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1.
Int J Mol Sci ; 21(20)2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33086574

RESUMO

We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.


Assuntos
Cisplatino/farmacologia , Neoplasias/terapia , Radioimunoterapia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/patologia , Resultado do Tratamento
2.
Nucl Med Commun ; 41(7): 688-695, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32371673

RESUMO

OBJECTIVE: We previously reported In-labeled anti-cadherin17 (CDH17) IgG visualized CDH17-positive gastric cancer xenografts. Unfortunately, a long waiting time was required to obtain high-contrast images due to long blood retention (blood half-life: 26 h). To accelerate blood clearance, we have developed anti-CDH17 minibody (D2101 minibody) and evaluated the pharmacokinetics in gastric cancer mouse models. METHODS: Two different single chain Fvs (scFvs), D2101 mutant and D2111, were developed from each parental IgG. The binding ability to CDH17 and stability in plasma were evaluated. D2101 minibody, constructed based on D2101 mutant scFv, was labeled with Cu (Cu-D2101 minibody), and the in-vitro and in-vivo properties were evaluated by cell ELISA, biodistribution experiments, and PET imaging in mice bearing CDH17-positive AGS and CDH17-negative MKN74 tumors. RESULTS: D2101 mutant and D2111 scFvs showed similar affinities to CDH17. D2101 mutant scFv was more stable than D2111 scFv in plasma. No loss of binding affinity of the D2101 minibody by chelate conjugation and radiolabeling procedures was observed. The biodistribution of Cu-D2101 minibody showed high uptake in AGS tumors and low uptake in MKN74. The blood half-life of Cu-D2101 minibody was 6.5 h. Improved blood clearance of Cu-D2101 minibody provided high tumor-to-blood ratios compared with the previous results of parental IgG in AGS xenograft mice. PET studies showed consistent results with biodistribution studies. CONCLUSIONS: Cu-D2101 minibody exhibited higher tumor-to-blood ratios at earlier time points than those of the radiolabeled parental IgG. Cu-D2101 minibody has potential as an immunoimaging agent for CDH17-positive tumors.


Assuntos
Transformação Celular Neoplásica , Radioisótopos de Cobre , Fragmentos de Imunoglobulinas/química , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/patologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo , Camundongos , Tomografia por Emissão de Pósitrons , Fatores de Tempo , Distribuição Tecidual
3.
Biotechnol Rep (Amst) ; 25: e00418, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31993343

RESUMO

Biparatopic fragment antibodies can overcome deficiencies in avidity of conventional antibody fragments. Here, we describe a technology for generating biparatopic antibodies through two-step targeting using a pair of polypeptides, SpyTag and SpyCatcher, that spontaneously react to form a covalent bond between antibody fragments. In this method, two antibody fragments, each targeting different epitopes of the antigen, are fused to SpyTag and to SpyCatcher. When the two polypeptides are serially added to the antigen, their proximity on the antigen results in covalent bond formation and generation of a biparatopic antibody. We validated the system with purified recombinant antigen. Results in antigen-overexpressing cells were promising although further optimization will be required. Because this strategy results in high-affinity targeting with a bipartite molecule that has considerably lower molecular weight than an antibody, this technology is potentially useful for diverse applications.

4.
Ann Nucl Med ; 34(1): 13-23, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605356

RESUMO

OBJECTIVE: Cadherin-17 (CDH17) is a transmembrane protein that mediates cell-cell adhesion and is frequently expressed in adenocarcinomas, including gastric cancer. CDH17 may be an effective diagnostic marker for the staging of gastric cancer. Here, we developed an 111In-labeled anti-CDH17 monoclonal antibody (Mab) as an imaging tracer and performed biodistribution and single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies using mice with CDH17-positive gastric cancer xenografts. CDH17 expression in gastric cancer specimens was also analyzed. METHODS: The cross-reactivity and affinity of our anti-CDH17 Mab D2101 was evaluated by surface plasmon resonance analysis and cell enzyme-linked immunosorbent assay, respectively. Biodistribution and SPECT/CT studies of 111In-labeled D2101 (111In-D2101) were performed. CDH17 expression in gastric cancer specimens was evaluated by immunohistochemistry. RESULTS: Surface plasmon resonance analysis revealed that D2101 specifically recognizes human CDH17, but not murine CDH17. The affinity of D2101 slightly decreased as a result of the radiolabeling procedures. The biodistribution study revealed high uptake of 111In-D2101 in tumors (maximum, 39.2 ± 9.5% ID/g at 96 h postinjection), but low uptake in normal organs, including the stomach. Temporal SPECT/CT imaging with 111In-D2101 visualized tumors with a high degree of tumor-to-nontumor contrast. Immunohistochemical analysis revealed that, compared with HER2, which is a potential marker of N-stage, CDH17 had a higher frequency of positivity in specimens of primary and metastatic gastric cancer. CONCLUSION: Our 111In-anti-CDH17 Mab D2101 depicted CDH17-positive gastric cancer xenografts in vivo and has the potential to be an imaging probe for the diagnosis of primary lesions and lymph-node metastasis in gastric cancer.


