Production of serine protease inhibitors by mutagenesis and their effects on the mortality of Aedes aegypti L. larvae

BACKGROUND: Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4’ selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1). METHODS: The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4. RESULTS: The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the μM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested. CONCLUSIONS: The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector

Novel pseudo-aspartic peptidase from the midgut of the tick Rhipicephalus microplus

The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudoaspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it’s non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it’s possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries

Baccharis dracunculifolia, the main source of green propolis, exhibits potent antioxidant activity and prevents oxidative mitochondrial damage

Baccharis dracunculifolia DC (Asteraceae) is the main botanical source used by honeybees to produce Brazilian green propolis whose hepatoprotective properties have been already described. in this work we investigated the protective effects of the glycolic extract of B. dracunculifolia (GEBd) against oxidative stress in isolated rat liver mitochondria (RLM). the GEBd was prepared by fractionated percolation using propylene glycol as solvent. the total phenols and flavonoids, which are substances with recognized antioxidant action, were quantified in GEBd and the phytochemical analysis was carried out by HPLC. GEBd exhibited significant scavenger activity towards DPPH radicals and superoxide anions in a concentration-dependent manner, and also a Fe2+ chelating activity. GEBd decreased the basal H2O2 generation and the Fe2+- or t-BuOOH-induced ROS production in isolated mitochondria. Lipid oxidation of mitochondrial membranes, protein thiol groups and GSH oxidation were also prevented by GEBd. This shows that B. dracunculifolia exhibit potent antioxidant activity protecting liver mitochondria against oxidative damage and such action probably contribute to the antioxidant and hepatoprotective effects of green propolis. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Mogi das Cruzes UMC, Ctr Interdisciplinar Invest Bioquim CIIB, Mogi Das Cruzes, SP, BrazilUniv Estadual Paulista UNESP, Inst Quim, São Paulo, BrazilFac Ciencias Farmaceut Ribeirao Preto FCFRP USP, São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, São Paulo, BrazilUniv Fed ABC UFABC, Ctr Ciencias Nat & Humanas CCNH, Santo Andre, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, São Paulo, BrazilFAPESP: 2008/01724-4FAPESP: 2008/07246-7CNPq: 301672/2009-1CNPq: 136255/2009-4Web of Scienc

The first serine protease inhibitor from Lasiodora sp. (Araneae: Theraphosidae) hemocytes

AbstractThis work reports, for the first time, the purification, characterization and antibacterial activity of an elastase inhibitor from Lasiodora sp. hemocytes (EILaH). The hemocyte extract inhibited chymotrypsin (22%), trypsin (44%), tissue plasminogen activator (52%), urokinase (58%) and human neutrophil elastase (99%). EILaH was purified by Trypsin-Sepharose column and RP-HPLC. SDS-PAGE of EILaH revealed a molecular mass of 8kDa and MALDI-TOF mass spectrometry revealed a single molecular mass of 8274Da. The amino terminal sequence determined was LPC(PF)PYQQELTC. The dissociation constant (Ki) for human neutrophil elastase was 0.32nM. Hemocyte extract exerted antibacterial effect on Bacillus subtilis and Enterococcus faecalis, while EILaH was only active against E. faecalis. Currently, Lasiodora sp. is undergoing a systematic review and this study contributes to molecular characterization of the genus. In addition, the results suggest that serine protease inhibitors expressed in Lasiodora sp. hemocytes may be involved in the defense against bacterial infection

Novel pseudo-aspartic peptidase from the midgut of the tick Rhipicephalus microplus

The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudoaspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it’s non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it’s possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries

