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1.
Ecotoxicol Environ Saf ; 194: 110402, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32151867

RESUMO

Sulfur (S) application in pakchoi (Brassica chinensis L.) cultivation is vital for reducing cadmium (Cd) accumulation in the plants. However, the mechanism of S application on Cd uptake and translocation in pakchoi is unclear. In this study, a hydroponic experiment was performed to investigate the effects of S application on Cd accumulation in pakchoi at one Cd concentration (50 µM, in comparison to the control condition, 0 µM) and three S levels (0, 2, 4 mM). The results showed that excessive S application (4 mM) reduced Cd accumulation and alleviated pakchoi growth inhibition caused by Cd stress in shoots and roots. With increased S application, the proportion of Cd in the vacuolar fraction and the proportion of NaCl-extractable Cd increased in roots. Additionally, S application increased the content of glutathione (GSH) and phytochelatins (PCs). The reduced Cd uptake and accumulation in pakchoi shoots could have been due to increased Cd chelation and vacuolar sequestration in roots. In addition, sufficient S application (2 mM) increased the expression of γ-glutamylcysteine synthetase (GSH1) and nicotinamide synthase (NAS) in roots, and excessive S application upregulated the expression of ATP sulfurylase (ATPS) and phytochelatin synthase (PCs). This study provides evidence for the mechanism of mitigating Cd toxicity in pakchoi and will be helpful for developing strategies to reduce Cd accumulation in the edible parts of pakchoi through S fertilizer application.


Assuntos
Brassica/efeitos dos fármacos , Cádmio/metabolismo , Poluentes do Solo/metabolismo , Sulfatos/farmacologia , Aminoaciltransferases/metabolismo , Transporte Biológico , Brassica/crescimento & desenvolvimento , Brassica/metabolismo , Cádmio/toxicidade , Fertilizantes/análise , Glutationa/metabolismo , Hidroponia , Modelos Teóricos , Fitoquelatinas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Poluentes do Solo/toxicidade , Sulfato Adenililtransferase/metabolismo , Sulfatos/metabolismo
2.
Cell Mol Life Sci ; 77(1): 81-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31728578

RESUMO

The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATTZnf451-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.


Assuntos
Quebras de DNA , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Animais , DNA/química , DNA/genética , DNA Topoisomerases Tipo II/química , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
3.
Chemosphere ; 240: 124902, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31563721

RESUMO

Eisenia fetida earthworm is an ecotoxicologically important test species to monitor various pollutants. However, there is a little knowledge about the effects of cadmium (Cd) on earthworms at the transcriptional level. Firstly, we exposed E. fetida to soils supplemented with different concentrations (10, 30, 60 mg/kg soil) of Cd. Moreover, we depicted the characterization of gene expressions with E. fetida using high-throughput profiling of gene expression. In addition, a comparison of the gene expression profiles between each Cd treatment group and the control group suggested that differential expressional genes (DEGs) mainly enriched in enzyme activity, metabolism, oxidative stress, regeneration and apoptosis pathways. 8 DEGs from these pathways had been selected randomly to confirm the data of RNA-seq. Among these DEGs, six genes (metallothionein-2, phytochelatin synthase 1a, CuZn superoxide dismutase, sex determining region Y-box 2, sex determining region Y-box 4b, TP53-regulated inhibitor of apoptosis 1-like) up-regulated and 2 genes (beta-1,4-endoglucanase, apoptosis-stimulating of p53 protein 2-like) down-regulated in response to Cd exposure. The alteration of them indicated that earthworms could reduce the toxicity and bioavailability of Cd in polluted soil ecosystems through different pathways. This work lays an important foundation for linking earthworm transcriptional level with the ecological risk of Cd in soil ecosystem.


