Otimização do meio de cultura para a produção de quitosanase por metarhizium anisopliae em cultivo descontínuo submerso

In this work a Plackett-Burman Design with 8 factors and 12 trials in 2 levels with 3 repetitions at the center point was used in order to investigate the influence of the concentration of chitosan, peptone, yeast extract, NaNO3, K2HPO4, KCl, MgSO4.7H2O and FeSO4 on chitosanase production by Metarhizium anisopliae. Runs were carried out using submerged discontinuous cultivation for enzyme production. The results of the Plackett & Burman Design showed that only two factors, chitosan concentration as well as FeSO4 had influence on chitosanolytic activity, while the increase in concentration of other factors not contributed significantly to the quitosanolítica activity. Cultivation medium optimization for enzyme production was carried out using a Composite Central Design, with the most important factors for chitosanolytic activity (chitosan and FeSO4), in accordance with Plackett & Burman Design, and keeping the other nutrients in their minimum values. On this other design, it was taken the highest limit in Plackett & Burman Design as the lowest limit (-1) to FeSO4 factor. The results showed that the enzyme production was favoured by increasing the chitosan concentration and by decreasing FeSO4. Maximum production for chitosanolytic activity was about 70.0 U/L and was reached in only 18 h of fermentation, a result about twenty-eight times greater than a former study using the same microorganism (about 2.5 U/L at 48 h)

Properties of a new fungal b-galactosidase with potential application in the dairy industry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production and action pattern of inulinase from Aspergillus Niger-245: hydrolysis of inulin from several sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Some Biochemical properties of polyphenoloxidase from spearmint (Mentha arvensis)

Polifenoloxidase (PPO, EC 1.14.18.1) extraída de folhas de Mentha arvensis foi isolada por precipitação com (NH4)2SO4 e diálise extensiva. Seu pH e temperatura ótimos variaram com o substrato. A PPO apresentou atividade com vários difenóis. Valores de Km foram 0,825; 0,928 e 7,41 mM para ácido caféico, 4-metilcatecol e catecol, respectivamente. Na inativação térmica, 50% da enzima foi inativada após 60 e 15 segundos a 70 e 75ºC, respectivamente. A medida de atividade residual mostrou um efeito estabilizante de sacarose a várias temperaturas e uma energia de ativação (Ea) para inativação aumentando com a concentração de sacarose de 0 a 40% (p/p). Valores de energias de ativação de 78,13; 80,37; 82,79 and 81,00 kJ/Mol foram encontradas para 0, 15, 30 e 40% de sacarose, respectivamente. A PPO foi inibida pelos ácidos ascórbico, benzóico, cinamico, ferulico, p-cumárico, protocatéquico, além de metabisulfito de sódio, resorcinol e pirogalol. Os valores de Ki mostram que o ácido ascórbico foi o mais efetivo inibidor. O tipo de inibição foi determinado para cada inibidor.

Análise dos traçados de ondas da agregação plaquetária em pacientes com doenças cardiovasculares em uso do ácido acetil salicílico comparados a doadores de sangue

A agregação plaquetária avalia a função das plaquetas através de diferentes vias de ativação plaquetária in vitro, fornecendo traçados de ondas equivalentes à propriedade física dessa agregação. Sabendo-se que a aspirina acetila irrever­sivelmente a enzima cicloxigenase prevenindo a geração de tromboxana A2, potente ativador da agregação plaquetária, esta droga tem sido analisada há mais de trinta anos na clínica médica em pacientes com doenças cardiovasculares como uma potente droga antitrombótica. Foram nossos objetivos obter traçados de ondas correspondentes às fases da agregação plaquetária para nossa padronização utilizando nosso grupo controle de doadores de sangue e compará-las com nosso grupo estudo, frente a diferentes agentes agonistas em diferentes concentrações: ADP 1µM e 3µM; AA, 0,5mM; ADR, 0,05 mg/mL, 0,025 mg/mL e 0,010 mg/mL. Os grupos analisados foram constituídos por 41 cardíacos e 40 doadores de sangue considerados controle. Dos cardíacos, 33 faziam uso regular do AAS na concentração de 200 mg/dia e oito na concentração de 100 mg/dia, sendo todos considerados hipertensos. A padronização da agregometria estava na dependência do encontro de traçados correspondentes a ondas de agregação, sendo esses obtidos em porcentagem no tempo de cinco minutos estandartizados pelo aparelho utilizado. Comparando os resultados entre os pacientes e o grupo controle, foi possível observar que, na presença dos agentes agregantes ADP 1µM e ADP 3 µM, ADR 0,05 mg/mL, 0,025mg/mL e 0,010 mg/mL, os pacientes apresentaram 1ª onda, mas não 2ª onda. Por sua vez, no caso do AA 0,5 mM não houve o encontro de traçados de ondas.

