RESUMEN
Neonicotinoids, such as acetamiprid (ACE), a pesticide used worldwide, are believed to be safe for human use. These molecules are structurally similar to nicotine, act as nicotinic acetylcholine receptor (nAChR) agonists, and were shown to be associated with neuromuscular and reproductive disorders, but these experiments were primarily performed in mature animals. In this study, the effects of ACE on the testes of immature mice were examined. The exposure of 3-week-old mice to ACE-containing water for 180 days led to a decrease in body weight and mildly affected spermatogenesis. Additionally, the expression of testosterone-metabolism genes, nAChR subunit genes, and proliferation-associated genes decreased in the testes of ACE-treated mice. Our results show that immature rodents may be less sensitive to ACE than mature ones, that mice may be more likely to accumulate ACE than rats, and that the development of disorders may be affected by the accumulation of ACE in the testes.
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Regulación de la Expresión Génica/efectos de los fármacos , Insecticidas/toxicidad , Neonicotinoides/toxicidad , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Insecticidas/metabolismo , Masculino , Ratones , Neonicotinoides/metabolismo , Receptores Nicotínicos/genética , Testosterona/metabolismo , Pérdida de Peso/efectos de los fármacosRESUMEN
PURPOSE: The N- and C-terminal regions of dynorphin (Dyn) A (1-17) activate opioid and N-methyl-D-aspartate receptors, respectively. Earlier studies demonstrated that Dyn-converting enzyme cleaved Dyn A (1-17) mainly at the Arg(6)-Arg(7) bond, resulting in the production of N- and C-terminal region peptide fragments, and that this enzyme was not inhibited by a mixture of the three peptidase inhibitors (PIs) amastatin (A), captopril (C), and phosphoramidon (P). The purpose of the present study was to evaluate antinociceptive potential and toxicity with intracerebroventricular administration of Dyn A (1-17) or (1-13) under pretreatment with a mixture of A, C, and P and/or Dyn-converting enzyme inhibitor (p-hydroxymercuribenzoate). METHODS: Peptide fragments from Dyn A (1-17) following incubation with membrane preparation under pretreatment with a mixture of the three PIs was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Infusion of drugs and peptides into the third ventricle in rats was performed via indwelling cannulae. Induction of antinociception and toxicity by Dyn A (1-17), Dyn A (1-13), Dyn A (1-6), or Dyn A (7-17) were determined by the tail-flick test and induction of barrel rotation, respectively. The effects of the PIs on antinociception and toxicity were evaluated by a dose-response study and a comparison of differences among various combinations of Dyn A (1-17) or Dyn A (1-13) and the three PIs and p-hydroxymercuribenzoate. RESULTS: MALDI-TOF-MS analysis identified Dyn A (1-6) and Dyn A (1-10) fragments as products following incubation of Dyn A (1-17) with membrane preparation of rat midbrain under pretreatment with a mixture of the three PIs. Pretreatment with a mixture of the three PIs produced an approximately 30-fold augmentation in antinociception induced by low-dose intracerebroventricular administration of Dyn A (1-17) or (1-13) in a µ-, δ- and κ-opioid receptor antagonist-reversible manner, but without signs of toxicity such as barrel rotation in the rat. Dyn A (1-17)-induced antinociception and toxicity was greater than that of Dyn A (1-6), Dyn A (1-13), or Dyn A (7-17) at the same dose. Dyn A (1-17)-induced antinociception and toxicity under pretreatment with various combinations of the three PIs and p-hydroxymercuribenzoate was greater than that with a mixture of the three PIs alone. CONCLUSION: These findings suggest that administration of a mixture of the three PIs increases Dyn A (1-17)- or (1-13)-induced antinociception under physiological conditions without toxicity.
