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1.
Artigo em Inglês | MEDLINE | ID: mdl-33848241

RESUMO

Pixel-level 2D object semantic understanding is an important topic in computer vision and could help machine deeply understand objects (e.g. functionality and affordance) in our daily life. However, most previous methods directly train on correspondences in 2D images, which is end-to-end but loses plenty of information in 3D spaces. In this paper, we propose a new method on predicting image corresponding semantics in 3D domain and then projecting them back onto 2D images to achieve pixel-level understanding. In order to obtain reliable 3D semantic labels that are absent in current image datasets, we build a large scale keypoint knowledge engine called KeypointNet, which contains 103,450 keypoints and 8,234 3D models from 16 object categories. Our method leverages the advantages in 3D vision and can explicitly reason about objects self-occlusion and visibility. We show that our method gives comparative and even superior results on standard semantic benchmarks.

2.
iScience ; 23(11): 101684, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33196019

RESUMO

Cancer cells alter their nutrition metabolism to cope the stressful environment. One important metabolism adjustment is that cancer cells activate glutaminolysis in response to the reduced carbon from glucose entering into the TCA cycle due to inactivation of several enzymes in glycolysis. An important question is how the cancer cells coordinate the changes of glycolysis and glutaminolysis. In this report, we demonstrate that the pyruvate kinase inactive dimer PKM2 facilitates activation of glutaminolysis. Our experiments show that growth stimulations promote PKM2 dimer. The dimer PKM2 plays a role in regulation of glutaminolysis by upregulation of mitochondrial glutaminase I (GLS-1). PKM2 dimer regulates the GLS-1 expression by controlling internal ribosome entry site (IRES)-dependent c-myc translation. Growth stimulations promote PKM2 interacting with c-myc IRES-RNA, thus facilitating c-myc IRES-dependent translation. Our study reveals an important linker that coordinates the metabolism adjustment in cancer cells.

3.
Plant Genome ; 13(2): e20019, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33016609

RESUMO

Mangroves is an umbrella term for plants located across the tropics and sub-tropics that live in the coastal region, between the sea and the land. All mangroves evolved from terrestrial plants, providing the opportunity to assess convergence, as well as the lineage-specific features, at the genetic level. In this study, we compared chloroplast genomes from 21 mangrove species, covering main phylogenetic clades. We demonstrate that chloroplast gene order, content, and genome size is largely conserved in mangroves. The exceptions are loss of the photosystem I gene psaZ in Acanthus ilicifolius and inversion of the ribosomal protein gene rpl23 in Avicennia germinans. The repeat content of mangrove chloroplast varied between species, but was conserved within species of the same order. Sequence diversity analysis revealed that the IR (invert repeat) region was highly conserved compared to the SC (single-copy) region in most phylogenetic clades, except clade core leptosporangiates (ferns). The ribosomal protein gene rps7 was under positive selection in Kandelia obovato, Rhizophora stylosa, Bruguiera sexangular and Rhizophora mangle, a monophyletic branch of clade fabids, while no evidence of positive selection was found in other mangrove lineages. Taken together, our data suggests that convergent evolutionary dynamics leaves no significant signal on the plastid genome of mangroves. The complete chloroplast genomes provided in this study shed light on the evolution of these important plastids and provides a valuable resource for further research efforts.


Assuntos
Avicennia , Genomas de Plastídeos , Rhizophoraceae , Avicennia/genética , Tamanho do Genoma , Filogenia , Rhizophoraceae/genética
4.
Biomed Res Int ; 2020: 8731857, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32462024