Assuntos
Caderinas/imunologia , Imunoconjugados/química , Imunoconjugados/imunologia , Radioisótopos de Índio/química , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/patologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoconjugados/farmacocinética , Marcação por Isótopo , Metástase Linfática , Camundongos , Estadiamento de Neoplasias , Distribuição Tecidual
5.
J Biochem ; 164(1): 65-76, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29924367

RESUMO

Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody-antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2 kcal mol-1 of free energy. Collectively, our results suggest that antibodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role.


Assuntos
Anticorpos/química , Peptídeos/química , Reações Antígeno-Anticorpo , Calorimetria , Ligação de Hidrogênio , Modelos Moleculares , Peptídeos/isolamento & purificação , Redobramento de Proteína
6.
Artigo em Inglês | MEDLINE | ID: mdl-29474162

RESUMO

Cadherin-17 (CDH17) is highly expressed in gastric cancer and is thus considered to be a good target for antibody therapy. CDH17 is classified as a nonclassical cadherin, in that it is composed of seven extracellular cadherin domains. We generated anti-CDH17 monoclonal antibodies (mAbs) which recognize the extracellular domain of CDH17. Competitive assay using AGS, a gastric cancer cell line, cells revealed that five selected anti-CDH17 mAbs recognize different epitopes on CDH17. As AGS cells were shown to exhibit broad expression pattern of CDH17 by flow cytometry, we separated three clones with a low (10,000/cell), medium (50,000/cell), and high (200,000/cell) expression level, designating them as AGSlow, AGSmed, and AGShigh, respectively. The mAbs, coupled with saporin, exhibited effective cytotoxicity to AGShigh, but poor cytotoxicity to AGSlow. By contrast, the immunotoxin cocktail using the three clones D2101, D2005, and D2008, which recognize different epitopes, exhibited efficient cytotoxicity, even to the AGSlow group. The effect of the immunotoxin cocktail is synergistic, as the combination index was demonstrated to be below 1.0, as calculated by the method of Chou and Talalay using CalcuSyn software. These results suggest that the immunotoxin cocktail targeted to multiple epitopes has synergistic effects on low expression level cells, which expand the applicable range of immunotoxin therapy for cancer.


Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Caderinas/imunologia , Sinergismo Farmacológico , Epitopos/imunologia , Imunotoxinas/farmacologia , Neoplasias Gástricas/patologia , Biomarcadores Tumorais/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/metabolismo , Humanos , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Células Tumorais Cultivadas
7.
J Biochem ; 162(3): 203-210, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28637250

RESUMO

Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Antígenos/química , Epitopos/química , Humanos , Fragmentos de Imunoglobulinas/imunologia
8.
PLoS One ; 12(4): e0175452, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28394950

RESUMO

Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Células COS , Cricetulus , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Células HEK293 , Humanos , Hibridomas/metabolismo , Imunização , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Precursoras de Linfócitos B/imunologia , Prostaglandina D2/análogos & derivados , Prostaglandina D2/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Obstrução Ureteral/imunologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , beta-Arrestinas/metabolismo
9.
Anal Biochem ; 504: 41-9, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27095060

RESUMO

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface.


Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Baculoviridae/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Animais , Baculoviridae/genética , Células CHO , Cricetulus , Humanos , Cinética , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Proteínas Roundabout
10.
Nature ; 526(7573): 397-401, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26416735

RESUMO

The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.


