Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Optimizing the Synthesis of AZN

Azulenyl nitrone (AZN) is a bright green compound that can be used to stain different compounds, including plastics. When these stained plastics are irradiated, as they commonly are in the sterilization of medical devices, AZN changes color from green to red, constituting a permanent change. This would make obsolete the current methods of radioactive labeling and maintain the integrity of medical equipment. Although a method of synthesis is already in place, the aim was to improve the yield significantly and find a more efficient and cost-effective procedure. Last year, the procedure used resulted in 18 to 20% of AZN synthesized at the most favorable conditions. With that in mind, this year modifications were done in the hopes of improving the yield. The solvent was changed to a mixture of isopropanol and triethylamine, a stronger base, and a catalytic amount of N-tertbutyl hydroxylamine hydrochloride was used (around 4 equivalents). The reaction time was also increased to 7 days, rather than 2. After several trials, the samples were run through column chromatography and the average yield was 70%, a much more promising result than that obtained last year. There is still research to be done to improve the technicalities of the procedure, including altering the amounts of N-tertbutyl hydroxylamine hydrochloride to try and obtain similar data with fewer amounts. This portion of the research will be done in the second half of the year. In the meantime, however, a novel and more efficient method of synthesis has been established for the production of AZN that can be potentially commercialized.

Optimizing the Synthesis of AZN

Azulenyl nitrone (AZN) is a bright green compound that can be used to stain different compounds, including plastics. When these stained plastics are irradiated, as they commonly are in the sterilization of medical devices, AZN changes color from green to red, constituting a permanent change. This would make obsolete the current methods of radioactive labeling and maintain the integrity of medical equipment. Although a method of synthesis is already in place, the aim was to improve the yield significantly and find a more efficient and cost-effective procedure. Last year, the procedure used resulted in 18 to 20% of AZN synthesized at the most favorable conditions. With that in mind, this year modifications were done in the hopes of improving the yield. The solvent was changed to a mixture of isopropanol and triethylamine, a stronger base, and a catalytic amount of N-tertbutyl hydroxylamine hydrochloride was used (around 4 equivalents). The reaction time was also increased to 7 days, rather than 2. After several trials, the samples were run through column chromatography and the average yield was 70%, a much more promising result than that obtained last year. There is still research to be done to improve the technicalities of the procedure, including altering the amounts of N-tertbutyl hydroxylamine hydrochloride to try and obtain similar data with fewer amounts. This portion of the research will be done in the second half of the year. In the meantime, however, a novel and more efficient method of synthesis has been established for the production of AZN that can be potentially commercialized.

Micropartículas de quitosana estruturadas com aerosil®: estabilidade, adsorção, encapsulação e liberação de substâncias ativas

Biodegradable microspheres used as controlled release systems are important in pharmaceutics. Chitosan biopolymer represents an attractive biomaterial alternative because of its physicochemical and biological characteristics. Chitosan microspheres are expected to become promising carrier systems for drug and vaccine delivery, especially for non-invasive ways oral, mucosal and transdermal routes. Controlling the swelling rate and swelling capacity of the hydrogel and improving the fragile nature of microspheres under acidic conditions are the key challenges that need to be overcomed in order to enable the exploration of the full pharmaceutical potential use of these microparticles. Many studies have focused on the modification of chitosan microsphere structures with cross-linkers, various polymers blends and new organic-inorganic hybrid systems in order to obtain improved properties. In this work, microspheres made of chitosan and nanosized hydrophobic silica (Aerosil R972) were produced by a method consisting of two steps. First, a preparation of a macroscopically homogeneous chitosan-hydrophobic silica dispersion was prepared followed by spray drying. FTIR spectroscopy, X-ray powder diffraction, differential scanning calorimetry, thermal gravimetric analysis, scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (TEM) were used to characterize the microspheres. Also, the were conducted acid stability, moisture sorption capacity, release properties and biological assays. The chitosan-hydrophobic silica composite microspheres showed improved thermal degradation, lower water affinity, better acid stability and ability to retard rifampicin and propranolol hydrochloride (drug models) release under simulated physiological conditions. In vitro biocompatibility studies indicated low cytotoxicity and low capacity to activate cell production of the pro-inflammatory mediator nitric oxide. The results show here encourage further studies on the use of the new chitosan-hydrophobic silica composite microspheres as drug carrier systems via oral or nasal routes.

