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1.
Fish Shellfish Immunol ; 96: 152-160, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31794843

RESUMO

C-type lectins are Ca2+-dependent carbohydrate-binding proteins containing one or more carbohydrate-recognition domains (CRDs). C-type lectins play crucial roles in innate immunity, including nonself-recognition and pathogen elimination. In the present study, two C-type lectins (designated ReCTL-1 and ReCTL-2) were identified from the shrimp Rimicaris exoculata which dwells in deep-sea hydrothermal vents. The open reading frames of ReCTL-1 and ReCTL-2 encoded polypeptides of 171 and 166 amino acids respectively, which were both composed of a signal peptide and a single CRD. The key motifs determining the carbohydrate binding specificity of ReCTL-1 and ReCTL-2 were respectively Glu-Pro-Ala (EPA) and Gln-Pro-Asn (QPN), which were firstly discovered in R. exoculata. ReCTL-1 and ReCTL-2 displayed similar pathogen-associated molecular pattern (PAMP) binding features and they bound three PAMPs-ß-glucan, lipopolysaccharide and peptidoglycan-with relatively high affinity. In addition, both could efficiently recognize and bind Gram-positive bacteria, Gram-negative bacteria and fungi. However, ReCTL-1 and ReCTL-2 exhibited different microbial agglutination activities: ReCTL-1 agglutinated Staphylococcus aureus and Saccharomyces cerevisiae, while ReCTL-2 agglutinated Micrococcus luteus, Vibrio parahaemolyticus and V. fluvialis. Both ReCTL-1 and ReCTL-2 inhibited the growth of V. fluvialis. All these results illustrated that ReCTL-1 and ReCTL-2 could function as important pattern-recognition receptors with broad nonself-recognition spectra and be involved in immune defense against invaders, but their specificities are not the same. In addition, the two ReCTLs possessed different carbohydrate binding specificities from each other and from the classical pattern: ReCTL-1 with an EPA motif bound d-galactose and l-mannose, while ReCTL-2 with a QPN motif bound d-fucose and N-acetylglucosamine.

2.
Fish Shellfish Immunol ; 95: 220-226, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31586458

RESUMO

The leading cause of mortality due to colorectal cancer (CRC) is highly associated with the development of liver metastases. Recently, we described cGAMP that is closely related to the metastatic state wherein the progress of metastatic tumors is associated with favorable outcomes in a zebrafish xenograft model. cGAMP was administered and the expression levels of type-I interferons were induced amongst tumor tissues to illuminate the overall measure of the induced STING/STAT3 axis in colorectal liver metastases. Furthermore, cGAMP-STING dependent STAT3 activation resulted in the inhibition of tumor cell proliferation, viability, and invasion in vitro. The subtotal reduction in tumor growth attributed to a large number of infiltrating inflammatory cells in vivo. We showed that cGAMP inhibited migration through angiogenesis by up-regulating IL-2, TNF-α, and IFN-γ, whereas STAT3 down-regulation inhibited CXCL8, BCL-2, and VEGFA expression. The importance of cGAMP in inhibiting the invasion front of CRC confirmed that the cGAMP dependent activation of STING/STAT3 axis played a key role in the inhibition of tumor progression.

3.
Arch Biochem Biophys ; 671: 203-209, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31302140

RESUMO

Activation of the NLRP3 inflammasome plays an important role in high glucose- induced endothelial dysfunction in patients with type 2 diabetes mellitus (T2DM). Dulaglutide, a newly developed glucagon-like peptide-1 receptor (GLP-1R) agonist, has been approved for the management of T2DM. In the current study, we aimed to investigate whether dulaglutide possesses a protective effect against high glucose- induced activation of the NLRP3 inflammasome. Our results indicate that dulaglutide treatment prevented high glucose- induced generation of reactive oxygen species (ROS) and protein carbonyl, as well as the expression of NADPH oxidase 4 (NOX-4) in human umbilical vein endothelial cells (HUVECs). Dulaglutide treatment could inhibit high glucose- induced release of lactate dehydrogenase (LDH) and the expression of TXNIP. Dulaglutide suppressed high glucose- induced activation of NLRP3 inflammasome by reducing the expression of NLRP3, ASC, and cleaved caspase 1 (P10). Notably, dulaglutide treatment suppressed high glucose- induced maturation of IL-1ß and IL-18. Mechanistically, our findings indicate that SIRT1 was involved in this process by showing that knockdown of SIRT1 by transfection with SIRT1 siRNA abolished the inhibitory effects of dulaglutide on IL-1ß and IL-18 secretion via suppression of NLRP3, ASC, and p10. These data suggest that dulaglutide might serve as a potential drug for the treatment of cardiovascular complications in T2DM patients.

