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1.
Fish Shellfish Immunol ; 106: 876-886, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32800983

RESUMEN

Spiroplasma eriocheiris (S. eriocheiris) infection causes a significant economic loss in Penaeus vannamei (P. vannamei) culture industry. However, the response of P. vannamei hemocytes to S. eriocheiris infection has not been extensively studied. In this study, we conducted full-length transcriptome and long non-coding RNA (lncRNA) analyses of P. vannamei hemocytes by a challenge test with S. eriocheiris. Following assembly and annotation, there were 8077 high-quality unigenes. A total of 1168 differentially expressed genes (DEGs) were obtained, including 792 up-regulated and 376 down-regulated genes by differential expression analysis. Gene ontology (GO) enrichment analysis showed that the up-regulated DEGs were mainly clustered into immune system process, defense response, cell cycle and organelle organization. On the other hand, the down-regulated DEGs included that genes that were mainly clustered into metabolic processes related to organic compounds, metabolic process and cellular metabolic process. Protein-protein interaction (PPI) network analysis of DEGs indicated that the pivotal gene interactions were connected to stress response, immune system process and cell cycle. The lncRNA analysis identified multiple lncRNAs, which were highly co-expressed with the immune-related genes, such as lncRNA transcript-12631 and transcript-12631, suggesting that lncRNAs may be involved in the regulation of immune defense in shrimp hemocytes. Additionally, 20 hub unigenes and putative lncRNAs related to immune system were validated by quantitative real-time PCR (qRT-PCR), validating the reliability of RNA-Seq. This study revealed a close connection between the immune and metabolic systems of S. eriocheiris infected P. vannamei.


Asunto(s)
Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Penaeidae/genética , Penaeidae/inmunología , ARN Largo no Codificante/inmunología , Spiroplasma , Animales , Infecciones por Bacterias Gramnegativas/veterinaria , Hemocitos/inmunología , Penaeidae/microbiología , Transcriptoma
2.
Molecules ; 23(9)2018 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-30231526

RESUMEN

A previous study showed that intravenous immunoglobulin (IVIG) could preserve higher levels of biologically active lactone moieties of topotecan, 7-ethyl-10-hydroxycamptothecin (SN-38) and 10-hydroxycamptothecin at physiological pH 7.40. As one of camptothecin analogues (CPTs), the interaction of 7-ethylcamptothecin and IVIG was studied in vitro in this study. It was shown that the main binding mode of IVIG to 7-ethylcamptothecin was hydrophobic interaction and hydrogen bonding, which is a non-specific and spontaneous interaction. The hydrophobic antigen-binding cavity of IgG would enwrap the drug into a host-guest inclusion complex and prevent hydrolysis of the encapsulated drug, while the drug is adjacent to the chromophores of IgG and may exchange energy with chromophores and quench the fluorescence of the protein. Also, the typical ß-sheet structure of IVIG unfolded partially after binding to 7-ethylcamptothecin. Additionally, the binding properties of IVIG and six CPTs with different substituents at A-ring and/or B-ring including camptothecin, topotecan, irinotecan, 10-hydroxycamptothecin, 7-ethylcamptothecin and SN-38 were collected together and compared each other. Synergizing with anti-cancer drugs, IVIG could be used as a transporter protein for 7-ethylcamptothecin and other CPTs, allowing clinicians to devise new treatment protocols for patients.


Asunto(s)
Camptotecina/análogos & derivados , Inmunoglobulinas Intravenosas/química , Camptotecina/química , Camptotecina/metabolismo , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Inmunoglobulinas Intravenosas/metabolismo , Inmunoglobulinas Intravenosas/farmacocinética , Cinética , Modelos Moleculares , Conformación Molecular , Unión Proteica , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , Termodinámica
3.
Biotechnol Appl Biochem ; 62(3): 309-15, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25066911

