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1.
Fish Shellfish Immunol ; 106: 876-886, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32800983

RESUMO

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.


Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Penaeidae/genética , Penaeidae/imunologia , RNA Longo não Codificante/imunologia , Spiroplasma , Animais , Infecções por Bactérias Gram-Negativas/veterinária , Hemócitos/imunologia , Penaeidae/microbiologia , Transcriptoma
2.
Molecules ; 23(9)2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30231526

RESUMO

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.


Assuntos
Camptotecina/análogos & derivados , Imunoglobulinas Intravenosas/química , Camptotecina/química , Camptotecina/metabolismo , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas Intravenosas/metabolismo , Imunoglobulinas Intravenosas/farmacocinética , Cinética , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Termodinâmica
3.
Biotechnol Appl Biochem ; 62(3): 309-15, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25066911

RESUMO

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.


Assuntos
Clonagem Molecular/métodos , DnaB Helicases/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Inteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Escherichia coli/genética , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/isolamento & purificação , Humanos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação
4.
Calcif Tissue Int ; 95(2): 153-65, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24907907

RESUMO

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.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Osteogênese/fisiologia , Osteoporose/prevenção & controle , Vacinas/farmacologia , Absorciometria de Fóton , Envelhecimento , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Osteoporose/metabolismo , Peptídeos/imunologia , Vacinação , Microtomografia por Raio-X
5.
Appl Microbiol Biotechnol ; 98(1): 105-14, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24113827

RESUMO

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.


Assuntos
Anticorpos/genética , Anticorpos/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Animais , Anticorpos/isolamento & purificação , Células CHO , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Camundongos , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
Environ Sci Pollut Res Int ; 29(19): 28973-28983, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34994935

RESUMO

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.


Assuntos
Microbiota , Planárias , Poluentes Químicos da Água , Animais , Antioxidantes/metabolismo , Disbiose , Microplásticos/toxicidade , Estresse Oxidativo , Plásticos/metabolismo , RNA Ribossômico 16S/genética , Poluentes Químicos da Água/análise
7.
Front Plant Sci ; 13: 847853, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35432418

RESUMO

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.
Artigo em Inglês | MEDLINE | ID: mdl-37494283

RESUMO

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.


Assuntos
Tamaricaceae , Tamaricaceae/genética , Plantas Tolerantes a Sal/genética , Salinidade , Ecossistema , Genômica
9.
Front Plant Sci ; 13: 991197, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36147226

RESUMO

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.
Artigo em Inglês | MEDLINE | ID: mdl-35310642

RESUMO

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.
Artigo em Inglês | MEDLINE | ID: mdl-20118230

RESUMO

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.


Assuntos
Bacteriófagos/metabolismo , Biblioteca de Peptídeos , Proteoma , Proteômica/métodos , Antígenos/química , Biotina/química , Biotinilação , Proteínas Sanguíneas/química , Reagentes de Ligações Cruzadas/química , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Ligantes , Albumina Sérica/química , Estreptavidina/química
12.
Cell Discov ; 7(1): 103, 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34719679

RESUMO

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.
Artigo em Inglês | MEDLINE | ID: mdl-19995555

RESUMO

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.


Assuntos
Depressores do Apetite/farmacologia , Glicemia/efeitos dos fármacos , Hiperglicemia/metabolismo , Hipolipemiantes/farmacologia , Hormônios Peptídicos/farmacologia , Anilidas/farmacologia , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Ingestão de Alimentos/efeitos dos fármacos , Insulina/metabolismo , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso , Nucleobindinas , Hormônios Peptídicos/biossíntese , Hormônios Peptídicos/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores para Leptina/genética , Rosiglitazona , Tiazolidinedionas/farmacologia
14.
Sci Rep ; 10(1): 4614, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165650

RESUMO

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.


Assuntos
Perfilação da Expressão Gênica , Fenótipo , Pinellia/genética , Transcriptoma , Biologia Computacional/métodos , Metabolismo Energético , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Redes e Vias Metabólicas , Fotossíntese , Pinellia/metabolismo , Plantas Medicinais/genética
15.
Gene ; 726: 144193, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669647

RESUMO

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.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , MicroRNAs/genética , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas/genética , Fosfatase Alcalina/genética , Animais , Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Calcificação Fisiológica/genética , Células Cultivadas , Dioxigenases , Feminino , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese/genética , Regulação para Cima/genética
16.
Lab Chip ; 9(6): 756-60, 2009 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-19255656

RESUMO

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.


Assuntos
Géis/química , Análise Serial de Proteínas/instrumentação , Análise Serial de Proteínas/métodos , Silício/química , Microscopia Eletrônica de Varredura , Porosidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Propriedades de Superfície
17.
Int J Cancer ; 125(1): 124-32, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19350628

RESUMO

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.


Assuntos
Adenocarcinoma/irrigação sanguínea , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Endotélio Vascular/química , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Mamárias Experimentais/irrigação sanguínea , Proteínas de Neoplasias/uso terapêutico , Neovascularização Patológica/prevenção & controle , Transferência Adotiva , Inibidores da Angiogênese/uso terapêutico , Animais , Biotinilação , Movimento Celular , Eletroforese em Gel Bidimensional , Feminino , Citometria de Fluxo , Immunoblotting , Técnicas Imunoenzimáticas , Imunoglobulina G/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Ratos , Ratos Endogâmicos F344 , Vacinação
18.
Protein Expr Purif ; 65(2): 238-43, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19152836

RESUMO

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.


Assuntos
Aprotinina/isolamento & purificação , Aprotinina/farmacologia , Fibrinolisina/antagonistas & inibidores , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Aprotinina/biossíntese , Aprotinina/química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Cinética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteína SUMO-1/metabolismo , Solubilidade
19.
Biomacromolecules ; 10(4): 877-83, 2009 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-19275167

RESUMO

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.


Assuntos
DNA/química , DNA/metabolismo , Hirudinas/química , Hirudinas/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Trombina/metabolismo , Anticoagulantes/química , Anticoagulantes/metabolismo , Antitrombinas/química , Antitrombinas/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Ágar , Microscopia de Força Atômica , Modelos Químicos , Oligopeptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Termodinâmica , Trombina/química
20.
Genes (Basel) ; 10(7)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311183

RESUMO

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.


Assuntos
Arachis/genética , Genoma de Planta , Estudo de Associação Genômica Ampla , Genômica , RNA Longo não Codificante/genética , RNA de Plantas/genética , Biologia Computacional , Bases de Dados Genéticas , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos
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