RESUMO
Coal-based porous materials for supercapacitors were successfully prepared using Taixi anthracite (TXA) by multi-stage activation. The characterization and electrochemical tests of activated carbons (ACs) prepared in different stages demonstrated that the AC from the third-stage activation (ACIII) shows good porous structures and excellent electrochemical performances. ACIII exhibited a fine specific capacitance of 199 F g-1 at a current density of 1 A g-1 in the three-electrode system, with 6 mol L-1 KOH as the electrolyte. The specific capacitance of ACIII remained 190 F g-1 even despite increasing the current density to 5 A g-1, indicating a good rate of electrochemical performance. Moreover, its specific capacitance remained at 98.1% of the initial value after 5000 galvanostatic charge-discharge (GCD) cycle tests at a current density of 1 A g-1, suggesting that the ACIII has excellent cycle performance as electrode materials for supercapacitors. This study provides a promising approach for fabricating high performance electrode materials from high-rank coals, which could facilitate efficient and clean utilization of high-rank coals.
Assuntos
Carvão Vegetal/síntese química , Carvão Mineral/análise , Carvão Vegetal/química , Capacitância Elétrica , Eletroquímica/instrumentação , Eletrodos , Microscopia de Força Atômica , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
Antibacterial peptide CM4 (ABP-CM4) is a natural product isolated from the silkworm Bombyx mori. It is a small cationic peptide with broad-spectrum activities against harmful microorganisms and may be used as a novel food preservative. However, ABP-CM4 lacks tertiary structure in water-like solutions, which makes it more susceptible to proteases and labile when exposed to air. In this study, ß-cyclodextrin (ß-CD) was chosen to form an inclusion complex with ABP-CM4, which enhanced the physical and chemical properties of ABP-CM4 but did not decrease its antibacterial activity. The storage stability and susceptibility to proteinases of ABP-CM4 were apparently improved under the protection of ß-CD. This technology could also be widely applied to other AMPs as an antimicrobial system to be used in the food industry.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Frutas/efeitos dos fármacos , beta-Ciclodextrinas/química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Frutas/microbiologiaRESUMO
Objective: To investigate the correlation of the single nucleotide polymorphismsï¼SNPsï¼ rs1799930 and rs1799931 of the N-acetyltransferase 2 geneï¼ NAT2ï¼ with the risk of male infertility in Nanjing area. Methods: We made a case-control study of 636 cases of male idiopathic infertility and 442 normal fertile men as controls. We genotyped the two SNPs by Sequenom Mass Array, analyzed the correlation of different genotypes with male infertility using the logistic regression model, and determined the association of the linkage effect of the two SNPs with male infertility by haplotype analysis. Results: Statistically significant differences were found between the case and control groups in sperm concentrationï¼[32. 32 ± 45. 49] vs [72. 77 ± 45. 21] × 106/ ml, P < 0. 01ï¼,the percentage of progressively motile spermï¼[15. 29 ± 5. 06] vs [42. 02 ± 9. 04]%,P < 0. 01ï¼,and the level of follicle-stimulating hormoneï¼[14. 69 ± 12. 37] vs [4. 72 ± 2. 51] U / L,P < 0. 01), but not in other parameters. No correlation was observed between the frequencies of the two SNPs or alleles in different models and male infertility. Haplotype analysis suggested a linkage effect within rs1799930 and rs1799931ï¼D' = 0. 998,r2= 0. 05ï¼ but no evident correlation between male infertility and genotype combination. Conclusion: The SNPs rs1799930 and rs1799931 of the NAT2 gene were not found to be correlated with the risk of idiopathic infertility in men.