RESUMEN

Glucagon-like peptide-1 (GLP-1) plays an important role in the regulation of postprandial insulin release. Here, we used the split DnaB mini-intein system to produce recombinant human GLP-1/7-36 (rhGLP-1) in Escherichia coli. The C-terminal domain of DnaB mini-intein (IntC) was genetically fused at the N-terminus of rhGLP-1 to produce IntC-GLP-1. IntC-GLP-1 and N-terminal domain of DnaB mini-intein (IntN) protein were prepared in a denatured buffer of pH 8.0. IntC-GLP-1 was diluted 1:8 into the phosphate buffer of pH 6.6. IntN was added into the diluted solution of IntC-GLP-1 at the molar ratio of 1:2. Then, rhGLP-1 was released from IntC-GLP-1 via inducible C-terminal peptide-bond cleavage by shifting pH from 8.0 to 6.6 at 25 °C for 24-H incubation. Then, the supernatant was applied to a Ni-Sepharose column, and the pass through fraction was collected. About 5.34 mg of rhGLP-1 with the purity of 97% was obtained from 1 L of culture medium. Mass spectrometry showed the molecular weight of 3,300.45 Da, which was equal to the theoretical value of GLP-1/7-36. The glucose-lowering activity of rhGLP-1 was confirmed by the glucose tolerance test in mice. In conclusion, the reported method was an efficient strategy to produce rhGLP-1 without using enzyme or chemical reagents, which could also be used for other similar peptides.


Asunto(s)
Clonación Molecular/métodos , AdnB Helicasas/genética , Péptido 1 Similar al Glucagón/metabolismo , Inteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Escherichia coli/genética , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/aislamiento & purificación , Humanos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación
4.
Calcif Tissue Int ; 95(2): 153-65, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24907907

RESUMEN

The investigation of agents for the treatment of osteoporosis has been a long-standing effort. The Wnt pathway plays an important role in bone formation and regeneration, and expression of Wnt pathway inhibitors, Dickkopf-1 (DKK1), appears to be associated with changes in bone mass. Inactivation of DKK1 leads to substantially increased bone mass in genetically manipulated animals. DKK1-derived peptides (DDPs) were added to BMP2-stimulated MC3T3-E1 preosteoblastic cells in vitro to evaluate inhibitory activity of DDPs in MC3T3-E1 cell differentiation. Study was extended in vivo on old female mice to show whether or not inhibition of endogenous DKK1 biological activity using DDPs vaccination approach leads to increase of bone formation, bone density, and improvement of bone microstructure. We reported that synthetic DDPs were able to reduce alkaline phosphatase activity, prevent mineralization and inhibit the differentiation of MC3T3-E1 cells in vitro. Furthermore, vaccination with these DDPs in aged female mice 4 times for a total period of 22 weeks promoted bone mass and bone microstructure. 3D microCT and histomorphometric analysis showed that there were significant increase in bone mineral densities, improvement of bone microstructure and promotion of bone formation in the vaccinated mice, especially in the mice vaccinated with DDP-A and DDP-C. Histological and scanning electron microscopy image analysis also indicated that vaccination increased trabecular bone mass and significantly decreased fragmentation of bone fibers. Taken together, these preclinical results suggest that vaccination with DDPs represents a promising new therapeutic approach for the treatment of bone-related disorders, such as osteoporosis.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/inmunología , Osteogénesis/fisiología , Osteoporosis/prevención & control , Vacunas/farmacología , Absorciometría de Fotón , Envejecimiento , Animales , Western Blotting , Modelos Animales de Enfermedad , Femenino , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Osteoporosis/metabolismo , Péptidos/inmunología , Vacunación , Microtomografía por Rayos X
5.
Appl Microbiol Biotechnol ; 98(1): 105-14, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24113827