Assuntos
Cádmio/toxicidade , Oligoquetos/fisiologia , Poluentes do Solo/toxicidade , Aminoaciltransferases , Animais , Disponibilidade Biológica , Cádmio/análise , Ecossistema , Perfilação da Expressão Gênica , Metalotioneína/genética , Metalotioneína/metabolismo , Oligoquetos/efeitos dos fármacos , Oligoquetos/genética , Estresse Oxidativo/efeitos dos fármacos , Solo , Poluentes do Solo/análise , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
4.
Int J Mol Sci ; 21(1)2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31861343

RESUMO

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.


Assuntos
Cádmio/metabolismo , Inativação Metabólica , Redes e Vias Metabólicas , Metalotioneína/metabolismo , Fitoquelatinas/metabolismo , Caramujos/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases , Animais , Cromatografia Líquida de Alta Pressão , Perfilação da Expressão Gênica , Especificidade da Espécie , Transcriptoma
5.
Pathog Dis ; 77(7)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778176

RESUMO

Legionella species synthesize phosphatidylcholine (PC) in two independent pathways: the three-step methylation of phosphatidylethanolamine PMT pathway and the one-step PCS pathway, in which the Pcs enzyme catalyzes the reaction between choline and CDP-diacylglycerol to form PC. Legionella pcs genes encode highly hydrophobic proteins with phosphatidylcholine synthase activity, which contain up to eight transmembrane helices with N- and C-termini located inside the bacterial cell. The comparative analysis of nucleotide sequences of pcs showed that these genes share high sequence identity among members of the Legionellaceae family. Legionella pmtA genes involved in the PMT pathway encoded small cytosolic proteins with putative phosphatidylethanolamine N-methyltransferase activity. The pmtA genes identified in Legionella species had lower sequence identity to each other than the pcs genes. The phylogenetic tree constructed based on the pcs and pmtA gene sequences showed phylogenetic relatedness between Legionella spp. and other bacteria. The utilization of extracellular choline by the four Legionella species leads to changes not only in the lipid components but also in proteins, and the interactions between these components lead to changes in cell surface properties, which result in a decline in induction of proinflammatory cytokines (TNF-α and IL-6).


Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Colina/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Legionella/genética , Legionelose/metabolismo , Legionelose/microbiologia , Metiltransferases/genética , Genes Bacterianos , Variação Genética , Humanos , Legionella/química , Legionella/classificação , Metabolismo dos Lipídeos , Lipídeos/química , Espectroscopia de Ressonância Magnética , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Filogenia , Espectroscopia de Infravermelho com Transformada de Fourier
6.
Microb Genom ; 5(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31609685

RESUMO

Penicillin-non-susceptible Streptococcus pneumoniae (PNSP) were first detected in the 1960s, and are now common worldwide, predominantly through the international spread of a limited number of strains. Extant PNSP are characterized by mosaic pbp2x, pbp2b and pbp1a genes generated by interspecies recombinations, with the extent of these alterations determining the range and concentrations of ß-lactams to which the genotype is non-susceptible. The complexity of the genetics underlying these phenotypes has been the subject of both molecular microbiology and genome-wide association and epistasis analyses. Such studies can aid our understanding of PNSP evolution and help improve the already highly-performing bioinformatic methods capable of identifying PNSP from genomic surveillance data.


Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas/genética , Peptidil Transferases/genética , Infecções Pneumocócicas , Streptococcus pneumoniae/genética , Genoma Bacteriano/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Penicilinas/metabolismo , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/microbiologia
7.
Int J Antimicrob Agents ; 54(6): 673-680, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31479743