Assuntos
Arilamina N-Acetiltransferase/genética , Infertilidade Masculina/genética , Polimorfismo de Nucleotídeo Único , Alelos , Estudos de Casos e Controles , Genótipo , Haplótipos , Humanos , Modelos Logísticos , MasculinoRESUMO
A proliferation-inducing ligand (APRIL) is a critical member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the cDNA of cat APRIL (cAPRIL) was successfully amplified. Sequence analysis showed that the open reading frame (ORF) of cAPRIL contains a putative furin protease cleavage site (R-R-K-R), a conserved putative N-glycosylation site (Asn(124)), and two conservative cysteine residues (Cys(196) and Cys(211)). Real-time quantitative PCR (qPCR) analysis revealed that cAPRIL could be detected in various tissues. The phylogenetic analysis and predicted three dimensional (3D) structure revealed that it is similar to its counterparts. The extracellular soluble domain of the cAPRIL (csAPRIL) fragment was cloned into the expression vector pET43.1a. SDS-PAGE and Western blotting analysis indicated a high-level expression of csAPRIL protein in Escherichia coli BL21 (DE3). MTT assays revealed that purified recombinant csAPRIL protein was able to stimulate proliferation of mouse B-cells. These findings indicate that cAPRIL plays an important role in proliferation of B-cells and provide the basis for investigation on the roles of APRIL in this important domestic species.
Assuntos
Gatos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/genética , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/farmacologiaRESUMO
B-cell activating factor (BAFF) from the TNF family is critical for B-cell survival and maturation. In this study, we identified a Yangtze alligator (Alligator sinensis, Alligatoridae) BAFF cDNA, designated as asBAFF, using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The open reading frame of this cDNA encodes a 287-amino acid protein containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian and avian BAFF. The amino acid identity between biologically soluble asBAFF (assBAFF) and csBAFF, hsBAFF, and msBAFF is 94, 76, and 71%, respectively. Real-time quantitative PCR analysis showed that the asBAFF gene is strongly expressed in the spleen. Since BAFF is always expressed as inclusion bodies in bacteria, it is difficult to purify. To enhance the soluble expression of assBAFF in Escherichia coli, we fused the extracellular region of the asBAFF gene to a small ubiquitin-related modifier gene (SUMO). Purified assBAFF was able to promote the survival of splenic lymphocytes and co-stimulate the proliferation of mouse B cells with anti-mouse IgM. These findings suggest that asBAFF plays an important role in the survival and proliferation of Yangtze alligator B cells, and because it is evolutionarily highly conserved, functional cross-reactivity exists between mammalian and Yangtze alligator BAFF.
Assuntos
Jacarés e Crocodilos/genética , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Jacarés e Crocodilos/classificação , Sequência de Aminoácidos , Animais , Fator Ativador de Células B/química , Sequência de Bases , Proliferação de Células/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Linfócitos/citologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Baço/citologiaRESUMO
The interferon-γ-inducible lysosomal thiol reductase (GILT) has been demonstrated to play an important role in the processing and presentation of MHC class II restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, we cloned a GILT gene homolog from goldfish (designated gGILT), a kind of precious freshwater fish with high market value. The open reading frame of gGILT consists of 756 bases encoding a protein of 251 amino acids with an estimated molecular mass of 27.8 kDa and a theoretical isoelectric point of 5.24. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 10 conserved cysteines. RT-PCR results showed that gGILT and gIFN-γ (goldfish IFN-γ) mRNA were expressed in a tissue-specific manner and obviously up-regulated in splenocytes and the cells from head kidney after induction with LPS. Recombinant gGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Further study revealed that gGILT was capable of catalyzing the reduction of the interchain disulfide bonds from intact IgG. This study shows that gGILT may be involved in the immune response to bacteria challenge and maintain first line of innate immune defense at basal level in goldfish. It also provides the basis for investigating on the role of GILT using goldfish as an animal model.
Assuntos
Proteínas de Peixes/genética , Carpa Dourada/genética , Carpa Dourada/imunologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/metabolismo , Carpa Dourada/metabolismo , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de SequênciaRESUMO
B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation and immunoglobulin secretion. In this study, the yellow grouper (Epinephelus awoara) BAFF (designated EaBAFF) gene was cloned using RT-PCR and RACE (rapid amplification of cDNA ends) techniques. The full-length EaBAFF was 1442bp and contained an open reading frame of 780bp encoding a putative protein of 259 amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. The soluble BAFF (EasBAFF) had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-EasBAFF was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, the WST-8 assay indicated that EasBAFF was not only able to promote the survival/proliferation of yellow grouper splenic lymphocytes but also able to promote the survival/proliferation of mouse splenic B cells. Our findings may provide valuable information for research into the immune system of E. awoara and EasBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in fish.
