Preparação e caracterização de nanocarreadores de beta-lactose à base de pectina e lisozima / Preparation and characterization of beta-lactose nanocarriers based on pectin and lysozyme

O objetivo deste trabalho foi preparar e caracterizar nanocarreadores via auto-organização a partir da pectina de citros e lisozima para o encapsulamento da ß-lactose. Foram estudadas três condições de interação entre os biopolímeros variando a razão molar pectina/lisozima (3:1, 2:1, 1:1, 1:2 e 1:3), o pH e o tempo de aquecimento. A confirmação da interação foi determinada por espectroscopia no infravermelho por transformada de Fourier (FTIR) e por calorimetria de varredura diferencial (DSC). Os espectros de infravermelho evidenciaram que ligações de hidrogênio foram as principais forças envolvidas na formação dos nanocarreadores e sugeriram a ausência de ß-lactose livre na superfície das nanopartículas. Os termogramas evidenciaram que as nanopartículas formadas na presença de ß-lactose têm maior estabilidade térmica do que as nanopartículas sem ß-lactose. Para ambas as formulações estudadas, na presença e na ausência de ß-lactose, a formação das nanopartículas ocorreu entre os valores de pKa e ponto isoelétrico (pI) da pectina e lisozima, respectivamente, sendo a melhor razão de interação pectina/lisozima 1:2, em pH 10, a 80 ºC por 30 min. As nanopartículas foram formadas via auto-organização e todos as partículas apresentaram distribuição de tamanho homogênea, formato esférico, diâmetro inferior a 100 nm e carga superficial negativa. A morfologia e o tamanho das partículas pouco alteraram com a incorporação da -lactose. A eficiência de encapsulação (EE) da ß-lactose foi superior a 96% para as concentrações estudadas. Ensaios preliminares in vitro, em células epiteliais de câncer de cólon (HCT-116), evidenciaram que as nanopartículas formadas são capazes de adentrar no meio intracelular, possivelmente, por via endocitose

This work aimed to prepare and characterize nanocarriers via self-assembly using citrus pectin and lysozyme for ß-lactose encapsulation. Three interaction conditions between the biopolymers were studied, varying the pectin/lysozyme molar ratio (3:1, 2:1, 1:1, 1:2 and 1:3), pH and heating time. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) determined the interaction's confirmation. The infrared spectra showed that hydrogen bonds were the main forces involved in the formation of nanocarriers and suggested the absence of free ß-lactose on the surface of the nanoparticles. The thermograms showed that nanoparticles formed in the presence of ß-lactose have greater thermal stability than nanoparticles without ß-lactose. For both formulations studied, in the presence and absence of lactose, the formation of nanoparticles occurred between the pKa and isoelectric point (pI) values of pectin and lysozyme, respectively, with the best pectin/lysozyme interaction molar ratio 1:2, at pH 10, at 80 °C for 30 min. Nanoparticles were formed via self-assembly, and all particles presented homogeneous size distribution, spherical shape, diameter less than 100 nm, and negative surface charge. The morphology and size of the particles changed little with the incorporation of ß-lactose. The encapsulation efficiency (EE) of ß-lactose was higher than 96% for the concentrations studied. Preliminary in vitro assays in colon cancer epithelial cells (HCT-116) showed that the nanoparticles formed are capable of entering the intracellular medium, possibly via endocytosis

Immunocytochemical characterization of primary cell culture in canine transmissible venereal tumor / Caracterização imunocitoquímica de culturas primárias de tumor venéreo transmissível canino

