Gd(III) chelates as NMR probes of protein-protein interactions. Case study: rubredoxin and cytochrome c3

Inorganic Chemistry 50(21):10600-7

An NMR structural study of nickel-substituted rubredoxin

J Biol Inorg Chem (2010) 15:409–420 DOI 10.1007/s00775-009-0613-6

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

Biomol NMR Assign (2007) 1:81–83 DOI 10.1007/s12104-007-9022-3

Integrated study by NMR and X-ray Crystallography on the analysis of the molecular interactions in heme-binding proteins

Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural

Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica Estrutural

Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.

Development and application of NMR techniques to study water-in-CO2 microemulsions

Self-assembly is a phenomenon that occurs frequently throughout the universe. In this work, two self-assembling systems were studied: the formation of reverse micelles in isooctane and in supercritical CO2 (scCO2), and the formation of gels in organic solvents. The goal was the physicochemical study of these systems and the development of an NMR methodology to study them. In this work, AOT was used as a model molecule both to comprehensively study a widely researched system water/AOT/isooctane at different water concentrations and to assess its aggregation in supercritical carbon dioxide at different pressures. In order to do so an NMR methodology was devised, in which it was possible to accurately determine hydrodynamic radius of the micelle (in agreement with DLS measurements) using diffusion ordered spectroscopy (DOSY), the micellar stability and its dynamics. This was mostly assessed by 1H NMR relaxation studies, which allowed to determine correlation times and size of correlating water molecules, which are in agreement with the size of the shell that interacts with the micellar layer. The encapsulation of differently-sized carbohydrates was also studied and allowed to understand the dynamics and stability of the aggregates in such conditions. A W/CO2 microemulsion was prepared using AOT and water in scCO2, with ethanol as cosurfactant. The behaviour of the components of the system at different pressures was assessed and it is likely that above 130 bar reverse microemulsions were achieved. The homogeneity of the system was also determined by NMR. The formation of the gel network by two small molecular organogelators in toluene-d8 was studied by DOSY. A methodology using One-shot DOSY to perform the spectra was designed and applied with success. This yielded an understanding about the role of the solvent and gelator in the aggregation process, as an estimation of the time of gelation.

EFEITO DE DIFERENTES NÍVEIS DE PROTEÍNA E DE FIBRA BRUTA NA ALIMENTAÇÃO DE JUVENIS DE MATRINXÃ, Brycon cephalus

São apresentados os resultados de um experimento realizado com matrinxãs, Brycon cephalus,alimentados com nove dietas contendo três níveis de proteína (19, 25 e 31%) combinados com três níveis de fibra (cerca de 2, 10 e 20%). O peso final foi mais elevado para os peixes que receberam ração com maiores níveis de proteína bruta, mas não foi afetado pela elevação dos níveis de fibra bruta. Os níveis mais elevados de proteína na ração aumentaram o conteúdo de proteína da carcaça, e diminuíram o conteúdo de gordura e cinza, enquanto os peixes alimentados com as rações com os níveis mais elevados de fibra tinham mais proteína e cinza corporal e menores níveis de gordura. Os resultados sugerem que ingredientes com níveis elevados de fibra são bem utilizados pelo matrinxã, aumentando desta maneira a variedade de subprodutos agrícolas, normalmente ricos em fibra bruta, que podem ser usados na formulação de dietas práticas para esta espécie.

Safest level of tricaine methanesulfonate (ms-222) to induce anesthesia in juveniles of Matrinxã, Brycon cephalus

The use of MS-222 as an anesthetic for matrinxã juveniles was investigated. At dosage of 100 mg/L or lower fish did not achieve a complete anesthesia state. At 150 mg/L, MS-222 induced anesthesia within 36 seconds and recovered from a 10 minutes period of anesthesia within 5.2 min. Higher concentrations (200, 250 and 300 mg/L) anesthetized fish in lesser times, with the offset of mortality (16.7 and 33.3%) at the 200 and 300 mg/L MS-222 doses, respectively. The only significant differences observed in the hematological parameters, was for the glucose values in fish anesthetized with 250 and 300 mg/L. From the results, the recommended dose of MS-222 for handling matrinxã juveniles is 150 mg/L.