RESUMO

Mangroves are a group of plant species that occupy the coastal intertidal zone and are major components of this ecologically important ecosystem. Mangroves belong to about twenty diverse families. Here, we sequenced and assembled chloroplast genomes of 14 mangrove species from eight families spanning five rosid orders and one asterid order: Fabales (Pongamia pinnata), Lamiales (Avicennia marina), Malpighiales (Excoecaria agallocha, Bruguiera sexangula, Kandelia obovata, Rhizophora stylosa, and Ceriops tagal), Malvales (Hibiscus tiliaceus, Heritiera littoralis, and Thespesia populnea), Myrtales (Laguncularia racemosa, Sonneratia ovata, and Pemphis acidula), and Sapindales (Xylocarpus moluccensis). These chloroplast genomes range from 149 kb to 168 kb in length. A conserved structure of two inverted repeats (IRa and IRb, ~25.8 kb), one large single-copy region (LSC, ~89.0 kb), and one short single-copy region (SSC, ~18.9 kb) as well as ~130 genes (85 protein-coding, 37 tRNAs, and 8 rRNAs) was observed. We found the lowest divergence in the IR regions among the four regions. We also identified simple sequence repeats (SSRs), which were found to be variable in numbers. Most chloroplast genes are highly conserved, with only four genes under positive selection or relaxed pressure. Combined with publicly available chloroplast genomes, we carried out phylogenetic analysis and confirmed the previously reported phylogeny within rosids, including the positioning of obscure families in Malpighiales. Our study reports 14 mangrove chloroplast genomes and illustrates their genome features and evolution.

5.
Environ Technol ; : 1-11, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31870218

RESUMO

Tunneling slurry waste causes multiple problems, including environmental pollution, and requires transportation and a large landfill space, and therefore, it is important to find a method that can quickly separate water from tunneling slurry waste for metro construction. This paper proposes a practical method to improve the effect of slurry waste separation by implementing five laboratory tests. The results of these tests reflect that vacuum combined electro-osmosis is a suitable and practical method for treating tunneling slurry waste. The water content after treatment by vacuum combined horizontal electric field electro-osmosis method is not only lower than that after other methods but also close to the liquid limit, which fully meets the requirements of engineering transportation. However, vacuum and filter press dewatering cannot give full play to the drainage effect when the slurry permeability coefficient is too low. This combined technique can improve water separation from the slurry and works well in engineering applications.

6.
J Fungi (Basel) ; 6(1)2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31878043

RESUMO

Penicillium is an ascomycetous genus widely distributed in the natural environment and is one of the dominant fungi involved in the decomposition of mangroves, which can produce a variety of antitumor compounds and bioactive substances. However, in mangrove ecosystems there is no complete genome in this genus. In this study, we isolated a fungus strain named Penicillium variabile HXQ-H-1 from coast mangrove (Fujian Province, China). We generated a chromosome-level genome with total size of 33.32 Mb, scaffold N50 of 5.23 Mb and contig N50 of 96.74 kb. Additionally, we anchored about 95.91% assembly sequences into the longest seven scaffolds, and predicted 10,622 protein-coding genes, in which 99.66% could be annotated by eight protein databases. The secondary metabolites analysis reveals the strain has various gene clusters involving polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and terpene synthase that may have a largely capacity of biotechnological potential. Comparison genome analysis between Penicillium variabile and Talaromyces islandicus reveals a small difference in the total number of genes, whereas HXQ-H-1 has a higher gene number with COG functional annotation. Evolutionary relationship of Penicillum based on genome-wide data was carried out for the first time, showing the strain HXQ-H-1 is closely related to Talaromyces islandicus. This genomic resource may provide a new resource for development of novel bioactive antibiotics, drug candidates and precursors in Penicillium variabile.