Assuntos
Frutose/metabolismo , Transportador de Glucose Tipo 5/química , Transportador de Glucose Tipo 5/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Bovinos , Membrana Celular/metabolismo , Cristalografia por Raios X , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Frutose/química , Glucose/química , Glucose/metabolismo , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 5/genética , Modelos Moleculares , Mutação Puntual/genética , Conformação Proteica , Ratos , Sais/química , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Simportadores/química , Simportadores/metabolismo
11.
PLoS One ; 10(5): e0125468, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26017283

RESUMO

INTRODUCTION: ROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that 90Y-labeled anti-ROBO1 monoclonal antibody (90Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models. METHODS: For the biodistribution study, 111In-labeled anti-ROBO1 monoclonal antibody (111In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with 90Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out. RESULTS: As a result of the biodistribution study, we observed tumor uptake of 111In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of 111In-labeled anti-ROBO1. In the RIT study, 90Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur. CONCLUSIONS: These results suggest that RIT with 90Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC.


Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas do Tecido Nervoso/imunologia , Receptores Imunológicos/imunologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Radioisótopos de Ítrio/uso terapêutico , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Radioimunoterapia/métodos , Receptores Imunológicos/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/radioterapia , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Radioisótopos de Ítrio/química , Radioisótopos de Ítrio/farmacocinética , Proteínas Roundabout
12.
Biochim Biophys Acta ; 1844(11): 1920-1924, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25135856

RESUMO

The monoclonal antibody has become an important therapeutic in the treatment of both hematological malignancies and solid tumors. The recent success of antibody-drug conjugates (ADCs) has broadened the extent of the potential target molecules in cancer immunotherapy. As a result, even molecules of low abundance have become targets for cytotoxic reagents. The multi-pass membrane proteins are an emerging target for the next generation antibody therapeutics. One outstanding challenge is the difficulty in preparing a sufficient amount of these membrane proteins so as to be able to generate the functional antibody. We have pursued the expression of various membrane proteins on the baculovirus particle and the utilization of displayed protein for immunization. The strong antigenicity of the virus acts either as a friend or foe in the making of an efficient antibody against an immunologically tolerant antigen. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

13.
EJNMMI Res ; 4: 29, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25006547

RESUMO

BACKGROUND: ROBO1 is a membrane protein that functions in axon guidance. ROBO1 contributes to tumour metastasis and angiogenesis and may have potential as a target protein of immunotherapy because ROBO1 is specifically expressed at high levels in hepatocellular carcinoma. In this study, we examined biodistribution and radioimmunotherapy (RIT) using a radioisotope-labelled anti-ROBO1 monoclonal antibody (MAb) against hepatocellular carcinoma models. METHODS: ROBO1-positive HepG2 human hepatocellular carcinoma xenograft nude mice were used in this study. We conjugated anti-ROBO1 MAb with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and the conjugates were labelled with (111)In and (90)Y. To study biodistribution, the (111)In-DOTA-anti-ROBO1 MAb was injected into HepG2 xenograft mice via the tail vein. To evaluate any antitumour effect, a RIT study was performed, and the (90)Y-DOTA-anti-ROBO1 MAb was injected via the tail vein. Tumour volume, mouse weight, and blood cell count were periodically measured throughout the experiments. The tumours and organs of mice were collected, and a histopathological analysis was carried out. RESULTS: The tumour uptake of (111)In-anti-ROBO1 MAb in HepG2 xenograft mice was 15.0% ± 0.69% injected dose per gram at 48 h after injection. Immunotherapy with cold-anti-ROBO1 MAb (70 µg) did not cause a significant antitumour effect. RIT with 6.7 MBq of (90)Y-anti-ROBO1 MAb caused significant tumour growth suppression. Transient body weight loss and bone-marrow suppression were observed. Histopathological analyses of tumours revealed the fatal degeneration of tumour cells, significant reduction of the Ki-67 index, and an increase of the apoptosis index. Normal organs showed no significant injury, but a transient reduction of hematopoietic cells was observed in the spleen and in the sternal bone marrow. CONCLUSIONS: These results suggest that RIT with (90)Y-anti-ROBO1 MAb is a promising treatment for ROBO1-positive hepatocellular carcinoma.

