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1.
BMB Rep ; 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38919020

RESUMO

The heterotrimeric molecular motor kinesin-2 is involved in the microtubule-dependent transport of intracellular cargo. It consists of two distinct motor subunits (KIF3A, and KIF3B) and a non-motor subunit, kinesin-associated protein 3 (KAP3). The cargo-binding domain (CBD) at the carboxyl (C)-terminus of KIF3s plays an important role in the interaction with several different binding proteins. To identify the binding proteins for heterotrimeric kinesin-2, we performed a yeast two-hybrid screen and found a new interaction with Disables-1 (Dab1), the intracellular adaptor protein of reelin receptors. Dab1 bound to the CBD of KIF3A, but did not interact with the C-terminal domain of KIF3B, KIF5B, KIF17 or KAP3. The phosphotyrosine binding (PTB) domain-containing region of Dab1 is essential for the interaction with KIF3A. KIF3A interacted with GST-Dab1, and GST-CaMKIIα, but did not interact with GST-apolipoprotein E receptor 2 (ApoER2)-C or with GST alone. When co-expressed in HEK-293T cells, KIF3A co-precipitated with Dab1, but not with KIF5B. Dab1 and KIF3A were co-localized in cultured cells. We also identified deduced cell surface expression of ApoER2 in KIF3A dominant-negative cells. These results suggest that the KIF3A plays a role in the intracellular trafficking of ApoER2 to the cell surface.

2.
Biomed Rep ; 6(1): 46-50, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28123706

RESUMO

Kinesin 1 is a member of the kinesin superfamily proteins (KIFs) of microtubule-dependent molecular motor proteins that transport organelles and protein complexes in cells. Kinesin 1 consists of a homo- or hetero-dimer of kinesin heavy chains (KHCs), often, although not always, associated with two kinesin light chains (KLCs). KLCs are non-motor proteins that associate with many different binding proteins and cargoes, but their binding partners have not yet been fully identified. In the present study, a yeast two-hybrid system was used to identify proteins that interact with the tetratricopeptide repeat (TPR) domain of KLC1. The results of the current study revealed an interaction between the TPR domain of KLC1 and FUN14 domain-containing protein 1 (FUNDC1), which is a mitochondrial outer membrane protein mediating hypoxia-induced mitophagy. FUNDC1 bound to the six TPR motif-containing regions of KLC1 and did not interact with KIF5B (a motor subunit of kinesin 1) and KIF3A (a motor subunit of kinesin 2) in the yeast two-hybrid assay. The cytoplasmic amino N-terminal domain of FUNDC1 is essential for interaction with KLC1. When co-expressed in HEK-293T cells, FUNDC1 co-localized with KLC1 and co-immunoprecipitated with KLC1, but not KIF5B. Collectively, these results indicate that KLC1 may potentially compete with LC3, a key component for autophagosome formation, to interact with FUNDC1.

4.
Cell Immunol ; 289(1-2): 55-62, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24727157

RESUMO

Transglutaminase 2 (TG2) is a ubiquitous enzyme involved in diverse biological processes. Recently, its function in adaptive immune responses has begun to emerge. Its presence and functions in B cells and T cells, for example, have been reported. However, those in dendritic cells (DCs), the principal antigen-presenting cells, are as yet unexplored in murine system. In this study, we first investigated the expression of TG2 in murine bone marrow-derived DCs, and then compared the functioning of these cells in the presence or absence of this enzyme using wild-type (WT) and TG2(-/-) mice. We found that the WT DCs expressed TG2 both in the cytoplasm and on the cell surface, both of which were elevated after LPS stimulation. Unexpectedly, between WT and TG2(-/-) DCs, there were no remarkable differences in cytokine secretion, IL-10 and IL-12, and neither in the expression of surface molecules CD80, CD86, and MHC II, excepting a moderate decrease of CD40 expression on the TG2(-/-) DCs. However, when T cells were stimulated with TG2(-/-) DCs, they showed decreased levels of proliferation, CD69 and CD25 expression, and IFN-γ secretion. The addition of anti-TG2 antibody to the WT DC-T cell co-culture resulted in decreased T cell activation. By immunofluorescence staining, TG2 was observed at DC-T cell interface (contact point). Taken together, we propose that TG2 on the surface of DCs modulates the DC-T cell interaction.