RESUMO

Mechanisms underlying the emergence of daptomycin resistance in Staphylococcus aureus remain unclear. In this study, Staphylococcus aureus strain 3d0, isolated from a patient with bloodstream infection and belonging to the predominant Chinese hospital-associated methicillin-resistant S. aureus (MRSA) clone ST239, was serially passaged on gradient broth containing daptomycin for 34 days. The whole genomes of 3d0 and its serial passage strains were sequenced and compared. Five single nucleotide polymorphisms, four IS256 insertions, and one 39-bp insert occurred in the progress of daptomycin resistance acquisition. IS256 insertion in the mprF promoter region resulted in mprF overexpression. Two novel point mutations in mprF and walK, leading to amino acid substitutions in MprF (G299V and L473I) and WalK (L7Q and Y225N), were shown by allelic replacement experiments to increase the minimum inhibitory concentration (MIC) of daptomycin by 2-4 times. Allelic replacement of both mprF and walK in strain 3d0 increased the daptomycin MIC by 4-8-fold, indicating that mprF and walK mutations synergistically contribute to daptomycin non-susceptibility. Notably, these mutants acquired resistance without losing fitness and exhibited decreased expression of cell wall degradation-related genes. In conclusion, this study revealed novel mutations of MRSA daptomycin resistance acquisition in vitro as well as several novel mutations in walK and mprF, and includes the first in-depth analysis of the mprF promoter. This study sheds light on how MRSA may acquire daptomycin resistance during daptomycin treatment.


Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Antibacterianos/farmacologia , Sequência de Bases , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Mutagênese Insercional , Regiões Promotoras Genéticas
8.
ACS Chem Biol ; 14(10): 2185-2196, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31487148

RESUMO

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different cross-linking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Fluoresceínas/química , Corantes Fluorescentes/química , Oligopeptídeos/metabolismo , Peptidoglicano/metabolismo , Enterococcus faecium/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Peptidoglicano/química
9.
Drug Dev Res ; 80(8): 1136-1145, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31486108

RESUMO

Three natural naphthoquinones were screened to find new anti-virulence agents as inhibitors against sortase A from Staphylococcus aureus (SaSrtA) by quantifying the increase in fluorescence intensity upon substrate cleavage at various concentrations. The 5-hydroxy-1,4-naphthalenedione derivatives, juglone and plumbagin, demonstrated a potent inhibitory effect, with IC50 values of 1.78 µM, respectively, 16.71 µM. The related 2-hydroxy-1,4-naphthalenedione derivative, lawsone, demonstrated the selectivity of the chemical scaffold having no significant effect on SaSrtA. The experimental assay was reinforced by molecular docking experiments, antimicrobial, and toxicological studies. Molecular docking studies and the electrophilic character analysis suggest bonding to the enzyme active cysteine residue by a Michael addition reaction. None of the compounds had a significant effect on the concentration of total thiol proteins in the Daphnia magna toxicological assay after 24 hr exposure. Juglone and plumbagin moderately inhibited biofilm formation with no significant effect on bacterial growth of S. aureus, Enterococcus faecalis, and Staphylococcus epidermidis, indicating a selective anti-virulence profile.


Assuntos
Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Naftoquinonas/farmacologia , Staphylococcus aureus/crescimento & desenvolvimento , Aminoaciltransferases/química , Antibacterianos/química , Proteínas de Bactérias/química , Biofilmes/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/química , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Concentração Inibidora 50 , Modelos Moleculares , Simulação de Acoplamento Molecular , Naftoquinonas/química , Ligação Proteica , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento
10.
J Microbiol Biotechnol ; 29(10): 1603-1606, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31474099

RESUMO

Sortase A (SrtA), a type of transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall, is important in the virulence of gram-positive bacteria. Three compounds were isolated from marine-derived Streptomyces sp. MBTH32 using various chromatography techniques. The structures of these compounds were determined based on spectroscopic data and comparisons with previously reported data. Among the metabolites tested, lumichrome showed strong inhibitory activity against Staphylococcus aureus SrtA without affecting cell viability. The results of cell clumping activity assessment suggest the potential for using this compound to treat S. aureus infection by inhibiting SrtA activity.


Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibrinogênio/metabolismo , Staphylococcus aureus/patogenicidade , Streptomyces/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Flavinas/química , Flavinas/isolamento & purificação , Flavinas/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Mutação , Staphylococcus aureus/enzimologia , Streptomyces/metabolismo , Virulência/efeitos dos fármacos
11.
Mar Drugs ; 17(9)2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31438635

RESUMO

Four new peptides were isolated from the culture broths of the marine-derived fungi Aspergillus allahabadii and A. ochraceopetaliformis. Based on the results of chemical and spectroscopic analyses, two compounds (1 and 2) from A. allahabadii were determined to be cyclopentapeptides, while those from A. ochraceopetaliformis were a structurally-related cyclodepsihexapeptide (3) and its linear analog (4). In addition to the presence of a D-amino acid residue, the almost reversed sequence of peptides in 3 and 4, relative to those of the 1 and 2, is notable. These new compounds exhibited moderate inhibition against the enzyme sortase A as well as a weak inhibition against isocitrate lyase (2).


Assuntos
Antibacterianos/farmacologia , Aspergillus/química , Bactérias/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bactérias/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Cisteína Endopeptidases , Ensaios Enzimáticos , Sedimentos Geológicos/microbiologia , Isocitrato Liase/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação
12.
Acta Crystallogr D Struct Biol ; 75(Pt 8): 718-732, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31373571

RESUMO

Pili in Gram-positive bacteria are flexible rod proteins associated with the bacterial cell surface, and they play important roles in the initial adhesion to host tissues and colonization. The pilus shaft is formed by the covalent polymerization of major pilins, catalyzed by sortases, a family of cysteine transpeptidases. Here, X-ray structures of the major pilins from Clostridium perfringens strains 13 and SM101 and of sortase from strain SM101 are presented with biochemical analysis to detect the formation of pili in vivo. The major pilin from strain 13 adopts an elongated structure to form noncovalently linked polymeric chains in the crystal, yielding a practical model of the pilus fiber structure. The major pilin from strain SM101 adopts a novel bent structure and associates to form a left-handed twist like an antiparallel double helix in the crystal, which is likely to promote bacterial cell-cell interactions. A modeling study showed that pilin with a bent structure interacts favorably with sortase. The major pilin from strain SM101 was considered to be in an equilibrium state between an elongated and a bent structure through dynamic conformational change, which may be involved in pili-mediated colonization and sortase-mediated polymerization of pili.


Assuntos
Clostridium perfringens/química , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Aminoaciltransferases/química , Proteínas de Bactérias/química , Clonagem Molecular/métodos , Cristalografia por Raios X , Cisteína Endopeptidases/química , Escherichia coli/genética , Modelos Moleculares , Polimerização , Domínios Proteicos
13.
Proc Natl Acad Sci U S A ; 116(36): 18041-18049, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31427528

RESUMO

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.


Assuntos
Actinomyces , Adesinas Bacterianas , Aminoaciltransferases , Proteínas de Bactérias , Cisteína Endopeptidases , Fímbrias Bacterianas , Actinomyces/química , Actinomyces/genética , Actinomyces/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo
14.
Int Immunopharmacol ; 75: 105770, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31377588

RESUMO

Up-regulated glutaminyl cyclase (QC) plays crucial roles in the initiation of Alzheimer's disease (AD) and kinds of chronic diseases mediated by inflammation. QC is supposed as a novel target for the therapeutics of these diseases. Here, we explored the anti-inflammation effects of diphenyl conjugated imidazole (DPCI) derivatives which were previously designed, synthesized and evaluated as novel QC inhibitors for AD treatment in our lab. Behavioral tests, QC activity assay, histology and ELISA analysis were conducted on both AD and lipopolysaccharides (LPS)-induced inflammatory model mice. It was shown that behavioral and cognitive performance in AD mice treated with the selected compound DPCI-23 were enhanced notably. QC activity, the formation of pE-Aß and Aß plaques and the activation of astrocytes and microglia cells in AD mice brains were inhibited, and the levels of inflammatory factors such as IL-6, IL-1ß and TNF-α in serum were reduced remarkably. Furthermore, elevated QC activity in inflammatory mice brains was also inhibited, and levels of IL-1ß, IL-1ra, TNF-α and CCL2 in serum, kidneys and brains together with the activated astrocytes and microglia cells in brains were all repressed significantly after the treatment of DPCI-23. These findings observed in this research demonstrated the anti-inflammation potency of DPCI-23 in modal of AD and inflammation by inhibiting QC activity, and may contribute to the employment of QC inhibitors for the prevention and treatment of AD and other inflammatory diseases.