4.
J Biotechnol ; 296: 83-92, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30898688

RESUMO

Silkworms are an economically important insect.Silkworm pupae are also a nutrient-rich food and can be used as a pharmaceutical intermediate.The N-terminus of Aß includes 1-15 amino acid residues with a B cell surface antigen that is necessary to produce antibody and prevent the adverse reactions observed in response to the full Aß42 peptide. In this study, we used silkworm pupae to develop a safer vaccine for Alzheimer's disease (AD) patients. Aß15 peptide was fused with the cholera toxin B subunit (CTB) and expressed in silkworm pupae. Then, we tested an oral vaccine with the peptide expressed by silkworm pupae in a transgenic mouse model of AD. The results show that anti-Aß antibodies were induced, Aß deposition in the brain decreased, the content of malondialdehyde was lower than in the other group, and memory and cognition of the mice improved. These results suggest that the high-nutrient CTB-Aß15 silkworm pupa vaccine has a potential clinical application for the prevention of AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/genética , Toxina da Cólera/administração & dosagem , Vacinas/administração & dosagem , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/imunologia , Animais , Bombyx/química , Bombyx/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , Vacinas/genética , Vacinas/imunologia
5.
BMC Mol Biol ; 20(1): 5, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755162

RESUMO

BACKGROUND: GTPase-activating proteins (GAPs) with a TBC (Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases. The related crystal structure has been studied and reported by other members of our research group in 2017 (Chen et al. in Protein Sci 26(4):834-846, 2017). The protein crystal structure and sequencing data accession numbers in Protein structure database (PDB) are 5TUB (Shark TBC1D15 GAP) and 5TUC (Sus TBC1D15 GAP), respectively. In this paper, we analyzed the Rab-GAP specificity of TBC1D15 in the evolution and influence of key amino acid residue mutations on Rab-GAP activity. RESULTS: Sequence alignment showed that five arginine residues of the TBC1D15-GAP domain are conserved among the species Sus/Mus/Homo but have been replaced by glycine or lysine in Shark. A fragment activity assay was conducted by altering the five residues of Shark TBC1D15-GAP to arginine, and the corresponding arginine in TBC1D15 GAP domains from Sus and Homo species were mutated to resemble Shark TBC1D15-GAP. Our data revealed that the residues of G28, K45, K119, K122 and K221 in the Shark TBC1D15-GAP domain had a key role in determining the specificity for Rab7 and Rab11. Mutation of the five residues significantly altered the Shark TBC1D15-GAP activity. CONCLUSIONS: These results revealed that the substrate specificity of TBC1D15 has had different mechanisms across the evolution of species from lower-cartilaginous fish to higher mammals. Collectively, the data support a different mechanism of Shark TBC1D15-GAP in substrate selection, which provides a new idea for the development of Marine drugs.


Assuntos
Sequência Conservada , Evolução Molecular , Proteínas Ativadoras de GTPase/química , Tubarões/metabolismo , Proteínas rab de Ligação ao GTP/química , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/genética , Cristalografia por Raios X , Glicina/química , Glicina/genética , Humanos , Lisina/química , Lisina/genética , Camundongos , Mutação , Domínios Proteicos , Alinhamento de Sequência , Tubarões/genética , Especificidade por Substrato , Suínos , Proteínas rab de Ligação ao GTP/genética
6.
AMB Express ; 9(1): 14, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30701481

