ABSTRACT
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.
ABSTRACT
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
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.
ABSTRACT
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
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.
ABSTRACT
The UL49.5 gene of most herpesviruses is conserved and encodes glycoprotein N. However, the UL49.5 protein of duck enteritis virus (DEV) (pUL49.5) has not been reported. In the current study, the DEV pUL49.5 gene was first subjected to molecular characterization. To verify the predicted intracellular localization of gene expression, the recombinant plasmid pEGFP-C1/pUL49.5 was constructed and used to transfect duck embryo fibroblasts. Next, the recombinant plasmid pDsRed1-N1/glycoprotein M (gM) was produced and used for co-transfection with the pEGFP-C1/pUL49.5 plasmid to determine whether DEV pUL49.5 and gM (a conserved protein in herpesviruses) colocalize. DEV pUL49.5 was thought to be an envelope glycoprotein with a signal peptide and two transmembrane domains. This protein was also predicted to localize in the cytoplasm and endoplasmic reticulum with a probability of 66.7%. Images taken by a fluorescence microscope at different time points revealed that the DEV pUL49.5 and gM proteins were both expressed in the cytoplasm. Overlap of the two different fluorescence signals appeared 12 h after transfection and continued to persist until the end of the experiment. These data indicate a possible interaction between DEV pUL49.5 and gM.
Subject(s)
Animals , Ducks/virology , Genes, Viral/genetics , Mardivirus/genetics , Membrane Glycoproteins/genetics , Microscopy, Fluorescence , Phylogeny , Polymerase Chain Reaction/veterinary , Viral Envelope Proteins/geneticsABSTRACT
Objective To study the expression and role of galanin in the hypothalamus of rat with the drug-induced hyperprolactinemia.Methods Hyperprolactinemia was induced by daily intraperitoneal injections 50 mg/kg sulpiride solution.The protein in the hypothalamus of rat was extracted to determine the expression levels of galanin with Western Blot.The expression and colocalization of galanin and dopamine in model and control group were observed with immunofluorescence.Results The model group showed a significant increase of serum prolactin (PRL) level and a significant decrease of serum estradiol (E2) level,as compared to the control group ((15.74±2.49) ng/ml vs (10.25±1.29) ng/ml and (4.24±0.69)pg/ml vs (9.56±3.25) pg/ml,respectively,P＜0.05).Both the expression level of galanin and the number of neurons coexisting with galanin and dopamine were decreased in the hypothalamus of the hyperprolactinemia rat compared with the control group.Western Blot revealed that,compared to the control group,the sulpiride model group had a significant increase of galanin but not TH (0.405±0.112 vs 0.985±0.158,P＜0.05 and 0.871 ± 0.046 vs 0.890±0.054,P＞ 0.05,respectively).Conclusion Galanin expression level has decrease in the hypothalamus of the hyperprolactinemia rat,which contributes to the reduction of dopaminergic neurons.
ABSTRACT
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.
Subject(s)
Humans , Glutathione Transferase/genetics , JNK Mitogen-Activated Protein Kinases , Urinary Bladder Neoplasms/genetics , Carcinoma, Transitional Cell , PhenotypeABSTRACT
Vitiligo has been associated with various disorders including thyroid disease, type I diabetes, alopecia areatal and pernicious anemia. It has been purported to be caused by autoimmune response. Vitiligo and alopecia areata occurring in the same patient has often been found, but colocalization of these two diseases has been rarely reported. It is suggested that this concurrence can result from a nonspecific immune mechanism affecting not only the melanocyte but the epithelium of the hair follicle as well. Thus colocalization can occur due to costimulation of a helper T-cell-mediated immunologic response or through inactivation of a suppressor-mediated response and other composite mechanisms such as neurogenic factors, stress and infection. We report a 17-year-old Korean female who presented with a 1-year history of alopecia and depigmentation on the same site of the left eyebrow. We diagnosed this skin lesion as colocalization of vitiligo and alopecia areata by both clinical and histopathological findings.
Subject(s)
Adolescent , Female , Humans , Alopecia Areata , Alopecia , Anemia, Pernicious , Autoimmunity , Epithelium , Eyebrows , Hair Follicle , Melanocytes , Skin , Thyroid Diseases , VitiligoABSTRACT
Both vitiligo and psoriasis are common skin disorders, which rarely coexist. We report a case of psoriasis which developed on a vitiligo lesion in a 58-year-old woman. We also discuss the possible mechanism of co-localization of vitiligo and psoriasis, including the role of autoimmunity, Koebner phenomenon, and common neuropeptides.