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Analgésicos Opioides/toxicidad , Analgésicos/efectos adversos , Analgésicos/farmacología , Dinorfinas/toxicidad , Inhibidores de Proteasas/farmacología , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacología , Animales , Química Encefálica/efectos de los fármacos , Captopril/administración & dosificación , Captopril/farmacología , Relación Dosis-Respuesta a Droga , Dinorfinas/administración & dosificación , Dinorfinas/farmacología , Glicopéptidos/administración & dosificación , Glicopéptidos/farmacología , Inyecciones Intraventriculares , Masculino , Dimensión del Dolor/efectos de los fármacos , Péptidos/administración & dosificación , Péptidos/farmacología , Ratas , Ratas Wistar , Receptores Opioides/efectos de los fármacosRESUMEN
Liposome-encapsulated hemoglobin with high O2 -affinity (P50 O2 = 10 mm Hg, h-LEH) was reported to enhance tumor radiosensitivity. We hypothesize that targeted O2 delivery to tumor hypoxia by h-LEH may also enhance chemotherapy to suppress tumor growth and metastasis in mice. Doxorubicin (DXR; 0.5 or 2 mg/kg i.p.) or S-1 (4 or 8 mg/kg orally) alone or in combination with h-LEH (5 mL/kg i.v.) was administered for 2 weeks to C57BL/6N mice inoculated with Lewis Lung Carcinoma (LLC) in the leg. After the 2-week therapy in six treatment groups, mice were sacrificed for quantitative assessment of tumor growth and lung metastasis. The tumor was then evaluated for its expression of hypoxia-inducible factor-1α (HIF-1α) and matrix metallopoteinase-2 (MMP-2) activity. Combined use of h-LEH and chemotherapeutic agents (DXR or S-1) showed no additional enhancement on suppression of the tumor growth over the chemotherapeutic agent alone. However, the combination use of h-LEH significantly suppressed the number and total area of metastatic colonies in the lung compared with each chemotherapeutic agent alone. Although HIF-1α expression and MMP-2 activity in the original tumor was significantly suppressed in the groups of mice treated with either DXR or S-1 alone, the addition of h-LEH to either agent showed further enhancement of oxygen-mediated degradation of HIF-1α and suppression of MMP-2 activity. Although the addition of h-LEH to DXR or S-1 had little effect on original LLC tumor growth, it significantly enhanced suppression of lung metastasis in mice.
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Sustitutos Sanguíneos/uso terapéutico , Hemoglobinas/uso terapéutico , Liposomas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/uso terapéutico , Sustitutos Sanguíneos/farmacología , Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos , Femenino , Hemoglobinas/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Liposomas/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Metástasis de la Neoplasia/patología , Trasplante de NeoplasiasRESUMEN
The objective of the study is to investigate the safety, feasibility, and degradation profile of a novel Mg alloy-based bioresorbable coronary scaffold (JFK-PRODUCT BRS) with thin struts (110 µm). Polymer- or Mg alloy-based BRSs have not replaced nondegradable metal stents because of the higher prevalence of scaffold thrombosis and restenosis in clinical practice; these poor clinical outcomes were due to inadequate scaffold designs, including thick struts (more than 150 µm) and their inappropriate degradation processes. Fourteen healthy pigs received 17 JFK-PRODUCT BRSs in the coronary arteries and were sacrificed at 1, 6, 12, 18, and 26 months after implantation. Angiography, optical coherence tomography, microfocus X-ray computed tomography (µCT), scanning electron microscopy with energy-dispersive X-ray spectrometry (SEM-EDX), and histopathological evaluation were performed. The JFK-PRODUCT had a median percent late recoil of 11.28% at 1 month. The µCT observation confirmed that scaffold discontinuity reached 64.8% at 12 months with increased scaffold inner area thereafter, suggesting artery positive remodeling. The inflammation was mild, peaked at 18 months, and decreased thereafter. The SEM-EDX analysis demonstrated gradual degradation of the scaffold with formation of inorganic deposits, presumed to be calcium phosphates. It also revealed the disappearance of calcium phosphates at 26 months, achieving almost complete replacement of the scaffold by biocomponents. The current study demonstrated the safety and feasibility of JFK-PRODUCT with a lower acute recoil rate despite its thin struts. The scaffolds were almost completely disappeared at 26 months after implantation.
