N7Gua was formed. After treatment with 4-OHE2, rat mammary tissue contained 1.4 μmol of adduct/mol DNA–phosphate. In each case, the level of stable adducts was negligible. These results, complemented by other data, strongly support the hypothesis that CE-3,4-Q are endogenous tumor initiators.

Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

Identification and Stereospecificity of the First Three Enzymes of 3-Dimethylsulfoniopropionate Biosynthesis in a Chlorophyte Alga1

Many marine algae produce 3-dimethylsulfoniopropionate (DMSP), a potent osmoprotective compound whose degradation product dimethylsulfide plays a central role in the biogeochemical S cycle. Algae are known to synthesize DMSP via the four-step pathway, l-Met → 4-methylthio-2-oxobutyrate → 4-methylthio-2-hydroxybutyrate → 4-dimethylsulfonio-2-hydroxy-butyrate (DMSHB) → DMSP. Substrate-specific enzymes catalyzing the first three steps in this pathway were detected and partially characterized in cell-free extracts of the chlorophyte alga Enteromorpha intestinalis. The first is a 2-oxoglutarate-dependent aminotransferase, the second an NADPH-linked reductase, and the third an S-adenosylmethionine-dependent methyltransferase. Sensitive radiometric assays were developed for these enzymes, and used to show that their activities are high enough to account for the estimated in vivo flux from Met to DMSP. The activities of these enzymes in other DMSP-rich chlorophyte algae were at least as high as those in E. intestinalis, but were ≥20-fold lower in algae without DMSP. The reductase and methyltransferase were specific for the d-enantiomer of 4-methylthio-2-hydroxybutyrate in vitro, and both the methyltransferase step and the step(s) converting DMSHB to DMSP were shown to prefer d-enantiomers in vivo. The intermediate DMSHB was shown to act as an osmoprotectant, which indicates that the first three steps of the DMSP synthesis pathway may be sufficient to confer osmotolerance.

Single photon emission computerized tomography imaging of amphetamine-induced dopamine release in drug-free schizophrenic subjects.

The dopamine hypothesis of schizophrenia proposes that hyperactivity of dopaminergic transmission is associated with this illness, but direct observation of abnormalities of dopamine function in schizophrenia has remained elusive. We used a newly developed single photon emission computerized tomography method to measure amphetamine-induced dopamine release in the striatum of fifteen patients with schizophrenia and fifteen healthy controls. Amphetamine-induced dopamine release was estimated by the amphetamine-induced reduction in dopamine D2 receptor availability, measured as the binding potential of the specific D2 receptor radiotracer [123I] (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl) methyl]benzamide ([123I]IBZM). The amphetamine-induced decrease in [123I]IBZM binding potential was significantly greater in the schizophrenic group (-19.5 +/- 4.1%) compared with the control group (-7.6 +/- 2.1%). In the schizophrenic group, elevated amphetamine effect on [123I]IBZM binding potential was associated with emergence or worsening of positive psychotic symptoms. This result suggests that psychotic symptoms elicited in this experimental setting in schizophrenic patients are associated with exaggerated stimulation of dopaminergic transmission. Such an observation would be compatible with an abnormal responsiveness of dopaminergic neurons in schizophrenia.

NHC-Ligand Effectiveness in the Fluorine-Free Hiyama Reaction of Aryl Halides

1-Benzyl-3-(2-hydroxy-2-phenylethyl)imidazolium chloride (5), which is a precursor of an N-heterocyclic carbene ligand, in combination with palladium acetate, has been employed as an effective catalyst for the fluorine-free Hiyama reaction. A systematic study of the catalytic mixture, by a 32 factorial design, has revealed that both the amount of palladium and the Pd/NHC precursor ratio are important factors for obtaining good yields of the coupling products, indicating an interaction between them. The best catalytic system involves mixing 0.1 mol-% palladium acetate in a 1:5 ratio (Pd/salt 5), which allows the effective coupling of a range of aryl bromides and chlorides with trimethoxy(phenyl)silane. The Hiyama reactions are carried out in NaOH solution (50 % H2O w/w), at 120 °C under microwave irradiation during 60 min.