RESUMEN

A murine monoclonal antibody (mAb) 3C7 against integrin αIIbß3 was previously obtained as a potential antithrombotic agent in our laboratory. The epitope of 3C7 is a specific conformation of the αIIbß3 complex, but not either of the two subunits, which makes it different from abciximab, a supplementary antibody drug used in percutaneous coronary intervention which has a cross-reaction with other integrins sharing the ß3 subunit. To reduce the human anti-mouse antibody reactions of 3C7, the variable regions of this antibody were cloned and fused with the constant counterparts of human IgG1. Two vectors of light and heavy chains were constructed and co-transfected into CHO-dhfr(-) cells. The chimeric antibody c3C7 was purified and the properties of c3C7 were compared with 3C7. Identical to its parent antibody 3C7, c3C7 binds to the αIIbß3 complex, but not to either of the subunits. The K(d) value of c3C7 was in the same order of magnitude as 3C7 (1.570 ± 0.326 vs 0.780 ± 0.182 nmol/L). Human platelet aggregation induced by adenosine diphosphate was effectively inhibited by c3C7 in a dose-dependent manner. In conclusion, after the modification, c3C7 retained the properties of its parent mAb with no loss of its biological activity. Therefore, c3C7 has the potential to become a novel agent for the treatment of thrombosis.


Asunto(s)
Anticuerpos/genética , Anticuerpos/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Animales , Anticuerpos/aislamiento & purificación , Células CHO , Cricetinae , Cricetulus , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Ratones , Agregación Plaquetaria/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo
6.
Environ Sci Pollut Res Int ; 29(19): 28973-28983, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-34994935

RESUMEN

Planarians are widely used as water quality indicator species to provide early warning of harmful pollution in aquatic ecosystems. However, the impact of microplastics on freshwater planarians remains poorly investigated. Here we simulated waterborne microplastic exposure in the natural environments to examine the effect on the antioxidant defense system and microbiota in Dugesia japonica. The results showed that exposure to microplastics significantly changed the levels of antioxidant enzymes, including superoxide dismutase, catalase, and glutathione S-transferase, indicating that microplastic exposure induces oxidative stress in planarians. High-throughput 16S rRNA gene sequencing results revealed that exposure to microplastics altered the diversity, abundance, and composition of planarian microbiota community. At phylum level, the relative abundance of the dominant phyla Proteobacteria and Bacteroidetes changed significantly after microplastic exposure. At genus level, the abundance of dominant genera also changed significantly, including Curvibacter and unclassified Chitinophagales. Predictive functional analysis showed that the microbiota of microplastic-exposed planarians exhibited an enrichment in genes related to fatty acid metabolism. Overall, these results showed that microplastics can cause oxidative stress and microbiota dysbiosis in planarians, indicating that planarians can serve as an indicator species for microplastic pollution in freshwater systems.


Asunto(s)
Microbiota , Planarias , Contaminantes Químicos del Agua , Animales , Antioxidantes/metabolismo , Disbiosis , Microplásticos/toxicidad , Estrés Oxidativo , Plásticos/metabolismo , ARN Ribosómico 16S/genética , Contaminantes Químicos del Agua/análisis
7.
Front Plant Sci ; 13: 847853, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35432418

RESUMEN

Soil salinization is a major environmental problem that seriously threatens the sustainable development of regional ecosystems and local economies. Fraxinus velutina Torr. is an excellent salt-tolerant tree species, which is widely planted in the saline-alkaline soils in China. A growing body of evidence shows that microRNAs (miRNAs) play important roles in the defense response of plants to salt stress; however, how miRNAs in F. velutina exert anti-salt stress remains unclear. We previously identified two contrasting F. velutina cuttings clones, salt-tolerant (R7) and salt-sensitive (S4) and found that R7 exhibits higher salt tolerance than S4. To identify salt-responsive miRNAs and their target genes, the leaves and roots of R7 and S4 exposed to salt stress were subjected to miRNA and degradome sequencing analysis. The results showed that compared with S4, R7 showed 89 and 138 differentially expressed miRNAs in leaves and roots, respectively. Specifically, in R7 leaves, miR164d, miR171b/c, miR396a, and miR160g targeting NAC1, SCL22, GRF1, and ARF18, respectively, were involved in salt tolerance. In R7 roots, miR396a, miR156a/b, miR8175, miR319a/d, and miR393a targeting TGA2.3, SBP14, GR-RBP, TCP2/4, and TIR1, respectively, participated in salt stress responses. Taken together, the findings presented here revealed the key regulatory network of miRNAs in R7 responding to salt stress, thereby providing new insights into improving salt tolerance of F. velutina through miRNA manipulation.