Assuntos
Fator Ativador de Células B/genética , Proteínas de Peixes/genética , Expressão Gênica , Perciformes/genética , Sequência de Aminoácidos , Animais , Fator Ativador de Células B/classificação , Fator Ativador de Células B/metabolismo , Linfócitos B/metabolismo , Western Blotting , Clonagem Molecular , Proteínas de Peixes/metabolismo , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Perciformes/metabolismo , Filogenia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Baço/citologia , TranscriptomaRESUMO
APRIL is a member of the tumor necrosis factor (TNF) family of ligands that mediate tumor cells proliferation as well as survival, depending on the cellular context. In this report, we present a novel method to obtain soluble human APRIL in Escherichia coli using the elastin-like polypeptide and SUMO (ELP-SUMO) tags. The fusion protein with ELP-SUMO tag was expressed in a soluble form at 15°C. After purification based on inverse transition cycling (ITC) method, the purified ELP-SUMO-hAPRIL fusion protein was subsequently cleaved by SUMO protease to release mature hAPRIL. Following affinity chromatography, the target protein was re-purified with high purity. Finally, about 4.8mg recombinant hAPRIL was obtained from 1l bacterial culture with no less than 85% purity. The molecular mass (Mr) of the recombinant hAPRIL was confirmed by MALDI-TOF MS as Mr 16,314. The purified hAPRIL exhibits biological activity on Jurkat cells. It is the first report on soluble production of hAPRIL in E. coli using ELP-SUMO tag.
Assuntos
Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/isolamento & purificação , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Clonagem Molecular , Elastina/química , Elastina/genética , Elastina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Células Jurkat , Espectrometria de Massas , Peptídeos/química , Peptídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteína SUMO-1/química , Proteína SUMO-1/genética , Solubilidade , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Antibacterial peptides (ABPs) with cancer-selective toxicity have received much more attention as alternative chemotherapeutic agents in recent years. However, the basis of their anticancer activity remains unclear. The modification of cell surface glycosylation is a characteristic of cancer cells. The present study investigated the effect of glycosylation, in particular sialic acid, on the anticancer activity of ABPs. We showed that aurein 1.2, buforin IIb and BMAP-28m exhibited selective cytotoxicity toward MX-1 and MCF-7 breast cancer cells. The binding activity, cytotoxicity and apoptotic activity of ABPs were enhanced by the presence of O-, N-glycoproteins, gangliosides and sialic acid on the surface of breast cancer cells. Among N-, O-glycoproteins and ganglioside, O-glycoproteins almost had the strongest effect on the binding and cytotoxicity of the three peptides. Further, up-regulation of hST6Gal1 in CHO-K1 cells enhanced the susceptibility of cells to these peptides. Finally, the growth of MX-1 xenograft tumors in mice was significantly suppressed by buforin IIb treatment, which was associated with induction of apoptosis and inhibition of vascularization. These data demonstrate that the three peptides bind to breast cancer cells via an interaction with surface O-, N-glycoproteins and gangliosides. Sialic acids act as key glycan binding sites for cationic ABP binding to glycoproteins and gangliosides. Therefore, glycosylation in breast cancer cells plays an important role in the anticancer activity of ABPs, which may partly explain their cancer-selective toxicity. Anticancer ABPs with cancer-selective cytotoxicity will be promising candidates for anticancer therapy in the future.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Peptídeos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células CHO/efeitos dos fármacos , Cricetulus , Feminino , Gangliosídeos/metabolismo , Glicoproteínas/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Células MCF-7/efeitos dos fármacos , Camundongos , Camundongos Nus , Ácido N-Acetilneuramínico/metabolismo , Peptídeos/metabolismo , Proteínas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
TNFSF13 is one of the tumor necrosis factor (TNF) superfamily members that plays important roles in immune homeostasis and proliferation or apoptosis of certain tumor cell lines. This report describes the development of Xenopus laevis TNFSF13 as a model to study its important role in relation to immunological diseases. In brief, TNFSF13 from Xenopus laevis (designated XlTNFSF13) was first amplified by RT-PCR and rapid amplification of cDNA end (RACE) techniques. Bioinformatics analyses revealed the gene structure, three-dimensional structure, and evolutionary relationships. Real-time quantitative PCR (QPCR) analysis identified the tissue distribution of XlTNFSF13 in the major visceral organs. The recombinant plasmid SUMO-XsTNFSF13 was expressed in E. coli Rosseta (DE3). Subsequently, the recombinant protein purified through Ni-NTA affinity chromatography was analyzed by SDS-PAGE and confirmed by Western blot analysis. Laser scanning confocal microscopy analysis revealed the binding activity of pSUMO-XsTNFSF13 to the surface of B cells. WST-8 assays further indicated that purified XsTNFSF13 could cause the survival/proliferation of B cells. In conclusion, we underscore that as a model organism for human disease, Xenopus laevis has been widely used in molecular biology research. Yet while TNFSF13 research in mammalian, fish (e.g., zebrafish), mouse, and human is widely available, studies in the amphibian species are limited. The latter area of OMICS and integrative biology scholarship is directly informed with the present study, with a view to implications for the future study of human immunological diseases.