Immunochemistry with anti-vimentin, anti-lysozyme, anti-alpha 1 antitrypsin, anti-CD3 and anti-CD79α antibodies has been used for characterization of primary cell culture in the transmissible venereal tumor (TVT). Samples for primary cell culture and immunohistochemistry assays were taken from eight dogs with cytological and clinical diagnosis of TVT. To validate the immunochemical results in the primary cell culture of TVT, a chromosome count was performed. For the statistical analysis, the Mann-Whitney test with p<0.05 was used. TVT tissues and culture cells showed intense anti-vimentin immunoreactivity, lightly to moderate immunoreactivity for anti-lysozyme, and mild for anti-alpha-antitrypsin. No marking was achieved for CD3 and CD79α. All culture cells showed chromosomes variable number of 56 to 68. This is the first report on the use of immunocytochemical characterization in cell culture of TVT. Significant statistic difference between immunochemistry in tissue and culture cell was not established, what suggests that the use of this technique may provide greater certainty for the confirmation of tumors in the primary culture. This fact is particularly important because in vitro culture of tumor tissues has been increasingly used to provide quick access to drug efficacy and presents relevant information to identify potential response to anticancer medicine; so it is possible to understand the behavior of the tumor.(AU)

Os anticorpos anti-vimentina, anti-lisozima, anti-alfa 1 antitripsina, anti-CD3 e anti-CD79α foram empregados para a caracterização de culturas primárias de tumor venéreo transmissível canino (TVT). Amostras para cultura primária e imuno-histoquímica foram coletadas de oito cães com diagnóstico clínico e citológico de TVT. Para validar o resultado inmunocitoquímico nas culturas de TVT foi realizada a contagem de cromossomos. Para a análise estatística o teste de Mann-Whitney foi empregado a um nível de significância de p<0.05. As culturas e os tecidos de TVT apresentaram intensa reatividade para vimentina, moderada a leve para Lisozima, moderada para alfa-antitripsina e não houve marcação para CD3 e CD79α. Finalmente, todas as culturas apresentaram números de cromossomos que variaram de 56 a 68. Este é o primeiro relato que apresenta o uso da immunocitoquímica para a caracterização de culturas de TVT. Assim, e devido ao fato de se observar semelhança entre a imunomarcação em células e tecidos, sugere-se que o uso desta técnica possa auxiliar na confirmação de culturas primárias do tumor, fato muito importante porque a utilização da cultura do tumor pode permitir o acesso a informação relevante sobre resposta potencial a um tratamento e conhecimento do comportamento biológico do tumor.(AU)

Oxidação de resíduos de triptofano em proteínas: formação de ligação cruzada ditriptofano e implicações patofisiológicas / Oxidation of tryptophan residues in proteins: formation of the ditryptophan cross-link and pathophysiological implications

Apesar de extensa investigação das modificações oxidativas irreversíveis sofridas pelas proteínas in vitro e in vivo, os produtos formados pela oxidação de resíduos de triptofano ainda permanecem apenas parcialmente conhecidos. Recentemente, nosso grupo caracterizou uma ligação cruzada de ditriptofano produzida pela recombinação de radicais hSOD1-triptofanila gerados pelo ataque do radical carbonato produzido durante a atividade peroxidásica da enzima superóxido dismutase humana (hSOD1). Neste trabalho, examinamos se a ligação ditriptofano pode ser formada em outras proteínas, além da hSOD1 e por outros oxidantes, além do radical carbonato. A lisozima da clara do ovo e a beta cristalino bovina foram utilizadas como alvos de oxidação. A lisozima foi utilizada por ser uma enzima pequena (129 aminoácidos) e de estrutura bem conhecida, contendo seis resíduos de Trp. Os resultados mostraram que o radical carbonato, gerado enzimatica ou fotoliticamente, promove a oxidação, dimerização e inativação da lisozima. Os principais produtos de oxidação caracterizados por análise de nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano e N-formilquinurenina juntamente com um dímero de lisozima (lisozima-Trp28-Trp28-lisozima) e um hetero dímero lisozima-hSOD1 (lisozima-Trp28-Trp32-hSOD1), ambos ligados por uma ligação ditriptofano. Também demonstramos que a irradiação da lisozima com luz UVC leva à formação do dímero lisozima-Trp28-Trp28-lisozima. Em consequência, resolvemos tratar a beta cristalino bovina com radical carbonato gerado fotoliticamente ou com luz UVC, e a proteína também sofreu oxidação, dimerização e agregação. Os principais produtos de oxidação caracterizados por nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano, N-formilquinurenina, DOPA e um dímero de beta cristalino (ßB2-Trp151-Trp151-ßB2). A irradiação com luz UVC também levou à formação de um dímero intra-cadeia, caracterizado como ßA2-Trp78-Trp81. Quando a beta cristalino foi irradiada com um simulador de luz solar (UVA e UVB) também foi possível observar um dímero, caracterizado como ßA2-Trp150-Trp150-ßA2. A presença de produtos de oxidação de resíduos de Trp, dentre eles a ligação cruzada ditritpofano, também foi avaliada in vivo, utilizando o cristalino de pacientes que foram submetidos a cirurgia para remoção de catarata. Beta, alfa e gama cristalino foram as principais proteínas identificadas nas frações solúvel e insolúvel do cristalino. A principal modificação pós-traducionais identificada foi deamidação. Um alto conteúdo de resíduos de metionina e triptofano oxidados foram identificados nas proteínas presentes na fração insolúvel. Os principais produtos de oxidação de Trp identificados por nano-ESI-Q-TOF-MS/MS foram quinurenina e N-formilquinurenina. A presença de dímeros covalentes no cristalino com catarata foi confirmada por análises de massas. A completa caracterização desses dímeros (ßB1-Trp127-Trp127-ßB1 e ßB1-Trp193-Trp193-ßB1) confirmou que as cadeias polipeptídicas foram ligadas por uma ligação ditriptofano. Em síntese, nossos dados demonstraram que o radical carbonato e a luz UV podem produzir dímeros de ditriptofano em diferentes proteínas. Também, a presença da ligação cruzada de ditriptofano in vivo (catarata humana) foi pela primeira vez detectada

