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1.
Methods Mol Biol ; 2555: 181-194, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306087

RESUMO

The discovery of new enzymes is strongly enabled by the implementation of high-throughput screening methods to detect enzymatic activity in single organisms or clone expression libraries, or to benchmark their performances against known prototypes. In this chapter, a number of methods, applicable at high-throughput scale, are described that allow the screening and characterization of enzymes relevant to biotechnology, particularly, ester-hydrolases (esterases, lipases, phospholipases, and polyester hydrolases).


Assuntos
Esterases , Lipase , Esterases/metabolismo , Lipase/metabolismo , Fosfolipases , Ensaios de Triagem em Larga Escala/métodos
2.
Methods Mol Biol ; 2555: 167-179, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306086

RESUMO

Metagenomic screening is a widely applied biotechnological approach for screening of novel industrial enzymes. The traditional method of metagenomic screening is based on the functional analyses of heterologously expressed environmental genes in a suitable host, which is the bottleneck of this method. To avoid limitation from the clone-dependent system, an in vitro expression technology has been developed in combination with next-generation sequencing and bioinformatics. First, the sequence profile of a target enzyme, e.g., poly(ethylene terephthalate) esterase in this protocol, is constructed according to the sequences of well-characterized enzymes. Then, the sequence screening is performed with this computationally generated profile among all available metagenomic databases. Afterwards, the candidate genes are synthesized and expressed in vitro with RNA polymerase and translation machinery from special cell extract. Finally, such in vitro produced enzymes are directly applied for the functional analyses. Comparing to the traditional screening methods, this in vitro screening technology can not only save time and materials, but also be easily developed for high-throughput screening with an automatic pipetting robot.


Assuntos
Esterases , Metagenoma , Esterases/genética , Esterases/metabolismo , Metagenômica , Polietilenotereftalatos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala
3.
Int J Mol Sci ; 23(21)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36362119

RESUMO

Proteases are abundant in prokaryotic genomes (~10 per genome), but their recovery encounters expression problems, as only 1% can be produced at high levels; this value differs from that of similarly abundant esterases (1-15 per genome), 50% of which can be expressed at good levels. Here, we design a catalytically efficient artificial protease that can be easily produced. The PluriZyme EH1AB1 with two active sites supporting the esterase activity was employed. A Leu24Cys mutation in EH1AB1, remodelled one of the esterase sites into a proteolytic one through the incorporation of a catalytic dyad (Cys24 and His214). The resulting artificial enzyme, EH1AB1C, efficiently hydrolysed (azo)casein at pH 6.5-8.0 and 60-70 °C. The presence of both esterase and protease activities in the same scaffold allowed the one-pot cascade synthesis (55.0 ± 0.6% conversion, 24 h) of L-histidine methyl ester from the dipeptide L-carnosine in the presence of methanol. This study demonstrates that active sites supporting proteolytic activity can be artificially introduced into an esterase scaffold to design easy-to-produce in-one protease-esterase PluriZymes for cascade reactions, namely, the synthesis of amino acid esters from dipeptides. It is also possible to design artificial proteases with good production yields, in contrast to natural proteases that are difficult to express.


Assuntos
Esterases , Peptídeo Hidrolases , Esterases/metabolismo , Peptídeo Hidrolases/metabolismo , Endopeptidases/metabolismo , Domínio Catalítico/genética , Ésteres/metabolismo , Concentração de Íons de Hidrogênio
4.
Viruses ; 14(10)2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36298681

RESUMO

Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. The balance of the opposing activities of the surface spike (S) and hemagglutinin-esterase (HE) glycoproteins controls BCoV avidity, which is critical for interspecies transmission and host adaptation. Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S. Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12 nucleotide insertion in the receptor-binding domain (RBD) of the HE gene similar to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12 nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and could bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding. The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.


