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Objective:To demonstrate the polymorphism of α chain of bovine major histocompatibility complex(BoLA)classⅠmolecule and domain binding constant chain(Ii).Methods:Total 75 BoLA Iα genes were obtained from three Huaibei cattle and analyzed by molecular biology software;the genes of typical BoLA Iα domains and Ii were cloned,and then inserted into prokaryotic expression plasmid.After induced protein expression;the domains of BoLA Ⅰα chain binding to Ii were detected by pull-down meth-od and Western blot.The recombinant eukaryotic expression plasmids were constructed and the co-localization of BoLA Iα segments with Ii was observed by laser confocal microscopy.Results:Firstly,it was found that there were at least 5 kinds of BoLA Iα in the cloned gene sequence,which were highly polymorphic and they were mainly distributed in the antigen peptide binding region(PBR)of BoLA Ⅰ(α1α2)and cytoplasmic region.Secondly,the prokaryotic recombinant plasmids containing BoLA Ⅰα1α2α3,BoLA Ⅰα1α2 or BoLA Ⅰα 3 were constructed,then they were respectively induced to express and purified,in which,the BoLA Ⅰα1α2α3 and BoLA Ⅰα1α2 had the activity of binding to Ii.Finally,in 293T cells BoLA Ⅰα1α 2α3 or BoLA Ⅰα1α2 was found that could co-localize with Ii,while a single BoLA Ⅰα3 could not.Conclusion:BoLA Ⅰα gene is highly polymorphic.BoLA Ⅰα1α2 is a func-tional fragment that binds to Ii and co-locates intracellular.
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Fragile X syndrome (FXS) is a leading genetic cause of autism intellectual disorder and autism spectrum disorder (ASD), with either limited treatment options or incurable. Fragile X-related gene 1 (FXR1) is a homolog of the Fragile X mental retardation gene 1 (FMR1), the causative gene of FXS, and both are highly homologous and functionally identical. In FXS, both PI3K (AKT/mTOR signaling pathway) and ERK1/2 (MAPK signaling pathway) expression levels were abnormal. Dual speci?city phosphatase 6 (DUSP6) is a member of the mitogen-activated protein kinases (MAPKs) that participates in the crosstalk between the two signaling systems of MEK/ERK and mTOR. By interacting with multiple nodes of MAPK and PI3K/AKT signaling pathways (including the mTOR complex), DUSP6 regulates cellular growth, proliferation, metabolism and participates in pathological processes of cancer and cognitive impairment. However, whether there is an interaction between FXR1P and DUSP6 and the effects of DUSP6 on the growth of SK-N-SH cells remains elusive. As demonstrated by our results, FXR1P was identified in the cytoplasm and nucleus of SK-N-SH cells co-localized with DUSP6, which might have regulated ERK1/2 signaling pathways in SK-N-SH cells. To a certain extent, FXR1P may reverse the negative regulation of ERK1/2 by DUSP6. Moreover, we discovered that not only does DUSP6 inhibit proliferation, but it also promotes the apoptosis of SK-N-SH cells.
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Aim To investigate the role of aberrant cytokeratin 18(CK18) expression in breast cancer metastasis, anrl to elucidate the mechanism by identif¬ying its target.Methods The expression of CK.18 in human breast cancer tissues and cells was determined using immunohistochemical staining and Western blot, respectively.CK18 expression in human breast cancer MCF-7 cells was effectively down-regulated by shRNA, and its effect on breast cancer metastasis was further determined by scratch wound healing assay.The co-lo- cation of CK18 and non-muscle II A ( NMIIA) in MCF-7 cells was examined using double immunofluo¬rescence staining.The effect of CK18 down-regulation on the levels of NMIIA and c-Abl-ERK signaling was quantified by Western blot.Results Lower CK18 lev¬els was found in metastatic than that in primary breast cancer tissues and in highly invasive MDA-MB-231 than that in MCF-7 cells.CK.18 down-regulation pro¬moted the wound repair ability of MCF-7 cells 72h after scratch.CK18 and NMIIA were shown to co-locate in cytoplasm of MCF-7 cells.Moreover, down-regulation of CK18 increased NMIIA expression and activated the c-Abl-ERK signaling pathway in MCF-7 cells.Con¬clusions Down-regulation of CK18 could promote me¬tastasis of breast cancer, which is related to increased NMIIA expression and the activation of c-Abl-ERK sig¬naling pathway.