Difficult macrocyclizations: New strategies for synthesizing highly strained cyclic tetrapeptides

Cyclic tetrapeptides are an intriguing class of natural products. To synthesize highly strained cyclic tetrapeptides; we developed a macrocyclization strategy that involves the inclusion of 2-hydroxy-6-nitrobenzyl (HnB) group at the N-terminus and in the middle of the sequence. The N-terminal auxiliary performs a ring closure/ring contraction role, and the backbone auxiliary promotes cis amide bonds to facilitate the otherwise difficult ring contraction. Following this route, the all-L cyclic tetrapeptide cyclo-[Tyr-Arg-Phe-Ala] was successfully prepared.

The influence of dietary sodium chloride, Arginine : Lysine ratio, and methionine source on apparent ileal digestibility of arginine and lysine in acutely heat-stressed broilers

The present study was carried out to determine the ileal digestibility of Arg and Lys in acutely heatstressed broilers using diets varying in Arg:Lys ratio, NaCl concentration, and Met Source. Male broilers were maintained at 22degreesC from 21 to 33 d of age and then at 32degreesC from 33 to 38 d of age. From 28 to 38 d of age, birds were fed a diet with an Arg:Lys ratio of 1.05 and 3 g of supplemental NaCl/kg of diet with or without L-arg free base to increase the Arg:Lys to 1.35, and with or without 3 g/kg of additional NaCl. Methionine was supplied as equimolar amounts of DL-Met or 2-hydroxy-4-(methylthio)-butanoic acid in a 2 x 2 x 2 design. At 38 d of age, digesta were collected from the terminal ileum, and amino acid analyses were conducted on feed and digesta samples and compared with acid-insoluble ash (dietary celite) to calculate the apparent ileal digestibilities of Lys and Arg. Increasing the NaCl concentration and the presence of HMB significantly decreased the digestibility of both Arg and Lys, whereas increasing the Arg:Lys ratio increased the digestibility of only Arg but did increase BW gain (P = 0.08). An interaction between dietary NaCl and Arg:Lys ratio as well as the 3-way interaction suggested that dietary NaCl could affect the apparent ileal digestibility of Arg and Lys at certain Arg:Lys ratios and the response may be influenced by the Met source.

Synthesis of 'difficult' peptides free of aspartimide and related products, using peptoid methodology

We developed an efficient, cost effective strategy for Fmoc-based solid phase synthesis of 'difficult' peptides and/or peptides containing Asp/Asn-Gly sequences, free of aspartimide and related products, using a peptoid methodology for the preparation of N-substituted glycines.