Subject(s)
Female , Humans , Middle Aged , Autoimmunity , Neuropeptides , Psoriasis , Skin , VitiligoABSTRACT
Lichen planus and vitiligo are common skin disorders, which rarely coexist. We report a case of colocalization of lichen planus and vitiligo in a 42-year-old woman. Furthermore, herein, we discuss the possible mechanism of colocalization of lichen planus and vitiligo, including the role of actinic damage in the initiation of lymphocytic infiltrates of lichen planus in vitiliginous skin.
Subject(s)
Adult , Female , Humans , Actins , Lichen Planus , Lichens , Skin , VitiligoABSTRACT
The role of GABA or glycine as an inhibitory neurotransmitter is well established, and GABAergic or glycinergic neurons appear to play an important role in the mammalian retinas. It has been reported that certain amacrine, bipolar, displaced amacrine and ganglion cells are consistently labeled with anti-GABA or anti-glycine antisera in the mammalian retinae so far, and it has been suggested that colocalization of GABA and glycine within the retinal neurons could be common in the mammalian retina by recent immunecytochemical and electrophysiological studies. This study was conducted to localize GABAergic and glycinergic neurons and to define whether GABA and glycine are colocalized within same retinal neurons of the rat retina by immunocytochemical method using anti-GABA and anti-glycine antisera. The results were as follows : 1. GABAergic neurons of the rat retina were amacrine, interplexiform, bipolar, displaced amacrine and ganglion cells, and processes of GABAergic neurons formed dense networks with distinct two bands in the inner plexiform layer. 2. Glycinergic neurons were amacrine, bipolar, displaced amacrine and ganglion cells,and their processes were evenly distributed as dense networks through whole inner plexiform layer. 3. 28.5% of GABA immunoreactive amacrine cells and 9.8% of GABA immunoreactive bipolar cells located in the inner nuclear layer,and 11.9% of labeled neurons located in the ganglion cell layer showed glycine immunoreactivity in the rat retina. These results demonstrate that GABA and glycine, major inhibitory neurotransmitters, are colocalized within certain amacrine and displaced amacrine cells, and a few bipolar cells, and that neurons synthesizing and utilizing both GABA and glycine as their neurotransmitters may play an unique role in the visual processing in the rat retina.
Subject(s)
Animals , Rats , Amacrine Cells , GABAergic Neurons , gamma-Aminobutyric Acid , Ganglion Cysts , Glycine , Immune Sera , Neurons , Neurotransmitter Agents , Retina , Retinal NeuronsABSTRACT
Objective:To investigate the intracellular localization and association of chicken major histocompatibility complex class Ⅰsubunits with invariant chain.Methods:At first,the sequences of MHCⅠ ? and ?2m subunits were amplified with RT-PCR.Secondly,we constructed the eukaryotic expression plasmids of MHCⅠ ? and MHCⅠ?2m that was fused with red or enhanced green fluorescent protein,respectively.The recombinant plasmids of MHC Ⅰsubunits and pEGFP-C1-Ii that could express enhanced green fluorescent protein were transiently transfected into COS-7 cells with Lipofectamine 2000.Immunofluorescence microscopy was carried out to detect the expression and intracellular localization of Ii and MHC Ⅰsubunits,and immunoprecipitation was used for analyzing their association.Results:The colocalization was found in endocytic compartments when GFP-Ii,MHCⅠ?-RFP and MHCⅠ?2m-RFP were co-expressed in COS-7 cells.Immunoprecipitation of Ii showed that GFP-Ii co-isolated with MHCⅠ?-GFP and MHCⅠ?2m-GFP subunits when COS-7 cells were transiently transfected with pEGFP-C1-Ii,pEGFP-N1-MHCⅠ? and pEGFP-N1-MHCⅠ?2m.Conclusion:Chicken Ii can not effectively associate with single ? or ?2m subunits from chicken MHC Ⅰmolecules,but Ii and the integrated MHC Ⅰmolecule can form the complexes that are colocalized in endomembrane system of COS-7 cells.