Luiz H. Catalani

Direct UV photocrosslinking of poly(N-vinyl-2-pyrrolidone) (PVP) to produce hydrogels

Hydrogels for biomedical purposes, made from synthetic polymers as starting materials and free of co-adjuvant molecules, have been produced almost exclusively by high-energy radiative processes. On the other hand, UV photocrosslinking of such materials has been used in conjunction of monomers and/or photoinitiators. This work was addressed to the analysis of poly(N-vinyl-2-pyrrolidone) (PVP) submitted to direct photocrosslinking in aqueous solution, using low pressure Hg lamp (lem ¼ 254 nm). The process efficiency was evaluated, and the properties of the hydrogel formed were determined. The product thus formed has similar micro- and macroscopic properties, as compared to hydrogels produced by high-energy radiation and presents no cytotoxicity. These results demonstrated the viability of using this method as a versatile alternative to hydrogel production, broadening the possibility of its production where high-energy radiation facilities are not available.

Synthesis and swelling behavior of xanthan-based hydrogels

In this work xanthan chains were crosslinked by esterification reaction at 165 °C either in the absence or in the presence of citric acid. Higher crosslinking density was obtained using citric acid, as evidenced by its lower swelling degree. Tensiometry, a very precise and sensitive technique, was applied to study swelling rates and diffusion mechanisms of water, which was initially quasi-Fickian, controlled by wicking properties, changing to Fickian or Anomalous, depending on hydrogel composition. Hydrogels swelling degree increased at high pH values, due to electrostatic repulsion and ester linkages rupture. Equilibrium swelling degree was affected by salts, depending on gel composition and kind of salt. Effects could be explained by interaction between ions and polymeric chains, EPA/EPD ability of water or osmotic gradient.

Oxidation of melatonin and its catabolites, N1‐acetyl‐N 2‐formyl‐5‐methoxykynuramine and N1‐acetyl‐5‐methoxykynuramine, by activated leukocytes

Abstract:  N 1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) and N1‐acetyl‐5‐methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)‐activated leukocytes. An HPLC‐based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse‐phase column using acetonitrile/H2O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV‐VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5–10% (from 18‐hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide‐dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

Superoxide-dependent oxidation of melatonin by myeloperoxidase.

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.

Supramolecular cationic tetraruthenated porphyrin induces single-strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in DNA in the presence of light.

Abstract— The aim of this investigation is the evaluation of DNA interaction of with tetraruthenated porphyrin (TRP) and of DNA damage in the presence of light. Direct‐fluorescence and electronic absorption measurements after incubation of DNA with TRP indicate strong binding between pBR322 DNA or calf thymus DNA with the modified porphyrin. Exposure of pBR322 DNA to TRP (up to 3 μM) and light leads to single‐strand break formation as determined by the conversion of the supercoiled form (form I) of the plasmid into the nicked circular form (form II). Oxidative DNA base damage was evaluated by the detection of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo) after irradiation of calf thymus DNA in the presence of the TRP. The data demonstrated a dose and time dependence with each type of DNA damage. These data indicate (1) a specificity of the binding mode and (2) type I and II photoinduced mechanisms leading to strand scission activity and 8‐oxodGuo formation. Accordingly, singlet molecular oxygen formation, after TRP excitation, was confirmed by near‐infrared emission. From these investigations a potential application of TRP in photodynamic therapy is proposed.

Charge generation, charge transport, and residual charge in the electrospinning of polymers: A review of issues and complications

Electrospinning has become a widely implemented technique for the generation of nonwoven mats that are useful in tissue engineering and filter applications. The overriding factor that has contributed to the popularity of this method is the ease with which fibers with submicron diameters can be produced. Fibers on that size scale are comparable to protein filaments that are observed in the extracellular matrix. The apparatus and procedures for conducting electrospinning experiments are ostensibly simple. While it is rarely reported in the literature on this topic, any experience with this method of fiber spinning reveals substantial ambiguities in how the process can be controlled to generate reproducible results. The simplicity of the procedure belies the complexity of the physical processes that determine the electrospinning process dynamics. In this article, three process domains and the physical domain of charge interaction are identified as important in electrospinning: (a) creation of charge carriers...