Assuntos
Comunicação Celular/imunologia , Células Dendríticas/imunologia , Proteínas de Ligação ao GTP/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Transglutaminases/metabolismo , Animais , Anticorpos/imunologia , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2/biossíntese , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Antígenos CD40/biossíntese , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/enzimologia , Proteínas de Ligação ao GTP/genética , Antígenos de Histocompatibilidade Classe II/biossíntese , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Lectinas Tipo C/biossíntese , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Knockout , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética
5.
Immunobiology ; 219(7): 554-64, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24698552

RESUMO

Vitamin C has been found to stimulate dendritic cells (DCs) to secrete more IL-12 and thereby drive naïve CD4(+) T cells to differentiate into Th1 cells. In the present study, we evaluated the effect of these vitamin C-treated DCs on CD8(+) T cell differentiation both in vitro and in vivo. Mouse bone marrow-derived DCs were prepared in the presence of GM-CSF and IL-15. With vitamin C treatment, these DCs, when LPS-stimulated, secreted more IL-12p70 and IL-15 than did untreated DCs. And when co-cultured with T cells, they yielded a higher frequency of IFN-γ(+) CD8(+) T cells. Moreover, we found that administering vitamin C-treated and tumor lysate-loaded DCs into mice yielded a higher frequency of CD44(high) CD62L(low) CD8(+) effector and effector memory T cells, which showed an increased ex vivo killing effect of the tumor cells. These DCs also elicited enhanced protective effects against inoculated tumor cells, most probably by way of the increased cytotoxic T cells, as was revealed by the decreased growth of the inoculated tumor cells in these mice. This ex vivo vitamin C treatment effect on DCs can be considered as a strategy for boosting DC vaccination potency against tumors.


Assuntos
Ácido Ascórbico/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Memória Imunológica/imunologia , Animais , Ácido Ascórbico/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/transplante , Citometria de Fluxo , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Imunoterapia Adotiva/métodos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-15/imunologia , Interleucina-15/metabolismo , Selectina L/imunologia , Selectina L/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Análise de Sobrevida , Vacinação/métodos , Vitaminas/imunologia , Vitaminas/farmacologia
6.
Free Radic Biol Med ; 67: 81-90, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24184603

RESUMO

Given the involvement of oxidative stress in liver-disease- or hepato-toxicant-induced hepatic damage and fibrosis, antioxidants are an effective preventive and therapeutic tool. The beneficial results of vitamin C, one of the physiological antioxidants, have been observed both in experimental animals and in humans. However, most of these studies have been concerned with supplementary vitamin C; the effects of under vitamin C insufficiency, which humans sometimes confront, have not been substantially investigated. In the present study, we established a vitamin C-insufficient animal model (half-to-normal serum vitamin C concentration) with gulo(-/-) mice that cannot synthesize vitamin C, and induced hepatotoxicity by means of thioacetamide (TAA) injections twice a week for 18 weeks. Additionally, we explored the direct effects of vitamin C both on immortalized human hepatic stellate LX-2 cells and on rat primary hepatic stellate cells. Vitamin C insufficiency resulted in a decreased survival rate and increased serum markers for hepatocyte damage, such as alanine aminotransferase and aspartate aminotransferase. Concomitantly, the levels of reactive oxygen species (ROS) and lipid peroxides in the liver were increased. Histological examinations of the vitamin C-insufficient liver revealed increases in collagen fiber deposition and activated-hepatic-stellate-cell number. Vitamin C, when directly applied to the LX-2 cells as well as the rat primary hepatic stellate cells, suppressed not only proliferation but hydrogen peroxide-induced collagen expression as well. In conclusion, vitamin C insufficiency exacerbated TAA-induced hepatotoxicity. These effects seem to be mainly from insufficient scavenging of ROS in the liver, and possibly in part, by directly affecting hepatic stellate cells.


Assuntos
Deficiência de Ácido Ascórbico/metabolismo , Ácido Ascórbico/administração & dosagem , L-Gulonolactona Oxidase/genética , Cirrose Hepática/metabolismo , Alanina Transaminase/sangue , Animais , Deficiência de Ácido Ascórbico/complicações , Deficiência de Ácido Ascórbico/genética , Deficiência de Ácido Ascórbico/patologia , Aspartato Aminotransferases/sangue , Colágeno/biossíntese , Colágeno/genética , Expressão Gênica , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , L-Gulonolactona Oxidase/deficiência , Peroxidação de Lipídeos/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/complicações , Cirrose Hepática/genética , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Tioacetamida
7.
Immunol Lett ; 147(1-2): 18-28, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22658978