8.
Gigascience ; 122022 12 28.
Artículo en Inglés | MEDLINE | ID: mdl-37494283

RESUMEN

BACKGROUND: The woody halophyte Tamarix chinensis is a pioneer tree species in the coastal wetland ecosystem of northern China, exhibiting high resistance to salt stress. However, the genetic information underlying salt tolerance in T. chinensis remains to be seen. Here we present a genomic investigation of T. chinensis to elucidate the underlying mechanism of its high resistance to salinity. RESULTS: Using a combination of PacBio and high-throughput chromosome conformation capture data, a chromosome-level T. chinensis genome was assembled with a size of 1.32 Gb and scaffold N50 of 110.03 Mb. Genome evolution analyses revealed that T. chinensis significantly expanded families of HAT and LIMYB genes. Whole-genome and tandem duplications contributed to the expansion of genes associated with the salinity adaptation of T. chinensis. Transcriptome analyses were performed on root and shoot tissues during salt stress and recovery, and several hub genes responding to salt stress were identified. WRKY33/40, MPK3/4, and XBAT31 were critical in responding to salt stress during early exposure, while WRKY40, ZAT10, AHK4, IRX9, and CESA4/8 were involved in responding to salt stress during late stress and recovery. In addition, PER7/27/57/73 encoding class III peroxidase and MCM3/4/5/7 encoding DNA replication licensing factor maintained up/downregulation during salt stress and recovery stages. CONCLUSIONS: The results presented here reveal the genetic mechanisms underlying salt adaptation in T. chinensis, thus providing important genomic resources for evolutionary studies on tamarisk and plant salt tolerance genetic improvement.


Asunto(s)
Tamaricaceae , Tamaricaceae/genética , Plantas Tolerantes a la Sal/genética , Salinidad , Ecosistema , Genómica
9.
Front Plant Sci ; 13: 991197, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36147226

RESUMEN

Yellowhorn (Xanthoceras sorbifolium) is an oil-bearing tree species growing naturally in poor soil. The kernel of yellowhorn contains valuable fatty acids like nervonic acid. However, the genetic basis underlying the biosynthesis of valued fatty acids and adaptation to harsh environments is mainly unexplored in yellowhorn. Here, we presented a haplotype-resolved chromosome-scale genome assembly of yellowhorn with the size of 490.44 Mb containing scaffold N50 of 34.27 Mb. Comparative genomics, in combination with transcriptome profiling analyses, showed that expansion of gene families like long-chain acyl-CoA synthetase and ankyrins contribute to yellowhorn fatty acid biosynthesis and defense against abiotic stresses, respectively. By integrating genomic and transcriptomic data of yellowhorn, we found that the transcription of 3-ketoacyl-CoA synthase gene XS04G00959 was consistent with the accumulation of nervonic and erucic acid biosynthesis, suggesting its critical regulatory roles in their biosynthesis. Collectively, these results enhance our understanding of the genetic basis underlying the biosynthesis of valuable fatty acids and adaptation to harsh environments in yellowhorn and provide foundations for its genetic improvement.