Time evolution of ultrasmall gold nanoparticle−protein interactions

To date, much effort has been devoted toward the study of protein corona formation onto large gold nanoparticles (GNPs). However, the protein corona concept breaks down for GNPs in the ultrasmall size regime (<3 nm), and, as a result, our understanding of ultrasmall GNP (usGNP)−protein interactions remains incomplete. Herein, we used anionic usGNPs and six different proteins as model systems to systematically investigate usGNP−protein interactions, with particular focus on the time evolution and long-term behavior of complex formation. The different proteins comprised chymotrypsin (Cht), trypsin (Try), thrombin (Thr), serum albumin (HSA), cytochrome c (Cyt c), and factor XII (FXII). We used a range of biochemical and biophysical methods to estimate binding affinities, determine the effects of usGNPs on protein structure and function, assess the reversibility of any protein structural and functional changes, and evaluate usGNP−protein complex stability. Among the main findings, we observed that prolonged (24 h) - but not short-term (10 min) - interactions between proteins and usGNPs permanently altered protein function, including enzyme activities (Try, Thr, and FXIIa), peroxidase-like activity (Cyt c), and ligand-binding properties (HSA). Remarkably, this occurred without any large-scale loss of the native global conformation, implying time-dependent effects of usGNPs on local protein conformation or dynamics. We also found that both short-(10 min) and long-term (24 h) interactions between proteins and usGNPs yielded short-lived complexes, i.e., there was no time-dependent “hardening” of the interactions at the binding interface as usually seen with large GNPs. The present study increases our fundamental understanding of nano-bio interactions in the ultrasmall size regime, which may assist the safe and effective translation of usGNPs into the clinic.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)2021/00685-

Isolation and characterization of Bothrops jararaca snake venom proteins that induce thrombocytopenia

Bothrops jararaca snake venom (BjV) induces thrombocytopenia in human patients and experimental models. Although various proteins that activate or inhibit platelets in vitro and ex vivo have been isolated and characterized from BjV, it is not known which ones account for the development of thrombocytopenia in vivo. In previous studies, we showed that BjV incubated with catalytic inhibitors of metalloproteinases or serine proteinases, and/or anti-botrocetin antibodies, still evoked platelet consumption in rats and mice. Thus, we initiated the isolation, identification and characterization of BjV toxins responsible for inducing thrombocytopenia in vivo, using ultrafiltration and liquid chromatography, and in vivo and ex vivo testing of fractions.Fil: Sachetto, Ana Tereza Azevedo. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Peichoto, María Elisa. Ministerio de Salud. Instituto Nacional de Medicina Tropical; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rosa, Jaqueline Gomes. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilFil: Torquato, Ricardo José Soares. Universidade do Sao Paulo. Departamento de Bioquímica; BrasilFil: Tanaka, Aparecida Sadae. Universidade do Sao Paulo. Departamento de Bioquímica; BrasilFil: Santoro, Marcelo Larami. Governo do Estado de Sao Paulo. Secretaria da Saude. Instituto Butantan; BrasilXV Congress of the Brazilian Society of ToxinologySão PauloBrasilBrazilian Society of Toxinolog

Glial cells modulate heparan sulfate proteoglycan (HSPG) expression by neuronal precursors during early postnatal cerebellar development

Cerebellum controls motor coordination, balance, eye movement, and has been implicated in memory and addiction. As in other parts of the CNS, correct embryonic and postnatal development of the cerebellum is crucial for adequate performance in the adult. Cellular and molecular defects during cerebellar development can lead to severe phenotypes, such as ataxias and tumors. Knowing how the correct development occurs can shed light into the mechanisms of disease. Heparan sulfate proteoglycans are complex molecules present in every higher eukaryotic cells and changes in their level of expression as well as in their structure lead to drastic functional alterations. This work aimed to investigate changes in heparan sulfate proteoglycans expression during cerebellar development that could unveil control mechanisms. Using real time RT-PCR we evaluated the expression of syndecans, glypicans and modifying enzymes by isolated cerebellar granule cell precursors, and studied the influence of soluble glial factors on the expression of those genes. We evaluated the possible involvement of Runx transcription factors in the response of granule cell precursors to glial factors. Our data show for the first time that cerebellar granule cell precursors express members of the Runx family and that the expression of those genes can also be controlled by glial factors. Our results also show that the expression of all genes studied vary during postnatal development and treatment of precursors with glial factors indicate that the expression of heparan sulfate proteoglycan genes as well as genes encoding heparan sulfate modifying enzymes can be modulated by the microenvironment, reflecting the intricate relations between neuron and glia. (C) 2010 ISDN. Published by Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Investiga Institutos de PesquisaUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilInvestiga Inst Pesquisa, BR-13084791 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilWeb of Scienc