RESUMEN
Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.
Asunto(s)
Apoptosis , Núcleo Celular/enzimología , Enfermedad de Huntington/enzimología , Linfocitos/enzimología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Núcleo Celular/ultraestructura , Citoplasma/enzimología , Citoplasma/ultraestructura , Activación Enzimática/genética , Células HEK293 , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Linfocitos/ultraestructura , Fosforilación/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Ácido Fítico/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
SCT from HLA-identical sibling donors is generally associated with an excellent survival in FA patients if performed prior to the development of MDS or leukemia. However, the optimal conditioning regimen has not been defined. We report here our experience with 15 Japanese FA patients who underwent HLA-matched sibling donor SCT. The aim of this study is to compare radiation-based conditioning to Flu-based conditioning for FA patients in a Japanese population where the T-cell somatic mosaicism is higher than in the Caucasian population. Eight patients (a-group) received a radiation-based conditioning (500-600 cGy of thoracoabdominal/TBI) with CY dose modification (20-120 mg/kg), and ATG; two patients exhibited rejection. Seven patients (b-group) received CY (40 mg/kg), 150-180 mg/m(2) of Flu, and ATG. Durable engraftment was demonstrated in all patients. In FA patients, Flu-based conditioning may allow stable engraftment in matched sibling donor transplantation without radiation, even in patients with T-cell somatic mosaicism.
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Trasplante de Médula Ósea , Trasplante de Células Madre de Sangre del Cordón Umbilical , Anemia de Fanconi/cirugía , Mosaicismo , Trasplante de Células Madre , Acondicionamiento Pretrasplante/métodos , Adolescente , Niño , Preescolar , Quimerismo , Ciclofosfamida/uso terapéutico , Anemia de Fanconi/genética , Femenino , Estudios de Seguimiento , Rechazo de Injerto/prevención & control , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Inmunosupresores/uso terapéutico , Lactante , Masculino , Linfocitos T , Resultado del Tratamiento , Vidarabina/análogos & derivados , Vidarabina/uso terapéutico , Adulto JovenRESUMEN
HOX genes expressed in a specific spatial and temporal manner play a crucial role in determining the body plan during the early development of vertebrates. In adult tissues, many HOX genes participate in normal hematopoiesis and carcinogenesis. We previously found that overexpression of the homeobox gene HOXD3 alters expression levels of cell adhesion molecules in human cancer cell lines. Here, we have investigated whether HOXD3 expression is related to the cell adhesion processes during mouse development focusing on dorsal midline cells or roof-plate cells of the neural tube and neural crest cells. We created transgenic mouse embryos, in which HOXD3 is expressed in the dorsal midline under the control of the Wnt1 regulatory element, and analyzed these embryos at embryonic day 10.5-13.5. In HOXD3-expressing transgenic embryos, although neural crest-derived structures in the trunk region appeared to be normal, striking abnormalities were found in the neural tube. In transgenic embryos expressing the lacZ gene under the control of the Wnt1 regulatory element, expression of lacZ was restricted to roof-plate cells within the neural tube. By contrast, in HOXD3-expressing transgenic embryos, expression of HOXD3 was not only located in the dorsal neural tube, but also had spread inside the ventricular zone in more ventral regions of the neural tube. These findings show that the HOXD3 transgene is expressed more broadly than the Wnt1 gene is normally expressed. Expression of both Wnt1 and Msx1, marker genes in the roof plate, was further extended ventrally in HOXD3-expressing embryos than in normal embryos, suggesting that expression of the HOXD3 transgene expands the roof plate ventrally within the neural tube. In the ventricular zone of HOXD3-expressing embryos at embryonic day 10.5, we observed an increase in the number of mitotic cells and failure of interkinetic nuclear migration of progenitor cells. Furthermore, in HOXD3-expressing embryos at embryonic day 12.5, the ventricular zone, in which progenitor cells became more loosely connected to each other, was composed of a large number of cells that did not express N-cadherin. Our results indicate that expression of HOXD3 is closely associated with modulation of cell-adhesive properties during embryonic development.