10.
Front Plant Sci ; 13: 842726, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35310642

RESUMEN

Fraxinus velutina Torr with high salt tolerance has been widely grown in saline lands in the Yellow River Delta, China. However, the salt-tolerant mechanisms of F. velutina remain largely elusive. Here, we identified two contrasting cutting clones of F. velutina, R7 (salt-tolerant), and S4 (salt-sensitive) by measuring chlorophyll fluorescence characteristics (Fv/Fm ratio) in the excised leaves and physiological indexes in roots or leaves under salt treatment. To further explore the salt resistance mechanisms, we compared the transcriptomes of R7 and S4 from leaf and root tissues exposed to salt stress. The results showed that when the excised leaves of S4 and R7 were, respectively, exposed to 250 mM NaCl for 48 h, Fv/Fm ratio decreased significantly in S4 compared with R7, confirming that R7 is more tolerant to salt stress. Comparative transcriptome analysis showed that salt stress induced the significant upregulation of stress-responsive genes in R7, making important contributions to the high salt tolerance. Specifically, in the R7 leaves, salt stress markedly upregulated key genes involved in plant hormone signaling and mitogen-activated protein kinase signaling pathways; in the R7 roots, salt stress induced the upregulation of main genes involved in proline biosynthesis and starch and sucrose metabolism. In addition, 12 genes encoding antioxidant enzyme peroxidase were all significantly upregulated in both leaves and roots. Collectively, our findings revealed the crucial defense pathways underlying high salt tolerance of R7 through significant upregulation of some key genes involving metabolism and hub signaling pathways, thus providing novel insights into salt-tolerant F. velutina breeding.

11.
J Biol Chem ; 285(13): 9367-9372, 2010 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-20118230

RESUMEN

To find a suitable ligand from a complex antigen system is still a mission to be accomplished. Here we have explored a novel "library against proteome" panning strategy for ligand screening and antigen purification from a complex system using phage-displayed antibody technology. Human plasma proteome was targeted for phage library panning. During the process, the panning was carried out in solution, using a biotin/streptavidin beads separation system, for three rounds. Nine monoclonal phages, bound tightly to a number of unknown plasma proteins, were selected from the last round, six of which were directly employed as cross-linked matrices to purify their corresponding antigens from the plasma. The proteins isolated by G5 and E1 matrices were identified as amyloid protein and apolipoprotein A-I precursor, respectively. The results demonstrated that it was feasible to simultaneously obtain a number of ligand phages for various antigens, including low abundant proteins in a non-comparative proteome-wide system.


Asunto(s)
Bacteriófagos/metabolismo , Biblioteca de Péptidos , Proteoma , Proteómica/métodos , Antígenos/química , Biotina/química , Biotinilación , Proteínas Sanguíneas/química , Reactivos de Enlaces Cruzados/química , Ensayo de Inmunoadsorción Enzimática , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/química , Ligandos , Albúmina Sérica/química , Estreptavidina/química
12.
Cell Discov ; 7(1): 103, 2021 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-34719679

RESUMEN

Epidemiological studies have demonstrated that the genetic factors partly influence the development of same-sex sexual behavior, but most genetic studies have focused on people of primarily European ancestry, potentially missing important biological insights. Here, we performed a two-stage genome-wide association study (GWAS) with a total sample of 1478 homosexual males and 3313 heterosexual males in Han Chinese populations and identified two genetic loci (rs17320865, Xq27.3, FMR1NB, Pmeta = 8.36 × 10-8, OR = 1.29; rs7259428, 19q12, ZNF536, Pmeta = 7.58 × 10-8, OR = 0.75) showing consistent association with male sexual orientation. A fixed-effect meta-analysis including individuals of Han Chinese (n = 4791) and European ancestries (n = 408,995) revealed 3 genome-wide significant loci of same-sex sexual behavior (rs9677294, 2p22.1, SLC8A1, Pmeta = 1.95 × 10-8; rs2414487, 15q21.3, LOC145783, Pmeta = 4.53 × 10-9; rs2106525, 7q31.1, MDFIC, Pmeta = 6.24 × 10-9). These findings may provide new insights into the genetic basis of male sexual orientation from a wider population scope. Furthermore, we defined the average ZNF536-immunoreactivity (ZNF536-ir) concentration in the suprachiasmatic nucleus (SCN) as lower in homosexual individuals than in heterosexual individuals (0.011 ± 0.001 vs 0.021 ± 0.004, P = 0.013) in a postmortem study. In addition, compared with heterosexuals, the percentage of ZNF536 stained area in the SCN was also smaller in the homosexuals (0.075 ± 0.040 vs 0.137 ± 0.103, P = 0.043). More homosexual preference was observed in FMR1NB-knockout mice and we also found significant differences in the expression of serotonin, dopamine, and inflammation pathways that were reported to be related to sexual orientation when comparing CRISPR-mediated FMR1NB knockout mice to matched wild-type target C57 male mice.

