Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biochem Biophys Res Commun ; 549: 105-112, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33667707

RESUMO

Escherichia coli is one of the most popularly used hosts to produce recombinant proteins. Most recombinant proteins are produced in the cytoplasm and periplasm, requiring multiple steps to extract and purify recombinant proteins. The Serratia marcescens Lip system (LipB-LipC-LipD) is a type 1 secretion system that selectively secretes LipA from the intracellular to extracellular space in a single step. This study aimed to establish a secretory production system for nanobodies, camelid-derived small molecule antibody fragments, using the S. marcescens Lip system. Surprisingly, E. coli harboring only LipC, a membrane fusion protein of the Lip system, could secrete an anti-green fluorescent protein (GFP)-Nb, a nanobody against GFP, without the addition of a long amino acid sequence. The LipC-based secretion system recognized the Val-Thr-Val sequence at the C-terminus of the nanobody. Finally, Strep-tagged anti-GFP-Nb was purified from culture supernatants of E. coli harboring LipC by Strep-affinity chromatography at a final yield of >5 mg per liter of culture supernatant. These results potently supported that the S. marcescens LipC-based secretion system has the potential to establish an efficient secretory production system for nanobodies.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Serratia marcescens/metabolismo , Anticorpos de Domínio Único/biossíntese , Sequência de Aminoácidos , Animais , Antígenos/metabolismo , Camelus , Meios de Cultura , Proteínas de Fluorescência Verde/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/isolamento & purificação
2.
Biochem Biophys Res Commun ; 552: 9-16, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33740666

RESUMO

Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling.


Assuntos
Receptores do Fator Natriurético Atrial/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células HEK293 , Células HeLa , Humanos , Ligantes , Proteínas Musculares/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de Transcrição/farmacologia
3.
Biomolecules ; 10(10)2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33096942

RESUMO

Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Citrus/química , Neoplasias Cutâneas/tratamento farmacológico , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Frutas/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
4.
Biochem Biophys Res Commun ; 519(2): 344-350, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31514996

RESUMO

Although we recently reported that sudachitin (5,7,4'-trihydroxy-6,8,3'-trimethoxyflavone), a polymethoxyflavone isolated from the peel of Citrus sudachi, can induce apoptosis in human keratinocyte HaCaT cells, the mechanism underlying its action remains unclear. In this study, we explored the mechanisms underlying sudachitin-induced apoptosis in HaCaT cells. Sudachitin activated p38MAPK and inhibited ERK1/2, whereas another polymethoxyflavone, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), activated ERK1/2. The p38MAPK inhibitor SB203580 significantly attenuated sudachitin-induced heat shock protein 27 phosphorylation, downstream of p38MAPK, and subsequent apoptosis, indicating that sudachitin induces apoptosis via the p38MAPK pathway. Additionally, sudachitin inhibited serum- and EGF-stimulated Raf-1-ERK1/2 activation, and blocked EGF-induced cell migration and proliferation in HaCaT cells. These results suggest that small structural differences in polymethoxyflavones can induce different cellular responses by altering the regulation of MAPK activities and that sudachitin may be a potential candidate for developing new drugs for skin diseases such as psoriasis.


Assuntos
Apoptose/efeitos dos fármacos , Citrus/química , Flavonoides/farmacologia , Glicosídeos/farmacologia , Queratinócitos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/metabolismo , Humanos
5.
Biosci Biotechnol Biochem ; 82(12): 2064-2071, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30185129

