Rhizopus arrhizus ucp1295 como fonte econômica para produção de biopolímeros funcionais quitina e quitosana utilizando substratos renováveis / Rhizopus arrhizus ucp1295 as economic source for production of functional biopolymers chitin and chitosan using renewable substrates

Neste trabalho foi investigada a produção de quitina e quitosana por Rhizopus arrhizus UCP 1295 isolado do solo da Caatinga do Estado de Pernambuco, Brasil, utilizando o efluente industrial de doces e milhocina como substratos de baixo custo, considerando a versatilidade de aplicação das biomoléculas. O micro-organismo foi cultivado em diferentes concentrações dos substratos efluente da indústria de doces e milhocina (CSL) em diferentes valores de pH, de acordo com um planejamento fatorial completo 23. Após 96 h de fermentação, a biomassa produzida foi liofilizada e submetida ao tratamento com álcali- ácido-. Os polissacarídeos extraídos foram caracterizados por espectroscopia por transformada de Fourier (FTIR) na região do infravermelho. A maior produção de biomassa (14,11 g/L) foi obtida na condição 6 (8% de efluente industrial de doces, 5% de milhocina e pH 5), enquanto os maiores rendimentos de quitina (169,3 mg/g) e quitosana (239,1 mg/g) foram obtidos em meio contendo 4% de efluente da indústria de doces, sem milhocina, nas condições 3 (pH 7) e 1 (pH 5), respectivamente. A quitina apresentou grau de acetilação de 71,4% e a quitosana de 86,0%, de desacetilação, respectivamente. Além disso, foi demonstrado que o efluente industrial de balas e milhocina são substratos renováveis e alternativos na formulação de novos meios de produção de quitina e quitosana. A versatilidade das biomoléculas deve-se as suas propriedades bioquímicas únicas, como biocompatibilidade, biodegradabilidade, não toxicidade, capacidade de formar filmes e aplicações industriais promissoras

Fonsecaea pedrosoi Sclerotic Cells: Secretion of Aspartic-Type Peptidase and Susceptibility to Peptidase Inhibitors

Fonsecaea pedrosoi is a dematiaceous fungus and the main causative agent of chromoblastomycosis that is a chronic disease usually affecting the human skin and subcutaneous tissues, which causes deformations and incapacities, being frequently refractory to available therapies. A typical globe-shaped, multiseptated and pigmented cells, known as sclerotic cells, are found in the lesions of infected individuals. In the present work, we have investigated the production of aspartic-type peptidase in F. pedrosoi sclerotic cells as well as the effect of peptidase inhibitors (PIs) on its enzymatic activity and viability. Our data showed that sclerotic cells are able to secrete pepstatin A-sensible aspartic peptidase when grown under chemically defined conditions. In addition, aspartic PIs (ritonavir, nelfinavir, indinavir, and saquinavir), which are clinically used in the HIV chemotherapy, significantly decreased the fungal peptidase activity, varying from 55 to 99%. Moreover, sclerotic cell-derived aspartic peptidase hydrolyzed human albumin, an important serum protein, as well as laminin, an extracellular matrix component, but not immunoglobulin G and fibronectin. It is well-known that aspartic peptidases play important physiological roles in fungal cells. With this task in mind, the effect of pepstatin A, a classical aspartic peptidase inhibitor, on the F. pedrosoi proliferation was evaluated. Pepstatin A inhibited the fungal viability in both cellular density- and drug-concentration manners. Moreover, HIV-PIs at 10 μM powerfully inhibited the viability (>65%) of F. pedrosoi sclerotic cells. The detection of aspartic peptidase produced by sclerotic cells, the parasitic form of F. pedrosoi, may contribute to reveal new virulence markers and potential targets for chromoblastomycosis therapy

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics

Arrabidaea chica Hexanic Extract Induces Mitochondrion Damage and Peptidase Inhibition on Leishmania spp.

Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasite’s ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents

The Natural Alkaloid Tryptanthrin Induces Apoptosis-like Death in <i>Leishmania</i> spp.

Leishmaniasis is a vector-borne disease against which there are no approved vaccines, and the treatment is based on highly toxic drugs. The alkaloids consist of a chemical class of natural nitrogen-containing substances with a long history of antileishmanial activity. The present study aimed at determining the antileishmanial activity and in silico pharmacokinetic and toxicological potentials of tryptanthrin alkaloid. The anti-Leishmania amazonensis and anti-L. infantum assays were performed against both promastigotes and intracellular amastigotes. Cellular viability was determined by parasites’ ability to grow (promastigotes) or differentiate (amastigotes) after incubation with tryptanthrin. The mechanisms of action were explored by mitochondrion dysfunction and apoptosis-like death evaluation. For the computational pharmacokinetics and toxicological analysis (ADMET), tryptanthrin was submitted to the PreADMET webserver. The alkaloid displayed anti-promastigote activity against L. amazonensis and L. infantum (IC50 = 11 and 8.0 μM, respectively). Tryptanthrin was active against intracellular amastigotes with IC50 values of 75 and 115 μM, respectively. Mitochondrial membrane depolarization was observed in tryptanthrin-treated promastigotes. In addition, parasites undergoing apoptosis-like death were detected after 18 h of exposure. In silico ADMET predictions revealed that tryptanthrin has pharmacokinetic and toxicological properties similar to miltefosine. The results presented herein demonstrate that tryptanthrin is an interesting drug candidate against leishmaniasis

Melanin from Fonsecaea pedrosoi Induces Production of Human Antifungal Antibodies and Enhances the Antimicrobial Efficacy of Phagocytes

Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients’ sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients’ lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses

Coconut Husk Fiber Proanthocyanidins: Ecofriendly Corrosion Inhibitor, Ecotoxicity and Biocide Activity

Volume 8 Issue 2 (February 201