Despite extensive investigation of irreversible oxidative modifications suffered by proteins in vitro and in vivo, the products formed by oxidation of tryptophan residues remain partially characterized. Our group recently described a ditryptophan cross-link produced by recombination of hSOD1-tryptophanyl radicals generated by attack of the carbonate radical produced during the peroxidase activity of the human superoxide dismutase (hSOD1) enzyme. Here, we examine whether the ditryptophan cross-link can be produced in others proteins besides the hSOD1 and by other oxidants, in addition to the carbonate radical. The egg white lysozyme and bovine beta crystalline were used as targets. Lysozyme was used because it is a small enzyme (129 amino acids) with a well-known structure, containing six Trp residues. The results showed that the carbonate radical, generated enzymatically or photolytically, promotes lysozyme oxidation, inactivation and dimerization. The major oxidation products characterized by nano-ESI-Q-TOF-MS/MS analysis were hydroxy-tryptophan and N-formylkynurenine together with a dimer of lysozyme (lysozyme-Trp28-Trp28-lysozyme) and a hetero dimer hSOD1-lysozyme (lysozyme-Trp28-Trp32-hSOD1), both bound by a ditryptophan cross-link. Also, it was demonstrated that lysozyme irradiation with UVC light leads to the formation of the dimer lysozyme-Trp28-Trp28-lysozyme. In view of these results, we decided to treat beta crystalline bovine with photolytically generated carbonate radical and UVC. Beta crystalline also suffered oxidation, dimerization and aggregation. The major oxidation products characterized were hydroxy-tryptophan, N-formylkynurenine, DOPA and a beta crystalline dimer (ßB2-Trp151-Trp151-ßB2) by nano-ESI-Q-TOF-MS/MS. Irradiation with UVC light also led to the formation of an intra-chain dimer, which was characterized as ßA2-Trp78-Trp81. When beta crystalline was irradiated with a solar simulator (UVA and UVB), it was also possible to observe a dimer which was characterized as ßA2-Trp150-Trp150-ßA2. The presence of oxidized tryptophan products, including the ditryptophan cross-link, was also evaluated in vivo in the lenses of patients submitted to cataract removal. Beta, alpha and gamma crystalline were the main proteins identified in soluble and insoluble fractions of the lenses. The main post translational modification identified was deamidation. A high content of oxidized methionine and tryptophan residues were identified in proteins present in the insoluble fraction. The main tryptophan oxidation products identified by nano-ESI-Q-TOF-MS/MS were kynurenine and N-formylkynurenine. The presence of covalent dimers in the lenses with cataract was demonstrated by mass analysis. Full MS/MS characterization of the dimers ßB1-Trp127-Trp127-ßB1 and ßB1-Trp193-Trp193-ßB1 confirmed that they were linked by a ditryptophan bond. In summary, our data demonstrate that the carbonate radical and UV light can produce ditryptophan dimers in different proteins. Also, the presence of the ditryptophan cross-link was first detected in vivo (human cataract)

