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1.
Int J Biol Macromol ; 138: 890-902, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31344413

RESUMO

The present work aimed to evaluate the antioxidant efficacy of beta-1,3-glucan binding/recognition protein against oxidative stress-induced Saccharomyces cerevisiae. Beta-1,3-glucan binding/recognition protein was attained from the Paratelphusa hydrodromus (Phß-GBP) using laminarin coupled Sepharose CL-6B column. The structural characteristics of Phß-GBP were analyzed through circular dichroism (CD), fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance spectroscopy (1H NMR) analysis. CD spectrum showed the occurrence of α-helix (4.5%), ß-sheets (23.6%), ß-turn (17.2%) and random coils (54.8%). FTIR confirms the occurrence of amide and aromatic compounds whereas 1H NMR predicts the secondary structures and presence of amino acids in the Phß-GBP. In vitro radical scavenging analysis disclose that Phß-GBP has the potential to scavenge DPPH (73%), peroxyl radicals (81%) and hydrogen peroxide (56%) at 100µg/ml concentration. Reactive oxygen species production, lipid peroxidation, cell death, and DNA damage were decreased in the Phß-GBP pretreated S. cerevisiae. In silico protein-protein interaction was performed between the ß-GBP-glutathione reductase and ß-GBP-catalase A. The interaction study reveals that glutathione reductase and catalase A interacts with ß-GBP to reduce the oxidized glutathione and remove free radicals. This finding demonstrates that Phß-GBP may act as a good antioxidant which protects from human pathologies linked with oxidative stress.


Assuntos
Amidinas/farmacologia , Antioxidantes/farmacologia , Proteínas de Transporte/farmacologia , Lectinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Amidinas/química , Antioxidantes/química , Proteínas de Transporte/química , Dano ao DNA/efeitos dos fármacos , Depuradores de Radicais Livres/farmacologia , Lectinas/química , Peroxidação de Lipídeos/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Mapeamento de Interação de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise Espectral , Relação Estrutura-Atividade
2.
Toxicol In Vitro ; 59: 312-321, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31158490

RESUMO

Precision-cut intestinal slices (PCIS) is an ex vivo culture technique that found its applications in toxicology, drug transport and drug metabolism testing, as well as in fibrosis research. The main limiting factor of PCIS as experimental model is the relatively short viability of tissue slices. Here, we describe a strategy for extending the life-span of PCIS during culture using medium that is routinely used for growing intestinal organoids. Mouse and rat PCIS cultured in standard medium progressively showed low ATP/protein content and severe tissue degradation, indicating loss of tissue viability. In turn, organoid medium, containing epithelial growth factor (EGF), Noggin and R-spondin, maintained significantly higher ATP/protein levels and better preserved intestinal architecture of mouse PCIS at 96 h. In contrast, organoid medium that additionally contained Wnt, had a clear positive effect on the ATP content of rat PCIS during 24 h of culture, but not on slice histomorphology. Our proof-of-concept study provides early evidence that employing organoid medium for PCIS culture improved tissue viability during extended incubation. Enabling lasting PCIS cultures will greatly widen their range of applications in predicting long-term intestinal toxicity of xenobiotics and elucidating their mechanism of action, among others.


Assuntos
Intestinos , Técnicas de Cultura de Tecidos , Trifosfato de Adenosina , Animais , Proteínas de Transporte/farmacologia , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Ratos Wistar , Nicho de Células-Tronco , Trombospondinas/genética , Proteína Wnt3A/farmacologia
3.
Thromb Haemost ; 119(7): 1124-1137, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31129912

RESUMO

BACKGROUND: The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. OBJECTIVE: This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. METHODS: Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. RESULTS: NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. CONCLUSION: NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.


Assuntos
Adenosina/metabolismo , Plaquetas/fisiologia , Colforsina/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptor A2A de Adenosina/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Adenilil Ciclases/metabolismo , Proteínas de Transporte/farmacologia , Células Cultivadas , Colágeno/metabolismo , AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Humanos , Indometacina/farmacologia , Peptídeos/farmacologia , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/agonistas , Transdução de Sinais , Tromboxano B2/metabolismo
4.
Dev Comp Immunol ; 98: 13-19, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30980872

RESUMO

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the function of frog PGRPs. In this study, a short-type PGRP (termed Xl-PGRP-S) gene was identified in the African clawed frog, Xenopus laevis. The predicted protein of Xl-PGRP-S contains several structural features known in PGRPs, including a typical PGRP domain and two closely spaced conserved cysteines. Xl-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in muscle. As a typical PRR, Xl-PGRP-S is inducible after peptidoglycan (PGN) stimulation, and has an ability to bind PGN. In addition, Xl-PGRP-S has been proven to have Zn2+-dependent amidase activity and antibacterial activity against Edwardsiella tarda. The present study represents the first discovery on the function of frog PGRPs, thus contributing to a better understanding of the functional evolution of PGRPs in early tetrapods.