7.
J Med Chem ; 61(9): 4155-4164, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29641204

RESUMO

Metabolic reprogramming of cancer cells is essential for tumorigenesis in which pyruvate kinase M2 (PKM2), the low activity isoform of pyruvate kinase, plays a critical role. Herein, we describe the identification of a nature-product-derived micheliolide (MCL) that selectively activates PKM2 through the covalent binding at residue cysteine424 (C424), which is not contained in PKM1. This interaction promotes more tetramer formation, inhibits the lysine433 (K433) acetylation, and influences the translocation of PKM2 into the nucleus. In addition, the pro-drug dimethylaminomicheliolide (DMAMCL) with similar properties as MCL significantly suppresses the growth of leukemia cells and tumorigenesis in a zebrafish xenograft model. Cell-based assay with knock down PKM2 expression verifies that the effects of MCL are dependent on PKM2 expression. DMAMCL is currently in clinical trials in Australia. Our discovery may provide a valuable pharmacological mechanism for clinical treatment and benefit the development of new anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Leucemia/patologia , Proteínas de Membrana/metabolismo , Sesquiterpenos de Guaiano/farmacologia , Hormônios Tireóideos/metabolismo , Acetilação/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Carcinogênese/efeitos dos fármacos , Proteínas de Transporte/química , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/química , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína , Especificidade por Substrato , Hormônios Tireóideos/química , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
8.
Nat Commun ; 9(1): 1702, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29703940

RESUMO

Mei (Prunus mume) is an ornamental woody plant that has been domesticated in East Asia for thousands of years. High diversity in floral traits, along with its recent genome sequence, makes mei an ideal model system for studying the evolution of woody plants. Here, we investigate the genetic architecture of floral traits in mei and its domestication history by sampling and resequencing a total of 351 samples including 348 mei accessions and three other Prunus species at an average sequencing depth of 19.3×. Highly-admixed population structure and introgression from Prunus species are identified in mei accessions. Through a genome-wide association study (GWAS), we identify significant quantitative traits locus (QTLs) and genomic regions where several genes, such as MYB108, are positively associated with petal color, stigma color, calyx color, and bud color. Results from this study shed light on the genetic basis of domestication in flowering plants, particularly woody plants.


Assuntos
Flores/genética , Genoma de Planta/genética , Fenótipo , Prunus/genética , Locos de Características Quantitativas/genética , Mapeamento Cromossômico , Domesticação , Estudo de Associação Genômica Ampla , Filogenia , Análise de Sequência de RNA
9.
Plant J ; 92(3): 452-468, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28849613

RESUMO

Allotetraploid oilseed rape (Brassica napus L.) is an agriculturally important crop. Cultivation and breeding of B. napus by humans has resulted in numerous genetically diverse morphotypes with optimized agronomic traits and ecophysiological adaptation. To further understand the genetic basis of diversification and adaptation, we report a draft genome of an Asian semi-winter oilseed rape cultivar 'ZS11' and its comprehensive genomic comparison with the genomes of the winter-type cultivar 'Darmor-bzh' as well as two progenitors. The integrated BAC-to-BAC and whole-genome shotgun sequencing strategies were effective in the assembly of repetitive regions (especially young long terminal repeats) and resulted in a high-quality genome assembly of B. napus 'ZS11'. Within a short evolutionary period (~6700 years ago), semi-winter-type 'ZS11' and the winter-type 'Darmor-bzh' maintained highly genomic collinearity. Even so, certain genetic differences were also detected in two morphotypes. Relative to 'Darmor-bzh', both two subgenomes of 'ZS11' are closely related to its progenitors, and the 'ZS11' genome harbored several specific segmental homoeologous exchanges (HEs). Furthermore, the semi-winter-type 'ZS11' underwent potential genomic introgressions with B. rapa (Ar ). Some of these genetic differences were associated with key agronomic traits. A key gene of A03.FLC3 regulating vernalization-responsive flowering time in 'ZS11' was first experienced HE, and then underwent genomic introgression event with Ar , which potentially has led to genetic differences in controlling vernalization in the semi-winter types. Our observations improved our understanding of the genetic diversity of different B. napus morphotypes and the cultivation history of semi-winter oilseed rape in Asia.