Estudio cualitativo del efecto del consumo de tintura madre de Allium sativum L. en la concentración de lisozima y proteínas totales en niños menores de 6 años con infecciones respiratorias agudas (I.R.A.) en el consultorio de Hualqui, VIII Región, Chile / Qualitative study of the effect on consumption of the mother tincture of Allium sativum (garlic) in the lysozyme concentration and total protein in children under 6 years old with acute respiratory infection in the Hualqui Medic´s office, VIII Region of Chile

Sampled population was children under 6 years with acute respiratory infection and the sample were obtained from sputum. The aim was to determine the changes in the concentrations ob both, lisozyme and total protein, before and after the intervention with the garlic mother tincture. It was a pilot study quantitative and through a system of nonrandomness simple of the probabilistic a sample of 25 individuals for determining if you belong to the treatment group (mother tincture) or control (placebo). The results indicate a decrease in the concentration of lysozyme and total proteins in the treatment group between 3 to 5 days after initiated treatment, on the other hand the control group showed an increase in the measurements. Only the treatment group showed positive changes in type symptomatical of the disease. Mother Tincture of garlic is a phytotherapeutic alternative excellent for effectively combat acute respiratory infections in children.

La población muestreada fueron niños menores de 6 años con I.R.A y la muestra fue obtenida de la expectoración. El objetivo fue determinar los cambios en la concentraciones de lisozima y proteínas totales, antes y después de la intervención con la tintura madre de ajo. Fue un estudio cuantitativo experimental y a través de un sistema de aleatoriedad simple del tipo probabilístico se toma una muestra de 25 individuos para determinar si pertenecerán al grupo tratamiento (tintura madre) o control (placebo). Los resultados obtenidos indican una disminución en la concentración de lisozima y proteínas totales del grupo tratamiento entre los 3 a 5 días después de iniciado el tratamiento, en cambio el grupo control reveló un aumento en las mediciones. Solamente el grupo tratamiento evidenció cambios positivos de tipo sintomatológico de la enfermedad. La tintura madre de ajo es una excelente alternativa fitoterapeútica para el combate eficaz contra las infecciones respiratorias agudas en niños.

Effect of lysozyme or nisin on survival of some bacteria treated with high pressure at subzero temperature

The aim of this work was to examine the inactivation of some Gram-positive and Gram-negative bacteria exposed to the pressure of 193 MPa at -20 ºC in the presence of lysozyme or nisin at concentration of 400 mg/ml. The highest effect of pressure at subzero temperature and lysozyme was found with pressure sensitive Pseudomonas fluorescens; viable cells of this strain were not detected in 1 ml of sample after combined treatment. The action of pressure at subzero temperature and lysozyme or nisin against Escherichia coli led to synergistic reduction by 0.7 or 1.6 log cycles, respectively, while it was practically insignificant for two Staphylococcus aureus strains. Viability loss of E. coli and S. aureus occurred during storage for 20 h of the samples at 37 and 5 ºC, which were previously pressurized with lysozyme or nisin. The synergistic effect of pressure and nisin at pH 5 against E. coli cells just after the pressure treatment was lower than that at pH 7, however, the extent of the lethal effect after storage was higher.