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Adhesión Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Tubo Neural/embriología , Proteína Wnt1/fisiología , Animales , Cadherinas/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Operón Lac/genética , Ratones , Ratones Transgénicos , Cresta Neural/metabolismo , Tubo Neural/metabolismo , Defectos del Tubo Neural/metabolismoRESUMEN
Four fully human monoclonal antibodies (MAbs) to Entamoeba histolytica intermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 of E. histolytica showed that XEhI-20 {immunoglobulin G2(kappa) [IgG2(kappa)]} and XEhI-28 [IgG2(kappa)] were specific to Igl1, XEhI-B5 [IgG2(kappa)] was specific to Igl2, and XEhI-H2 [IgM(kappa)] was reactive with both Igls. Gene analyses revealed that the V(H) and V(L) germ lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge with E. histolytica trophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/aislamiento & purificación , Entamoeba histolytica/inmunología , Lectinas/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antiprotozoarios/uso terapéutico , Células CHO , Adhesión Celular , Membrana Celular/química , Cricetinae , Cricetulus , Citoplasma/química , Citometría de Flujo , Inmunización Pasiva , Inmunoglobulina G/inmunología , Región Variable de Inmunoglobulina/genética , Absceso Hepático/prevención & control , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADN , Vacuolas/químicaRESUMEN
Fms-related tyrosine kinase 3-internal tandem duplications (FLT3-ITD) are strongly associated with the refractory nature of acute myeloid leukemia (AML) by the standard combined chemotherapy. FLT3-ITD-expressing murine and human myeloid cell lines, HF6/FLT3-ITD and K562/FLT3-ITD cells, respectively, were developed in order to clarify whether FLT3-ITD is involved in the resistance to cytotoxic agents in AML. Both of these cell lines were specifically resistant to the pyrimidine analogue cytosine arabinoside (ara-C), an essential agent for AML, accompanied by the downregulation of equilibrative nucleoside transporter 1 (ENT1), a transporter responsible for the cellular uptake of ara-C. The ENT1 promoter activity and the cellular uptake of ara-C were reduced in K562/FLT3-ITD cells, and rescued by pretreating the cells with PKC412, a FLT3 inhibitor. In addition, the expression of hypoxia inducible factor 1 alpha subunit (HIF1A) transcripts was upregulated in K562/FLT3-ITD cells, and the induction of HIF-1alpha reduced the promoter activity of ENT1 gene in K562 cells. Taken together, these findings suggest that FLT3-ITD specifically induces ara-C resistance in leukemic cells by the repression of ENT1 expression, possibly through the upregulation of HIF-1alpha, while also partially accounting for the poor prognosis of AML with FLT3-ITD due to resistance to the standard chemotherapy protocols which include ara-C.