Assuntos
Doenças dos Bovinos , Infecções por Coronavirus , Coronavirus Bovino , Humanos , Bovinos , Animais , Hemaglutininas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Mutação , Glicoproteínas/genética , Esterases/genética , Esterases/metabolismo , Nucleotídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/genética
5.
Molecules ; 27(20)2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36296710

RESUMO

The aim of the present study is to investigate the phytochemical composition of tiger nut (TN) (Cyperus esculentus L.) and its neuroprotective potential in scopolamine (Scop)-induced cognitive impairment in rats. The UHPLC-ESI-QTOF-MS analysis enabled the putative annotation of 88 metabolites, such as saccharides, amino acids, organic acids, fatty acids, phenolic compounds and flavonoids. Treatment with TN extract restored Scop-induced learning and memory impairments. In parallel, TN extract succeeded in lowering amyloid beta, ß-secretase protein expression and acetylcholine esterase (AChE) activity in the hippocampus of rats. TN extract decreased malondialdehyde levels, restored antioxidant levels and reduced proinflammatory cytokines as well as the Bax/Bcl2 ratio. Histopathological analysis demonstrated marked neuroprotection in TN-treated groups. In conclusion, the present study reveals that TN extract attenuates Scop-induced memory impairments by diminishing amyloid beta aggregates, as well as its anti-inflammatory, antioxidant, anti-apoptotic and anti-AChE activities.


Assuntos
Disfunção Cognitiva , Cyperus , Fármacos Neuroprotetores , Animais , Ratos , Escopolamina/efeitos adversos , Cyperus/química , Fármacos Neuroprotetores/uso terapêutico , Antioxidantes/metabolismo , Acetilcolina/metabolismo , Peptídeos beta-Amiloides/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteína X Associada a bcl-2/metabolismo , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Malondialdeído/metabolismo , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Extratos Vegetais/metabolismo , Flavonoides/metabolismo , Aminoácidos/metabolismo , Ácidos Graxos/metabolismo , Citocinas/metabolismo , Esterases/metabolismo
6.
Biochemistry (Mosc) ; 87(9): 932-939, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36180989

RESUMO

The autotransporter AT877 from Psychrobacter cryohalolentis belongs to the family of outer membrane proteins containing N-terminal passenger and C-terminal translocator domains that form the basis for the design of display systems on the surface of bacterial cells. It was shown in our previous study that the passenger domain of AT877 can be replaced by the cold-active esterase EstPc or the tenth domain of fibronectin type III (10Fn3). In order to increase efficiency of the 10Fn3 surface display in Escherichia coli cells, four deletion variants of the Fn877 hybrid autotransporter were obtained. It was demonstrated that all variants are present in the membrane of bacterial cells and facilitate binding of the antibodies specific against 10Fn3 on the cell surface. The highest level of binding is provided by the variants Δ239 and Δ310, containing four and seven beta-strands out of twelve that comprise the structure of the translocator domain. Using electrophoresis under semi-native conditions, presence of heat modifiability in the full-size Fn877 and its deletion variants was demonstrated, which indicated preservation of beta structure in their molecules. The obtained results could be used to optimize the bacterial display systems of 10Fn3, as well as of other heterologous passenger domains.


Assuntos
Escherichia coli , Sistemas de Secreção Tipo V , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Escherichia coli/genética , Escherichia coli/metabolismo , Esterases/metabolismo , Fibronectinas/metabolismo , Proteínas de Membrana/metabolismo , Psychrobacter , Sistemas de Secreção Tipo V/metabolismo
7.
Plant Physiol Biochem ; 191: 67-77, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36195034

RESUMO

Ammonium promotes rice P uptake and reutilization better than nitrate, under P starvation conditions; however, the underlying mechanism remains unclear. In this study, ammonium treatment significantly increased putrescine and ethylene content in rice roots under P deficient conditions, by increasing the protein content of ornithine decarboxylase and 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase compared with nitrate treatment. Ammonium treatment increased rice root cell wall P release by increasing pectin content and pectin methyl esterase (PME) activity, increased rice shoot cell membrane P release by decreasing phosphorus-containing lipid components, and maintained internal P homeostasis by increasing OsPT2/6/8 expression compared with nitrate treatment. Ammonium also improved external P uptake by regulating root morphology and increased rice grain yield by increasing the panicle number compared with nitrate treatment. The application of putrescine and ethylene synthesis precursor ACC further improved the above process. Our results demonstrate for the first time that ammonium increases rice P acquisition, reutilization, and homeostasis, and rice grain yield, in a putrescine- and ethylene-dependent manner, better than nitrate, under P starvation conditions.


