New inhibitors of homoserine dehydrogenase from Paracoccidioides brasiliensis presenting antifungal activity.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the genus Paracoccidioides spp., which mainly affects workers in rural regions of Latin America. Although the antifungal agents currently available for the treatment of PCM are effective in controlling the disease, many months are needed for healing, making the side effects and drug interactions relevant. In addition, conventional treatments are not able to control the sequelae left by PCM, even after the cure, justifying the search for new therapeutic options against PCM. In this context, the enzyme homoserine dehydrogenase of P. brasiliensis (PbHSD) was used to screen a library of natural products from the Zinc database using three different docking programs, i.e. Autodock, Molegro, and CLC Drugdiscovery Workbench. Three molecules (Zinc codes 2123137, 15967722, and 20611644) were better ranked than the homoserine substrate (HSE) and were used for in vitro trials of the minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MCF). All three molecules presented a fungicidal profile with MICs/MCFs of 8, 32, and 128 µg mL-1, respectively. The two most promising molecules presented satisfactory results with wide therapeutic ranges in the cytotoxicity assays. Molecular dynamics simulations of PbHSD indicated that the ligands remained bound to the protein by a common mechanism throughout the simulation. The molecule with the lowest MIC value presented the highest number of contacts with the protein. The results presented in this work suggest that the molecule Zinc2123137 may be considered as a hit in the development of new therapeutic options for PCM.

Purification, structural and biophysical characterisation of the major seminal plasma protein from Texel rams.

Spermadhesins are a group of low molecular weight proteins present in seminal plasma. In Texel rams, they represent more than 70% of the seminal plasma proteins. Although their functions have not yet been fully clarified, there is much discussion about the role of these proteins in maintaining sperm viability during and after the semen freezing process. This work sought to isolate the major component of the seminal plasma from rams of the Texel breed (O. aries SPD2) and to evaluate its structural and biophysical characteristics in order to better understand its role in spermatic viability. The protein was isolated by centrifugation and ion exchange chromatography and its biophysical properties were evaluated by circular dichroism spectrometry. Molecular dynamics simulations of the modelled protein compared to the homologous bovine protein were also carried out. The results showed that O. aries SPD2 has a transition temperature (Tm) of 65 °C and a ΔHm of 322.5 kJ mol-1, similar to the results for other spermadhesins described in the literature. The estimated composition of the secondary structure elements for the native protein is in agreement with that observed for the theoretical model. Its structural characteristics were preserved in simulations at temperatures of 27 °C and 40 °C, as was the case for bull spermadhesin. Taken together, these results suggest that the major component of the spermadhesins of Texel rams (O. aries SPD2) may play an important role in maintaining the viability of spermatozoa in fresh semen as well as after thawing.

Mutations in catalase-peroxidase KatG from isoniazid resistant Mycobacterium tuberculosis clinical isolates: insights from molecular dynamics simulations.

The current multidrug therapy for tuberculosis (TB) is based on the use of isoniazid (INH) in combination with other antibiotics such as rifampin, ethambutol and pyrazinamide. Literature reports have shown that Mycobacterium tuberculosis, the causative agent of TB, has become resistant to this treatment by means of point mutations in the target enzymes of these drugs, such as catalase-peroxidase (KatG). By means of equilibrium molecular dynamics in the presence of the ligand, this work evaluated ten point mutations described in the enzyme KatG that are related to resistance to INH . The results showed that the resistance mechanism is related to stereochemical modifications at the N-terminal domain of the protein, which restrict INH access to its catalytic site, not involving mechanisms of electrostatic nature. These results show insights that can be useful for the identification of new anti-TB drugs which may be able to circumvent this mechanism of resistance.

Atropine is more efficient than ZM241385 to reduce a drastic level of fade caused by cisatracurium at 50 Hz / Atropina é mais eficiente que ZM241385 para reduzir o intenso nível de fadiga causada por cisatracurium a 50 Hz

The effects of atropine (non selective muscarinic antagonist) and ZM241385 (A2A receptors antagonist) in the cisatracurium-induced drastic (100%) level of fade at 50 Hz (10 s) (100% Fade) were compared in the phrenic nerve-diaphragm muscle preparations of rats indirectly stimulated at a physiological tetanic frequency (50 Hz). The lowest dose and the instant cisatracurium caused 100% Fade were investigated. Cisatracurium induced 100% Fade 15 min after being administered. Atropine reduced the cisatracurium-induced 100% Fade, but the administration of ZM241385 separately, or combined with atropine, did not cause any effect in the cisatracurium-induced 100% Fade. Data indicate that the simultaneous blockage of M1 and M2 muscarinic receptors on motor nerve terminal by atropine is more efficient than the blockage of presynaptic A2A receptors for a safer recovery of patients from neuromuscular blockade caused by cisatracurium.