Characterization of tear film protein in rabbits following exposure to SR90 B-radiation

Purpose To assess the changes in tear composition in rabbits following exposure to Sr90 beta - radiation. Methods The damage induced by beta - radiation from Sr90 applicator, at doses of 20 Gy, 40 Gy and 60 Gy in weekly sitting of 10 Gy to treated left eye and the right untreated one of 25 there-month-old New Zealand white rabbits was assessed. An additional ten rabbits were used as a control. Tears collected after 24 hours were measured for protein content and lysozyme concentration. The lysozyme molecular structure was studied through the use of column chromatography; cellogel paper electrophoresis and radial diffusion technique was utilized for the demonstrated doses. The delayed effects of beta-radiation on tear lysozyme after one and three months' post exposure was also studied. All samples were analyzed six times and the mean result obtained. Results The radiation damage process increased by increasing the exposure doses and this phenomenon still propagated after the study delay periods which reported no remarks for repair. A significant decrease in the lysozyme concentration for the treated and untreated eyes was observed after the dose of 60 Gy [-61.46%] for the treated eye and [-70.64%] for the untreated one. Conclusions Although irradiation has proved to be a valuable tool in controlling eye disease, with excellent survival rates, nevertheless, side effects of radiation form serious limitations for the applicabilities of this technique

Prenatal development of human major salivary glands. Histological and immunohistochemical characteristics with reference to adult and neoplastic salivary glands

No abstract available.

Dosagem de lisozima na lágrima de recém-nascidos / Lysozyme concentration in tears of newborn babies

A concentraçäo de lisozima lacrimal foi medida pelo método da lisoplaca em 47 recém-nascidos divididos em 3 grupos; 22 de termo, 11 de termo com baixo peso e 14 prematuros. A concentraçäo de lisozima foi maior no grupo de termo comparando-se com o grupo de baixo peso e prematuros e seus valores aumentaram com peso e idade gestacional dos recém-nascidos

Ammiotic fluid lysozyme activity in fetal distress

Lysozyme activity was m,easured in amniotic fluid samples from 90 pregnant women with gestacional age ranging from 30 to 41 weeks. Twenty-nine samples were from high-risk subjects with different pathologies and signs of fetal distress. The control group consisted of 20 normal and 41 pathological pregnant women, whose disorders included Rh isoimmunization, diabetes, systemic arterial hypertension and pre-eclampsia without signs of fetal distress. amniotic fluid lysozyme levels in normal controls were similar to those detected in abnormal pregnant women without signs of fetal distress (x = 156.0 vs 131.8 microng/ml for 43-37 weeks of gestation), with a tendency toward higher values as pregnancy progressed to term in high-risk pregnant women with signs of fetal distress, regardless of neonate birth weight, than in subjects showing no such sugns (x = 40.3 and x = 25.4 microng/ml at 34-41 weeks of gestation, respectively). These data support the possibility of using amniotic fluid lysosyme activity levels as an indicator of fetal distress

Influência da bromohexine sobre os níveis de lisozima da secreçäo lacrimal, o teste de rosa bengala e os sintomas oculares na síndrome de Sjögren / Influence of bromohexine on tear lysozyme level, rose-bengal test and ocular symptoms in Sjogren's syndrome

Foram estados, em 21 pacientes portadoras de Síndrome de Sjögren primária ou secundária, a influência da bromohexine sobre os níveis de lisozima da secreçäo lacrimal, teste de rosa bengala e sintomas oculares. Realizamos estudo cego com administraçäo de placebo (um comprimido por dia) e bromohexine (um comprimido de 48 mg por dia), durante 2 períodos consecutivos de 3 semanas (21 dias), com avaliaçöes do nível de lisozima lacrimal, sintomas oculares e teste de rosa bengala no dia antecedente ao ínicio do tratamento e após o uso de placebo (22§) e de bromohexine (44§ dia). Näo houve influência estatisticamente significante da bromohexine sobre os parâmetros analisados

Hydrolytic enzymes in macrophages from leprosy patients in presence of Mycobacterium leprae.

Presence of Mycobacterium leprae in association with in vitro cultured macrophages, from bacillary negative long term treated lepromatous leprosy patients, induces reduced level of protein and lowering of hydrolytic enzymes like p-glucuronidase, Lysozyme and Lactic dehydrogenase. Alkaline phosphatase, on the other hand is increased. In the macrophages from normal healthy individuals or tuberculoid leprosy patients, presence of M.leprae increases both protein and levels of all the above enzymes. This observation shows that macrophages from lepromatous leprosy patients are unable to manifest in presence of M. leprae, the key enzymes involved in degradation of complex biological entities phagocytosed by the cells.