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Antimetabolitos Antineoplásicos/farmacología , Citarabina/farmacología , Tranportador Equilibrativo 1 de Nucleósido/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Tranportador Equilibrativo 1 de Nucleósido/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Regiones Promotoras GenéticasRESUMEN
Thrombospondin (TSP) 2 interacts with matrix metalloproteinases (MMPs) and matrix serine proteases such as plasminogen activator (PA). Malignant melanoma is an aggressive human neoplasm showing aggressive metastatic features. We examined the effects of TSP2 gene introduction in the human malignant melanoma cell line A375. We established three clones transfected with human TSP2 (A375/TSP2). The in vitro invasiveness was remarkably suppressed (42-61%) in the TSP2-transfectants, while growth properties were preserved. The A375/TSP2 showed significantly decreased liver metastatic potential (liver weight: 3.88+/-0.30 g in A375/TSP2, 7.07+/-0.67 g in vector-transfectant (A375/V), p<0.01, Mann-Whitney U test) in super immuno-deficient mice (NOD/SCID/gammacnull, NOG). The PA inhibitor-1 (PAI-1) and PAI-2 mRNAs were significantly overexpressed in A375/TSP2. The increased activities of PAI-1 and PAI-2 were confirmed by reverse zymography. The vascularity of metastatic lesions was significantly decreased in A375/TSP2 (vascular density: 0.62+/-0.15% in A375/TSP2, 4.96+/-0.61% in A375/V, p<0.01, Welch test). These results suggest that TSP2 suppresses hematogenous metastasis through microenvironment-modification including PAI up-regulation and anti-vascularization in human malignant melanoma.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Neoplasias Hepáticas Experimentales/prevención & control , Melanoma Experimental/prevención & control , Trombospondinas/genética , Animales , Northern Blotting , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Neoplasias Hepáticas Experimentales/genética , Masculino , Melanoma Experimental/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Neovascularización Patológica , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 2 de Activador Plasminogénico/genética , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Carbon tetrachloride (CCl4) is known to induce liver damage. Animal experiments with CCl4 injections have revealed many findings, especially mechanisms of liver damage and liver regeneration. Recently, proteomic approaches have been introduced in various studies to evaluate the quantitative and qualitative changes in the comprehensive proteome level. The aim of this research is to elucidate the key protein for liver damage, liver protection and liver regeneration by using proteomic techniques. 50 % (v/v) CCl4 in corn oil was administered intraperitoneally to adult male rats at a dose of 4ml/kg body weight. Approximately 24h after the injection, the liver was removed and extracted proteins were analyzed with cleavable isotope coded affinity tag (cICAT) reagents, two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). A twelvefold increase in D-dopachrome tautomerase (DDT) was indicated. This enzyme has been reported to be involved in the biosynthesis of melanin, an antioxidant. According to the histological analysis, melanin levels were increased in un-damaged hepatocytes of CCl4-treated rats. These results suggest that the increase in DDT is a response to liver damage, accelerates melanin biosynthesis and protects the liver from oxidative stress induced by CCl4.
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Intoxicación por Tetracloruro de Carbono/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Oxidorreductasas Intramoleculares/metabolismo , Hígado/enzimología , Animales , Western Blotting , Peso Corporal/efectos de los fármacos , Células COS , Intoxicación por Tetracloruro de Carbono/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/genética , Vectores Genéticos , Hígado/patología , Masculino , Espectrometría de Masas , Melaninas/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , TransfecciónRESUMEN
Lipofuscin is an intracellular aggregate of highly oxidized proteins that cannot be digested in the ubiquitin-proteasome system and accumulate mainly in lysosomes, especially in aged cells and pathological conditions. However, no systematic study has evaluated the cardiac accumulation of lipofuscin during human ageing and sudden cardiac death (SCD). Age estimation in unidentified bodies and postmortem SCD diagnosis are important themes in forensics. Thus, we aimed to elucidate their correlations with myocardial lipofuscin accumulation. We collected 76 cardiac samples from autopsy patients aged 20-97 years. After histopathological examination, myocardial lipofuscin was measured using its autofluorescence. Lipofuscin accumulated mainly in the perinuclear zone, and its accumulation rate positively correlated with chronological ageing (r = 0.82). Meanwhile, no significant change in lipofuscin level was observed with different causes of death, including SCD. There was also no significant change in lipofuscin level in relation to body mass index, serum brain natriuretic peptide level, or heart weight. Moreover, we performed LC3 and p62 immunoblotting to evaluate autophagic activity, and no change was observed in ageing. Therefore, lipofuscin accumulation more directly reflects chronological ageing rather than human cardiac pathology. Our study reveals the stability and utility of cardiac lipofuscin measurement for age estimation during autopsy.