RESUMO

Tansglutaminase 2 (TG2) mediates post-translational modifications of proteins that are involved in a variety of biological processes. Previous reports suggest an involvement of TG2 in adaptive immune responses. However, little has been elucidated in this regard. We explored, in this study, the role of TG2 in humoral immune response to keyhole limpet hemocyanin (KLH) using TG2(-/-) C57BL/6 mice. After primary and secondary immunization with KLH, the serum titer of the antigen-specific antibody was higher in the TG2(-/-) mice than in the wild-type mice. Not only the amount of the specific antibody was increased, but also the affinity of the antibody was estimated as higher in these mice. The TG2(-/-) spleen showed an enhanced germinal center response with higher percentages of GL7(+) germinal center B cells and B220(low) CD138(high) plasma cells. In addition, germinal center B cells from TG2(-/-) mice showed an increased expression of B lymphocyte induced maturation protein-1 (Blimp-1) as well as activation-induced cytidine deaminase (AID). Our results, in sum, indicate a regulatory role of TG2 in humoral immune response to a protein antigen, probably by way of modulating the expression level of proteins related to humoral immune reposes.


Assuntos
Formação de Anticorpos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Citidina Desaminase/genética , Proteínas de Ligação ao GTP/metabolismo , Fatores de Transcrição/genética , Transglutaminases/metabolismo , Animais , Anticorpos/sangue , Anticorpos/imunologia , Afinidade de Anticorpos/imunologia , Formação de Anticorpos/genética , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Hemocianinas/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genética
8.
Cell Immunol ; 266(2): 192-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21074755

RESUMO

Vitamin C has been reported to shift immune responses toward Th1. In this study, we evaluated whether this effect was by way of dendritic cells. Murine dendritic cells (DCs) were prepared from bone marrow precursors. DCs treated with vitamin C secreted an increased amount of IL-12p70 after activation with LPS. These cells rendered naïve T cells to secrete more Th1 cytokine, IFN-γ, and less Th2-cytokine, IL-5 in the culture supernatants. Vitamin C-treatment also increased phosphorylation of p38 and ERK1/2 in DCs. p38 inhibitor in culture media suppressed the effect of vitamin C to elevate IL-12p70 secretion. In contrast, ERK inhibitor elevated IL-12p70 secretion. In summary, vitamin C taken up into DCs increased IL-12p70 secretion of these cells by modulating the activation of signal molecules, and thus shifted immune responses toward Th1. These data provide us a new insight on the role of vitamin C in modulating immune responses.


Assuntos
Ácido Ascórbico/farmacologia , Células Dendríticas/efeitos dos fármacos , Interleucina-12/metabolismo , Linfócitos T/efeitos dos fármacos , Células Th1/imunologia , Animais , Células Cultivadas , Células Dendríticas/imunologia , Interferon gama/imunologia , Interleucina-5/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/imunologia , Espécies Reativas de Oxigênio/análise , Linfócitos T/imunologia
9.
Anat Cell Biol ; 43(1): 25-35, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21190002

RESUMO

Vitamin C, one of essential micronutrients, has been reported to modulate the humoral immune responses in some mammals. We investigated whether vitamin C might modulate this response in mice by directly affecting B cells. Splenic B cells were isolated and activated by CD40- and B cell receptor-ligation in vitro. The cells were cultured with a pretreatment of vitamin C from 0 to 1 mM of concentrations. Vitamin C slightly increased apoptosis of B cells dose-dependently and behaved as an antioxidant. We found that in vivo administration of vitamin C by intraperitoneal injection affected isotype switching as previously reported: the titer of antigen-specific IgG1 antibody was decreased, while that of IgG2a was unaffected. Somewhat different from those observed in vivo, in vitro exposure to vitamin C slightly decreased isotype switching to IgG1 and increased isotype switching to IgG2a. Pretreatment with vitamin C in the safe range did not affect either proliferation of cultured B cells or the expression of CD80 and CD86 in those cells. Taken together, in vivo results suggest that vitamin C acts to modulate isotype switching in the mouse. However, because of our in vitro results, we suggest that the modulation exerted by vitamin C in vivo is by indirectly affecting B cells, perhaps by directly influencing other immune cells such as dendritic cells.