13.
Biochem Biophys Res Commun ; 391(1): 1039-42, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19995555

RESUMEN

Nesfatin-1 is recently reported as a satiety molecule to suppress food intake via the melanocortin signaling in hypothalamus when injected centrally and peripherally. Here we report that nesfatin-1 is also anti-hyperglycemic. It was found that the intravenous injection of nesfatin-1 significantly reduced blood glucose in hyperglycemic db/db mice. This anti-hyperglycemic effect of nesfatin-1 was time-, dose-, insulin-dependent and peripheral.


Asunto(s)
Depresores del Apetito/farmacología , Glucemia/efectos de los fármacos , Hiperglucemia/metabolismo , Hipolipemiantes/farmacología , Hormonas Peptídicas/farmacología , Anilidas/farmacología , Animales , Proteínas de Unión al Calcio , Proteínas de Unión al ADN , Ingestión de Alimentos/efectos de los fármacos , Insulina/metabolismo , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso , Nucleobindinas , Hormonas Peptídicas/biosíntesis , Hormonas Peptídicas/genética , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores de Leptina/genética , Rosiglitazona , Tiazolidinedionas/farmacología
14.
Sci Rep ; 10(1): 4614, 2020 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-32165650

RESUMEN

Pinellia (Pinellia ternata (Thunb.) Breit.), as important medicinal plant, has been used to treat various ailments for a long time. The sixteen ploid plant (2n = 16 * 13 = 208) Pinellia T2Plus line was obtained from an octoploid (2n = 8 * 13 = 104) T2 line by chromosome-doubling technique. Compared with T2 line, the content of various medicinal components (polysaccharide, guanosine, adenosine and ephedrine) was increased in T2Plus line. In this study, the transcriptome of T2 line and T2Plus line were characterized by RNA sequencing (RNA-seq) technology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis on differential expressed unigenes (DEGs) revealed that multiple metabolic pathway were enriched significantly, such as 'Starch and sucrose metabolism', 'Purine metabolism', 'Photosynthesis' and six transcription factors (MYB, WRKY, bHLH, lateral organ boundaries domain (LBD), homeodomain-zipper (HD-ZIP) and Ethylene-responsive factor (ERF)) play a key role in difference of transcriptome between T2 line and T2Plus line. These metabolic pathways and transcription factors may play an important role in the difference of medicinal components and epigenetic features between these two Pinellia cultivars. This conclusion provides a robust theoretical basis for the mechanism of the formation of medicinal ingredients in Pinellia cultivars.


Asunto(s)
Perfilación de la Expresión Génica , Fenotipo , Pinellia/genética , Transcriptoma , Biología Computacional/métodos , Metabolismo Energético , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Redes y Vías Metabólicas , Fotosíntesis , Pinellia/metabolismo , Plantas Medicinales/genética
15.
Gene ; 726: 144193, 2020 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-31669647