Assuntos
Doença de Alzheimer/imunologia , Aminoaciltransferases/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Imidazóis/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Rim/efeitos dos fármacos , Rim/imunologia , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos
15.
Rev Esp Quimioter ; 32 Suppl 3: 3-10, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364335

RESUMO

Ceftobiprole, a novel last generation parenteral cephalosporin, has an extended spectrum of activity, notably against methicillin-resistant Staphylococcus aureus (MRSA), ampicillin-susceptible enterococci, penicillin-resistant pneumococci, Enterobacterales and susceptible Pseudomonas aeruginosa. It exerts an inhibitory action on essential peptidoglycan transpeptidases, interfering with cell wall synthesis. The inhibitory action of ceftobiprole through binding to abnormal PBPs like PBP2a in methicillin-resistant staphylococci and PBP2b and PBP2x in the case of ß-lactam-resistant pneumococci, ultimately leads to rapid bacterial cell death. In the case of Enterobacterales, ceftobiprole retains activity against narrow spectrum ß-lactamases but is hydrolysed by their extended-spectrum counterparts, overexpressed Amp C, and carbapenemases. It is also affected by certain efflux pumps from P. aeruginosa. For anaerobic bacteria, ceftobiprole is active against Gram-positive Clostridioides difficile and Peptococcus spp. and Gram-negative Fusobacterium nucleatum but not against Bacteroides group or other anaerobic Gram-negatives. In in vitro studies, a low propensity to select for resistant subpopulations has been demonstrated. Currently, ceftobiprole is approved for the treatment of community-acquired pneumonia and hospital-acquired pneumonia with the exception of ventilator-associated pneumonia. Ceftobiprole's place in therapy appears to lie mainly in its combined activity against Gram-positive organisms, such as S. aureus and S. pneumoniae alongside that against Gram-negative organisms such as P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Aminoaciltransferases/antagonistas & inibidores , Antibacterianos/metabolismo , Cefalosporinas/metabolismo , Infecções Comunitárias Adquiridas/tratamento farmacológico , Endopeptidases/efeitos dos fármacos , Enterobacteriaceae/efeitos dos fármacos , Enterococcus/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Resistência às Penicilinas , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia
16.
Mol Oral Microbiol ; 34(5): 219-233, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31342653

RESUMO

Sortase A contributes to adhesion and biofilm formation of Streptococcus mutans by anchoring surface proteins like P1 onto the cell wall, and few other functional characterization has been annotated to this protein and its coding gene srtA. In this study we investigated that whether srtA deletion would affect S. mutans virulence determinants in addition to adhesion and further explored whether these effects were caused due to changes in S. mutans genomic transcription. We used acid-killing assays, glycolytic rate assessments, and exopolysaccharide (EPS) formation tests to detect whether srtA deletion influenced S. mutans acid tolerance/production and glucan formation. Comparisons between RNA-sequencing data from both the exponential and stationary phases of UA159 and the srtA-deleted strain were made to determine the impact of srtA knockout on S. mutans genomic transcription. Results of our assays indicated that S. mutans aciduricity was enhanced in the srtA deleted strain when bacterial cells were directly subjected to pH 2.8, but the enhancement was repressed when the acid tolerance response was induced in advance. The srtA mutation strain exhibited reduced EPS formation in mature biofilms. SrtA deletion led to pleiotropic changes in the S. mutans transcriptome with a growth phase-dependent pattern. The affected genes mainly included those involved in aciduricity, carbohydrate transport, and EPS formation. It was concluded that S. mutans srtA exhibited multiple effects on the virulence traits of this pathogen, including acid tolerance and glucan formation, and that these alterations could be partially due to transcriptional changes upon loss of srtA.