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Metabolic engineering is a powerful tool to increase many valuable metabolites through enhancing pathways or introducing exogenous pathways from other organisms. As the complexity of the targeted structure increases, many problems arise when the host suffers from flux imbalance and some toxic effects. An emerging approach to solve these problems is the use of synthetic scaffolds to co-localize key enzymes and metabolites of the synthetic pathways, enhance the metabolic flux and limit the interaction between intermediate products in the host cell. Although many scaffolds made of proteins and nucleic acids have been explored and applied to a variety of research to the heterogeneous synthesis of multiple metabolites, success is rather limited. The precise assembly of synthetic scaffolds remains a difficult task. In this review, we summarized the application of synthetic scaffolds in metabolic engineering, and outlined the main principle of scaffold designs, then highlighted the current challenges in their application.
Subject(s)
Metabolic Engineering , Metabolic Networks and Pathways , Proteins , Synthetic BiologyABSTRACT
Objective: To observe the expression characteristics and co-localization of exogenous TRIM22 and HIV capsid protein p24 in glioma cells. Methods: The vectors of pEGFP-N3-TRIM22 or pDsRed1-p24 were transfected into U-251 glioma cells respectively to examine the expression of TRIM22-EGFP or p24-DsRed1 by confocal microscopy. Moreover, we used a confocal z-stacking program to achieve series of optical sections and to rebuild 3-D images by ImageJ 1.50i software to detect the expression characteristics of p24-DsRed1 in U-251 cells. In the end, the vectors of pEGFP-N3-TRIM22 and pDsRed1-p24 were co-transfected into U-251 cells to detect the co-localization between TRIM22 and p24 by confocal microscopy. Results: Confocal microscopy results showed that TRIM22-EGFP or p24-Dsred1 was localized to the cell body as well as to protuberance in U-251 cells, and 3D structural reconstruction showed that p24-Dsred1 could be transferred to foot processes of U-251 cells. Simultaneously, confocal microscopy results also showed that TRIM22 and p24 could be co-localized and their combination could be released by budding in U-251 cells co-transfected with pEGFP-N3-TRIM22 and pDsRed1-p24. Conclusion: TRIM22 co-localized to HIV capsid protein p24 and their combination can be released by budding in glioma cells.
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Bryconamericus is a highly diverse group of characid fishes, being cytogenetic a valuable tool for the delimitation of species. Bryconamericus aff. iheringii (Upper Uruguay/Lower Paraná), B. coeruleus (Upper Paraná), B. cf. ecai e B. cf. eigenmanni (Upper Uruguay) were studied cytogenetically, and presented 2n=52 chromosomes, with interpopulational/interspecific variation of karyotype and fundamental number. Heterochromatin was evidenced in pericentromeric, telomeric and interstitial regions, and it was shown to be an important cytogenetic marker. Single nucleolar organizing regions (NORs) were found in B. cf. eigenmanni, B. cf. ecai and B. aff. iheringii (Lower Paraná), and multiple in B. aff. iheringii (Upper Uruguay) and B. coeruleus, with occurrence of two patterns for the first species, and three for the second. The 5S/18S rDNA-FISH confirmed the location of the NORs and showed single 5S rDNA cistrons only in B. aff. iheringii (Lower Paraná), evidencing the dispersion of both genes, often co-located, in the karyotype of the others species. The data of this work contribute for the delimitation of the species of the genus. Co-localization of ribosomal genes may represent a plesiomorphic condition for the group, and their dispersion suggest the occurrence of duplication, pseudogeneization and transposition events mediated by mobile genetic elements.(AU)