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Envejecimiento/metabolismo , Muerte Súbita Cardíaca , Lipofuscina/metabolismo , Miocardio/metabolismo , Adulto , Anciano , Envejecimiento/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miocardio/patologíaRESUMEN
Podoplanin, a transmembrane glycoprotein, is overexpressed in certain types of tumors and induces platelet aggregation by binding to C-type lectin-like receptor 2 (CLEC-2) on the platelet membrane. Activated platelets release granule components, which in turn, trigger epithelial-mesenchymal transition and confer invasive capacity to the tumor cells. Therefore, blocking the podoplanin-CLEC-2 interaction by a small-molecule compound is a potential therapeutic strategy to prevent cancer metastasis and invasion. To effectively identify such inhibitory compounds, we have developed a pull-down-based inhibitory compound screening system. An immunoglobulin Fc domain-CLEC-2 fusion protein was used as a bait to capture podoplanin derived from podoplanin-overexpressing HeLa cells in the presence and absence of the test compound. The protein complex was then pulled down using protein A beads. To shorten the turnaround time, increase throughput, and decrease the workload for the operators, centrifugal filter units were employed to separate free and bound podoplanin, instead of using customary aspiration-centrifugation washing cycles. Slot blotting was also utilized in lieu of gel electrophoresis and electrical transfer. Thus, the use of our pull down screening system could facilitate the effective selection of potential inhibitor compounds of the podoplanin-CLEC-2 interaction for cancer therapy. Importantly, our methodology is also applicable to targeting other protein-protein interactions.
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Evaluación Preclínica de Medicamentos/métodos , Lectinas Tipo C/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Células HeLa , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Unión Proteica , Proteínas RecombinantesRESUMEN
Coenzyme Q10 (CoQ10) is essential for ATP production in the mitochondria, and is an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, but none have yet been isolated and characterized. Here we purified a CoQ10-binding protein from human urine and identified it to be saposin B, a housekeeping protein necessary for sphingolipid hydrolysis in lysosomes. We confirmed that cellular saposin B binds CoQ10 in human sperm and the hepatoma cell line HepG2 by using saposin B monoclonal antibody. The molar ratios of CoQ10 to saposin B were estimated to be 0.22 in urine, 0.003 in HepG2, and 0.12 in sperm. We then confirmed that aqueous saposin B extracts CoQ10 from hexane to form a saposin B-CoQ10 complex. Lipid binding affinity to saposin B decreased in the following order: CoQ10>CoQ9>CoQ7>>alpha-tocopherol>>cholesterol (no binding). The CoQ10-binding affinity to saposin B increased with pH, with maximal binding seen at pH 7.4. On the other hand, the CoQ10-donating activity of the saposin B-CoQ10 complex to erythrocyte ghost membranes increased with decreasing pH. These results suggest that saposin B binds and transports CoQ10 in human cells.
RESUMEN
Migraine attacks alter various molecules that might be related to the pathophysiology of migraine, such as serotonin, calcitonin gene-related peptide, and nitric oxide. The underlying pathophysiology of migraine is as yet unclear. We explored key proteins related to the pathogenesis of migraine here. Serum was collected from two patients with migraine with aura (MA) and seven patients with migraine without aura (MO) during attack-free periods and migraine attacks. Samples were analyzed using 2-dimensional gel electrophoresis. Nineteen protein spots were altered between the attack-free versus migraine attack periods. Mass spectrometric analysis was performed to identify the proteins within each of the 19 altered spots. Thirty-six proteins were significantly altered in samples collected during attack-free periods versus migraine attacks. The protein with the statistically most significant MASCOT/Mowse score (268±112) among lipoproteins was apolipoprotein (ApoE). In the MA and MO groups, ApoE protein levels were significantly higher during migraine attack than during the attack-free period (p<0.05). ApoE protein levels were also significantly increased in the MA group during the attack-free period compared to healthy controls and patients with tension type headaches (p<0.01). Migraine alters ApoE levels, especially in MA. ApoE might play an important role in the pathophysiology of migraine, and may act as a diagnostic biomarker of migraine.