RESUMO

The unique antimicrobial mechanism of antimicrobials make them a promising substitute for antibiotics for fighting drug-resistant bacteria. Both melittin and thanatin have antimicrobial bioactivity. However, thanatin does not inhibit the growth of Staphylococcus aureus. Melittin can inhibit S. aureus and has strong hemolytic activity. In the present study, the mutant fragments of melittin and thanatin were combined by flexible peptides to form a novel hybrid peptide, which was synthesized based on the secondary and tertiary structure prediction. The hybrid peptide inhibited S. aureus with a hemolytic concentration of above 45 µmol/L and inhibition rate in SMMC-7721 cells of 19.14%. The hybrid antimicrobial peptide, which was designed by the combination of α-helix and ß-lamellar antimicrobial peptides, showed that both types of peptides did not interact with each either on spatial structure or biological activities, thereby providing a novel idea for the design of artificial antimicrobial peptides.

7.
Gene ; 683: 210-215, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30316925

RESUMO

Glucose transport into skeletal muscle is mediated by the principal glucose transporter protein, GLUT4, which can be transported from intracellular vesicles to the cytoplasmic membrane, and the translocation of GLUT4 vesicles requires a variety of Rab proteins. Previously we reported a new type of TBC1D15 from C. plagiosum with Rab-GAP activity for Rab7. Here we reported that TBC1D15 regulated glucose uptake by affecting the translocation of GLUT4 through late endosomal pathway. When TBC1D15 was knocked out by CRISPR/Cas9, a significant reduction in 2-NBDG uptake was observed, and the total amount of GLUT4 was significantly reduced in TBC1D15-/- cells compared to that in WT cells. Furthermore, concentrated distribution of Rab7 in Lamp1-decorated late endosome/lysosome and an increase in co-localization between GLUT4 and Rab7 was observed in TBC1D15-/- cells. These results suggested that TBC1D15 served as a master regulator in GLUT4 translocation and further affected GLUT4-mediated glucose uptake.


Assuntos
Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Animais , Endossomos/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HeLa , Humanos , Transporte Proteico , Ratos , Proteínas rab de Ligação ao GTP/metabolismo
8.
Biomed Pharmacother ; 106: 491-498, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29990837

RESUMO

In the pathogenesis of diabetes mellitus (DM), islet microvasculares are severely damaged due to glucolipotoxicity and other reasons. Vascular endothelial growth factor (VEGF) is an indispensable and specific angiogenic factor in the pathogenesis and treatment of diabetic islet microvascular disease. Mesenchymal stem cells (MSCs) are regarded as a promising treatment of diabetes because of their immunosuppressive effect and multipotential differentiation potency. In this study, we tested whether MSCs over-expressing VEGF conditioned medium (MSC-VEGF-CM) could ameliorate pancreatic islet endothelial cells (MS-1) dysfunction induced by a common diabetic inducer palmitate (PA). We found that cell survival and migration were restrained by PA and partly repaired by the pro-protected of MSC-VEGF-CM. Meanwhile, PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathways were also up-regulated. Though apoptosis-related proteins, caspase-3 and caspase-9, had no significantly suppressed between MSC-VEGF-CM and MSC-CM alone, the expression levels of vascular surface factors such as CD31, VE-cadherin, occludin and ICAM-1, were remarkably up-regulated by the pro-protected of MSC-VEGF-CM. Our data suggested that MSC-VEGF-CM had therapeutic effect on the PA-induced dysfunction through the re-activation of PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathways.


Assuntos
Meios de Cultivo Condicionados/metabolismo , Angiopatias Diabéticas/enzimologia , Células Endoteliais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ácido Palmítico/toxicidade , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Angiopatias Diabéticas/patologia , Angiopatias Diabéticas/cirurgia , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Transplante de Células-Tronco Mesenquimais , Camundongos , Comunicação Parácrina , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética
9.
AMB Express ; 8(1): 84, 2018 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-29785529