Assuntos
Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Doenças do Sistema Imunitário/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologia , Sequência de Aminoácidos , Animais , Fator Ativador de Células B/biossíntese , Linfócitos B/imunologia , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Clonagem Molecular/métodos , Biologia Computacional/métodos , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Doenças do Sistema Imunitário/imunologia , Camundongos , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Distribuição Tecidual , Proteínas de Xenopus/biossíntese , Xenopus laevisRESUMO
The tumor necrosis factor superfamily (TNFSF) proteins are cytokines involved in many biological processes. In this study, the TNF superfamily member 14 (TNFSF14, LIGHT) has been isolated from zebrafish Danio rerio (designated zLIGHT). The full-length open reading frame (ORF) of zLIGHT cDNA consists of 708 bp encoding a protein of 235 amino acids. The zLIGHT open reading frame (ORF) genomic sequence consists of three introns and four exons, is about 9.9 kb in size. Real-time quantitative PCR (qPCR) analysis suggested that zLIGHT was predominantly expressed in zebrafish spleen. The soluble LIGHT (zsLIGHT) had been cloned into the pSUMO vector, SDS-PAGE and Western blotting analysis confirmed that the recombinant protein SUMO-zsLIGHT was efficiently expressed in Escherichia coli BL21 (DE3). Laser scanning confocal microscopy analysis showed that SUMO-zsLIGHT could bind to its receptors on T cells. LIGHT is involved in many important biological effects, including up-regulating proinflammatory chemokines, cytokines, inducing cell death, apoptosis, and enhancing T cell survival. Zebrafish may conduct as a model animal for further research on LIGHT.
Assuntos
Regulação da Expressão Gênica/fisiologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Peixe-ZebraRESUMO
In mammals, interferon-γ-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. Here, we reported the cloning of a GILT gene homologue from zebrafish (zGILT), a tropical freshwater fish. The full-length cDNA of zGILT gene is 768 nucleotides (nt) encoding a protein of 255 amino acids (aa), with a putative molecular weight of 28.33 kDa. The deduced protein is highly homologous to that of fish and mammalian GILTs and shares 57.1% sequence identity to that of Atlantic salmon and 55.7-21.6% sequence identity to that of various mammals. The deduced protein possesses all the main features characteristic of known GILT proteins including the signature sequence CQHGX2ECX2NX4C spanning residues 117-132, CXXC motif at residues 72-75, one potential sites for N-linked glycosylation at residual positions 54. The zGILT expression is obviously up-regulated in spleen and kidney after immunization with LPS although it also is constitutively expressed in heart, liver, muscle and intestine, suggesting that zGILT may be involved in the immune response to bacterial challenge. The soluble recombinant protein was successfully purified using Ni-nitrilotriacetic acid resin. Recombinant His-zsGILT appeared on SDS-PAGE in the ranges of their estimated size of 18.94-kDa. After purification, further study revealed that zsGILT was capable of catalyzing the reduction of the interchain disulfide bonds intact IgG. These results will allow for further investigation to unravel the role of this key enzyme in class II MHC-restricted antigen processing and to use zebrafish as an in vivo model for related studies.