Síntese, caracterização e estudo da associação em meio aquoso de copolímeros responsivos de carboximetilcelulose-g-Jeffamine®

This thesis presents the synthesis, characterization and study of the associative behaviour in aqueous media of new responsive graft copolymers, based on carboxymethylcellulose as the water-soluble backbone and Jeffamine® M-2070 e Jeffamine® M-600 (commercial polyetheramines) as the thermoresponsive grafts with high cloud point temperatures in water. The synthesis was performed on aqueous medium, by using 1-ethyl-3- (3-(dimethylamino)-propyl)carbodiimide hydrochloride and N-hydroxysuccinimide as activators of the reaction between carboxylategroupsfrom carboxymethylcellulose and amino groups from polyetheramines. The grafting reaction was confirmed by infrared spectroscopy and the grafting percentage by 1H NMR. The molar mass of the polyetheramines was determined by 1H NMR, whereas the molar mass of CMC and graft copolymers was determined by static light scattering. The salt effect on the association behaviour of the copolymers was evaluated in different aqueous media (Milli-Q water, 0.5M NaCl, 0.5M K2CO3 and synthetic sea water), at different temperatures, through UV-vis, rheology and dynamic light scattering. None of the copolymers solutions, at 5 g/L, turned turbid in Milli-Q water when heated from 25 to 95 °C, probably because of the increase in hydrophibicity promoted by CMC backbone. However, they became turbid in the presence of salts, due to the salting out effect, where the lowest cloud point was observed in 0.5M K2CO3, which was attributed to the highest ionic strength in water, combined to the ability of CO3 2- to decrease polymer-solvents interactions. The hydrodynamic radius and apparent viscosity of the copolymers in aqueous medium changed as a function of salts dissolved in the medium, temperature and copolymer composition. Thermothickening behaviour was observed in 0.5M K2CO3 when the temperature was raised from 25 to 60°C. This performance can be attributed to intermolecular associations as a physical network, since the temperature is above the cloud point of the copolymers in this solvent.

Nd isotopes an geochemistry of bulk deep sea sediments of the Atlantic Ocean

Nd isotopes preserved in fossil fish teeth and ferromanganese crusts have become a common tool for tracking variations in water mass composition and circulation through time. Studies of Nd isotopes extracted from Pleistocene to Holocene bulk sediments using hydroxylamine hydrochloride (HH) solution yield high resolution records of Nd isotopes that can be interpreted in terms of deep water circulation, but concerns about diagenesis and potential contamination of the seawater signal limit application of this technique to geologically young samples. In this study we demonstrate that Nd extracted from the > 63 µm, decarbonated fraction of older Ocean Drilling Program (ODP) sediments using a 0.02 M HH solution produces Nd isotopic ratios that are within error of values from cleaned fossil fish teeth collected from the same samples, indicating that the HH-extractions are robust recorders of deep sea Nd isotopes. This excellent correlation was achieved for 94 paired fish teeth and HH-extraction samples ranging in age from the Miocene to Cretaceous, distributed throughout the north, tropical and south Atlantic, and composed of a range of lithologies including carbonate-rich oozes/chalks and black shales. The strong Nd signal recovered from Cretaceous anoxic black shale sequences is unlikely to be associated with ferromanganese oxide coatings, but may be derived from abundant phosphatic fish teeth and debris or organic matter in these samples. In contrast to the deep water Nd isotopic signal, Sr isotopes from HH-extractions are often offset from seawater values, suggesting that evaluation of Sr isotopes is a conservative test for the integrity of Nd isotopes in the HH fraction. However, rare earth elements (REE) from the HH-extractions and fish teeth produce distinctive middle REE bulge patterns that may prove useful for evaluating whether the Nd isotopic signal represents uncontaminated seawater. Alternatively, a few paired HH-extraction and cleaned fish teeth samples from each site of interest can be used to verify the seawater composition of the HH-extractions. The similarity between isotopic values for the HH-extraction and fish teeth illustrates that the extensive cleaning protocol applied to fish teeth samples is not necessary in typical, carbonate-rich, deep sea sediments.

Seawater carbonate chemistry, Mg/Ca, Sr/Ca, oxygen production and calcification rate during experiments with coral Pocillopora damicornis, 2011