RESUMO

The abuse of antibiotics and following rapidly increasing of antibiotic-resistant pathogens is the serious threat to our society. Natural products from microorganism are regarded as the important substitution antimicrobial agents of antibiotics. We isolated a new strain, Bacillus sp. GFP-2, from the Chiloscyllium plagiosum (Whitespotted bamboo shark) intestine, which showed great inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria. Additionally, the growth of salmon was effectively promoted when fed with inactivated strain GFP-2 as the inhibition agent of pathogenic bacteria. The genes encoding antimicrobial peptides like LCI, YFGAP and hGAPDH and gene clusters for secondary metabolites and bacteriocins, such as difficidin, bacillibactin, bacilysin, surfactin, butirosin, macrolactin, bacillaene, fengycin, lanthipeptides and LCI, were predicted in the genome of Bacillus sp. GFP-2, which might be expressed and contribute to the antimicrobial activities of this strain. The gene encoding ß-1,3-1,4-glucanase was successfully cloned from the genome and this protein was detected in the culture supernatant of Bacillus sp. GFP-2 by the antibody produced in rabbit immunized with the recombinant ß-1,3-1,4-glucanase, indicating that this strain could express ß-1,3-1,4-glucanase, which might partially contribute to its antimicrobial activities. This study can enhance a better understanding of the mechanism of antimicrobial activities in genus Bacillus and provide a useful material for the biotechnology study in antimicrobial agent development.

10.
J Chem Inf Model ; 58(5): 1066-1073, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29672052

RESUMO

Protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme, is reported as an important therapeutic target in leukemia and lymphoma. In the present study, based on the combination of virtual screening and biochemical validations, we discovered a series of small-molecule inhibitors targeting PRMT5. Among those, DC_Y134 exhibited the most potent activity with IC50 value of 1.7 µM and displayed good selectivity against other methyltransferases. Further treatment with DC_Y134 inhibited the proliferation of several hematological malignancy cell lines by causing cell cycle arrest and apoptosis. Western blot assays indicated that DC_Y134 reduced the cellular symmetrically dimethylated levels. In addition, we analyzed the binding mode of DC_Y134 through molecular docking, which revealed that DC_Y134 occupies the binding site of substrate arginine and explained the selectivity of this inhibitor. Taken together, compound DC_Y134 could be used to elucidate the biological roles of PRMT5 and serve as a lead compound for treatment of hematologic malignancies.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Interface Usuário-Computador
11.
Fish Shellfish Immunol ; 78: 238-247, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29678793

RESUMO

Galectins are ß-galactoside binding lectins that play crucial roles in innate immunity in vertebrates and invertebrates through their conserved carbohydrate-recognition domains (CRDs). In the present study, single- and four-CRD-containing galectins were identified in oyster Crassostrea gigas (designated CgGal-2 and CgGal-3). The open reading frames (ORFs) of CgGal-2 and CgGal-3 encode polypeptides of 200 and 555 amino acids, respectively. All CRDs of CgGal-3 include two consensus motifs essential for ligand-binding, and a novel motif is present in CgGal-2. Pathogen-associated molecular pattern (PAMP) profiles were determined for recombinant rCgGal-2 and rCgGal-3, and rCgGal-2 displayed low binding affinity for PAMPs, while rCgGal-3 bound various PAMPs including glucan, lipopolysaccharide (LPS), and peptidoglycan (PGN) with relatively high affinity. Furthermore, rCgGal-2 and rCgGal-3 exhibited different microbe binding profiles; rCgGal-2 bound to Gram-negative bacteria (Escherichia coli and Vibrio vulnificus) and fungi (Saccharomyces cerevisiae and Pichia pastoris), while rCgGal-3 bound to these microbes but also to Gram-positive bacteria (Micrococcus luteus). In addition, rCgGal-3 possessed microbial agglutinating activity and coagulation activity against fungi and erythrocytes, respectively, but rCgGal-2 lacked any agglutinating activity. Carbohydrate binding specificity analysis showed that rCgGal-3 specifically bound D-galactose. Furthermore, rCgGal-2 and rCgGal-3 functioned as opsonin participating in the clearance against invaders in C. gigas. Thus, CgGal-2 with one CRD and CgGal-3 with four CRDs are new members of the galectin family involved in immune responses against bacterial infection. Differences in the organisation and amino acid sequences of CRDs may affect their specificity and affinity for nonself substances.