Assuntos
Compostos de Amônio , Oryza , Compostos de Amônio/metabolismo , Compostos de Amônio/farmacologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , Esterases/metabolismo , Etilenos/metabolismo , Lipídeos , Nitratos/metabolismo , Ornitina Descarboxilase/metabolismo , Oryza/metabolismo , Oxirredutases/metabolismo , Pectinas/metabolismo , Fósforo/metabolismo , Raízes de Plantas/metabolismo , Putrescina/metabolismo
8.
Sci Rep ; 12(1): 17712, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36271284

RESUMO

Transcriptional analysis of the network of transcription regulators and target pathways in exposed organisms may be a hard task when their genome remains unknown. The development of hundreds of qPCR assays, including primer design and normalization of the results with the appropriate housekeeping genes, seems an unreachable task. Alternatively, we took advantage of a whole transcriptome study on Rhinella arenarum larvae exposed to the organophosphorus pesticides azinphos-methyl and chlorpyrifos to evaluate the transcriptional effects on a priori selected groups of genes. This approach allowed us to evaluate the effects on hypothesis-selected pathways such as target esterases, detoxifying enzymes, polyamine metabolism and signaling, and regulatory pathways modulating them. We could then compare the responses at the transcriptional level with previously described effects at the enzymatic or metabolic levels to obtain global insight into toxicity-response mechanisms. The effects of both pesticides on the transcript levels of these pathways could be considered moderate, while chlorpyrifos-induced responses were more potent and earlier than those elicited by azinphos-methyl. Finally, we inferred a prevailing downregulation effect of pesticides on signaling pathways and transcription factor transcripts encoding products that modulate/control the polyamine and antioxidant response pathways. We also tested and selected potential housekeeping genes based on those reported for other species. These results allow us to conduct future confirmatory studies on pesticide modulation of gene expression in toad larvae.


Assuntos
Clorpirifos , Praguicidas , Animais , Azinfos-Metil , Clorpirifos/metabolismo , Praguicidas/farmacologia , Larva , Transcriptoma , Compostos Organofosforados/farmacologia , Antioxidantes/metabolismo , Bufo arenarum/metabolismo , Esterases/metabolismo , Poliaminas/metabolismo , Fatores de Transcrição/metabolismo
9.
Int J Biochem Cell Biol ; 153: 106316, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36280040

RESUMO

Notum, which belongs to the α/ß hydrolase family, is a deacylated extracellular protein that regulates the Wnt signaling pathway. Studies have found that Notum participates in the progression of colorectal cancer and hepatocellular carcinoma, but its role in oral squamous cell carcinoma (OSCC) is currently unclear. This study aimed to explore the role of Notum in regulating OSCC and further reveal the underlying mechanisms. Various approaches including bioinformatics analysis, enzyme-linked immunosorbent assay and immunohistochemical staining were used to detect the expression of Notum in OSCC cells and tissues. Cell counting kit-8 assay, clone formation assay, wound healing assay, transwell assay and in-gel zymography assay were explored to evaluate the regulation of Notum in OSCC proliferation and migration. Hoechst 33342/PI assay, cell immunofluorescence, flow cytometry and in vivo tumorigenesis experiment were applied for OSCC apoptosis. Real-time quantitative polymerase chain reaction analysis was performed for mRNA level while western blotting was conducted to detect protein expression. The results showed that Notum was highly expressed in OSCC tissues and cells, and Notum promoted the proliferation and migration of OSCC cells while it inhibited their apoptosis. Furthermore, signaling pathway analysis showed that Notum led to potential pro-survival of OSCC through crosstalk between sonic hedgehog (Shh) and Wnt/ß-catenin signaling via phosphorylation of glycogen synthase kinase-3 beta. These results will help to elucidate the mechanism and also provide new ideas for targeted treatment of OSCC.