Assuntos
Brassica napus/genética , Brassica/genética , Variação Genética , Genoma de Planta/genética , Genômica , Sequência de Aminoácidos , Evolução Biológica , Cruzamento , Sequenciamento de Nucleotídeos em Larga Escala , Fenótipo , Poliploidia , Alinhamento de Sequência , Análise de Sequência de DNA
10.
Gigascience ; 6(6): 1-5, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28475810

RESUMO

Rhodiola crenulata, a well-known medicinal Tibetan herb, is mainly grown in high-altitude regions of the Tibet, Yunnan, and Sichuan provinces in China. In the past few years, increasing numbers of studies have been published on the potential pharmacological activities of R. crenulata, strengthening our understanding into its putitive active ingredient composition, pharmacological activity, and mechanism of action. These findings also provide strong evidence supporting the important medicinal and economical value of R. crenulata. Consequently, some Rhodiola species are becoming endangered because of overexploitation and environmental destruction. However, little is known about the genetic and genomic information of any Rhodiola species. Here we report the first draft assembly ofthe R. crenulata genome, which was 344.5 Mb (25.7 Mb Ns), accounting for 82% of the estimated genome size, with a scaffold N50 length of 144.7 kb and a contig N50 length of 25.4 kb. The R. crenulata genome is not only highly heterozygous but also highly repetitive, with ratios of 1.12% and 66.15%, respectively, based on the k-mer analysis. Furthermore, 226.6 Mb of transposable elements were detected, of which 77.03% were long terminal repeats. In total, 31 517 protein-coding genes were identified, capturing 86.72% of expected plant genes in BUSCO. Additionally, 79.73% of protein-coding genes were functionally annotated. R. crenulata is an important medicinal plant and also a potentially interesting model species for studying the adaptability of Rhodiola species to extreme environments. The genomic sequences of R. crenulata will be useful for understanding the evolutionary mechanism of the stress resistance gene and the biosynthesis pathways of the different medicinal ingredients, for example, salidroside in R. crenulata.


Assuntos
Genoma de Planta , Rhodiola/genética , Análise de Sequência de DNA/métodos , Elementos de DNA Transponíveis , Tamanho do Genoma , Medicina Tradicional Tibetana , Anotação de Sequência Molecular
11.
Wound Repair Regen ; 24(2): 328-36, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-26808610

RESUMO

Neutrophils infiltration/activation following wound induction marks the early inflammatory response in wound repair. However, the role of the infiltrated/activated neutrophils in tissue regeneration/proliferation during wound repair is not well understood. Here, we report that infiltrated/activated neutrophils at wound site release pyruvate kinase M2 (PKM2) by its secretive mechanisms during early stages of wound repair. The released extracellular PKM2 facilitates early wound healing by promoting angiogenesis at wound site. Our studies reveal a new and important molecular linker between the early inflammatory response and proliferation phase in tissue repair process.


Assuntos
Neovascularização Fisiológica , Neutrófilos/metabolismo , Piruvato Quinase/metabolismo , Cicatrização/fisiologia , Ferimentos e Lesões/enzimologia , Ferimentos e Lesões/patologia , Animais , Proliferação de Células , Modelos Animais de Doenças , Matriz Extracelular/patologia , Imuno-Histoquímica , Inflamação/enzimologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia
12.
J Immunol ; 195(2): 661-71, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26085683

