Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide.

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

Panorama of Bacterial Infections Caused by Epidemic Resistant Strains.

Antimicrobial resistance (AMR) represents a critical obstacle to public health worldwide, due to the high incidence of strains resistant to available antibiotic therapies. In recent years, there has been a significant increase in the prevalence of resistant epidemic strains, associated with this, public health authorities have been alarmed about a possible scenario of uncontrolled dissemination of these microorganisms and the difficulty in interrupting their transmission, as nosocomial pathogens with resistance profiles previously considered sporadic. They become frequent bacteria in the community. In addition, therapy for infections caused by these pathogens is based on broad-spectrum antibiotic therapy, which favors an increase in the tolerance of remaining bacterial cells and is commonly associated with a poor prognosis. In this review, we present the current status of epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococcus (VRE), MDR Mycobacterium tuberculosis, extended-spectrum ß-lactamase-producing Enterobacterales (ESBL), Klebsiella pneumoniae carbapenemase (KPC), and-New Delhi Metallo-beta-lactamase-producing Pseudomonas aeruginosa (NDM).

The enzymes in COVID-19: A review.

COVID-19 brought a scientific revolution since its emergence in Wuhan, China, in December 2019. Initially, the SARS-CoV-2 virus came to attention through its effects on the respiratory system. However, its actions in many other organs also have been discovered almost daily. As enzymes are indispensable to uncountable biochemical reactions in the human body, it is not surprising that some enzymes are of relevance to COVID-19 pathophysiology. Past evidence from SARS-CoV and MERS-CoV outbreaks provided hints about the role of enzymes in SARS-CoV-2 infection. In this setting, ACE-2 is an enzyme of great importance since it is the cell entry receptor for SARS-CoV-2. Clinical data elucidate patterns of enzymatic alterations in COVID-19, which could be associated with organ damage, prognosis, and clinical complications. Further, viral mutations can create new disease behaviors, and these effects are related to the activity of enzymes. This review will discuss the main enzymes related to COVID-19, summarizing the findings on their role in viral entry mechanism, the consequences of their dysregulation, and the effects of SARS-CoV-2 mutations on them.

Characterization of brain acetylcholinesterase of bentonic fish Hoplosternum littorale: Perspectives of application in pesticides and metal ions biomonitoring.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a serine hydrolase, whose main function is to modulate neurotransmission at cholinergic synapses. It is, therefore, the primary target of some pesticides and heavy metals. Its inhibition in aquatic organisms has been used as an indicator of the presence of these pollutants in water bodies. The present study aimed to characterize physicochemical and kinetic parameters of brain AChE in the benthic fish Hoplosternum littorale and to analyze the in vitro effects of pesticides (dichlorvos, diazinon, chlorpyrifos, parathion-methyl, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron, novaluron and pyriproxyfen) and metal ions (As3+, Cd2+, Cu2+, Fe2+, Mn2+, Mg2+, K+, Pb2+, Hg2+, Zn2+) investigating the potential of this enzyme as environmental biomarker based on current regulations. Specific substrates and inhibitors have indicated AChE to be the predominant cholinesterase (ChE) in the brain of H. littorale. Peak activity was observed at pH 8.0 and 30 °C. The enzymatic activity is otherwise moderately thermostable (≈ 50% activity at 45 °C). The enzyme can reduce the activation energy of acetylthiocholine hydrolysis reaction to 8.34 kcal mol-1 while reaching a rate enhancement of 106. Among the pesticides under study, dichlorvos presented an IC50 value below the maximum concentrations allowed by legislation. This study presents the first report on the inhibition of brain AChE activity from Siluriformes by the pesticides novaluron and pyriproxyfen. Mercury ion also exerted a strong inhibitory effect on its enzymatic activity. The H. littorale enzyme thus has the potential to function as an in vitro biomarker for the presence of the pesticide dichlorvos as well as mercury in areas of mining and industrial discharge.

Trypsin purification using magnetic particles of azocasein-iron composite.

This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.