Assuntos
Esterases , Neoplasias Bucais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Esterases/genética , Esterases/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Via de Sinalização Wnt/genética
10.
Pestic Biochem Physiol ; 187: 105173, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36127039

RESUMO

Declines of the monarch butterfly population have prompted large-scale plantings of milkweed to restore the population. In North America, there are >73 species of milkweed to choose from for these nationwide plantings. However, it is unclear how different milkweed species affect monarch caterpillar physiology, particularly detoxification enzyme activity and gene expression, given the highly variable cardenolide composition across milkweed species. Here, we investigate the effects of a high cardenolide, tropical milkweed species and a low cardenolide, swamp milkweed species on pyrethroid sensitivity as well as detoxification enzyme activity and expression in monarch caterpillars. Caterpillars fed on each species through the fifth-instar stage and were topically treated with bifenthrin after reaching this final-instar stage. Esterase, glutathione S-transferase, and cytochrome P450 monooxygenase activities were quantified as well as the expression of selected esterase, glutathione S-transferase, ABC transporter, and cytochrome P450 monooxygenase transcripts. There were no significant differences in survival 24 h after treatment with bifenthrin. However, bifenthrin significantly increased glutathione S-transferase activity in caterpillars feeding on tropical milkweed and significantly decreased esterase activity in caterpillars feeding on tropical and swamp milkweed. Significant differential expression of ABC transporter, glutathione S-transferase, and esterase genes was observed for caterpillars feeding on tropical and swamp milkweed and not receiving bifenthrin treatment. Furthermore, significant differential expression of glutathione S-transferase and esterase genes was observed for bifenthrin-treated and -untreated caterpillars feeding on tropical milkweed relative to swamp milkweed. These results suggest that feeding on different milkweed species can affect detoxification and development mechanisms with which monarch caterpillars rely on to cope with their environment.


Assuntos
Asclepias , Borboletas , Inseticidas , Piretrinas , Transportadores de Cassetes de Ligação de ATP , Animais , Asclepias/metabolismo , Borboletas/genética , Cardenolídeos/metabolismo , Esterases/genética , Esterases/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Inseticidas/metabolismo , Inseticidas/toxicidade , Oxigenases de Função Mista/metabolismo , Piretrinas/metabolismo , Piretrinas/toxicidade
11.
Pharmacol Res Perspect ; 10(5): e01009, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36121122

RESUMO

The anti-cocaine monoclonal antibody, h2E2, is a candidate for treating cocaine-use disorder. h2E2 binds to and sequesters cocaine in the plasma compartment, effectively decreasing cocaine concentrations in the brains of rats and mice. Despite the binding of cocaine to h2E2, plasma cocaine concentrations decline rapidly in rodents over time, but there was a drastic decrease in the urinary elimination of cocaine in the presence of h2E2. Since cocaine is not being renally excreted, the apparent disappearance of cocaine from the plasma must be explained by either metabolism or distribution. However, binding of cocaine to h2E2 may restrict the availability of cocaine for hydrolysis by endogenous esterases. Therefore, the antibody would be expected to extend the elimination half-life of cocaine. In contrast, previous studies reported h2E2 as having no effect on the rate of cocaine clearance. It is important to examine the ultimate clearance of the cocaine to ascertain its half-life and potential for re-intoxication. Therefore, we investigated the effects of h2E2 on cocaine hydrolysis in vitro and on cocaine metabolism and disposition in vivo over a 6-h time course. The spontaneous and enzyme-mediated in vitro hydrolysis of cocaine was drastically decreased in the presence of h2E2 in vitro. Additionally, in mice, h2E2 significantly increased the distribution and elimination half-lives of cocaine relative to vehicle controls over an extended time course. Therefore, we concluded that h2E2 slowing the distribution and elimination of cocaine is the most appropriate explanation for the initial disappearance of cocaine from the plasma in vivo.


Assuntos
Cocaína , Animais , Anticorpos Monoclonais Humanizados , Encéfalo/metabolismo , Cocaína/metabolismo , Esterases/metabolismo , Taxa de Depuração Metabólica , Camundongos , Ratos
12.
ACS Nano ; 16(9): 14644-14657, 2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36048539