Atividade da 6-fosfogliconato desidrogenase em deficientes de glicose-6-fosfato desidrogenase

As enzimas G6PD e 6PGD são responsáveis pela geração do aporte de NADPH, necessário para a detoxificação dos agentes oxidantes produzidos pelo estresse oxidativo metabólico nos eritrócitos. Devido à alta prevalência de deficiência de G6PD na população mundial, principalmente de origem negróide africana, muitos estudos têm sido realizados na tentativa de conhecer melhor a atuação destas enzimas. O objetivo deste estudo foi avaliar a atividade enzimática da 6PGD, nos deficientes de G6PD, para verificar a existência de aumento da atividade desta enzima, correlacionando com um possível aumento do número de reticulócitos ou presença de alterações da série vermelha. A pesquisa em 2.657 indivíduos do sexo masculino resultou em 97 deficientes de G6PD, determinando uma prevalência de 3,65% para a região de Bauru (SP), com atividade enzimática média de G6PD de 1,74 UI.g Hb-1. min-1 a 37ºC, 14,4% da atividade da G6PD normal. A atividade enzimática média da 6PGD foi de 9,5 UI.g Hb-1. min-1 a 37ºC, estando aumentada em 47,4% dos deficientes de G6PD. Os resultados não confirmaram que a hipótese do aumento da atividade enzimática da 6PGD, em deficientes de G6PD, seja decorrente da presença de um número aumentado de reticulócitos na corrente circulatória, faixa etária ou alterações eritrocitométricas que denotem anemia. O mais provável é que a hemólise autolimitada, imposta pelos processos oxidativos, preserve os eritrócitos mais jovens, que possuem atividade enzimática mais elevada, uma vez que naturalmente ocorre diminuição da atividade destas enzimas com o envelhecimento celular.

Efeito da temperatura de estocagem de leveduras de panificação sobre a atividade da glicerol-3-fosfato desidrogenase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

O complexo nuclear vestibular do sagui (callithrix jacchus): caracterização citoarquitetônica e neuroquímica

To the vertebrates, maintain body balance against the gravitational field and be able to orient themselves in the environment are fundamental aspects for survival, in which the participation of vestibular system is essential. As part of this system, the vestibular nuclear complex is the first central station that, by integrating many information (visual, proprioceptive), and the vestibular, assumes the lead role in maintaining balance. In this study, the vestibular nuclear complex was evaluated in relation to its cytoarchitecture and neurochemical content of cells and axon terminals, through the techniques of Nissl staining and immunohistochemistry for neuronal specific nuclear protein (NeuN), glutamate (Glu), substance P (SP), choline acetyltransferase (ChAT) (enzyme that synthesizes acetylcholine-Ach) and glutamic acid decarboxylase (GAD) (enzyme that synthesizes gamma-amino butyric acid-GABA). The common marmoset (Callithrix jacchus) was used as experimental animal, which is a small primate native from the Atlantic Forest in the Brazilian Northeast. As results, the Nissl technique, complemented by immunohistochemistry for NeuN allowed to delineate the vestibular nucleus superior, lateral, medial and inferior (or descending) in the brain of the common marmoset. Neurons and terminals immunoreactive to Glu and ChAT and only immunoreactive terminals to SP and GAD were seen in all nuclei, although in varying density. This study confirms the presence in the vestibular nuclei of the common marmoset, of Glu and SP in terminals, probably from the first order neurons of vestibular ganglion, and of GABA in terminals, presumably from Purkinge cells of the cerebellum. Second-order neurons of the vestibular nuclei seem to use Glu and Ach as neurotransmitters, judging by their expressive presence in the cell bodies of these nuclei in common marmosets, as reported in other species

Ativação local da rota da quinurenina por moduladores inflamatórios durante a meningite bacteriana.