Assuntos
Apresentação de Antígeno/imunologia , Regulação da Expressão Gênica/imunologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Análise de Variância , Animais , Apresentação de Antígeno/genética , Sequência de Bases , Western Blotting , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica/genética , Imunoglobulina G/metabolismo , Rim/metabolismo , Lipopolissacarídeos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da Espécie , Baço/metabolismo , Peixe-Zebra/imunologiaRESUMO
Here we describe the identification of the hedgehog Erinaceus europaeus homologue of a proliferation-inducing ligand (APRIL) of the TNF family (designated heAPRIL). Hedgehog APRIL contains two cysteine residues (Cys(196) and Cys(211)), a furin protease cleavage site and a conserved putative N-glycosylation site (Asn(124)). Real-time quantitative PCR (qPCR) analysis revealed that heAPRIL could be detected in various tissues. MTT assays and flow cytometric analysis revealed that Nus-hesAPRIL and hesAPRIL could promote the survival/proliferation of splenic B cells. Laser scanning confocal microscopy analysis showed GFP-hesAPRIL could successfully bind to the APRIL receptors of lymphocytes.
Assuntos
Regulação da Expressão Gênica , Ouriços/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Aminoácidos/metabolismo , Animais , Linfócitos B/citologia , Proliferação de Células , Sobrevivência Celular , Clonagem Molecular , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Baço/citologia , Homologia Estrutural de Proteína , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismoRESUMO
To investigate the effects of human anti-BAFF scFv-Fc against the hsBAFF, ICR mice were randomly divided into six groups: control, hsBAFF (1 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + Ab (1 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + Ab (2 mg x kg(-1)), hsBAFF (1 mg x kg(-1)) + human IgG (1 mg x kg(-1)) and hsBAFF (1 mg x kg(-1)) + human IgG (2 mg x kg(-1)) groups. The effects of scFv-Fc administration on the proliferation of B lymphocytes were evaluated using an MTT assay. The titres of antibody in the serum and B lymphocytes differentiation were assessed by ELISA and flow cytometry, respectively. The results showed that administration of scFv-Fc to mice injected with hsBAFF significantly prevented human BAFF-induced increases in splenic B cell numbers and serum immunoglobulin levels. Furthermore, this fully human antibody would avoid inducing the human anti-mouse antibody (HAMA) response when used in humans. These findings suggest that the compact antibody may be useful in therapeutic or diagnostic application of the BAFF-associated autoimmune diseases in human.
Assuntos
Fator Ativador de Células B/imunologia , Proliferação de Células , Fragmentos Fc das Imunoglobulinas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Fator Ativador de Células B/metabolismo , Linfócitos B/citologia , Peso Corporal , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Camundongos , Camundongos Endogâmicos ICR , Distribuição Aleatória , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/metabolismo , Baço/citologiaRESUMO
In the present study, the cDNA of Père David's deer APRIL (miAPRIL) was successfully amplified, and nucleotide sequence analysis showed that the open reading frame (ORF) of miAPRIL consists of 753 bases encoding 250-amino acids. Phylogenetic analysis exhibited high homology with the even-toed ungulates (artiodactyla). The extracellular soluble domain of the miAPRIL (misAPRIL) fragment was cloned into the expression vector pET43.1a and transformed into Escherichia coli BL21 (DE3) for recombinant expression. SDS-PAGE and western blotting analysis indicated that misAPRIL protein expression was successfully achieved. Indirect immunofluorescence staining demonstrated that misAPRIL has the ability to bind to the surface of Père David's deer lymphocytes. Flow cytometry analysis revealed that misAPRIL was able to enhance lymphocytes survival in a dose-dependent manner.
Assuntos
Cervos/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Animais , Clonagem Molecular , DNA Complementar/genética , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Linfócitos/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/biossínteseRESUMO
The increasing resistance of bacteria and fungi to currently available antibiotics is a major concern worldwide, leading to enormous efforts to develop new antibiotics with new modes of actions. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as a promising candidate for a new antibiotic. For pharmaceutical applications, a large quantity of antimicrobial peptides needs to be produced economically. In this communication, the progress in the structural characteristics, heterologous production, and biological evaluation of ABP-CM4 are reviewed.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Fungos/efeitos dos fármacos , Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Pichia/genética , Pichia/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the Japanese sea perch (Lateolabrax japonicus) homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS) (designated LjBAFF). The cDNA contains an open reading frame (ORF) of 783 nucleotides that are translated into a predicted 260 amino acid protein. Like most known BAFFs, Japanese sea perch BAFF contains three cysteine residues (Cys(123), Cys(218) and Cys(232)) which are conserved in the aligned BAFF sequences and a furin protease cleavage site (R-K-K-R). Real-time quantitative PCR (qPCR) analysis revealed that LjBAFF could be detected in various tissues and predominantly expressed in lymphoid tissue spleen. The soluble BAFF (LjsBAFF) had been cloned into a pET28a vector to express the recombinant protein. The His-LjsBAFF was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. After purification, MTT assays and flow cytometric analysis revealed that LjsBAFF could promote the survival/proliferation of splenic B cells in vitro. Furthermore, bacterially expressed LjsBAFF induced the selective expansion of B cells in the spleen when administered to young mice. Our results suggest that like its mammalian counterparts, LjsBAFF plays an important role in the survival and/or proliferation of B cells.