Os efeitos de atropina e ZM241385 (antagonistas de receptores A2A) no drástico (100%) nível de fadiga (100% Fadiga) produzido pelo cisatracúrio a 50 Hz (10 s) foram comparativamente investigados em preparações nervo frênico músculo diafragma isolado de ratos indiretamente estimuladas com a frequência fisiológica tetanizante de 50 Hz. A menor dose e o instante no qual cisatracúrio causou 100% Fadiga foram pesquisados. O cisatracúrio induziu 100% Fadiga 15 min depois de ser administrado. A atropina reduziu a fadiga de 100% causada pelo cisatracúrio, mas ZM241385, ou a administração combinada de atropina com ZM241385, não causou qualquer efeito na 100% Fadiga produzida pelo cisatracúrio. Os dados indicam que o bloqueio simultâneo dos receptores muscarínicos M1 e M2 no terminal nervoso motor pela atropina é mais eficiente do que o bloqueio dos receptores pré-sinápticos A2A no auxilio da recuperação mais segura do bloqueio da transmissão neuromuscular causada por cisatracúrio.

Estudo morfométrico do plexo mientérico do duodeno de ratos submetidos ao alcoolismo / Morphometric study of duodenum myenteric plexus in rats submitted to alcoholism

O etanol é uma molécula que se difunde facilmente pelas membranas celulares e no citosol leva a produção de compostos tóxicos que podem alterar as proteínas celulares e o DNA. Assim, o objetivo deste estudo foi avaliar os efeitos do etanol sobre os parâmetros corporais e a morfometria dos neurônios mientéricos do duodeno de ratos submetidos ao alcoolismo crônico. Foram utilizados 14 Rattus norvegicus, mantidos durante 16 semanas, divididos em dois grupos: Grupo controle (n=7), que recebeu ração padronizada e água ad libitum, e Grupo experimental (n=7), que recebeu a mesma ração e aguardente de cana em concentrações crescentes ad libitum. Ao final do período, os ratos foram anestesiados para a realização da laparatomia e retirada do duodeno, que foi submetido à realização de preparados de membrana e corados com Giemsa. Os dados foram submetidos ao teste estatístico t (Student) com nível de significância de 5%. Os animais alcoolizados apresentaram médias inferiores na ingesta líquida, sólida, crescimento e peso corporal, sendo que a diferença entre as médias foi significativa (p<0,005). Nos animais do GE a área do pericário foi 40,6% menor que o GC, do núcleo 70,5% menor no GE e do citosol 26% menor no GE, sendo a diferença estatisticamente significante para as três regiões celulares observadas (p<0,05). Logo, o consumo crônico de etanol por 16 semanas interferiu na ingestão e ganho de peso dos animais e provocou redução na área do pericário, núcleo e citosol dos neurônios mientéricos do duodeno de ratos.

Ethanol is a molecule that diffuses easily through cellular membranes, and in cytosol leads to the production of toxic compounds that can alter cellular proteins and DNA. Thus, the aim of this study was to evaluate the effects of ethanol on body parameters and morphometry of duodenum myenteric neurons of rats submitted to chronic alcoholism. We used 14 Rattus norvegicus, which were kept for 16 weeks, divided into two groups: control group (n = 7), which received standard feed and water ad libitum, and experimental group (n = 7), which received the same feed and sugar cane brandy in increasing concentrations ad libitum. At the end of the period, the rats were anesthetized for the realization of laparotomy and duodenum removal, which was submitted to preparations of membrane and stained with Giemsa. The data were submitted to statistical test t (Student) with a significance level of 5%. The intoxicated animals showed lower average in liquid and solid intake, growth and body weight, and the difference between both groups average was significant (p <0.005). In animals of the EG, the area of pericardium was 40.6% lower than the CG, the nucleus was 70.5% lower in the EG, and cytosol was 26% lower in the EG. The difference is statistically significant for all the three cellular regions observed (p <0.05). Therefore, the chronic consumption of ethanol for 16 weeks interfered with intake and weight gain of animals, and caused a reduction in the area of perikarion, nucleus and cytosol of duodenum myenteric neurons in rats.