RESUMEN

OBJECTIVE: miR-199a-5p was increased during osteoblast differentiation, which may target and regulate TET2, a gene attracted a lot of attention in the osteoblast differentiation in the past few years. However, the role of miR-199a-5p in osteoblast differentiation by targeting TET2 is not established. METHODS: The correlation between miR-199a-5p and TET2 was verified through dual luciferase reporter assay, and their expressions in human bone marrow stromal cells (hBMSCs) during the osteoblast differentiation were detected. hBMSCs were transfected with TET2 siRNA, miR-199a-5p mimic or/and TET2 CRISPR activation plasmid., and then prepared for the induction of osteoblast differentiation, followed by alkaline phosphatase (ALP) and alizarin red staining, qRT-PCR and Western blotting. In vivo, ovariectomized (OVX) mice were injected with agomir-miR-199a-5p, antagomiR-199a-5p or/and TET2 siRNA to calculate the BMD and BV/TV ratio of mice, as well as to measure the expressions of osteogenesis-related genes in bone tissues. RESULTS: A gradual increase of miR-199a-5p was observed in hBMSCs during the induction of osteoblast differentiation, while TET2 expression was decreased. Besides, miR-199a-5p was reduced in the bone tissue of OVX mice, while TET2 was up-regulated. In addition, overexpression of miR-199a-5p and inhibition of TET2 augmented ALP activity in hBMSCs, with the enhanced calcification and the up-regulated expressions of Runx2, OSX and OCN, which also increased the quality of bone in OVX mice accompanying the enhancement BV/TV ratio, BMD and osteogenesis-related genes. CONCLUSION: MiR-199a-5p may promote the osteoblast differentiation and prevent OVX-induced osteoporosis by targeting TET2.


Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , MicroARNs/genética , Osteoblastos/metabolismo , Proteínas Proto-Oncogénicas/genética , Fosfatasa Alcalina/genética , Animales , Células de la Médula Ósea/metabolismo , Huesos/metabolismo , Calcificación Fisiológica/genética , Células Cultivadas , Dioxigenasas , Femenino , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Osteogénesis/genética , Regulación hacia Arriba/genética
16.
Lab Chip ; 9(6): 756-60, 2009 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-19255656

RESUMEN

A proof-of-concept study demonstrated the feasibility of a novel gel-pad microarray on porous silicon chips, by initiation of an atom transfer radical propagation (ATRP) polymerisation of (polyethylene glycol) methacrylate (PEGMA) with surface Si-H species, stepwise chemical conversions of the gel membrane to an NTA-Ni2+/histidine-tagged protein system, and matrix-assisted laser desorption/ionisation mass spectroscopy (MALDI MS) and fluorescence detections.


Asunto(s)
Geles/química , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Silicio/química , Microscopía Electrónica de Rastreo , Porosidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Propiedades de Superficie
17.
Int J Cancer ; 125(1): 124-32, 2009 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-19350628

RESUMEN

Angiogenesis is critical for tumor growth and metastasis. Tumor tissues induce the expression of angiogenesis-associated proteins on endothelial surface that can be targeted for tumor immunotherapy. In our study, the rat tumor endothelial proteins (EP) were isolated in situ via biotinylation of tumor vascular endothelial luminal surface followed by streptavidin affinity chromatography. The isolated tumor EP contained numerous up-regulated angiogenesis-associated endothelial proteins. The administration of these tumor EP as a vaccine to mice reduced the microvessel density in subcutaneous primary LLC tumors, delayed spontaneous LLC tumor metastasis and prolonged post-surgery life span. T lymphocytes from tumor EP-vaccinated mice lysed human umbilical vascular endothelial cells, but not tumor cells in vitro, in a dose-dependent manner. Furthermore, adoptive transfer of antitumor EP antibodies in vivo targeted to tumor endothelium and inhibited spontaneous LLC tumor metastasis. This study provides a successful preclinical exploration of the active immunotherapy for tumor by targeting tumor angiogenesis.


Asunto(s)
Adenocarcinoma/irrigación sanguínea , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Endotelio Vascular/química , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Mamarias Experimentales/irrigación sanguínea , Proteínas de Neoplasias/uso terapéutico , Neovascularización Patológica/prevención & control , Traslado Adoptivo , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Biotinilación , Movimiento Celular , Electroforesis en Gel Bidimensional , Femenino , Citometría de Flujo , Immunoblotting , Técnicas para Inmunoenzimas , Inmunoglobulina G/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Conejos , Ratas , Ratas Endogámicas F344 , Vacunación
18.
Protein Expr Purif ; 65(2): 238-43, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19152836