Assuntos
Proteínas de Transporte/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Edwardsiella tarda/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Peptidoglicano/metabolismo , Filogenia , Ligação Proteica , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/farmacologia , Xenopus laevis/metabolismo , Zinco/metabolismo
5.
Protein Pept Lett ; 26(6): 414-422, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-30919769

RESUMO

BACKGROUND: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). OBJECTIVE: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. METHODS: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. RESULTS: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. CONCLUSION: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.


Assuntos
Antifúngicos/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Quitina/química , Xenorhabdus/química , Animais , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Escherichia coli/genética , Inseticidas/química , Larva/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Ligação Proteica
6.
Nat Commun ; 10(1): 1023, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833574

RESUMO

Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor, with approximately 25% of DIPGs harboring activating ACVR1 mutations that commonly co-associate with H3.1K27M mutations. Here we show that in vitro expression of ACVR1 R206H with and without H3.1K27M upregulates mesenchymal markers and activates Stat3 signaling. In vivo expression of ACVR1 R206H or G328V with H3.1K27M and p53 deletion induces glioma-like lesions but is not sufficient for full gliomagenesis. However, in combination with PDGFA signaling, ACVR1 R206H and H3.1K27M significantly decrease survival and increase tumor incidence. Treatment of ACVR1 R206H mutant DIPGs with exogenous Noggin or the ACVR1 inhibitor LDN212854 significantly prolongs survival, with human ACVR1 mutant DIPG cell lines also being sensitive to LDN212854 treatment. Together, our results demonstrate that ACVR1 R206H and H3.1K27M promote tumor initiation, accelerate gliomagenesis, promote a mesenchymal profile partly due to Stat3 activation, and identify LDN212854 as a promising compound to treat DIPG.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Astrocitoma/metabolismo , Neoplasias do Tronco Encefálico/metabolismo , Genoma Humano/genética , Glioma/metabolismo , Histonas/metabolismo , Receptores de Ativinas Tipo I/genética , Animais , Astrocitoma/tratamento farmacológico , Astrocitoma/genética , Astrocitoma/patologia , Neoplasias do Tronco Encefálico/tratamento farmacológico , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Proteínas de Transporte/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Histonas/genética , Humanos , Camundongos , Mutação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
7.
Planta ; 249(5): 1503-1519, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30706136

RESUMO

MAIN CONCLUSION: Chitin-binding proteins behave as storage and antifungal proteins in the seeds of Moringa oleifera. Moringa oleifera is a tropical multipurpose tree. Its seed constituents possess coagulant, bactericidal, fungicidal, and insecticidal properties. Some of these properties are attributed to a group of polypeptides denominated M. oleifera chitin-binding proteins (in short, Mo-CBPs). Within this group, Mo-CBP2, Mo-CBP3, and Mo-CBP4 were previously purified to homogeneity. They showed high amino acid similarity with the 2S albumin storage proteins. These proteins also presented antimicrobial activity against human pathogenic yeast and phytopathogenic fungi. In the present study, the localization and expression of genes that encode Mo-CBPs and the biosynthesis and degradation of the corresponding proteins during morphogenesis and maturation of M. oleifera seeds at 15, 30, 60, and 90 days after anthesis (DAA) and germination, respectively, were assessed. The Mo-CBP transcripts and corresponding proteins were not detected at 15 and 30 days after anthesis (DAA). However, they accumulated at the latter stages of seed maturation (60 and 90 DAA), reaching the maximum level at 60 DAA. The degradation kinetics of Mo-CBPs during seed germination by in situ immunolocalization revealed a reduction in the protein content 48 h after sowing (HAS). Moreover, Mo-CBPs isolated from seeds at 60 and 90 DAA prevented the spore germination of Fusarium spp. Taken together, these results suggest that Mo-CBPs play a dual role as storage and defense proteins in the seeds of M. oleifera.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Quitina/metabolismo , Moringa oleifera/metabolismo , Moringa oleifera/fisiologia , Sementes/metabolismo , Sementes/fisiologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Fusarium/efeitos dos fármacos , Germinação/fisiologia
8.
Microbes Infect ; 21(1): 40-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29981934

RESUMO

Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host-mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms_AcpM) and a strain transformed with an empty vector (Ms_Vec). Ms_AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms_AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms_Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection.


