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1.
Pharmaceuticals (Basel) ; 17(4)2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38675422

RESUMO

Lycii Radicis Cortex (LRC) is a traditional medicine in East Asia with various beneficial effects, including antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, and anti-depressant properties. However, its potential effects on skeletal muscle atrophy have not been studied. In this study, the protective effects of LRC extract (LRCE) on dexamethasone (DEX)-induced muscle atrophy were investigated in C2C12 myotubes and mice. We evaluated the effect of LRCE on improving muscle atrophy using a variety of methods, including immunofluorescence staining, quantitative polymerase chain reaction (qPCR), Western blot, measurements of oxidative stress, apoptosis, ATP levels, and muscle tissue analysis. The results showed that LRCE improved myotube diameter, fusion index, superoxide dismutase (SOD) activity, mitochondrial content, ATP levels, expression of myogenin and myosin heavy chain (MHC), and reduced reactive oxygen species (ROS) production in dexamethasone-induced C2C12 myotubes. LRCE also enhanced protein synthesis and reduced protein degradation in the myotubes. In mice treated with DEX, LRCE restored calf thickness, decreased mRNA levels of muscle-specific RING finger protein 1 (MuRF1) and atrogin-1, and increased insulin-like growth factor 1 (IGF-1) mRNA level. Moreover, LRCE also repaired gastrocnemius muscle atrophy caused by DEX. Although human studies are not available, various preclinical studies have identified potential protective effects of LRCE against muscle atrophy, suggesting that it could be utilized in the prevention and treatment of muscle atrophy.

2.
Int J Biol Macromol ; 255: 128313, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37995783

RESUMO

Tyrosinase-mediated protein conjugation has recently drawn attention as a site-specific protein modification tool under mild conditions. However, the tyrosinases reported to date act only on extremely exposed tyrosine residues, which limits where the target tyrosine can be located. Herein, we report a tyrosinase from Streptomyces avermitilis (SaTYR), that exhibits a much higher activity against tyrosine residues on the protein surface than other tyrosinases. We determined the crystal structure of SaTYR and revealed that the enzyme has a relatively flat and shallow substrate-binding pocket to accommodate a protein substrate. We demonstrated SaTYR-mediated fluorescence dye tagging and PEGylation of a surface tyrosine residue that was unreacted by other tyrosinases with an approximately 95.2 % conjugation yield in 1 h. We also present a structural rationale that considers the steric hindrance from adjacent residues and surrounding structures along with the extent of solvent exposure of residues, as necessary when determining the optimal positions for introducing target tyrosine residues in SaTYR-mediated protein modification. The study demonstrated that the novel tyrosinase, SaTYR, extends the scope of tyrosinase-mediated protein modification, and we propose that site-specific tyrosine conjugation using SaTYR is a promising strategy for protein bioconjugation in various applications.


Assuntos
Monofenol Mono-Oxigenase , Streptomyces , Monofenol Mono-Oxigenase/metabolismo , Proteínas/metabolismo , Tirosina/química
3.
Int J Mol Sci ; 24(3)2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36768200

RESUMO

Skeletal muscle atrophy occurs when protein degradation exceeds protein synthesis and is associated with increased circulating glucocorticoid levels. Salvia plebeia R.Br. (SPR) has been used as herbal remedy for a variety of inflammatory diseases and has various biological actions such as antioxidant and anti-inflammatory activities. However, there are no reports on the effects of SPR and its bioactive components on muscle atrophy. Herein, we investigated the anti-atrophic effect of SPR and rosmarinic acid (RosA), a major compound of SPR, on dexamethasone (DEX)-induced skeletal muscle atrophy in C2C12 myotubes. Myotubes were treated with 10 µM DEX in the presence or absence of SPR or RosA at different concentrations for 24 h and subjected to immunocytochemistry, western blot, and measurements of ROS and ATP levels. SPR and RosA increased viability and inhibited protein degradation in DEX-treated C2C12 myotubes. In addition, RosA promoted the Akt/p70S6K/mTOR pathway and reduced ROS production, and apoptosis. Furthermore, the treatment of RosA significantly recovered SOD activity, autophagy activity, mitochondrial contents, and APT levels in DEX-treated myotubes. These findings suggest that SPR and RosA may provide protective effects against DEX-induced muscle atrophy and have promising potential as a nutraceutical remedy for the treatment of muscle weakness and atrophy.


Assuntos
Dexametasona , Fibras Musculares Esqueléticas , Humanos , Dexametasona/efeitos adversos , Dexametasona/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Músculo Esquelético/metabolismo , Ácido Rosmarínico
4.
BMC Biotechnol ; 22(1): 21, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927722

RESUMO

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes nosocomial infections and often exhibits antibiotic resistance. Therefore, the development of an accurate method for detecting P. aeruginosa is required to control P. aeruginosa-related outbreaks. In this study, we established an enzyme-linked immunosorbent assay method for the sensitive detection of three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and multidrug-resistant ATCC BAA-2108. We produced a recombinant antibody (rAb) against P. aeruginosa V-antigen (PcrV), which is a needle tip protein of the type III secretion system of P. aeruginosa using mammalian cells with high yield and purity, and confirmed its P. aeruginosa binding efficiency. The rAb was paired with commercial anti-P. aeruginosa Ab for a sandwich ELISA, resulting in an antigen-concentration-dependent response with a limit of detection value of 230 CFU/mL. These results suggest that the rAb produced herein can be used for the sensitive detection of P. aeruginosa with a wide range of applications in clinical diagnosis and point-of-care testing.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias , Ensaio de Imunoadsorção Enzimática , Humanos , Mamíferos , Infecções por Pseudomonas/diagnóstico
5.
RSC Adv ; 12(25): 15643-15651, 2022 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-35685704

RESUMO

The determination of the molecular weight (MW) of a protein using high-resolution mass spectrometry (MS) is a crucial tool used to confirm whether the protein was correctly expressed and adequately purified. However, a non-volatile buffer is normally used for protein purification and storage. Therefore, a pre-treatment step using ultrafiltration (UF) is required to exchange the buffer with a volatile buffer prior to the introduction of the protein sample into the MS equipment. This pre-treatment step is time-consuming. In this study, a trap column-based pre-treatment method applied in a nano-LC system was developed for rapid and convenient analysis of the MW of proteins. First, the trap column system was compared with the conventional UF treatment system and non-treatment system using bovine serum albumin. Subsequently, the trap column system was applied to analyze the MW of commercially available and lab-synthesized recombinant proteins. The intensity of the base peak and signal-to-noise ratio of the trap column-based pre-treated protein were higher than those of the UF-treated protein. Moreover, the entire automated procedure of the trap column-based system was conducted within 20 min, which confirms its use in versatile and accurate protein identification.

6.
ACS Omega ; 7(11): 9690-9700, 2022 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35350310

RESUMO

Staphylococcus aureus is a major resistant pathogen in clinical practice. Due to the increasing number of infections, rapid and sensitive detection of antibiotic-resistant S. aureus as well as antibiotic-sensitive S. aureus is important for the prevention and control of infectious diseases. In this study, we produced recombinant antibodies against S. aureus from mammalian human embryonic kidney 293 Freestyle cells with high yield and purity. These recombinant antibodies showed high binding affinity and low detection limit in both indirect and sandwich enzyme-linked immunosorbent assays for the detection of methicillin-resistant S. aureus and methicillin-sensitive S. aureus. These results suggest that the recombinant antibodies produced herein can be used for the accurate detection of S. aureus with a wild range of applications in medical diagnosis, food safety, and drug discovery.

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