Assuntos
Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Percas/genética , Sequência de Aminoácidos , Animais , Fator Ativador de Células B/farmacologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Expressão Gênica , Contagem de Linfócitos , Camundongos , Dados de Sequência Molecular , Percas/imunologia , Filogenia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Distribuição TecidualRESUMO
Bone morphogenetic proteins (BMPs) are cytokines from the TGF-ß superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of human BMP-14. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography. After the SUMO-BMP14 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 45 mg recombinant hBMP-14 was obtained from 1 litre bacterial culture with no less than 95% purity. The purified hBMP-14 dimer was over 90% purity and could induce the expression of alkaline phosphatase activity in C2C12 cells in a dose-dependent manner. Thus the SUMO-mediated peptide expression and purification system potentially could be employed for the production of other homodimeric proteins.
Assuntos
Escherichia coli/genética , Expressão Gênica , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Escherichia coli/metabolismo , Fator 5 de Diferenciação de Crescimento/isolamento & purificação , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/isolamento & purificação , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
In this study, an interferon-γ-inducible-lysosomal thiol reductase (GILT) homologue has been cloned and identified from South African clawed frog Xenopus laevis (designated XlGILT). The open reading frame (ORF) of XlGILT consists of 771 bases encoding a protein of 256 amino acids with an estimated molecular mass of 28.76kDa and a theoretical isoelectric point of 5.12. The N-terminus of the XlGILT was found to have a putative signal peptide with a cleavage site amino acid position at 15-16. SMART analysis showed that the XlGILT contained a GILT active-site C(69)GGC(72) motif and a GILT signature motif C(114)QHGKEECIGNLIETC(129). The expression levels of XlGILT mRNA were higher in spleen and peripheral blood mononuclear cells (PBMCs), moderate in liver, intestine, heart and kidney, and lower in lung. The XlGILT mRNA expression was significantly up-regulated in spleen in vivo and PBMCs in vitro after LPS stimulation. The soluble X. laevis GILT (XlsGILT) was inserted into a pET28a vector and expressed in BL21 (DE3) cells as a His-tag fusion enzyme. After purification, further study revealed that XlsGILT was capable of catalyzing the reduction of the interchain disulfide bonds intact IgG. These results will allow for further investigation to unravel the role of this key enzyme in class II MHC-restricted antigen processing and to use X. laevis as an in vivo model for related studies.
Assuntos
Expressão Gênica , Lisossomos/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Vetores Genéticos , Mucosa Intestinal/metabolismo , Rim/metabolismo , Leucócitos Mononucleares/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Lisossomos/genética , Dados de Sequência Molecular , Miocárdio/metabolismo , Fases de Leitura Aberta/genética , Oxirredutases/biossíntese , Oxirredutases/genética , Proteínas Recombinantes de Fusão/biossíntese , Análise de Sequência de DNA , Baço/metabolismoRESUMO
Full-length cDNA encoding TWEAK and Fn14 from bovine was isolated. We used bioinformatics to analyze the gene structure, function, evolutionary relationships, and predicted three-dimensional structure of proteins and binding sites. Real-time quantitative PCR (Q-PCR) analysis revealed that both TWEAK and Fn14 are constitutively expressed in various tissues in bovine. Bovine TWEAK and Fn14 interaction analysis by yeast two-hybrid. Our results suggest that the TWEAK-Fn14 pathway is evolutionarily highly conserved. It will be helpful for investigation on the biological role of the TWEAK/Fn14 system in this important animal model. Furthermore, it provides insight into the molecular evolution of the emerging TWEAK and Fn14 families.