Activation of the kynurenine (KYN) pathway (KP) by modulators of immune system has been observed during several neurological diseases. Here we assessed the association of chemo-/cytokine levels with the concentration of KP metabolites in cerebrospinal fluid (CSF) and plasma samples from patients with bacterial meningitis (BM). All samples were collected from 42 patients diagnosed with acute bacterial meningitis (ABM), aseptic meningitis, tuberculous meningitis and patients without infection neurological disorders. CSF and plasma concentration of metabolites from the KP was assessed by high pressure liquid chromatography (HPLC) and cytokines and chemokines by Bio-plex 200 suspension array system. Concentrations of the KP metabolites KYN and kynurenic acid (KYNA) were significantly higher in CSF of patients with ABM compared to other groups. Tryptophan (TRP), anthranilic acid (AA), 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) did not show statistical significance, although some of them presented a good accumulation during ABM. The expression of TNF-alpha, IL-6, IL-1beta, IFN-gamma, IL-10, IL-1 receptor antagonist (IL-1Ra), MIP-1alpha, MIP-1beta, MCP-1 and G-CSF was about 100-fold higher in CSF from ABM patients than other infected groups. In all CSF and plasma samples, the concentration of IL-2, IL-12(p70), IL-4, IL-8 and GM-CSF was not significant. ABM still showed significant concentrations of IL-6, IL-10, IL-1Ra and MCP-1 in plasma samples. Based on the comparison of KP metabolites concentrations between plasma and CSF samples we conclude that the activation of the tryptophan pathway upon BM occurs within the brain. This increase in KP metabolites is most due to activation of the KP by molecules as IFN-gamma and TNF-alpha in response to infection.

Análise das mudanças no perfil protéico durante o estresse oxidativo in vivo e atuação da MutY-Glicosilase em respostas celulares

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Análise imuno-histoquímica das metaloproteinases da matriz ( MMP-1,MMP-2 e MMP-9) na doença periodontal

The tissular destruction found in periodontal diseases is caused mainly by components of the host that have its production stimulated by the products of the microorganisms present on the plaque. Matrix Metalloproteinases (MMPs), a class of enzymes involved both in physiologic and pathologic extracellular matrix degradation are considered the main responsible for the characteristic tissular loss in periodontal disease, and the understanding of how this happens can have a series of beneficial implications for prevention, diagnosis and treatment of this illness. The aim of this work was to study the immunohistochemical expression of MMP-1, MMP-2, and MMP-9 in fragments of gingival biopsies with clinical diagnose of periodontal disease. MMP-1 has expressed significantly more than the others MMPs in gingivitis both in the epithelium (p=0,0008) and connective tissue (p=0,0049). In periodontitis, both MMP-1 and MMP-9 has expressed significantly more than MMP-2 in the epithelium (p<0,0001) and in the connective tissue (p=0,0002). MMP-1 and MMP-9 presented more expression in periodontitis than in gingivitis but MMP-1 only in the connective tissue (p=0,03) and MMP-9 in the epithelium (p=0,003) and in the connective tissue (p=0,04). In conclusion, these results indicate that the MMP-1 presents high expression in every stages of the periodontal diseases, and increases its expression in the connective tissue when the gingivitis evolves to periodontitis. Therefore, it may have an important role in connective tissue degradation and bone loss observed in disease, since early, in gingivitis, until late stages, in periodontitis, of the periodontal disease. MMP-9 has expressed more in periodontitis than in gingivitis, both in epithelium and in connective tissue. It means that this enzyme may have some importance in the progression of gingivitis to periodontitis by acting in bone resorption observed in this desease

Os núcleos dopaminérgicos do mesencéfalo do mocó (kerodon rupestris): caracterização citoarquitetônica e por imunoistoquímica para tirosina-hidroxilase