Reactive processing of polyolefins using antioxidant systems

Various monoacrylic compounds containing a hindered phenol function (e.g.3,5-di-tert.-butyl-4-hydroxy benzyl alcohol, DBBA and vinyl-3-[3',5'-di-tert.-butyl-4-hydroxy phenyl] propionate, VDBP), and a benzophenone function (2-hydroxy-4-[beta hydroxy ethoxy] benzophenone, HAEB) were synthesised and used as reactive antioxidants (AO's) for polypropylene (PP). These compounds were reacted with PP melt in the presence of low concentration of a free radical generator such a peroxide (reactive processing) to produce bound-antioxidant concentrates. The binding reaction of these AO's onto PP was found to be low and this was shown to be mainly due to competing reactions such as homopolymerisation of the antioxidant. At high concentrations of peroxide, higher binding efficiency resulted, but, this was accompanied by melt degradation of the polymer. In a special reactive processing procedure, a di- or a trifunctional reactant (referred to as coagent), e.g.tri-methylol propane tri-acrylate, Tris, and Divinyl benzene, DVB, were used with the antioxidant and this has led to an enhanced efficiency of the grating reaction of antioxidants on the polymer in the melt. The evidence suggests that this is due to copolymerisation of the antioxidants with the coagent as well as grafting of the copolymers onto the polymer backbone. Although the 'bound' AO's containing a UV stabilising function showed lower overall stabilisation effect than the unbound analogues before extraction, they were still much more effective when subjected to exhaustive solvent extraction. Furthermore, a very effective synergistic stabilising activity when two reactive AO's containing thermal and UV stabilising functions e.g. DBBA and HAEB, were reactively processed with PP in the presence of a coagent. The stabilising effectiveness of such a synergist was much higher than that of the unbound analogues both before and after extraction. Analysis using the GPC technique of concentrates containing bound-DBBA processed in the presence of Tris coagent showed higher molecular weight (Mn), compared to that of a polymer processed without the coagent, but was still lower than that of the control processed PP with no additives. This indicates that Tris coagent may inhibit further melt degradation of the polymer. Model reactions of DBBA in liquid hydrocarbon (decalin) and analysis of the products using FTIR and NMR spectroscopy showed the formation of grafted DBBA onto decalin molecules as well as homopolymerisation of the AO. In the presence of Tris coagent, copolymerisation of DBBA with the Tris inevitably occured; which was followed by grafting of the copolymer onto the decalin, FTIR and NMR results of the polymer concentrates containing bound-DBBA processed with and without Tris, showed similar behaviour as the above model reactions. This evidence supports the effect of Tris in enhancing the efficiency of the reaction of DBBA in the polymer melt. Reactive procesing of HAEB in polymer melts exhibited crosslinking formation In the early stages of the reaction, however, in the final stage, the crosslinked structure was 'broken down' or rearranged to give an almost gel free polymer with high antioxidant binding efficiency.

The behaviour of antioxidants in polyolefins under conditions of U.V. exposure

The effects of antioxidants and stabilizers on the oxidative degradation of polyolefins (low density polyethylene [LDPE] and polypropylene [PPJ have been studied after subjecting to prior high temperature processing treatments. The changes in the both chemical and physical properties of unstabilized polymers occurring during processing were found to be strongly dependent on the amount of oxygen present in the mixer. Subsequent thermal and photo-oxidation showed very similar characteristics and the chromophore primarily responsible for:both thermo and photooxidative degradation of unstabilized polymers was found to be hydroperoxide formed during processing. Removal of hydroperoxide by heat treatment in an inert atmosphere although increasing ketonic carbonyl concentration, markedly decreased the rate of photo-oxidation, introducing an induction period similar to that of an unprocessed sample. It was concluded that hydroperoxides are the most important initiators in normally processed polymers during the early stages of photo-oxidation. Antioxidants such as metal dithiocarbamates which act by destroying peroxides into non-radica1 products were found to be efficient melt stabilizers for polyolefins and effective UV stabilizers during the initial photo-oxidation stage, whilst a phenolic antioxidant, n-octadecyl-3-(3',5'-di-terbutyl 4'hydroxypheny1) propionate (Irganox 1076) retarded photo-oxidation rate in the later stages. A typical 'UV absorber' 2-hydroxy-4-octyloxy-benzophenone (HOBP) has a minor thermal antioxidant action but retarded photo-oxidation at all stages. A substituated piperidine derivative, Bis [2.2.6.6-tetramethylpiperidlnyl-4] sebacate (Tinuvin 770) behaved as an pro-oxidant during thermal oxidation of polyolefins but was an effective stabilizer against UV light. The UV absorber, HOBP synergised effectively with both peroxide decomposing antioxidants (metal dithiocarbamates) and a chain-breaking antioxidant (Irganox 1076) during photo-oxidation of the poymers studed whereas the combined effect was additive during thermal oxidation. By contrast, the peroxide decornposers and chain-breaking antioxidant (Irganox 1076) which were effective synergists during thermal oxidation of LDPE· were antagonistic during photo-oxidation. The mechanisms of these processes are discussed.