High concentrations of the melatonin metabolite, N1-acetyl-N 2-formyl-5-methoxykynuramine, in cerebrospinal fluid of patients with meningitis: a possible immunomodulatory mechanism

Abstract:  We evaluated the presence of the melatonin metabolite N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK), in cerebrospinal fluid (CSF) of patients with viral meningitis (n = 20) and control samples (n = 8) and correlate AFMK levels with inflammatory markers such as cellularity, protein, tumor necrosis factor (TNF)‐α, interleukin (IL)‐8 and IL‐1β levels. A portion of the CSF was extracted with dichloromethane (1:5) and analyzed by high‐performance liquid chromatography (HPLC) under standardized conditions for AFMK. AFMK was detected in 16 of 20 CSF samples of patients with viral meningitis; the concentration of AFMK was found to be above the quantification limit (50 nmol/L) in six of these samples. AFMK was not detected in any of the eight control samples. The samples were classified into groups according to AFMK levels: undetectable (<10 nmol/L, group I), detectable but below the quantification limit (< 50 nmol/L, group II), and quantified (>50 nmol/L, group III). Group II presented the highest levels of proteins and IL‐8, whereas group III showed the lowest levels of the inflammatory parameters. This study supports our hypothesis that inflammation favors the formation of AFMK and that this compound has immunomodulatory activity in vivo.

Oxidation of melatonin and its catabolites, N-1-acetyl-N (2)-formyl-5-methoxykynuramine and N-1-acetyl-5-methoxykynuramine, by activated leukocytes

Abstract:  N 1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) and N1‐acetyl‐5‐methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)‐activated leukocytes. An HPLC‐based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse‐phase column using acetonitrile/H2O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV‐VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5–10% (from 18‐hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide‐dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

Interferon-gamma independent oxidation of melatonin by macrophages

Abstract: Mononuclear phagocytes appear to synthesize kynurenine‐like products from the oxidation of biologically active indole compounds including melatonin, catalyzed by interferon (IFN)‐γ‐inducible enzyme indoleamine 2,3‐dioxygenase (IDO). Concanavalin A (Con A) is a plant lectin that induces interferon‐gamma (IFN‐γ) production by T cells. In this study we investigated whether Con A‐primed peritoneal macrophages are able to oxidize melatonin to N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK). The AFMK production was accompanied by chemiluminescence. It was found that Con A‐primed but not resident macrophages produce AFMK. Surprisingly, Con A‐primed macrophages from IFN‐γ‐deficient mice were as effective as macrophages from IFN‐γ‐sufficient mice in oxidizing melatonin. Moreover, addition of an inhibitor of IDO (1‐methyltryptophan) did not affect melatonin oxidation. Con A‐primed but not resident macrophages have a significant content of myeloperoxidase (MPO) and inhibition of MPO by azide completely blocked chemiluminescence and AFMK production. Thus, our findings provide evidence that melatonin oxidation by macrophages may occur through a mechanism dependent of MPO and independent of IFN‐γ and IDO activity.

Studies on the Intramolecular Electron Transfer Catalyzed Thermolysis of 1,2-Dioxetanes

Abstract This work reports the synthesis and the chemiluminescence properties of the dioxetanes: 4-ethyl-4-methyl-3-(3-methoxyphenyl)-1,2-dioxetane (I), 4-ethyl-4-methyl-3-(3-tert-butyldimethylsilyloxyphenyl)-1,2-dioxetane (II), 4,4-dimethyl-3-(3-methoxybenzyl)-1,2-dioxetane (III) and 4,4-dimethyl-3-(3-tert-butyldimethylsilyloxybenzyl)-1,2-dioxetane (IV). While in the thermal decomposition of I–IV preferential formation of triplet excited states is observed, in the presence of fluoride ions the decomposition rate constants of II and IV increase drastically and singlet excited states are formed with high quantum yields. These results are discussed based on the CIEEL (‘Chemically Initiated Electron Exchange Luminescence’) mechanism where the decomposition of the dioxetanes should be initiated by an intramolecular electron transfer from the phenolate ion (generated by fluoride catalyzed deprotection of the silyloxy group), either directly bound to the peroxidic ring (II) or separated from it by a methylene bridge (IV).