RESUMO

CD47, a self recognition marker expressed on tissue cells, interacts with immunoreceptor SIRPα expressed on the surface of macrophages to initiate inhibitory signaling that prevents macrophage phagocytosis of healthy host cells. Previous studies suggested that cells may lose surface CD47 during aging or apoptosis to enable phagocytic clearance. In the current study, we demonstrate that the level of cell surface CD47 is not decreased, but the distribution pattern of CD47 is altered, during apoptosis. On nonapoptotic cells, CD47 molecules are clustered in lipid rafts forming punctates on the surface, whereas on apoptotic cells, CD47 molecules are diffused on the cell surface following the disassembly of lipid rafts. We show that clustering of CD47 in lipid rafts provides a high binding avidity for cell surface CD47 to ligate macrophage SIRPα, which also presents as clusters, and elicits SIRPα-mediated inhibitory signaling that prevents phagocytosis. In contrast, dispersed CD47 on the apoptotic cell surface is associated with a significant reduction in the binding avidity to SIRPα and a failure to trigger SIRPα signal transduction. Disruption of plasma membrane lipid rafts with methyl-ß-cyclodextrin diffuses CD47 clusters, leading to a decrease in the cell binding avidity to SIRPα and a concomitant increase in cells being engulfed by macrophages. Taken together, our study reveals that CD47 normally is clustered in lipid rafts on nonapoptotic cells but is diffused in the plasma membrane when apoptosis occurs; this transformation of CD47 greatly reduces the strength of CD47-SIRPα engagement, resulting in the phagocytosis of apoptotic cells.


Assuntos
Antígenos de Diferenciação/imunologia , Apoptose/efeitos da radiação , Antígeno CD47/imunologia , Células Epiteliais/efeitos da radiação , Macrófagos/efeitos da radiação , Receptores Imunológicos/imunologia , Animais , Antígenos de Diferenciação/genética , Apoptose/efeitos dos fármacos , Sítios de Ligação , Antígeno CD47/química , Antígeno CD47/genética , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/efeitos da radiação , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/efeitos dos fármacos , Fagocitose/efeitos da radiação , Cultura Primária de Células , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Receptores Imunológicos/genética , Transdução de Sinais , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/efeitos da radiação , Raios Ultravioleta , beta-Ciclodextrinas/farmacologia
13.
J Cell Biochem ; 116(8): 1595-601, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25649741

RESUMO

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine (RX-5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX-5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of (3) H-labeled RX-5902 to Y593 phospho-p68 RNA helicase. We further demonstrated RX-5902 inhibited the ß-catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX-5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the ß-catenin pathway, such as c-Myc, cyclin D1 and p-c-Jun. Therefore, our study indicates that the inhibition of Y593 phospho-p68 helicase - ß-catenin interaction by direct binding of RX-5902 to Y593 phospho-p68 RNA helicase may contribute to the anti-cancer activity of this compound.


Assuntos
Antineoplásicos/farmacologia , RNA Helicases DEAD-box/metabolismo , Neoplasias/tratamento farmacológico , Piperazinas/farmacologia , Quinoxalinas/farmacologia , beta Catenina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , RNA Helicases DEAD-box/química , Humanos , Neoplasias/metabolismo , Fosforilação , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
J Biol Chem ; 289(37): 25812-21, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25070887

RESUMO

It is long known that pyruvate kinase isoform M2 (PKM2) is released into the circulation of cancer patients. The PKM2 levels in patients have been suggested as a diagnostic marker for many types of cancers. However, it is not known how PKM2 is released in the blood, and whether the circulating PKM2 has any physiological function(s) in tumor progression. In this report, we demonstrate that PKM2 in the blood facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes tumor angiogenesis by increasing endothelial cell proliferation, migration, and cell-ECM adhesion. Only the dimeric PKM2 possess the activity in promoting tumor angiogenesis, which is consistent with the observations that PKM2 in circulation of cancer patients is a dimer form.


Assuntos
Proteínas de Transporte/sangue , Proliferação de Células/genética , Proteínas de Membrana/sangue , Neovascularização Patológica/patologia , Isoformas de Proteínas/sangue , Hormônios Tireóideos/sangue , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Glicólise/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Neoplasias/sangue , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Neovascularização Patológica/sangue , Ensaios Antitumorais Modelo de Xenoenxerto
15.
J Biol Chem ; 288(22): 15971-9, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23576436

RESUMO

Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.