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Apolipoproteínas E/sangre , Biomarcadores/sangre , Migraña con Aura/sangre , Migraña sin Aura/sangre , Adulto , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Persona de Mediana Edad , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: We have previously found that Wnt signaling is activated in mesothelioma cells. To clarify the effect of blocking Wnt signaling in mesothelioma, the expression of dishevelled (Dvl), an intermediator of Wnt signaling, was down-regulated by a reformed type of small interfering RNA (siRNA), stealth RNAi, which can reduce the cytotoxic interferon response unlike conventional siRNA. MATERIALS AND METHODS: Mesothelioma cell lines were transfected with stealth RNAi of Dvl, and cell growth and colony formation were examined. The synergistic effect on cell growth of Dvl stealth RNAi and cisplatin in combination was evaluated. RESULTS: Dvl stealth RNAi down-regulated the expression of Dvl-3 in mesothelioma cells and induced cell cycle aberration which caused suppression of cell growth. Colony formation was also suppressed. Dvl stealth RNAi and cisplatin in combination suppressed cell growth synergistically. CONCLUSION: Our data suggest that inhibition of Wnt signaling leads to significant antitumor effects.
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Proteínas Adaptadoras Transductoras de Señales/genética , Cisplatino/farmacología , Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Fosfoproteínas/genética , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Procesos de Crecimiento Celular , Terapia Combinada , Proteínas Dishevelled , Regulación hacia Abajo , Humanos , Mesotelioma/metabolismo , Mesotelioma/patología , Fosfoproteínas/biosíntesis , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
The pituitary tumor-transforming gene (PTTG) is a homolog of yeast Securin, which arrests the activation of Separin to induce sister chromatid separation in the transition from metaphase to anaphase. Pituitary tumor-transforming gene is also known to induce angiogenesis during pituitary tumorigenesis. It has not been clarified whether PTTG functions as a cytoplasmic or a nuclear protein. Our immunohistochemical study indicated that PTTG is localized in the cytoplasm of pituitary tumor cells. In the present study, confocal laser scanning microscopy (CLSM) analysis of human pituitary adenomas and immunoelectron microscopy of the mouse pituitary cell line, AtT-20, demonstrated the localization of PTTG in the Golgi apparatus and vesicles. Secreted PTTG was detected by immunoblotting from culture medium of mouse pituitary tumor cell lines. Our results suggested that PTTG is a secretory protein produced by pituitary tumor cells. In addition, PTTG may exert autocrine and/or paracrine functions as a newly proposed important pathway for the action of PTTG.
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Adenoma/metabolismo , Línea Celular Tumoral/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Hipofisarias/metabolismo , Adenoma/patología , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Línea Celular Tumoral/patología , Expresión Génica , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Ratones , Microscopía Confocal , Microscopía Inmunoelectrónica , Proteínas de Neoplasias/genética , Neoplasias Hipofisarias/patología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SecurinaRESUMEN
OBJECTIVE: Numerous monoclonal antibodies have been developed for the purpose of medical treatments, including cancer treatment. For clinical application, the most useful are human-derived antibodies. In this study, we tried to prepare designed antigen-specific antibodies of completely human origin using immunodeficient mouse. METHODS: Nonobese diabetic/severe combined immunodeficient/IL-2 receptor gamma null mouse (NOG) mouse was used to reconstitute the human immune system with umbilical cord blood hematopoietic stem cells (CB-NOG mouse) and to prepare human-derived Her-2-epitope-specific antibodies. Hybridoma lines were prepared by fusing the human myeloma cell line Karpas707H. RESULTS: Serum of immunized NOG mouse contained human-derived immunoglobulin M (IgM) antibodies specific for a short peptide sequence of 20 amino acids, including the epitope peptide of apoptotic Her-2 antibody CH401. Hybridoma lines were successfully prepared with spleen B cells obtained from the immunized CB-NOG mouse. One of these cell lines produced human IgM against the epitope peptide that can recognize surface Her-2 molecule. CONCLUSION: We could produce human-derived IgM antibody against Her-2 epitope peptide in CB-NOG mouse, succeeding in generation of human hybridoma-secreting IgM against a given peptide.