Assuntos
Apoptose/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/patologia , Mycobacterium tuberculosis/química , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Células Cultivadas , Feminino , Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/microbiologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/fisiologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
9.
J Virol ; 93(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30305350

RESUMO

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprisingly, the majority of the genes identified are neither associated with innate or adaptive immunity nor associated with any antiviral activity. Confirmatory studies of eight of the genes validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together, these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules.IMPORTANCE Norovirus is one of the leading causes of food-borne illness worldwide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed, we performed genome-wide CRISPR activation screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 49 genes that could block murine norovirus replication in human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data are a resource for those studying noroviruses, and we provide a robust approach to identify novel antiviral genes.


Assuntos
Antivirais/farmacologia , Infecções por Caliciviridae/genética , Proteínas de Transporte/farmacologia , Redes Reguladoras de Genes , Norovirus/fisiologia , Animais , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/virologia , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Norovirus/efeitos dos fármacos , Ativação Transcricional , Ubiquitina-Proteína Ligases , Regulação para Cima , Internalização do Vírus , Replicação Viral/efeitos dos fármacos
10.
Nat Protoc ; 14(1): 28-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30470820

RESUMO

The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.


Assuntos
Técnicas de Cultura de Células , Endoderma/citologia , Células Epiteliais/citologia , Fundo Gástrico/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Antro Pilórico/citologia , Ativinas/farmacologia , Benzamidas/farmacologia , Proteínas de Transporte/farmacologia , Diferenciação Celular , Colágeno/química , Meios de Cultura/química , Meios de Cultura/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Combinação de Medicamentos , Endoderma/efeitos dos fármacos , Endoderma/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator 10 de Crescimento de Fibroblastos/farmacologia , Fator 4 de Crescimento de Fibroblastos/farmacologia , Fundo Gástrico/metabolismo , Humanos , Laminina/química , Especificidade de Órgãos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Proteoglicanas/química , Antro Pilórico/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
11.
Blood Adv ; 2(24): 3627-3636, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30573565

RESUMO

Platelet secretion is central to physiological and pathophysiological platelet function. SNAP23 has long been implicated as being a principal SNARE protein regulating platelet granule secretion, although this has not been definitively demonstrated in genetic models. Here, using a platelet-specific conditional SNAP23 knockout mouse, we show that absence of SNAP23 results in complete ablation of dense granule, α granule, and lysosomal secretion. Measured granule cargo content and granule numbers were normal, suggesting SNAP23 regulates fusion of granules with the extracellular membrane, rather than granule loading or formation. A macrothrombocytopenia was also observed, which, combined with ablation of secretion, resulted in a pronounced bleeding defect in a tail bleed assay and almost complete ablation of arterial and venous thrombosis. The macrothrombocytopenia was not due to reduced megakaryopoiesis but instead likely was due to the increased loss of platelets through bleeding, consistent with an increase in platelet total RNA content indicating a greater number of reticulated platelets. The data definitively show SNAP23 to be critical for granule release of any kind from platelets, irrespective of stimulus, and this is the first single gene to be shown to be universally essential for exocytosis in platelets.


Assuntos
Plaquetas/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Trombose/patologia , Animais , Plaquetas/patologia , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Grânulos Citoplasmáticos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Proteínas Qb-SNARE/deficiência , Proteínas Qc-SNARE/deficiência , Trombopoetina/farmacologia , Trombose/prevenção & controle , Trombose Venosa/patologia , Trombose Venosa/prevenção & controle
12.
Sci Rep ; 8(1): 16847, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442953