Bryconamericus é um grupo altamente diverso de caracídeos, sendo a citogenética uma valiosa ferramenta para a delimitação de espécies. Bryconamericus aff. iheringii (Alto Uruguai/Baixo Paraná), B. coeruleus (Alto Paraná), B. cf. ecai e B. cf. eigenmanni (Alto Uruguai) foram estudados citogeneticamente, e apresentaram 2n=52 cromossomos, com variação interpopulacional/interespecífica de cariótipo e número fundamental (NF). Heterocromatinas foram evidenciadas nas regiões pericentromérica, telomérica e intersticial, e mostrou-se um importante marcador citogenético. Regiões organizadores de nuclcéolos (RONs) simples foram encontradas em B. cf. eigenmanni, B. cf. ecai e B. aff. iheringii (Baixo Paraná), e múltiplas em B. aff. iheringii (Alto Uruguai) e em B. coeruleus, com a ocorrência de dois padrões de localização para a primeira espécie, e três para a segunda. A FISH-DNAr 5S/18S confirmou a localização das RONs e mostrou cístrons simples de DNAr 5S apenas em B. aff. iheringii (Baixo Paraná), evidenciando a dispersão de ambos os genes, muitas vezes co-localizados, no cariótipo das demais espécies. Os dados deste trabalho contribuem para a delimitação das espécies do gênero. A co-localização dos genes ribossomais pode representar uma condição plesiomórfica para o grupo, e sua dispersão sugere a ocorrência de eventos de duplicação, pseudogenização e transposição mediada por elementos genéticos móveis.(AU)
Subject(s)
Gene Transfer, Horizontal , Cytogenetics/methods , Characidae/genetics , DNA, Ribosomal , Genetic MarkersABSTRACT
Previously developed Asn-Gly-Arg (NGR) peptide-modified multifunctional poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG)-based nanoparticles (TPIC) have been considered to be promising carriers for the co-delivery of DNA and doxorubicin (DOX). As a continued effort, the aim of the present study was to further evaluate the interaction between TPIC and human umbilical vein endothelial cells (HUVEC) to better understand the cellular entry mechanism. In the present investigation, experiments relevant to co-localization, endocytosis inhibitors and factors influencing the internalization were performed. Without any treatment, there was no co-localization between aminopeptidase N/CD13 (APN/CD13) and caveolin 1 (CAV1). However, co-localization between CD13 and CAV1 was observed when cells were incubated with an anti-CD13 antibody or TPIC. As compared with antibody treatment, TPIC accelerated the speed and enhanced the degree of co-localization. TPIC entered HUVEC not only together with CD13 but also together with CAV1. However, this internalization was not dependent on the enzyme activity of CD13 but could be inhibited by methyl--eyclodextfin (MCD), further identifying the involvement of caveolae-mediated endocytosis (CvME). This conclusion was also verified by endocytosis inhibitor experiments.
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Transitional cell carcinoma (TCC) of urinary bladder belongs to glutathione S-transferase P1 (GSTP1) overexpressing tumors. Upregulated GSTP1 in TCC is related to apoptosis inhibition. This antiapoptotic effects of GSTP1 might be mediated through protein:protein interaction with c-Jun NH2-terminal kinase (JNK). Herein, we analyzed whether a direct link between GSTP1 and JNK exists in TCC. The presence of GSTP1/JNK complexes was analyzed by immunoprecipitation and Western blotting in 20 TCC specimens, obtained after surgery. Co-localization of GSTP1 and JNK was also investigated in the 5637 TCC cell line by immunofluorescence confocal microscopy. By means of immunoprecipitation we show for the first time the presence of GSTP1/JNK complexes in all TCC samples studied. A co-localization of GSTP1 and JNK was also demonstrated in the 5637 TCC cell line by means of confocal microscopy. Protein-protein interactions, together with co-localization between GSTP1 and JNK provide evidence that GSTP1 most probably inhibits apoptosis in TCC cells by non-covalent binding to JNK.