RESUMEN

Aprotinin is a Kunitz-type inhibitor with a relatively broad specificity. It has been shown to be clinically useful for the management of hemorrhagic complications. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant aprotinin and a human aprotinin analogue (cloned form human cDNA library). Both fusion proteins were overexpressed mainly as inclusion bodies in Escherichia coli and accounted for approximately 28% of the total cell proteins. After purification by Ni-Sepharose affinity chromatography and renaturation, the fusion proteins were cleaved with SUMO protease 1. Aprotinin and its analogue were separated from the fusion partner by the subtractive chromatography using Ni-Sepharose and then further purified with CM-cellulose. Kinetic studies demonstrated that the amidolytic activity of plasmin was competitively inhibited by recombinant aprotinin with a K(i) of 8.6+/-2.4 nM, which was similar to the K(i) (7.5+/-2.7 nM) of natural aprotinin. The K(i) of human aprotinin analogue was 22.7+/-6.5 nM. The expression strategy described in this study allows convenient high yield and easy purification of small recombinant protease inhibitors with complete native sequences.


Asunto(s)
Aprotinina/aislamiento & purificación , Aprotinina/farmacología , Fibrinolisina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Aprotinina/biosíntesis , Aprotinina/química , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca de Genes , Humanos , Cinética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteína SUMO-1/metabolismo , Solubilidad
19.
Biomacromolecules ; 10(4): 877-83, 2009 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-19275167

RESUMEN

The recently arising antithrombin drug, angiomax, was successfully conjugated with a 5'-amino oligonucleotide through click chemistry. This oligo-angiomax conjugate was assembled into a two-dimensional DNA lattice with other oligonucleotides together. Besides the plane sheet of DNA lattices, an interesting angiomax-involved DNA tubing structure, constructed by 40 to 50 angiomax stripes which are parallel to the longitudinal axis of the tube, was also imaged. After incubation of thrombins with the angiomax-involved DNA lattice, the binding of thrombins to arrayed angiomax peptides was observed. Finally a chromogenic substrate bioassay was employed to estimate the antithrombin activities as assembled oligo-angiomax DNA lattice approximately 1.1, oligo-angiomax approximately 2.7 angiomax. The functionalized DNA lattices have the potential to be used as a powerful platform for investigation of biomolecular interactions such as drug-protein, protein-protein, DNA-RNA, and DNA-protein interactions in the nano- and subnanoscales.


Asunto(s)
ADN/química , ADN/metabolismo , Hirudinas/química , Hirudinas/metabolismo , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Trombina/metabolismo , Anticoagulantes/química , Anticoagulantes/metabolismo , Antitrombinas/química , Antitrombinas/metabolismo , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Agar , Microscopía de Fuerza Atómica , Modelos Químicos , Oligopéptidos/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Termodinámica , Trombina/química
20.
Genes (Basel) ; 10(7)2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31311183

RESUMEN

Long non-coding RNAs (lncRNAs) are involved in various regulatory processes although they do not encode protein. Presently, there is little information regarding the identification of lncRNAs in peanut (Arachis hypogaea Linn.). In this study, 50,873 lncRNAs of peanut were identified from large-scale published RNA sequencing data that belonged to 124 samples involving 15 different tissues. The average lengths of lncRNA and mRNA were 4335 bp and 954 bp, respectively. Compared to the mRNAs, the lncRNAs were shorter, with fewer exons and lower expression levels. The 4713 co-expression lncRNAs (expressed in all samples) were used to construct co-expression networks by using the weighted correlation network analysis (WGCNA). LncRNAs correlating with the growth and development of different peanut tissues were obtained, and target genes for 386 hub lncRNAs of all lncRNAs co-expressions were predicted. Taken together, these findings can provide a comprehensive identification of lncRNAs in peanut.


Asunto(s)
Arachis/genética , Genoma de Planta , Estudio de Asociación del Genoma Completo , Genómica , ARN Largo no Codificante/genética , ARN de Planta/genética , Biología Computacional , Bases de Datos Genéticas , Estudio de Asociación del Genoma Completo/métodos , Genómica/métodos
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