Flavones and related compounds as inhibitors of protein tyrosine kinases

Aberrant tyrosine protein kinase activity has been implicated in the formation and maintenance of malignancy and so presents a potential target for cancer chemotherapy. Quercetin, a naturally occuring flavonoid, inhibits the tyrosine protein kinase encoded by the Rous sarcoma virus but also exhibits many other effects. Analogues of this compound were synthesised by the acylation of suitable 2-hydroxyacetophenones with appropriately substituted aromatic (or alicyclic) acid chlorides, followed by base catalysed rearrangement to the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones. Acid catalysed ring closure furnished flavones. The majority of the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones were shown by NMR to exist in the enol form. This was supported by the crystal structure of 1-(2-hydroxy-4-methoxyphenyl)-3-phenylpropan-1,3-dione. In contrast, 1.(4,6-dimethoxy-2-hydroxyphenyl)-3-phenylpropan-1,3-dione did not exhibit keto-enol tautomerism in the NMR spectrum and was shown in its crystal structure to assume a twisted conformation. Assessment of the biological activity of the analogues of quercetin was carried out using whole cells and the kinase domain of the tyrosine protein kinase encoded by the Abelson murine leukaemia virus, ptab150 kinase. Single cell suspension cultures and clonogenic potential of murine fibroblasts transformed by the Abelson Murine leukaemia virus (ANN-1 cells) did not indicate the existence of any structure activity relationship required for cytotoxicity or cytostasis. No selective toxicity was apparent when the `normal' parent cell line, (3T3), was used to assess the cytotoxic potential of quercetin. The ICS50 for these compounds were generally in the region of 1-100M. The potential for these compounds to inhibit ptab150 kinase was determined. A definite substitution requirement emerged from these experiments indicating a necessity for substituents in the A ring or in the 3-position of the flavone nucleus. Kinetic data showed these inhibitors to be competitive for ATP.

Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

Synthesis of Iodinated Naphthoquinones Using Morpholine-Iodine Complex

artículo (arbitrado)--Universidad de Costa Rica. Escuela de Química, 2004. Este documento es privado debido a limitaciones de derechos de autor.

Novos monômeros obtidos a partir do metacrilato de glicidila, bisfenol A e 4, 4 isopropilidenodicicloexanol : síntese, caracterização estrutural e propriedades de compósitos para utilização em resinas de restauração dental

Nowadays, composite resins are the direct restorative materials more important in dental clinical performance, due to their versatility and aesthetic excellence. Bis-GMA (2,2-bis[4(2-hydroxy-3-metacryloxypropoxy)phenil]propane) is the base monomer more frequently used in restorative composite resins. However, this monomer presents some disadvantages, such as high viscosity and two aromatic rings in its structure that can promote allergic reactions to the humans. In this work, the main purpose was to synthesize new monomers from glycidyl methacrylate to use in dental restorative materials. Structural characterization of the monomers was carried out through FTIR and NMR 1H, and eight composites were produced from the new monomers, by addition of silane-treated alumino silicate particles (inorganic filler) and a photocuring system (camphorquinone and ethyl 4-dimethylaminebenzoate). The composites were analyzed by environmental scanning electronic microscopy and the water sorption and solubility, compressive strength and elastic modulus were determined. A commercial composite resin [Z100 (3M)] was used to comparison effect. The new composites presented general characteristics similar to the commercial ones; however, they didn t present the properties expected. This behavior was attributed to the lower degree of monomer reaction and to the granulometry and size distribution of the mineral filler in the polymeric matrix

4-Hydroxy-4,4-diphenylbutan-2-one

The mol­ecules of the title compound, C16H16O2, display an intra­molecular O—HO hydrogen bond between the hydroxyl donor and the ketone acceptor. Inter­molecular C—Hπ inter­actions connect adjacent mol­ecules into chains that propagate parallel to the ac diagonal. The chains are arranged in sheets, and mol­ecules in adjacent sheets inter­act via inter­molecular O—HO hydrogen bonds.