RESUMO

As it is closely associated with tumor proliferation, metastasis, and the immunosuppressive microenvironment, the dysfunctional Hippo pathway has become an extremely attractive target for treating multiple cancers. However, to date, the corresponding chemotherapeutic nanomedicines have not been developed. Herein, a supramolecular self-delivery nanomedicine with in situ transforming capacity was tailor-constructed for Hippo-pathway restoration, and its inhibitory effect against tumor growth and metastasis was investigated in a highly aggressive triple-negative breast cancer (TNBC) model. Stimulated by overexpressed glutathione (GSH) and esterase in cancer cells, the self-assembled nanomedicine transformed from inactive nanospheres to active nanofibers conjugating tyrosvaline and spatiotemporally synchronously released the covalently linked flufenamic acid in situ, together activating the maladjusted Hippo pathway by simultaneously acting on different targets upstream and downstream. The transcriptional expression of Yes-associated protein (YAP) and related growth-promoted genes were significantly reduced, finally significantly repressing the proliferation and metastasis of cancer cells. Additionally, the Hippo-pathway restoration showed an excellent radiosensitization effect, making the targeted therapy combined with radiotherapy display a prominent synergistic in vivo anticancer effect against TNBC. This work reports a specifically designed smart nanomedicine to restore the function of the Hippo pathway and sensitize radiotherapy, providing an attractive paradigm for targeted drug delivery and cancer combination therapy.


Assuntos
Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Esterases/metabolismo , Esterases/uso terapêutico , Ácido Flufenâmico/uso terapêutico , Glutationa/metabolismo , Via de Sinalização Hippo , Humanos , Nanomedicina , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Microambiente Tumoral , Proteínas de Sinalização YAP
13.
J Vector Borne Dis ; 59(2): 145-153, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36124480

RESUMO

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60-75kDa and ~80-95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.


Assuntos
Aedes , Arginina Quinase , Dengue , Aedes/genética , Animais , Arginina Quinase/metabolismo , Esterases/metabolismo , Esterases/farmacologia , Raios gama , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacologia , Masculino , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/farmacologia , Ubiquitinas/metabolismo , Ubiquitinas/farmacologia
14.
World J Microbiol Biotechnol ; 38(12): 217, 2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36070019

RESUMO

Cold-adapted esterases have potential industrial applications. To fulfil the global continuous demand for these enzymes, a cold-adapted esterase member of family VI from Lysinibacillus sp. YS11 was cloned on pET-28b (+) vector and expressed in E. coli BL21(DE3) Rosetta cells for the first time. The open reading frame (654 bp: GenBank MT120818.1) encodes a polypeptide (designated EstRag: 217 amino acid residues). EstRag amino acid sequence has conserved esterase signature motifs: pentapeptide (GFSQG) and catalytic triad Ser110-Asp163-His194. EstRag 3D predicted model, built with LOMETS3 program, showed closest structural similarity to PDB 1AUO_A (esterase: Pseudomonas fluorescens); TM-align score program inferences. Purified EstRag to 9.28-fold, using Ni2+affinity agarose matrix, showed a single protein band (25 kDa) on SDS-PAGE, Km (0.031 mM) and Kcat/Km (657.7 s-1 mM-1) on p-NP-C2. Temperature and pH optima of EstRag were 35 °C and 8.0, respectively. EstRag was fully stable at 5-30 °C for 120 min and at pH(s) 8.0-10.0 after 24 h. EstRag activity (391.46 ± 0.009%) was impressively enhanced after 30 min preincubation with 5 mM Cu2+. EstRag retained full stability after 30 min pre-incubation with 0.1%(v/v) SDS, Triton X-100, and Tween-80. EstRag promising characteristics motivate performing guided evolution and industrial applications prospective studies.


Assuntos
Bacillaceae , Esterases , Álcalis , Bacillaceae/genética , Bacillaceae/metabolismo , Temperatura Baixa , Detergentes/química , Detergentes/farmacologia , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Esterases/metabolismo , Estudos Prospectivos
15.
Chemosphere ; 307(Pt 4): 136215, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36041517