RESUMO

A variety of polyphenols have been isolated from plants, and their biological activities have been examined. Sudachitin (5,7,4'-trihydroxy-6,8,3'-trimethoxyflavone) is a polymethoxyflavone that is isolated from the peel of Citrus sudachi. Although we previously reported that sudachitin possesses an anti-inflammatory activity, its other biological activities are not yet understood. In this study, we report a novel biological activity of sudachitin, which selectively induced apoptosis in human keratinocyte HaCaT cells. Another polymethoxyflavone, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), promoted autophagy but not apoptosis in HaCaT cells. On the other hand, 3'-demethoxysudachitin (5,7,4'-trihydroxy-6,8-dimethoxyflavone) failed to induce apoptosis and autophagy. These results show that three polymethoxyflavones have different effects on apoptosis and autophagy in HaCaT cells. Understanding the structure and biological activity of polymethoxyflavones may lead to the discovery of potential candidates for cancer drug development without significant toxic side effects. Abbreviations: ROS: reactive oxygen species; DMSO: dimethyl sulfoxide; MTT: 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; MAPK: mitogen-activated protein kinase.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Citrus/química , Flavonas/farmacologia , Flavonoides/farmacologia , Glicosídeos/farmacologia , Queratinócitos/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Flavonas/química , Flavonoides/química , Glicosídeos/química , Humanos , Queratinócitos/citologia , Relação Estrutura-Atividade
6.
Beilstein J Nanotechnol ; 6: 1010-5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25977872

RESUMO

We propose a novel patterning technique for gold nanoparticles on substrates that combines a chemical reaction with electron beam irradiation. First, gold nanoparticles are placed in a two-dimensional arrangement on the substrate. Then, particular nanoparticles are fixed on the substrate by irradiation with a focused electron beam to produce a desired pattern. Finally, the unfixed nanoparticles are removed. Using this technique, an array of gold nanoparticles, for example, in the form of a line or patterned over an area, are prepared on the substrate. This technique could contribute to the fabrication of plasmonic devices and other applications that require the controlled placement of gold nanoparticles on substrates.

7.
J Phys Chem B ; 112(3): 918-22, 2008 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-18161961

RESUMO

Sonoluminescence spectra from argon-saturated NaCl solution were measured in the concentration range of 0.5-4 M at the frequency of 138 kHz. The line broadening of sodium atom emission was observed at various acoustic powers in the range from 1.8 to 16.2 W. The sodium D line showed a maximum intensity at a NaCl concentration of 2 M, which corresponded to the maximum production of OH radicals estimated by KI dosimetry. The effects of the addition of a small amount of ethanol on the line width and intensity were closely investigated at various acoustic powers. The sodium line width increases with ethanol concentration and also with power, whereas the line intensity is strongly quenched with increasing ethanol concentration. The results conclusively show that the sodium emission occurs in the gas phase within bubbles. The line broadening is due to interactions with high-pressure argon, and the maximum relative density of gas at bubble collapse was estimated to be 59.5 from the comparison with spectroscopic data. Further line broadening and quenching upon the addition of ethanol arise from collisions with gaseous products obtained from the decomposition of ethanol. The mechanism of sodium excitation is inferred to be as follows. Sodium ions enter bubbles as droplets, and salts are formed because of the high temperature within bubbles. Sodium atoms are generated by the dissociation of salts and then undergo electronic excitation by OH and H radicals.

8.
J Vet Med Sci ; 68(12): 1251-5, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17213692

RESUMO

Blood examinations and genotyping of Factor XI (F11) were performed in growth retardation Japanese Black cattle and their dams. Genotyping of F11 revealed that the recessive homozygous and heterozygous genotype frequencies were 5.2% and 50.0% in the Claudin-16 (CL-16) deficiency group (n=58), 0% and 14.2% in the renal dysplasia group (n=7), 0% and 26.1% in the non-CL-16 deficiency nephritis group (n=23), 8.9% and 46.7% in the hypogenesis syndrome group (n=45), 6.2% and 25.0% in the neonatal weak calf syndrome group (n=32), 9.1% and 38.6% in the respective dams group (n=44), 0% and 23.1% in the normal cattle group (n=13), and 5.9% and 38.2% in total (n=222), respectively. These results showed that the carrier rate of F11 deficiency was high in Japanese Black cattle, and that the CL-16 deficiency, hypogenesis syndrome, neonatal weak calf syndrome, and dams groups had a large amount of recessive homozygous genotype than the other groups. No abnormal bleeding was observed clinically in the present study, and 4 of the recessive homozygous dams showed normal growth and parturition.


Assuntos
Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/genética , Deficiência do Fator XI/veterinária , Heterozigoto , Animais , Bovinos , Fator XI/genética , Deficiência do Fator XI/epidemiologia , Deficiência do Fator XI/genética , Feminino , Genótipo , Japão/epidemiologia , Masculino
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...