The Sr/Ca of aragonitic coral skeletons is a commonly used palaeothermometer. However skeletal Sr/Ca is typically dominated by weekly-monthly oscillations which do not reflect temperature or seawater composition and the origins of which are currently unknown. To test the impact of transcellular Ca2+ transport processes on skeletal Sr/Ca, colonies of the branching coral, Pocillopora damicornis, were cultured in the presence of inhibitors of Ca-ATPase (ruthenium red) and Ca channels (verapamil hydrochloride). The photosynthesis, respiration and calcification rates of the colonies were monitored throughout the experiment. The skeleton deposited in the presence of the inhibitors was identified (by 42Ca spike) and analysed for Sr/Ca and Mg/Ca by secondary ion mass spectrometry. The Sr/Ca of the aragonite deposited in the presence of either of the inhibitors was not significantly different from that of the solvent (dimethyl sulfoxide) control, although the coral calcification rate was reduced by up to 66% and 73% in the ruthenium red and verapamil treatments, respectively. The typical precision (95% confidence limits) of mean Sr/Ca determinations within any treatment was <±1% and differences in skeletal Sr/Ca between treatments were correspondingly small. Either Ca-ATPase and Ca channels transport Sr2+ and Ca2+ in virtually the same ratio in which they are present in seawater or transcellular processes contribute little Ca2+ to the skeleton and most Ca is derived from seawater transported directly to the calcification site. Variations in the activities of Ca-ATPase and Ca-channels are not responsible for the weekly-monthly Sr/Ca oscillations observed in skeletal chronologies, assuming that the specificities of Ca transcellular transport processes are similar between coral genera.

Transformation to "high grade" neuroendocrine carcinoma as an acquired drug resistance mechanism in EGFR-mutant lung adenocarcinoma.

Several different acquired resistance mechanisms of EGFR mutant lung adenocarcinoma to EGFR-tyrosine kinase inhibitor (TKI) therapy have been described, most recently transformation to small cell lung carcinoma (SCLC). We describe the case of a 46-year-old female with relapsed EGFR exon 19 deletion lung adenocarcinoma treated with erlotinib, and on resistance, cisplatin-pemetrexed. Liver rebiopsy identified an afatinib-resistant combined SCLC and non-small cell carcinoma with neuroendocrine morphology, retaining the EGFR exon 19 deletion. This case highlights acquired EGFR-TKI resistance through transformation to the high-grade neuroendocrine carcinoma spectrum and that that such transformation may not be evident at time of progression on TKI therapy.

Procoagulant state after raloxifene therapy in postmenopausal women

AZEVEDO, George Dantas de et al. Procoagulant state after raloxifene therapy in postmenopausal women. Fertility and Sterility, Estados Unidos, v.84, n.6, p.1680-1684, 2005

Raloxifene therapy does not affect uterine blood flow in postmenopausal women: a transvaginal Doppler study

AZEVEDO, George Dantas de et al. Raloxifene therapy does not affect uterine blood flow in postmenopausal women: a transvaginal Doppler study. Maturitas, Amsterdam, v.47, n.3, p.195-200, 2004

A cocaína e suas manifestações orofaciais

A cocaína é uma droga com ação estimulante no sistema nervoso central, extraída e refinada a partir da planta de coca (Erythroxylum coca). É característica por induzir o consumidor a um estado de hipervigilância reduzindo ao mesmo tempo, o cansaço e a fadiga. Este pó branco, cristalino, de sabor amargo, possui também um efeito anestésico local e vasoconstritor. As formas de apresentação mais comuns da droga são o cloridrato de cocaína e a cocaína crack. Esta droga destaca-se por ser o estimulante mais consumido na Europa com cerca de 3,4 milhões de consumidores estimados no ano de 2014. A prevalência do consumo desta droga em Portugal aumentou 0,3% de 2001, para 2012 na população geral (15-64 anos). Os estudos mais recentes em populações escolares (entre 2010 e 2011) evidenciaram, de um modo geral, o aumento da prevalência de consumo nesta população. Os efeitos adversos resultantes, tanto a nível físico como psíquico, são vários, sendo as manifestações orofaciais as que mais interferem na Qualidade de vida do toxicómano. As manifestações mais frequentes são as perfurações do septo nasal e palatino, bruxismo, gengivite, erosão dentária, xerostomia, cárie, lesões brancas atípicas e cefaleias em salva, tendo o Médico Dentista um papel importante no diagnóstico e tratamento destas lesões. A legislação, ao nível Europeu, sobre drogas procura uma uniformização das medidas aplicadas nos países membros, baseando-se no equilíbrio entre as sanções e o tratamento. Apesar das convenções das Nações Unidas sobre drogas limitarem o consumo de estupefacientes e substâncias psicotrópicas exclusivamente para fins médicos e científicos, cabe aos países signatários a liberdade de decisão das políticas a adoptar em matérias de infrações penais como a posse e o consumo ilegal.