RESUMO

In this study the effects of environmentally realistic concentrations of the antibiotics sulfamethoxazole (SMX) and oxytetracyclyne (OTC) on Lithobates catesbeianus tadpoles were evaluated, through the analyzes of the frequencies of micronucleus and nuclear abnormalities in erythrocytes, alterations in leucocytes, liver histopathology, and changes in hepatic esterase activities and oxidative stress biomarkers. The animals were exposed for 16 days at concentrations of 0 (control), 20, 90 and 460 ng L-1. No significant difference was found in the frequencies of micronucleus and nuclear abnormalities. The two highest concentrations of SMX and all concentrations of OTC caused a significant increase in the number of lymphocytes. A significant decrease in the number of neutrophils compared to the control group was observed for all concentrations tested of both antibiotics. Also, decrease in the activity of glutathione S-transferase and high histopathological severity scores, indicating liver damage, were found in tadpoles exposed to the two highest concentrations of SMX and all concentrations of OTC. The main changes in the liver histopathology were the presence of inflammatory infiltrate, melanomacrophages, vascular congestion, blood cells and eosinophils. Esterase activities were unchanged. Indeed, the two highest concentrations of OTC caused a reduction in the activities of superoxide dismutase and glucose 6-phosphate dehydrogenase, while the highest concentration inhibited the activity of glutathione peroxidase and increased protein carbonyl levels. These results evidences that environmentally realistic concentrations of SMX and OTC in aquatic environments are capable to significantly disrupt tadpoles' physiology, possibly affecting negatively their survival rate in natural environments.


Assuntos
Oxitetraciclina , Poluentes Químicos da Água , Animais , Antibacterianos/farmacologia , Biomarcadores/metabolismo , Esterases/metabolismo , Glucose/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Larva , Fígado/metabolismo , Oxitetraciclina/farmacologia , Fosfatos/metabolismo , Rana catesbeiana , Sulfametoxazol/metabolismo , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/metabolismo
16.
Chem Commun (Camb) ; 58(75): 10484-10487, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36040293

RESUMO

Histone deacetylases (HDACs) play crucial roles in the epigenetic regulation of gene expression. Here, we report CM-Bhc-SAHA, a novel caged HDAC inhibitor, genetically targeting cells of interest. Mammalian cells expressing porcine liver esterase led to the optochemical inhibition of endogenous HDAC activity when treated with CM-Bhc-SAHA and irradiated with 405 nm light.


Assuntos
Epigênese Genética , Inibidores de Histona Desacetilases , Animais , Esterases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Mamíferos/metabolismo , Suínos
17.
Biol Direct ; 17(1): 20, 2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-35978367

RESUMO

BACKGROUND: Recently, microRNAs (miRNAs), have been extensively investigated in diseases. The upregulated expression of miR-19b-3p has been validated in patients with hypertrophic cardiomyopathy. Nonetheless, it regulatory mechanism in myocardial infarction (MI) is still unclear. PURPOSE: This research aimed to investigate the role and molecular regulation mechanism of miR-19b-3p in MI. METHODS: QRT-PCR and western blot assays measured RNA and protein expression. Cell apoptosis were tested by flow cytometry and TUNEL assays. Cell viability was measured by trypan blue staining method. RIP and luciferase report assays examined gene interaction. The assays were performed under hypoxia condition. RESULTS: MiR-19b-3p was highly expressed in myocardial cell line H9C2, primary cardiomyocytes, and tissues from MI mouse model. MiR-19b-3p inhibition suppressed the apoptosis of cardiomyocytes. BC002059 could up-regulate ABHD10 through sequestering miR-19b-3p. BC002059 upregulation was observed to repress cell apoptosis. Rescue experiments demonstrated that miR-19b-3p overexpression abrogated the suppressive impact of BC002059 on the apoptosis of MI cells, and infarct size, area at risk as well as CK-MB and LDH release of MI mouse model tissues, which was further abolished via ABHD10 increment. CONCLUSION: MiR-19b-3p regulated by BC002059/ABHD10 axis promotes cell apoptosis in MI, which might provide a novel perspective for MI alleviation research.


Assuntos
Esterases/metabolismo , MicroRNAs , Infarto do Miocárdio , Animais , Apoptose/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Regulação para Cima
18.
Glycobiology ; 32(10): 826-848, 2022 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-35871440

RESUMO

The substitution and de-substitution of carbohydrate materials are important steps in the biosynthesis and/or breakdown of a wide variety of biologically important polymers. The SGNH hydrolase superfamily is a group of related and well-studied proteins with a highly conserved catalytic fold and mechanism composed of 16 member families. SGNH hydrolases can be found in vertebrates, plants, fungi, bacteria, and archaea, and play a variety of important biological roles related to biomass conversion, pathogenesis, and cell signaling. The SGNH hydrolase superfamily is chiefly composed of a diverse range of carbohydrate-modifying enzymes, including but not limited to the carbohydrate esterase families 2, 3, 6, 12 and 17 under the carbohydrate-active enzyme classification system and database (CAZy.org). In this review, we summarize the structural and functional features that delineate these subfamilies of SGNH hydrolases, and which generate the wide variety of substrate preferences and enzymatic activities observed of these proteins to date.