Assuntos
Crassostrea/genética , Galectina 2/genética , Galectina 3/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Crassostrea/imunologia , Fungos/fisiologia , Galectina 2/química , Galectina 2/imunologia , Galectina 3/química , Galectina 3/imunologia , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Filogenia , Alinhamento de Sequência
12.
Int J Syst Evol Microbiol ; 67(10): 4024-4031, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28893367

RESUMO

A Gram-staining-negative, aerobic, non-spore-forming, coccoid to rod shaped bacteria with prosthecate and flagellum, designated as HSF6T, was isolated from deep seawater samples collected from the South China Sea at depth of 2.5 km and subjected to a polyphasic taxonomic investigation. Colonies of strain HSF6T were 1-2 mm in diameter, smooth, circular, convex and yellow. Strain HSF6T was found to grow at 15-37 °C (optimum, 25-35 °C), pH 5.0-9.5 (optimum, pH 7.0-7.5) and with 0-8 % (w/v) NaCl (optimum, 2 %). Chemotaxonomic analysis showed the predominant respiratory quinone of strains HSF6T were ubiquinone-10, and the major fatty acids were C18 : 1ω7c, C16 : 0 and 11-methyl C18 : 1ω7c. The polar lipids were monoglycosyldiglyceride (MGDG), sulfo-quinovosyl diacylglycerol (SQDG), three unknown glycolipids (GL1-3) and five unknown lipids (L1-5). The DNA G+C content of strain HSF6T was determined to be 51.0 mol% with HPLC. The comparison of 16S rRNA gene sequence similarities show that strain HSF6T was related most closely to genus Parvularcula with similarity ranging from 91.0 to 91.8 %. The phylogenetic trees, using the 16S rRNA gene sequence, reconstructed with neighbour-joining, maximum-parsimony and maximum-likelihood methods showed that strain HSF6T constituted a separated branch in the family 'Parvularculaceae'. Differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain HSF6T is clearly distinct from validly published genera. On the basis of these features, we propose strain HSF6T (=MCCC 1K03223T=KCTC 52486T) represents a novel species of a novel genus with the name Hyphococcus flavus gen. nov., sp. nov.


Assuntos
Alphaproteobacteria/classificação , Filogenia , Água do Mar/microbiologia , Alphaproteobacteria/genética , Alphaproteobacteria/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química
13.
Fish Shellfish Immunol ; 70: 398-407, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28916358

RESUMO

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads.


Assuntos
Proteínas de Artrópodes/genética , Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Lectinas Tipo C/genética , Aglutinação , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Fungos/fisiologia , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Filogenia , Alinhamento de Sequência
14.
Artigo em Inglês | MEDLINE | ID: mdl-28822868

RESUMO

Liver is a vital organ present in animals for detoxification, protein synthesis, digestion and other functions and its powerful regenerative capacity is well known. C. plagiosum is an abundant fish that is representative of the cartilaginous class in the southeast coastal region of China and its liver accounts for >70% of the fish's visceral weight and contains many bioactive substances. MicroRNAs (microRNAs) play important roles in a wide range of biological processes in eukaryotes, including cell proliferation, differentiation, apoptosis. However, microRNAs in response to liver regeneration has not been well studied. This study aimed to identify the microRNAs that participate in liver regeneration and other liver-related diseases and to improve our understanding of the mechanisms of liver regeneration in sharks. To this end, normal and regenerating liver tissues from C. plagiosum were harvested 0, 3, 6, 12 and 24h after partial hepatectomy (pH) and were sequenced using the Illumina/Solexa platform. In total, 309 known microRNAs and 590 novel microRNAs were identified in C. plagiosum. There were many microRNAs differentially expressed in the normal and regenerating livers between time points. Using target prediction and GO analysis, most of the differentially expressed microRNAs were assigned to functional categories that may be involved in regulating liver regeneration, such as cell proliferation, differentiation and apoptosis. The microRNA expression profile of liver regeneration will pave the way for the development of effective strategies to fight against liver disease and other related disease.