Assuntos
Proliferação de Células , Multimerização Proteica/fisiologia , Proteínas Tirosina Quinases/metabolismo , Piruvato Quinase/metabolismo , Transdução de Sinais/fisiologia , Sítios de Ligação , Linhagem Celular , Humanos , Fosforilação/fisiologia , Proteínas Tirosina Quinases/genética , Piruvato Quinase/genética
16.
Proteins ; 70(3): 938-49, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17847085

RESUMO

Anti-ErbB2 antibodies targeting distinct epitopes can have different biological functions on cancer cells. A21 prepared by surface epitope masking (SEM) method is a tumor-inhibitory anti-ErbB2 monoclonal antibody. Previously we engineered a single chain chimeric antibody chA21 with potential for therapy of ErbB2-overexpressing tumors. Here, we mapped the A21 epitope on ErbB2 extracellular domain (ECD) by screening a combinatorial phage display peptide library, serial subdomain deletion, and mutagenesis scanning. X-ray crystal structure of the A21 scFv fragment at 2.1 A resolution was also determined. A molecular model of Ag-Ab complex was then constructed based on the crystal structures of the A21 scFv and ErbB2 ECD. Some of biological functions of the A21 mAb and its derivative antibodies including their tumor cell growth inhibition and effects on the expression, internalization, and phosphorylation of ErbB2 receptor were also investigated. The results showed that A21 recognized a conformational epitope comprising a large region mostly from ErbB2 extracellular subdomain I with several surface-exposed residues important for the binding affinity. These data provide unique functional properties of A21 that are quite different from two broadly used anti-ErbB2 mAbs, Herceptin and 2C4. It suggested that the A21 epitope may be another valuable target for designing new anti-ErbB2 therapeutics.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Antineoplásicos/química , Antineoplásicos/imunologia , Epitopos/química , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Antineoplásicos/farmacologia , Sítios de Ligação de Anticorpos , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Mapeamento de Epitopos , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Humanos , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única , Relação Estrutura-Atividade , Transfecção , Células Tumorais Cultivadas
17.
Biochim Biophys Acta ; 1769(9-10): 593-602, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17822787

RESUMO

HEC1 (highly expressed in cancer), which localizes to kinetochore in cell mitosis, plays an essential role in chromosome segregation for M phase progression. To clarify the mechanism of its transcriptional regulation, we searched out and isolated its 5'-flanking region. Mapping of this region identified that it is a TATA-less promoter and contains several putative binding sites for different transcription factors. The results from HeLa cells transfected with pGL3 luciferase reporter vectors containing progressive deletion of the HEC1 5'-flanking region demonstrated that two elements containing binding sites for cAMP responsive element binding (CREB) protein and activating transcription factor 4 (ATF4 or CREB2) are critical for transcriptional activity. Mutation of the two elements, not downstream E2F box, resulted in a significant reduction of the promoter activity. Gel shift and supershift assays also demonstrated specific binding of transcription factors to their putative binding sites. Furthermore, overexpression of either CREB or ATF4 enhanced the activation of the HEC1 promoter and overexpression of both of them had an additive effect on the activation of the HEC1 transcription. Conversely, overexpression of dominant negative mutants of either CREB or ATF4 resulted in downregulation of HEC1 mRNA significantly. Our study provided a new insight into a potential mechanism of how transcription factors of CREB family are involved in the regulation of kinetochore protein HEC1 in cancer-related cells.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/biossíntese , Divisão Celular/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/biossíntese , Transcrição Genética/fisiologia , Fator 4 Ativador da Transcrição/genética , Proteínas de Ciclo Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas do Citoesqueleto , Regulação para Baixo/genética , Células HeLa , Humanos , Cinetocoros/metabolismo , Mutação , Proteínas Nucleares/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Elementos de Resposta/fisiologia
18.
Vaccine ; 24(16): 3321-31, 2006 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-16472546