Assuntos
Carboidratos , Hidrolases , Biopolímeros/biossíntese , Biopolímeros/química , Carboidratos/biossíntese , Carboidratos/química , Esterases/química , Esterases/classificação , Esterases/metabolismo , Hidrolases/química , Hidrolases/classificação , Hidrolases/metabolismo , Conformação Proteica
19.
Bioorg Med Chem Lett ; 73: 128909, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35907608

RESUMO

Tyrosyl-DNA phosphodiesterase 1(TDP1) is a promising target for a new therapy in oncological disease as an adjunct to topoisomerase 1 (TOP1) drugs. In this paper, novel thiazolidin-4-one derivatives with a benzyl and monoterpene substituents were synthesized. Compounds with a monoterpene fragment attached via a phenyloxy linker were active against TDP1 with IC50 values in the 1 ÷ 3 µM range, while direct attachment of monoterpene moiety to the thiazolidin-4-one fragment had no activity. Molecular modelling predicted two plausible binding modes of the active compounds both effectively blocking access to the catalytic site of TDP. At non-toxic concentrations the active ligands potentiated the efficacy of the TOP1 poison topotecan in human cervical cancer HeLa cells, but not in non-cancerous HEK293A cells.


Assuntos
Inibidores de Fosfodiesterase , Diester Fosfórico Hidrolases , Esterases/metabolismo , Células HeLa , Humanos , Monoterpenos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade
20.
Pest Manag Sci ; 78(11): 4579-4588, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35837767

RESUMO

BACKGROUND: Due to the development of insecticide resistance in mosquitoes, with worldwide mosquito-borne diseases resurgence in recent years, recent advances in proteome technology have facilitated a proteome-wide analysis of insecticide resistance-associated proteins in mosquitoes. Understanding the complexity of the molecular basis of insecticide resistance mechanisms employed by mosquitoes will help in designing the most effective and sustainable mosquito control methods. RESULTS: After 30 generations, insecticide-selected strains showed elevated resistance levels to the cypermethrin used for selection. Proteome data allowed the detection of 2892 proteins, of which 2885 differentially expressed proteins (DEPs) achieved quantitative significances in four stages (egg, larvae, pupae, adult) of Culex pipiens pallens cypermethrin-resistant strain as compared to the susceptible strain. Among them, a significant enrichment of proteins, including cuticular proteins, enzymes involved in the detoxification (cytochrome P450, glutathione S-transferases, esterase, ATP-binding cassette) and some biological pathways (oxidative phosphorylation, hippo signalling) that are potentially involved in cypermethrin resistance, was observed. Thirty-one representative DEPs (cytochrome P450, glutathione S-transferase, cuticle protein) during Cx. pipiens pallens developmental stages were confirmed by a parallel reaction monitoring strategy. CONCLUSIONS: The present study confirmed the power of isobaric tags for relative and absolute quantification for identifying concomitantly quantitative proteome changes associated with cypermethrin in Cx. pipiens pallens. Proteome analysis suggests that proteome modifications can be selected rapidly by cypermethrin, and multiple resistance mechanisms operate simultaneously in cypermethrin-resistance of Cx. pipiens pallens, Our results interpret that an up-regulated expression of proteins and enzymes like cytochrome P450, glutathione S-transferases, esterase etc. has an impact in insecticide resistance. Previously neglected penetration resistance (cuticular proteins) may play an important role in the adaptive response of Cx. pipiens pallens to insecticides. This information may serve as a basis for future work concerning the possible role of these proteins in cypermethrin resistance in mosquito Cx. pipiens pallens. © 2022 Society of Chemical Industry.


Assuntos
Culex , Inseticidas , Piretrinas , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Esterases/metabolismo , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Inseticidas/metabolismo , Inseticidas/farmacologia , Proteoma/metabolismo , Piretrinas/metabolismo , Piretrinas/farmacologia , Transferases/metabolismo , Transferases/farmacologia
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