RESUMO

We have recently reported that green soybean cultivar, echigomidori, and not the yellow cultivar, fukuyutaka, is a rich source of hormone-like peptide leginsulin consisting of 37 amino acids (Leg_1_37, PDB 1JU8A) and its C-terminal glycine deletant, Leg_1_36. Green soybean is mature, but the color of the seedcoat and cotyledon remains green. Therefore, in this study, we examined the leginsulin content in different varieties of 11 colored soybeans (including green, yellow, red, brown and black) and edamame (immature soybean). Profile analysis of soybean constituents by LC-MS showed that Leg_1 (36 + 37) detected as a prominent peak in 3 green and 1 yellow soybean cultivar was the strongest contributor in principal component analysis, indicating Leg_1 is the most characteristic feature for distinguishing soybean cultivars. However, smaller amounts of leginsulin-like peptides, defined as Leg_2 and Leg_3, were detected in other samples. The cDNA sequences and LC-MS/MS analyses revealed that Leg_2 was a homologue of Leg_1 with three amino acid substitutions derived from SNPs, while Leg_3 was a Leg_1/Leg_2 paralog. Expression levels of Leg_1 were markedly higher than Leg_2 and Leg_3. Additionally, in glucose uptake assay, purified TRX-His-tag fused recombinant Leg_1_37 prepared by bacterial expression showed stronger insulin-like activities than other variants including Leg_2, Leg_3, and their Gly deletants in myotube-like differentiated L6 and C2C12 cells. These results suggest that dietary consumption of soybean seed, especially including a higher amount of Leg_1_37, could be useful for lowering of blood glucose.


Assuntos
Proteínas de Transporte/farmacologia , Insulinas/farmacologia , Peptídeos/farmacologia , Proteínas de Plantas/farmacologia , Soja/química , Albuminas , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diferenciação Celular/efeitos dos fármacos , DNA Complementar/genética , Etanol , Regulação da Expressão Gênica de Plantas , Insulinas/química , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Peptídeos/química , Extratos Vegetais/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ratos , Soja/genética
13.
Nat Microbiol ; 3(12): 1472-1485, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30478389

RESUMO

Plasma membrane integrity is essential for the viability of eukaryotic cells. In response to bacterial pore-forming toxins, disrupted regions of the membrane are rapidly repaired. However, the pathways that mediate plasma membrane repair are unclear. Here we show that autophagy-related (ATG) protein ATG16L1 and its binding partners ATG5 and ATG12 are required for plasma membrane repair through a pathway independent of macroautophagy. ATG16L1 is required for lysosome fusion with the plasma membrane and blebbing responses that promote membrane repair. ATG16L1 deficiency causes accumulation of cholesterol in lysosomes that contributes to defective membrane repair. Cell-to-cell spread by Listeria monocytogenes requires membrane damage by the bacterial toxin listeriolysin O, which is restricted by ATG16L1-dependent membrane repair. Cells harbouring the ATG16L1 T300A allele associated with inflammatory bowel disease were also found to accumulate cholesterol and be defective in repair, linking a common inflammatory disease to plasma membrane integrity. Thus, plasma membrane repair could be an important therapeutic target for the treatment of bacterial infections and inflammatory disorders.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Listeria monocytogenes/efeitos dos fármacos , Animais , Autofagia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Toxinas Bacterianas/toxicidade , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Colesterol/metabolismo , Modelos Animais de Doenças , Exocitose , Células HeLa , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Humanos , Listeria monocytogenes/metabolismo , Lisossomos , Masculino , Camundongos
14.
Stem Cell Reports ; 11(6): 1539-1550, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30472010

RESUMO

The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism.


Assuntos
Células Epiteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Absorção Intestinal , Intestino Delgado/citologia , Preparações Farmacêuticas/metabolismo , Animais , Biomarcadores/metabolismo , Células CACO-2 , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Dexametasona/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Imidazóis/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Absorção Intestinal/efeitos dos fármacos , Maleimidas/farmacologia , Camundongos , Piridinas/farmacologia , Trombospondinas/farmacologia , Proteína Wnt3A/farmacologia
15.
J Microbiol Methods ; 154: 19-24, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30291881

RESUMO

The purpose of this study was to establish a method for determining the bacteriolytic activity after separation of lysozyme-binding proteins from egg white. Lysozyme-binding proteins such as ovotransferrin and ovalbumin were separated by non-denaturing two-dimensional electrophoresis (2DE) and transferred to a membrane. The lysozyme activity of the separated and immobilized egg white proteins was assessed directly to produce a non-denaturing 3D map of the egg white proteins by incorporating an axis that combined each spot's lysozyme-activity with the non-denaturing 2DE pattern. Lysozyme-ovotransferrin and lysozyme-ovalbumin complexes could be reconstructed in vitro after the cathode end fraction containing lysozyme was added to purified ovotransferrin and ovalbumin, respectively. These complexes retained lysozyme activity even after separation by non-denaturing 2DE. Furthermore, when the lysozyme-ovotransferrin complex from egg white was extracted after separation by isoelectric focusing by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, the complex possessed bacteriolytic activity against both Bacillus subtilis and Escherichia coli. These methods can be applied to investigate protein complexes possessing bacteriolytic activity against a wide range of both Gram-positive and Gram-negative bacteria.