The 3-hydroxytyramine/dopamine (DA) is a monoamine of catecholamineric group and consists in the progenitor substantia of synthesis of noradrenaline and adrenaline, having the enzyme tyrosine hydroxylase as a regulator of this process. Nuclei of midbrain expressing DA are the retrorubral field (RRF, A8 group), the substantia nigra pars compacta (SNc, A9 group) and the ventral tegmental area (VTA, A10 group). These nuclei are involved in three complex circuitry called mesostriatal, mesocortical and mesolimbic, which are related directly with various behavioral manifestations such as motor control, reward signaling in behavioural learning, motivation and pathological manifestations of Parkinson s disease and schizophrenia. The aim of this study was describe the morphology of midbrain dopaminergic neurons (A8, A9 and A10) of the rock cavy (Kerodon rupestris), a rodent belonging to the family Caviidae typical of the Brazilian Northeast, which is being adopted as a model for neuroanatomical studies in laboratory of neuroanatomy of the Federal University of Rio Grande do Norte. Coronal sections of brains of the rock cavies were submitted to staining by Nissl s method and immunohistochemistry against tyrosine hydroxylase. The nuclear organization of the midbrain dopaminergic nuclei of the rock cavy is very similar to that found in other animals of the order Rodentia, except by the presence of the tail of substantia nigra, which was found only in the studied species. We concluded that the midbrain dopaminergic nuclei are phylogenetically stable among species, but we think to be it necessary to expand the studies about the particularity found the rock cavy, investigating its occurrence in other species of rodents or investigating its functional relevance

Participação do circuito dopaminérgico nas alterações do comportamento de medo inato de camundongos infectados pelo Toxoplasma gondii

The protozoan parasite Toxoplasma gondii transforms the innate aversion of rats for cat urine into a fatal attraction, that increases the likelihood of the parasite completing its life cycle in the cat s intestine. The neural circuits implicated in innate fear, anxiety, and learned fear all overlap considerably, raising the possibility, that T. gondii may disrupt all of these nonspecifically. In this study, we evaluated immunoreactivity for tyrosine hydroxylase (TH) in areas associated with innate fear of infected male swiss mice. The latent Toxoplasma infection converted the aversion of mice to feline odors into attraction. This loss of fear is remarkably specific, as demonstrated by Vyas et al (2007), because infection did not diminish learned fear, anxiety-like behavior, olfaction, or nonaversive learning. However, the neurochemical mechanism related to alterations in innate fear due to T. gondii infection remains poorly studied. 20 mice were inoculated with bradyzoites (25 cysts) from a Toxoplasma gondii (Me-49 strain). The brains were removed after 60 days, sectioned and processed for TH immunohistochemistry. The correlation between the amount of cysts per area and the densitometric analysis of neurotransmitter reactivity was low in the areas implicated in innate fear of infected animals, when comparated with noninfected controls

Participação da neurotransmissão dopaminergica no efeito hiperlocomotor do neuropeptideo S

Neuropeptide S (NPS) is an endogenous 20-aminoacid peptide which binds a G protein-coupled receptor named NPSR. This peptidergic system is involved in the modulation of several biological functions, such as locomotion, anxiety, nociception, food intake and motivational behaviors. Studies have shown the participation of NPSR receptors in mediating the hyperlocomotor effects of NPS. A growing body of evidence suggests the participation of adenosinergic, dopaminergic and CRF systems on the hyperlocomotor effects of NPS. Considering that little is known about the role of dopaminergic system in mediating NPS-induced hyperlocomotion, the present study aims to investigate the locomotor actions of intracerebroventricular (icv) NPS in mice pretreated with α-metil-p-tirosine (AMPT, inhibitor of dopamine synthesis), reserpine (inhibitor of dopamine vesicle storage) or sulpiride (D2 receptor antagonist) in the open field test. A distinct group of animals received the same pretreatments described above (AMPT, reserpine or sulpiride) and the hyperlocomotor effects of methylphenidate (dopamine reuptake inhibitor) were investigated in the open field. NPS and methylphenidate increased the mouse locomotor activity. AMPT per se did not change the locomotion of the animals, but it partially reduced the hyperlocomotion of methylphenidate. The pretreatment with AMPT did not affect the psychostimulant effects of NPS. Both reserpine and sulpiride inhibited the stimulatory actions of NPS and methylphenidate. These findings show that the hyperlocomotor effects of methylphenidate, but not NPS, were affected by the pretreatment with AMPT. Furthermore, methylphenidate- and NPS-induced hyperlocomotion was impaired by reserpine and sulpiride pretreatments. Together, data suggests that NPS can increase locomotion even when the synthesis of catecholamines was impaired. Additionally, the hyperlocomotor effects of NPS and methylphenidate depend on monoamines vesicular storaged, mainly dopamine, and on the activation of D2 receptors. The psychostimulant effects of NPS via activation of dopaminergic system display clinical significance on the treatment of diseases which involves dopaminergic pathways, such as Parkinson s disease and drug addiction