Assuntos
Aminoaciltransferases , Proteínas de Bactérias , Biofilmes , Cisteína Endopeptidases , Streptococcus mutans , Transcriptoma , Aminoaciltransferases/fisiologia , Proteínas de Bactérias/fisiologia , Cisteína Endopeptidases/fisiologia , Streptococcus mutans/patogenicidade , Virulência
17.
J Plant Physiol ; 240: 153011, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31357099

RESUMO

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na+/K+ ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H2O2) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H2O2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H2O2 scavenging, regulation of osmotic adjustment and ion homeostasis.


Assuntos
Aminoaciltransferases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Tabaco/fisiologia , Aminoaciltransferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relação Dose-Resposta a Droga , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Tabaco/genética
18.
Adv Mater ; 31(33): e1902462, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31265196

RESUMO

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though "smart" platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user-specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site-specifically modified proteins from hydrogels following Boolean logic. A sortase-mediated transpeptidation reaction is used to generate recombinant proteins C-terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli-labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two-input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli-sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Materiais Biocompatíveis/química , Cisteína Endopeptidases/química , Sistemas de Liberação de Medicamentos/métodos , Hidrogéis/química , Proteínas Recombinantes/química , Aminoaciltransferases/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Cisteína Endopeptidases/administração & dosagem , Dissulfetos/química , Liberação Controlada de Fármacos , Humanos , Luz , Oxirredução , Peptídeos/química , Fotólise , Polietilenoglicóis/química , Proteínas Recombinantes/administração & dosagem , Staphylococcus aureus/enzimologia
19.
ACS Chem Biol ; 14(8): 1836-1844, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31348637

RESUMO

Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species. The latter approach necessitates the creation of fusions whose behavior may not accurately reflect that of their unmodified counterparts. Here, we report a method for the characterization of protein internalization in real time through sortase-mediated, site-specific labeling of single-domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. Quenched probes of this type have been used to measure enzyme activity in complex environments and for different cell types, but not as a sensor of protein movement into living cells. This approach allows a quantitative assessment of the movement of proteins into protease-containing endosomes in real time in living cells. We demonstrate considerable variation in the rate of endosomal delivery for different cell surface receptors. We were also able to characterize the kinetics of influenza virus delivery to cathepsin-positive compartments, showing highly coordinated arrival in endosomal compartments. This approach should be useful for identifying proteins expressed on cells of interest for targeted endosomal delivery of payloads, such as antibody-drug conjugates or antigens that require processing.


Assuntos
Corantes Fluorescentes/química , Vírus da Influenza A/fisiologia , Proteínas de Membrana/metabolismo , Peptídeos/química , Rodaminas/química , Aminoaciltransferases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Cães , Endossomos/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Transporte Proteico , Internalização do Vírus
20.
Methods Mol Biol ; 2033: 67-80, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332748

RESUMO

The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding.In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFvanti-GPIIb/IIIa. Following this, our protocol illustrates the chemoenzymatic modification of the antibody at the C-terminus with an orthogonal click chemistry linker. This avoids any random attachment to the biologically active antigen binding site of the antibody. Finally, we describe the modification of a nanoparticle surface with scFv attachment via two methods: (1) direct Sortase-mediated conjugation; or (2) a two-step system which consists of scFv Sortase-mediated conjugation followed by strain promoted azide-alkyne cycloaddition. Finally, methodology is described to assess the successful assembly of targeted particles.


Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Imunoconjugados/genética , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/genética , Sequência de Aminoácidos/genética , Aminoaciltransferases/genética , Anticorpos/genética , Anticorpos/imunologia , Antígenos/imunologia , Azidas/química , Proteínas de Bactérias/genética , Química Click/métodos , Reação de Cicloadição/métodos , Cisteína/genética , Cisteína/imunologia , Cisteína Endopeptidases/genética , Humanos , Imunoconjugados/imunologia , Lisina/genética , Lisina/imunologia , Nanomedicina , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/imunologia
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