Assuntos
Antibacterianos/farmacologia , Proteínas de Transporte/farmacologia , Galinhas , Proteínas do Ovo/química , Proteínas do Ovo/isolamento & purificação , Muramidase/farmacologia , Animais , Compostos Azo/química , Bacillus subtilis/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Bacteriólise , Conalbumina/química , Conalbumina/farmacologia , Eletroforese em Gel Bidimensional/métodos , Escherichia coli/efeitos dos fármacos , Imobilização , Focalização Isoelétrica/métodos , Muramidase/química , Ovalbumina/química , Ovalbumina/farmacologia
16.
Biochem Biophys Res Commun ; 505(3): 905-909, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30309654

RESUMO

RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. RUNX1 function is under the tight control through posttranslational modifications, including phosphorylation and ubiquitination. We previously developed a luminescence-based binding assay (AlphaScreen) to systematically detect RUNX1-interacting E3 ubiquitin ligases. In this study, we showed that a nuclear ubiquitin ligase RNF38 induced ubiquitination of RUNX1. RNF38-induced RUNX1 ubiquitination did not promote RUNX1 degradation, but rather stabilized RUNX1 protein. We also found that RNF38 enhanced RUNX1-mediated transcriptional repression of the erythroid master regulator KLF1 in K562 cells. Consequently, RNF38 cooperated with RUNX1 to inhibit erythroid differentiation of K562 cells. Thus, our study identified RNF38 as a novel E3 ligase that modifies RUNX1 function without inducing its degradation.


Assuntos
Proteínas de Transporte/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Ubiquitinação/efeitos dos fármacos , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Humanos , Células K562 , Fatores de Transcrição Kruppel-Like , Estabilidade Proteica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/farmacologia
17.
Proc Natl Acad Sci U S A ; 115(45): 11625-11630, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30352848

RESUMO

Increasing age is the greatest known risk factor for the sporadic late-onset forms of neurodegenerative disorders such as Alzheimer's disease (AD). One of the brain regions most severely affected in AD is the hippocampus, a privileged structure that contains adult neural stem cells (NSCs) with neurogenic capacity. Hippocampal neurogenesis decreases during aging and the decrease is exacerbated in AD, but the mechanistic causes underlying this progressive decline remain largely unexplored. We here investigated the effect of age on NSCs and neurogenesis by analyzing the senescence accelerated mouse prone 8 (SAMP8) strain, a nontransgenic short-lived strain that spontaneously develops a pathological profile similar to that of AD and that has been employed as a model system to study the transition from healthy aging to neurodegeneration. We show that SAMP8 mice display an accelerated loss of the NSC pool that coincides with an aberrant rise in BMP6 protein, enhanced canonical BMP signaling, and increased astroglial differentiation. In vitro assays demonstrate that BMP6 severely impairs NSC expansion and promotes NSC differentiation into postmitotic astrocytes. Blocking the dysregulation of the BMP pathway and its progliogenic effect in vivo by intracranial delivery of the antagonist Noggin restores hippocampal NSC numbers, neurogenesis, and behavior in SAMP8 mice. Thus, manipulating the local microenvironment of the NSC pool counteracts hippocampal dysfunction in pathological aging. Our results shed light on interventions that may allow taking advantage of the brain's natural plastic capacity to enhance cognitive function in late adulthood and in chronic neurodegenerative diseases such as AD.