Assuntos
Regeneração Hepática/genética , Fígado/metabolismo , MicroRNAs/metabolismo , Tubarões/genética , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Fígado/fisiologia , Regeneração Hepática/fisiologia , MicroRNAs/análise , MicroRNAs/genética , Tubarões/metabolismo
15.
Chem Biol Drug Des ; 90(6): 1260-1270, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28636189

RESUMO

Protein arginine methylation, a post-translational modification critical for a variety of biological processes, is catalyzed by protein arginine N-methyltransferases (PRMTs). In particular, PRMT1 is responsible for over 85% of the arginine methylation in mammalian cells. Dysregulation of PRMT1 is involved in diverse pathological diseases including cancers. However, most current PRMT1 inhibitors are lack of specificity, efficacy, and bioavailability. Herein, a series of alkyl bis(oxy)dibenzimidamide derivatives were identified as selective PRMT1 inhibitors. Among them, the most potent compound corresponds to hexamidine (IC50  = 5.9 ± 1.7 µm), which is an antimicrobial agent. The binding between hexamidine and PRMT1 was further validated by thermal shift assays and nuclear magnetic resonance (NMR) experiments. Molecular docking and NMR assays indicated that hexamidine occupied the substrate binding pocket. Furthermore, hexamidine effectively blocked cell proliferation in cancer cell lines related to PRMT1 overexpression. Taken together, this study has provided a druggable scaffold targeting PRMT1 as well as a new way to repurpose old drugs which is a complementary tool for the discovery of new lead compounds.


Assuntos
Amidas/química , Inibidores Enzimáticos/química , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Amidas/metabolismo , Amidas/toxicidade , Benzamidinas/química , Benzamidinas/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Transferência Ressonante de Energia de Fluorescência , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
16.
Protein Sci ; 26(4): 834-846, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28168758

RESUMO

TBC1D15 belongs to the TBC (Tre-2/Bub2/Cdc16) domain family and functions as a GTPase-activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark-TBC1D15 and Sus-TBC1D15 belong to the same subfamily of TBC domain-containing proteins, and their GAP-domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost.


Assuntos
Proteínas de Peixes/química , Proteínas Ativadoras de GTPase/química , Guanosina Trifosfato/química , Complexos Multiproteicos/química , Proteínas rab de Ligação ao GTP/química , Animais , Cristalografia por Raios X , Proteínas de Peixes/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Complexos Multiproteicos/metabolismo , Estrutura Quaternária de Proteína , Tubarões , Suínos , Proteínas rab de Ligação ao GTP/metabolismo
17.
BMC Genomics ; 18(1): 201, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28231766

RESUMO

BACKGROUND: A transposable element (TE) is a DNA fragment that can change its position within a genome. Transposable elements play important roles in maintaining the stability and diversity of organisms by transposition. Recent studies have shown that approximately half of the genes in Bombyx mori are TEs. RESULTS: We systematically identified and analyzed the BmAGO2-associated TEs, which exceed 100 in the B. mori genome. Additionally, we also mapped the small RNAs associated with BmAGO2 in B.mori. The transposon Bm1645 is the most abundant TE associated with BmAGO2, and Bm1645-derived small RNAs represent a small RNA pool. We determined the expression patterns of several Bm1645-derived small RNAs by northern blotting, and the results showed there was differential expression of multiple small RNAs in normal and BmNPV-infected BmN cells and silkworms from various developmental stages. We confirmed that four TE-siRNAs could bind to BmAGO2 using EMSA and also validated the recognition sites of these four TE-siRNAs in Bm1645 by dual-luciferase reporter assays. Furthermore, qRT-PCR analysis revealed the overexpression of the four TE-siRNAs could downregulate the expression of Bm1645 in BmN cells, and the transcription of Bm1645 was upregulated by the downregulation of BmAGO2. CONCLUSIONS: Our results suggest Bm1645 functions as a source of small RNAs pool and this pool can produce many BmAGO2-associated small RNAs that regulate TE's expression.