Procoagulant state after raloxifene therapy in postmenopausal women

AZEVEDO, George Dantas de et al. Procoagulant state after raloxifene therapy in postmenopausal women. Fertility and Sterility, Estados Unidos, v.84, n.6, p.1680-1684, 2005

Raloxifene therapy does not affect uterine blood flow in postmenopausal women: a transvaginal Doppler study

AZEVEDO, George Dantas de et al. Raloxifene therapy does not affect uterine blood flow in postmenopausal women: a transvaginal Doppler study. Maturitas, Amsterdam, v.47, n.3, p.195-200, 2004

Produção de quitosana a partir de resíduos de camarão: análise do processo e da operação de secagem

A quitina é encontrada principalmente nos exoesqueletos de crustáceos, insetos e na parede celular de fungos. O biopolímero quitosana é obtido através da hidrólise alcalina da quitina. A despolimerização da quitosana é realizada para se obter um produto com valores baixos de massa molecular. O uso da quitosana em diversas áreas é diretamente relacionada com a massa molecular e o grau de desacetilação do polímero. Os objetivos deste trabalho foram o estudo da cinética de secagem de quitina em camada delgada utilizando um modelo difusivo, considerando a resistência externa à transferência de massa; a determinação do comportamento da massa molecular média viscosimétrica da quitosana, durante a secagem convectiva, em camada delgada; a otimização das etapas de desacetilação e despolimerização da quitosana. A quitina foi obtida de resíduos de camarão. Os experimentos da secagem de quitina e da quitosana foram em secador de bandejas, a 60°C, sendo que para a quitina foram utilizadas duas velocidades do ar de 0,5 e 1,5 m/s. A estimativa da viscosidade intrínseca foi através da equação de Huggins e a massa molecular da quitosana foi calculada pela equação de Mark-Houwink-Sakurada. As otimizações da reação de desacetilação e despolimerização foram realizadas utilizando a metodologia da superfície de resposta. Para a reação de desacetilação foram variados o tempo e a temperatura. Para a reação de despolimerização foram analisados a concentração de ácido clorídrico, a temperatura e o tempo de reação. O modelo difusivo com difusividade efetiva variável, utilizado para analisar a secagem de quitina, apresentou concordância com os dados experimentais, onde foi observado o efeito da resistência externa à transferência de massa, quando utilizada a menor velocidade do ar. A condição ótima da reação de desacetilação para massa molecular foi observada na temperatura de 130°C em 90 min, e correspondeu a massa molecular de 150 kDa e um grau de desacetilação de 90%. A operação de secagem da quitosana causou um aumento na massa molecular média viscosimétrica de 27% e este aumento foi linear com o tempo e a umidade do polímero, apresentando duas regiões. As condições da reação de despolimerização para alcançar 50 kDa foram à temperatura de reação de 65°C, concentração de ácido clorídrico de 35% v/v. Nestas condições a cinética de despolimerização foi de pseudo-primeira ordem, apresentando duas fases.

Effects of feeding carnitine and ractopamine on rainbow trout (Oncorhynchus mykiss, Walbaum 1972)

L-carnitine is required for the transfer of long-chain fatty acids from the cytosol to the mitochondrial matrix for 13-oxidation of them and ractopamine, beta adrenergic agonists, have potential stimulating lipolysis and altering rates of protein degradation and synthesis. Present study was carried out to improve lipid body oxidation and protein-sparing action of fish through addition of L-carnitine and ractopamine to diet of rainbow trout, Oncorhynchus mykiss, Walbaum 1972. An eight-week feeding trial was carried out to evaluate the effects of supplementation of tree levels of L-carnitine tartrate (0, 1 and 2 g/kg) and two levels of ractopamine hydrochloride (0 and 10 ppm) on growth performance, fillet muscle fatty acid compositions and blood biochemical parameters in 288 juvenile rainbow trout (130 g) at 3X2 factorial experimental design. Ractopamine and 1 g/kg carnitine improved the specific growth rate, feed conversion ratio, protein efficiency ratio and weight gain at the end of experiment. The protein and lipid contents of fillet muscle were affected by the inclusion of 10 mg/kg ractopamine in the diet, increasing crude protein and reducing crude fat (P<0.05) of fish fillet muscle. The highest protein and lowest fat contents of fish fillet were observed in diet that contains 2 g/kg carnitine plus ractopamine. Ractopamine and carnitine increased levels of albumin, total protein and globulin in fish blood serum, but carnitine increased triacylglycerol and cholesterol. Fatty acids compositions of fish fillet were also affected by ractopamine and carnitine. All fatty acids except for eicosapentaenoic acid and docosahexaenoic acid, were increased by dietary supplementation of ractopamine. Total saturated fatty acids were not affected by carnitine. Supplementation (P>0.05). However, total n-3 poly unsaturated fatty acids were reduced by carnitine supplementation. A significant interaction was observed between ractopamine and carnitine supplementation regarding the saturated (P<0.01) and n-3 poly unsaturated fatty acid (P<0.001) of fish fillet. This study shows that supplementation of 1 g/kg carnitine and 10 ppm ractopamine could improve performance of juvenile rainbow trout and their combination in diet results in protein increment, fat reduction and change in profile of fatty acids in fillet muscle.