Assuntos
Envelhecimento/genética , Doença de Alzheimer/tratamento farmacológico , Proteína Morfogenética Óssea 6/genética , Proteínas de Transporte/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Proteína Morfogenética Óssea 6/antagonistas & inibidores , Proteína Morfogenética Óssea 6/metabolismo , Diferenciação Celular , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Transdução de Sinais
18.
Fish Shellfish Immunol ; 83: 61-75, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30176334

RESUMO

In the present study, immunoenhancing effect of ß-1, 3 glucan binding protein based selenium nanowire (Phß-GBP-SeNWs) in common carp, Cyprinus carpio was assessed. Biological based selenium nanoform was synthesized, using crustacean immune molecule ß-GBP purified from the haemolymph of Paratelphusa hydrodromus. The morphological property of Phß-GBP-SeNWs was analyzed through TEM which reveals, the synthesized nanowire exhibits approximately 30-50 nm width with smooth surface. For this current study, fish were fed with experimental diet includes Phß-GBP, sodium selenite, selenomethionine and Phß-GBP-SeNWs supplemented diet at different concentrations (0.5 mg, 1 mg and 2 mg) for 30 days. The growth performance, cellular and humoral immune responses (myeloperoxidase, reactive oxygen species, alkaline phosphatase and lysozyme activity) and antioxidant enzymes (glutathione peroxidase and catalase activity) in the fish fed with Phß-GBP-SeNWs supplemented diet were significantly increased in dose-dependent manner, which was observed at two different interval period (15th and 30th day). Also, Phß-GBP-SeNWs supplemented diet fed fish gain resistant after challenged with aquatic pathogen Aeromonas hydrophila and the relative survival percentage was increased. Agar disc diffusion and BacLight assay clearly demonstrated the antibacterial property of plasma of fish fed with Phß-GBP-SeNWs supplemented diet against aquatic pathogen A. hydrophila, Vibrio parahaemolyticus and Vibrio alginolyticus. Moreover, confocal laser scanning microscopic analysis clearly showed that, Phß-GBP-SeNWs supplemented diet fed fish plasma was more efficient in disrupting the architecture of bacterial colonies and thereby reduced the thickness of biofilm. Thus, the present study indicates that, incorporation of Phß-GBP-SeNWs in the diet enhances the fish immune responses and disease resistance against aquatic pathogens.


Assuntos
Carpas/imunologia , Carpas/microbiologia , Proteínas de Transporte/farmacologia , Doenças dos Peixes/imunologia , Lectinas/farmacologia , Nanofios/química , Selenito de Sódio/farmacologia , Aeromonas hydrophila , Fosfatase Alcalina/metabolismo , Animais , Biofilmes , Resistência à Doença , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/prevenção & controle , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
19.
Physiol Rep ; 6(17): e13841, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30187671

RESUMO

Gαi2 , a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed Gαi2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gαi2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen-related peptide (CoRP), which are further known to enhance degranulation and activation of αIIb ß3-integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gαi2 and their wild-type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 µg/mL or 5 µg/mL) significantly upregulated PS-exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P-selectin, and αIIb ß3-integrin activation. These molecular alterations were significantly less pronounced in Gαi2 -deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gαi2 signaling in governing platelet activation and apoptosis.


Assuntos
Apoptose , Plaquetas/metabolismo , Degranulação Celular , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Proteínas de Transporte/farmacologia , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacologia , Trombina/farmacologia
20.
Cell Cycle ; 17(15): 1901-1916, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30109813

RESUMO

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the sensor inositol-requiring enzyme 1α (IRE1), an endoribonuclease that splices the mRNA of the transcription factor XBP1 (X-box-binding protein 1). To better understand the protein network that regulates the activity of the IRE1 pathway, we systematically screened the proteins that interact with IRE1 and identified a ribonuclease inhibitor called ribonuclease/angiogenin inhibitor 1 (RNH1). RNH1 is a leucine-rich repeat domains-containing protein that binds to and inhibits ribonucleases. Immunoprecipitation experiments confirmed this interaction. Docking experiments indicated that RNH1 physically interacts with IRE1 through its cytosolic RNase domain. Upon ER stress, the interaction of RNH1 with IRE1 in the ER increased at the expense of the nuclear pool of RNH1. Inhibition of RNH1 expression using siRNA mediated RNA interference upon ER stress led to an increased splicing activity of XBP1. Modulation of IRE1 RNase activity by RNH1 was recapitulated in a cell-free system, suggesting direct regulation of IRE1 by RNH. We conclude that RNH1 attenuates the activity of IRE1 by interacting with its ribonuclease domain. These findings have implications for understanding the molecular mechanism by which IRE1 signaling is attenuated upon ER stress.


Assuntos
Proteínas de Transporte/metabolismo , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Transporte/farmacologia , Linhagem Celular Transformada , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Modelos Moleculares , Domínios Proteicos , Proteoma , Processamento de RNA , Uromodulina/metabolismo
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