Assuntos
Proteínas Argonauta/genética , Bombyx/genética , Elementos de DNA Transponíveis , Regulação da Expressão Gênica , Pequeno RNA não Traduzido/genética , Animais , Bombyx/virologia , Mapeamento Cromossômico , Regulação para Baixo , Família Multigênica , Interferência de RNA , Reprodutibilidade dos Testes
18.
Int J Syst Evol Microbiol ; 67(5): 1169-1176, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28068219

RESUMO

A Gram-stain-negative, aerobic, non-spore-forming, non-motile, oval to rod-shaped, prosthecate bacterium, designated strain WM6T, was isolated from a seawater sample collected from the South China Sea at a depth of 150 m and subjected to a polyphasic taxonomic investigation. Cells of strain WM6T were approximately 0.5-0.6 µm in width and 0.8-1.2 µm in length, and colonies were smooth, circular, convex and whitish yellow. Strain WM6T was found to grow at 10-45 °C (optimum, 30 °C), at pH 6.5-9.0 (optimum, pH 7.5-8.5) and with 1-6 % (w/v) NaCl (optimum, 1-2 %). Chemotaxonomic analysis showed the predominant respiratory quinone and the major fatty acid of strains WM6T were ubiquinone-10 and C18 : 1ω7c, respectively. The polar lipids of strain WM6T were phosphatidylglycerol, glucuronopyranosyldiglyceride, monoglycosyldiglyceride, sulfo-quinovosyl diacylglycerol, seven unknown glycolipids and two unknown lipids. The DNA G+C content of strain WM6T was determined to be 59.8 mol% by HPLC. 16S rRNA gene sequence similarities showed that strain WM6T was related most closely to the genus Maricaulis with a similarity range from 92.3 to 93.8 %. Phylogenetic trees reconstructed with the neighbour-joining and maximum-likelihood methods using mega and maximum-likelihood methods using arb showed that strain WM6T constituted a separated branch in the family Hyphomonadaceae. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain WM6T is clearly distinct from any validly published genus. On the basis of these features, strain WM6T represents a novel species of a new genus with the name Hyphobacterium vulgare gen. nov., sp. nov. The type strain of Hyphobacterium vulgare is WM6T (=MCCC 1K03222T=KCTC 52487T).


Assuntos
Alphaproteobacteria/classificação , Filogenia , Água do Mar/microbiologia , Alphaproteobacteria/genética , Alphaproteobacteria/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , China , DNA Bacteriano/genética , Ácidos Graxos/química , Glicolipídeos/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química
19.
Fish Physiol Biochem ; 43(3): 791-802, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28054243

RESUMO

Liver regeneration is a complicated process, and understanding the regulatory mechanism will be helpful in the treatment of diseases associated with liver. In this study, the one-third liver resection model was established in Chiloscyllium plagiosum, and the whole transcriptome of the C. plagiosum was generated using the Illumina-Solexa sequencing platform. Differentially expressed genes were analyzed using bioinformatics methods and verified using quantitative real-time PCR (qRT-PCR). Using miRanda and TargetScan, we screened the microRNA library for miRNAs that target the glutathione S-transferase P1(GSTP1) gene. Dual-luciferase reporter assays were used to confirm binding between the miRNA and GSTP1. Finally, we used western blotting analysis to determine expression of the GSTP1 protein. As a result, 65,356 unigenes were obtained in normal and damaged liver tissues, with mean length of 955 bp. A total of 359 differentially expressed genes were acquired; 217 of which were upregulated, and 142 were downregulated, including the GSTP1 gene, following liver resection. The presence of the GSTP1 protein in C. plagiosum was shown for the first time. Luciferase reporter assay revealed that GSTP1 messenger RNA was targeted by ipu-miR-143. The discovery and differential expression analysis of GSTP1 in C. plagiosum will be a valuable resource to explain the molecular mechanism of GSTP1 regulation of liver repair.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Glutationa Transferase/metabolismo , Regeneração Hepática/fisiologia , Fígado/metabolismo , MicroRNAs/metabolismo , Tubarões/fisiologia , Regiões 3' não Traduzidas , Animais , DNA/genética , Glutationa Transferase/genética , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
20.
J Insect Physiol ; 91-92: 56-62, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27374983

RESUMO

Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated-particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H2O2-treated BmN cells and suppressing the apoptosis induced by H2O2. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm.


Assuntos
Bombyx/metabolismo , Proteínas de Insetos/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Western Blotting , Bombyx/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Larva/crescimento & desenvolvimento , Larva/metabolismo , Espectrometria de Massas em Tandem
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