Fauna parasitológica de Alevinos de Matrinchã Brycon cephalus (Günther, 1869) coletados nos Rios Negro e Solimões, na Amazônia central

Aproximadamente 2500 alevinos de matrinchã com comprimento padrão de 0,8 a 5,0cm foram coletados no lago Catalão, margem direita do rio Negro e no rio Solimões, próximo a ilha da Marchantaria, Estado do Amazonas. Uma subamostra de 136 peixes foi retirada para exames parasitológicos e o restante foi colocado em tanques em uma estação de piscicultura, durante 30 dias. Após esse período uma segunda amostra foi retirada e examinada. Dos 136 peixes: 49 (36%) estavam parasitados com metacercárias de trematódeos; 32 (23,5%) com nematóides Spirocamallanus inopinatus; 26 (19%) com monogenóideos Jainus amazonensis; 3 (2%) com protozoários Trichodina sp.; 1 (0,7%) com acantocéfalo Echinorhynchus sp. e 2 (1,5%) com estágios larvais de cestóides. Dos peixes mantidos nos tanques, após 30 dias foi examinada uma subamostra de 60 peixes: 42 (70%) estavam parasitados com J. amazonensis; 14 (23%) com S. inopinatus; 9 (15%) com metacercárias de trematódeos e 2 (3%) com Echinorhynchus sp. A intensidade média e abundância média foram maiores em J. amazonensis (342 e 65), seguido por cestóides (70 e 1), Trichodina sp. (13 e 0,3), trematódeos (5 e 2), S. inopinatus (1 e 0,3) e Echinorhynchus sp. (1 e 0,007). Na estação de piscicultura a intensidade média e abundância média também foram maiores para o J. amazonensis (222 e 115) em relação aos demais grupos. A espécie J. amazonensis apresentou maior intensidade média e menor prevalência na natureza. Houve variação na composição da fauna parasitógica e nos índices de infestação, após 30 dias de permanência na estação de piscicultura.

Composição corporal e alimentar do Matrinxã, Brycon cephalus (Günther, 1869), na Amazônia central

Foram estudados a dieta, a composição corporal e o valor de energia bruta do conteúdo estomacal, filé e fígado, bem como alguns frutos e sementes que fazem parte da dieta do matrinxã, relacionando-se estes parâmetros com as flutuações do nível da água durante um ciclo anual. Observou-se que a espécie se alimenta de sementes, frutos, flores, restos de vegetais e insetos, apresentando hábito alimentar onívoro. Estômagos vazios foram frequentes (68,5%) nos diferentes períodos hidrológicos. As análises proximais e valor da energia bruta do filé e fígado dos peixes analisados não apresentaram diferenças significativas entre os sexos (P>0,05). No entanto, observou-se diferenças entre os períodos de águas baixas e altas. As médias das composições centesimais e os valores de energia bruta do conteúdo estomacal, expressos com base na matéria seca revelaram, na enchente, um baixo teor de proteína (8,9%) e cinza (1,3%), alto valor de lipídios (70,2%) e energia (775 Kcal EB/100g). Na seca, o teor de proteína (24,9%) e cinza (6,3%) foram maiores, porém os teores de lipídios (13,2%) e de energia (416,9 Kcal EB 100g) foram mais baixos que na cheia. Foi verificado abundante depósito de gordura cavitária o ano todo, diminuindo em janeiro, mês em que o matrinxã realiza a desova.

Estocagem tecidual e utilização de lipídeos em Matrinxã, Brycon cephalus (Günther, 1869)

Os lipídeos exercem diversas funções biológicas, como constituintes de membranas, precursores de hormônios, e por seu alto conteúdo calórico, um eficiente composto para estoque de energia. Nos peixes, ao contrário dos mamíferos, os modelos de deposição lipídica são mais diversos. O objetivo deste trabalho foi avaliar a concentração de lipídeo total no plasma e nos tecidos somático e reprodutivo do matrinxã, Brycon cephalus, macho e fêmea, destacando alguns índices somáticos relacionados à dinâmica desse substrato. De outubro/97 a janeiro/99, foram amostrados, mensalmente, 8 a 12 peixes, totalizando 174 animais. Após anestesia, retirou-se sangue heparinizado para dosagem do triacilglicerol plasmático. Cada peixe foi mensurado e fígado, gônadas e gordura visceral retirados e pesados para cálculo do IHS (índice hepatossomático) IGS (índice gonadossomático) e IGVS (índice gorduro-viscerossomático). Os maiores teores do lipídeo total ocorreram no músculo vermelho, com aproximadamente 18%, seguido pelo fígado e gônadas com valores médios de 16,5%. O músculo branco apresentou o menor teor com 2,3%. A análise de variância dos IHS e IGVS mostrou que ambos apresentaram efeito significativo para bimestre, com índices mais baixos em períodos de temperaturas mais altas, e não significativo para sexo e a interação sexo e bimestre. O matrinxã estoca lipídeos em vários tecidos corpóreos incluindo gordura mesentérica, fígado, músculos e gônadas.