RESUMO

DNA vaccine represents an attractive approach to therapy of chronic hepatitis B virus (HBV) infection because of its ability to generate antigen-specific immunity; nevertheless, there is still a need to increase the potency of DNA vaccine. Mycobacterium tuberculosis heat shock protein70 (HSP70) has both chaperon and cytokine functions, and has been shown to act as an adjuvant when co-administered with peptide antigens or given as fusion proteins. Here we evaluated the effects of two truncated HSP70 molecules, N-terminal domain (HSP70(1-360), amino acids 1-360) and C-terminal domain (HSP70(359-610), amino acids 359-610) of mycobacterial HSP70, on the potency of antigen-specific immunity generated by a HBV DNA vaccination. We found that only the HSP70(359-610)-fused HBV DNA vaccination resulted in a significant increase in hepatitis B surface antigen (HBsAg)-specific humoral response, while the HSP70(1-360)- or the complete HSP70 molecule-fused vaccine did not. Moreover, HSP70(359-610)-fused DNA vaccine did not induce anti-HSP70 antibody. Interestingly, HSP70(359-610) not only enhanced HBsAg-specific cytotoxic lymphocytes (CTL) responses but also overcame the epitope suppression caused by L(d)-restricted epitope. Meanwhile, HSP70(369-610) mediated T helper (Th) cell balance towards Th1 pathway. In a HBV transgenic mouse model, the HSP70(359-610) fusion vaccine facilitated clearance of circulating HBsAg and down-regulation of HBV replication. These results suggested that the truncated mycobacterial HSP70 molecule, HSP70(359-610), might be a superior candidate to deliver the adjuvant function in HBV DNA vaccination instead of the complete HSP70 molecule.


Assuntos
Proteínas de Choque Térmico HSP70/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proliferação de Células , Citocinas/biossíntese , Feminino , Proteínas de Choque Térmico HSP70/genética , Hepatite B/terapia , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/genética , Vacinas contra Hepatite B/administração & dosagem , Vírus da Hepatite B/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mycobacterium tuberculosis/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Vacinas de DNA/administração & dosagem , Replicação Viral
19.
Protein Expr Purif ; 47(1): 249-57, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16403645

RESUMO

Anti-ErbB2 antibodies are used as convenient tools in exploration of ErbB2 functional mechanisms and in treatment of ErbB2-overexpressing tumors. When we employed the yeast Pichia pastoris to express an anti-ErbB2 single-chain antibody (scFv) derived from the tumor-inhibitory monoclonal antibody A21, the yield did not exceed 1-2 mg/L in shake flask cultures. As we considered that the poor codon usage bias may be one limiting factor leading to the inefficient translation and scFv production, we designed and synthesized the full-length scFv gene by choosing the P. pastoris preferred codons while keeping the G+C content at relatively low level. Codon optimization increased the scFv expression level 3- to 5-fold and up to 6-10 mg/L. Northern blotting further confirmed that the increase of scFv expression was mainly due to the enhancement of translation efficiency. Investigation of culture conditions revealed that the maximal cell growth and scFv expression were achieved at pH 6.5-7.0 with 2% casamino acids after 72 h methanol induction. Secreted scFv was easily purified (>95% homogeneous product) from culture supernatants in one step by using Ni2+ chelating affinity chromatography. The yield was approximately 10-15 mg/L. Functional studies showed that the A21 scFv could be internalized with high efficiency after binding to the ErbB2-overexpressing cells, suggesting this regent may prove especially useful for ErbB2-targeted immunotherapy.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Clonagem Molecular , Códon/biossíntese , Pichia , Receptor ErbB-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Códon/química , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Dados de Sequência Molecular , Pichia/genética
20.
Sheng Wu Gong Cheng Xue Bao ; 21(4): 590-6, 2005 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-16176098

RESUMO

Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.


Assuntos
Dissulfetos/imunologia , Escherichia coli/metabolismo , Receptor ErbB-2/biossíntese , Anticorpos Monoclonais/imunologia , Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Solubilidade , Transfecção
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