14.
J Neuropathol Exp Neurol ; 73(7): 714-28, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24918637

RESUMO

DJ-1, the product of a causative gene of a familial form of Parkinson disease, undergoes preferential oxidation of Cys106 (cysteine residue at position 106) under oxidative stress. Using specific monoclonal antibodies against Cys106 oxidized DJ-1 (oxDJ-1), we examined oxDJ-1 immunoreactivity in brain sections from DJ-1 knockout and wild-type mice and in human brain sections from cases classified into different Lewy body stages of Parkinson disease and Parkinson disease with dementia. Oxidized DJ-1 immunoreactivity was prominently observed in neuromelanin-containing neurons and neuron processes of the substantia nigra; Lewy bodies also showed oxDJ-1 immunoreactivity. Oxidized DJ-1 was also detected in astrocytes in the striatum, in neurons and glia in the red nucleus, and in the inferior olivary nucleus, all of which are related to regulation of movement. These observations suggest the relevance of DJ-1 oxidation to homeostasis in multiple brain regions, including neuromelanin-containing neurons of the substantia nigra, and raise the possibility that oxDJ-1 levels might change during the progression of Lewy body-associated neurodegenerative diseases.


Assuntos
Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos/química , Western Blotting , Neurônios Dopaminérgicos/fisiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Bulbo/citologia , Bulbo/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Neostriado/citologia , Neostriado/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Inclusão em Parafina , Peroxirredoxinas , Proteína Desglicase DJ-1 , Proteínas Recombinantes/química , Coloração pela Prata , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Substância Negra/citologia , Substância Negra/metabolismo
15.
Monoclon Antib Immunodiagn Immunother ; 32(4): 270-6, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23909421

RESUMO

Aquaporin-4 (AQP4), the most abundant water channel in the brain, plays a central role in water homeostasis, neuronal activity, and migration of astrocytes in the central nervous system. Recent studies have demonstrated that AQP4 is a target of an autoantibody specifically detected in an autoimmune neurologic disease called neuromyelitis optica. Here we have generated a monoclonal antibody (MAb) against the C-terminal region of AQP4 using a baculovirus expressing mouse AQP4 as an immunogen. This antibody (clone E5206) recognized both human and mouse AQP4s in a denaturing condition and was able to precipitate AQP4 from cell lysates of CHO cells stably expressing AQP4. Western blot analysis using deletion mutants revealed that the epitope was located within a region between Asp(303) and Leu(320) in the C-terminal tail of AQP4. Although clone E5206 could not be used for immunostaining when cells or tissues were fixed with 4% paraformaldehyde or 10% formalin, it could be used when cells were fixed with 10% trichloroacetic acid and when a formalin-fixed tissue section was pretreated with antigen-retrieval reagents. This MAb can be a valuable tool for analysis of AQP4 in a variety of physiological and pathophysiological contexts, in human tissues and organs as well as in rodent models, both in vitro and in vivo.


Assuntos
Anticorpos Monoclonais/imunologia , Aquaporina 4/imunologia , Encéfalo/metabolismo , Hibridomas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Formação de Anticorpos , Especificidade de Anticorpos , Western Blotting , Encéfalo/imunologia , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Recombinantes , Homologia de Sequência de Aminoácidos
16.
Hybridoma (Larchmt) ; 31(5): 325-32, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23098298

RESUMO

L-Asparaginase (ASNase) is important for the treatment of childhood acute lymphoblastic leukemia. ASNase sensitivity has been shown to correlate with the asparagine synthetase (ASNS) protein content in acute lymphoblastic leukemia cell lines. However, there have been few studies to determine ASNS protein levels in human leukemias, since no appropriate monoclonal antibody is available for such quantitative analysis. In this study, we report the generation of anti-ASNS monoclonal antibodies, which are applicable to flow cytometry and enzyme-linked immunosorbent assay. These monoclonal antibodies should provide a valuable tool for the quantification of ASNS protein level and estimation of ASNase-resistance in leukemia cells.


Assuntos
Anticorpos Monoclonais/imunologia , Aspartato-Amônia Ligase/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/imunologia , Baculoviridae/genética , Western Blotting , Linhagem Celular Tumoral , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vetores Genéticos , Humanos , Imunização , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
17.
Nature ; 482(7384): 237-40, 2012 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-22286059

RESUMO

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain. Here we report the raising of a mouse monoclonal antibody against human A(2A)AR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket, and describe the structure of A(2A)AR in complex with the antibody Fab fragment (Fab2838). This structure reveals that Fab2838 recognizes the intracellular surface of A(2A)AR and that its complementarity-determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active ß(2)-adrenergic receptor structure, but locks A(2A)AR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Agonismo Inverso de Drogas , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/imunologia , Animais , Anticorpos Monoclonais/imunologia , Regiões Determinantes de Complementaridade/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Ligantes , Camundongos , Modelos Moleculares , Opsinas/imunologia , Pichia , Conformação Proteica/efeitos dos fármacos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/imunologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química
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