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Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Epítopos de Linfocito B/inmunología , Hibridomas/inmunología , Inmunoglobulina M/inmunología , Péptidos/inmunología , Receptor ErbB-2/inmunología , Quimera por Trasplante/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Linfocitos B/citología , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Epítopos de Linfocito B/farmacología , Humanos , Hibridomas/citología , Inmunización , Inmunoglobulina M/uso terapéutico , Ratones , Ratones Mutantes , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/inmunología , Péptidos/farmacología , Quimera por Trasplante/genéticaRESUMEN
It is well known that polyether-based copolymers have good blood compatibility, although many mechanisms have been proposed to explain their favorable performance. Our objective in carrying out the present study was to obtain a better understanding of the effect of the (poly)ether segment on blood compatibility. Therefore, we synthesized poly(propylene glycol) (PPG)-based initiators for atom transfer polymerization, where the number of propylene glycol (PG) units in the PPG (Pn(PG) was varied from 1 to 94. Methyl methacrylate (MMA) was polymerized using the initiators, resulting in the formation of polyMMAs with a PG-based ether part at the polymer terminal. We mainly investigated the effects of Pn(PG) on the surface properties and platelet compatibility of the PPG-polyMMA. X-ray photoelectron spectroscopy and surface contact angle (CA) analysis revealed the exposure of the PG units at the surface of the polymer. The platelet compatibility of the polymers was improved compared with a commercial polyMMA, even when Pn(PG) = 1. These results suggest that PG units have an important influence on favorable blood compatibility, regardless of the Pn(PG) value. We also investigated protein adsorption behavior in terms of the amount and deformation of fibrinogen adsorbed on the polymer surface.
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Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Plaquetas/efectos de los fármacos , Fenómenos Químicos , Polímeros/química , Polimetil Metacrilato/química , Polimetil Metacrilato/farmacología , Glicoles de Propileno/química , Adsorción , Animales , Bovinos , Fibrinógeno/química , Humanos , Adhesividad Plaquetaria/efectos de los fármacos , Polimerizacion , Albúmina Sérica Bovina/química , Propiedades de Superficie , Agua/químicaRESUMEN
TAR DNA-binding protein 43 (TDP-43) has been identified as a major component of ubiquitin-positive inclusions in the brains and spinal cords of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) or amyotrophic lateral sclerosis (ALS). The phosphorylated C-terminal fragment of TDP-43 forms aggregates in the neuronal cytoplasm, possibly resulting in neuronal cell death in patients with FTLD-U or ALS. The inositol pyrophosphate known as diphosphoinositol pentakisphosphate (InsP7) contains highly energetic pyrophosphate bonds. We previously reported that inositol hexakisphosphate kinase type 2 (InsP6K2), which converts inositol hexakisphosphate (InsP6) to InsP7, mediates cell death in mammalian cells. Moreover, InsP6K2 is translocated from the nucleus to the cytosol during apoptosis. In this study, we verified that phosphorylated TDP-43 co-localized and co-bound with InsP6K2 in the cytoplasm of anterior horn cells of the spinal cord. Furthermore, we verified that cell death was augmented in the presence of cytoplasmic TDP-43 aggregations and activated InsP6K2. However, cells with only cytoplasmic TDP-43 aggregation survived because Akt activity increased. In the presence of both TDP-43 aggregation and activated InsP6K2 in the cytoplasm of cells, the expression levels of HSP90 and casein kinase 2 decreased, as the activity of Akt decreased. These conditions may promote cell death. Thus, InsP6K2 could cause neuronal cell death in patients with FTLD-U or ALS. Moreover, InsP6K2 plays an important role in a novel cell death pathway present in FTLD-U and ALS.