Feeding of the Brycon cephalus, Triportheus elongatus and Semaprochilodus insignis (osteichthyes, characiformes) larvae in Solimões/Amazonas River and floodplain areas.

Information on larval fish feeding is essential for understanding their trophic relations, including the management in conditions totally or partially controlled by humans. An experiment was designed to evaluate the larval diets of three commercially important species. Four varzea-lakes and the adjacent river were sampled with bongo and hand nets from January 1993 to November 1995. Larval diets were evaluated by length-classes and capture sites, and were tested by two factor ANOVA. The larvae were feeding in all habitats, except in the flooded forests. The three species had different diets, which varied with their length and lake. The rotifers were the main initial food item of the three species, replaced by fish larvae in Brycon cephalus, cladocerans in Triportheus elongatus and detritus in Semaprochilodus insignis. The increase of the ingestion limit, as the larvae grew, was higher than the increase in the consumed prey size for the three species.

Preliminary observations on the effect of sudden changes of temperature on survival of young Matrinxã (Brycon cephalus) under laboratory conditions

Matrinxã is a very promising amazonian fish for fish culture in Brazil. This study is aimed at determining the approximate tolerated temperature range in this species. Groups of ten young matrinxã specimens (15.1±0.8 cm average length and 58.3±10.3 g average weight) were subjected to 9 different temperatures for 24 hours without previous acclimation. Fish were transferred from an initial temperature of 27ºC to those ranging from 12 to 39ºC at 3ºC intervals. Both 12ºC and 39ºC temperatures were lethal for this species with 100% mortality rate. Following 2 minutes of exposure to 39ºC fish changed behavior, showing an increase in opercular movements and erratic swimming; mortality reached 100% after 18 minutes. At 12ºC, fish lost equilibrium immediately after exposure and started swimming erratically; after only 4 minutes fish became lethargic and remained immobile on the bottom of the tank. Total mortality was only evident following 24 hours. At 15ºC matrinxã lost equilibrium after 5 to 6 minutes of exposure but mortality was only 20% after 24 hours. Fish tolerated well temperatures ranging from 18 to 36ºC with 100% survival after 24 hours. This preliminary study suggests that temperatures between 18 and 36ºC are the approximate range normally tolerated by this species, although survival at other temperatures may be increased by gradually acclimating fish to the more severe increases or decreases in temperature. In addition, it indicates that matrinxã may be cultivated over a wide geographical area.

Avaliação de níveis protéicos para a nutrição de juvenis de matrinxã (Brycon cephalus)

O matrinxã (Brycon cephalus - Gunther, 1869), é uma espécie de peixe nativa da bacia Amazônica que tem despertado grande interesse entre pesquisadores e piscicultores de todo o Brasil. A crescente demanda pelo cultivo desta espécie em ambiente controlado se deve, principalmente, à sua pronta adaptação ao cativeiro e à aceitação de alimentos artificiais, tanto de origem vegetal quanto animal, ao lado do seu elevado valor comercial. Contudo, para ser um empreendimento comercial bem sucedido, o alimento a ser fornecido a esta espécie deve atender a suas necessidades em proteína e permitir elevados ganhos de peso em períodos curtos. O experimento foi conduzido na estação de aqüicultura da Embrapa Amazônia Ocidental. Foram testados cinco níveis de proteína bruta (16, 19, 22, 25 e 28%) em dietas isocalóricas (EB = 390 kcal/100g) em um delineamento experimental inteiramente casualizado com três repetições para cada tratamento. Os resultados obtidos após 210 dias de estudo mostraram que a dieta contendo 28 % de proteína bruta promoveu o maior ganho de peso, melhor conversão alimentar e mais alto crescimento corporal entre os níveis testados, indicando que este nível protéico atendeu satisfatoriamente às necessidades de proteína para esta espécie, nas condições deste experimento.