10.
Hybridoma (Larchmt) ; 29(2): 147-52, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20443707

RESUMO

A human monoclonal antibody can be a good method for tumor diagnosis and treatment. This study is aimed at the generation of human antibody fragments against urokinase plasminogen activator (uPA) known to be related to tumor metastasis using the naive human antibody phage display library. Three clones--A2, A8, and E4--were selected from 1 x 10(10) sized human naïve antibody phage library using BIAcore rescue and screen. Clone A8 was finally selected by flow cytometry against Hep3 and HT1080, uPA overexpressing tumor cell lines. A8 clone consisted of 324 bp lambda and 402 bp heavy chains. The affinity (K(D)) of purified A8 antibody fragments was 1.44 x 10(-8) M(-1). The antibody fragment was reacted with HT1080 in a dose-dependent manner but not reacted with LS513 normal fibroblast. In this study, uPA specific human monoclonal antibody fragment A8 was made with BIAcore selection. Selected A8 was bound specifically to uPA expressed on the tumor cell surface. Further study for the application of A8 antibody clones will be needed.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos de Cadeia Única/imunologia , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA/genética , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Anticorpos de Cadeia Única/genética , Ressonância de Plasmônio de Superfície
11.
Anat Cell Biol ; 43(4): 294-302, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21267403

RESUMO

Asthma is a Th2-dependent disease mediated by IgE and Th2 cytokines, and asthmatic patients suffer from oxidative stresses from abnormal airway inflammation. Vitamin C is a micro-nutrient functioning as an antioxidant. When administered at a mega-dose, vitamin C has been reported to shift immune responses toward Th1. Thus, we tried to determine whether vitamin C exerted beneficial effects in asthma animal model. Asthma was induced in mice by sensitizing and challenging with ovalbumin. At the time of challenge, 3~5 mg of vitamin C was administered and the effects were evaluated. Vitamin C did not modulate Th1/Th2 balance in asthma model. However, it decreased airway hyperreactivity to methacholine, decreased inflammatory cell numbers in brochoalveolar lavage fluid, and moderate reduction of perivascular and peribronchiolar inflammatory cell infiltration. These results suggest that vitamin C administered at the time of antigen challenge exerted anti-inflammatory effects. Further studies based on chronic asthma model are needed to evaluate a long-term effect of vitamin C in asthma. In conclusion, even though vitamin C did not show any Th1/Th2 shifting effects in this experiment, it still exerted moderate anti-inflammatory effects. Considering other beneficial effects and inexpensiveness of vitamin C, mega-dose usage of vitamin C could be a potential supplementary modality for the management of asthma.

12.
Immunobiology ; 214(4): 311-20, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19327547

RESUMO

Vitamin C is an essential micronutrient, which has been implicated in various biological processes, including immune response. In fact, in vivo administration of vitamin C modulates T cell proliferation and cytokine secretion. In this study, we analyzed the mechanism by which mouse T cells take up vitamin C, and whether this uptake directly affected T cell functions. T cells internalized more vitamin C when they were activated, due to enhanced glucose transporter (GLUT)-1 and GLUT-3 expression that persisted up to 48 h after activation. Blocking oxidation of ascorbic acid (the reduced form of vitamin C) in the culture medium with 1,4-dithio-threitol (DTT) almost completely inhibited the enhanced vitamin C uptake. The presence of vitamin C at low concentrations during in vitro T cell activation did not affect proliferation or cytokine secretion (IFN-gamma, TNF-alpha, or IL-4) in response to PMA/ionomycin. In contrast, high concentrations (0.25-0.5 mM) of vitamin C lowered cell viability, reduced thymidine uptake, and decreased cytokine secretion. In conclusion, activated T cells upregulated GLUT-1 and -3 to increase vitamin C uptake. They took up vitamin C mostly in its oxidized form, rarely in its reduced form. Application of vitamin C to T cells in vitro did not recapitulate previously reported in vivo responses to vitamin C, suggesting that in vivo, vitamin C modulates T cells indirectly through other components of the microenvironment.


Assuntos
Ácido Ascórbico/metabolismo , Ácido Desidroascórbico/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Linfócitos T/metabolismo , Animais , Ácido Ascórbico/química , Ácido Ascórbico/imunologia , Transporte Biológico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/imunologia , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Ácido Desidroascórbico/química , Ácido Desidroascórbico/imunologia , Ditiotreitol/farmacologia , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/imunologia , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/imunologia , Separação Imunomagnética , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução/efeitos dos fármacos , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Timidina/metabolismo
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