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Safe and noninvasive methods for breast cancer screening with improved accuracy are urgently needed. Volatile organic compounds (VOCs) in biological samples such as breath and blood have been investigated as noninvasive novel markers of cancer. We investigated volatile organic compounds in urine to assess their potential for the detection of breast cancer. One hundred and ten women with biopsy-proven breast cancer and 177 healthy volunteers were enrolled. The subjects were divided into two groups: a training set and an external validation set. Urine samples were collected and analyzed by gas chromatography and mass spectrometry. A predictive model was constructed by multivariate analysis, and the sensitivity and specificity of the model were confirmed using both a training set and an external set with reproducibility tests. The training set included 60 breast cancer patients (age 34-88 years, mean 60.3) and 60 healthy controls (age 34-81 years, mean 58.7). The external validation set included 50 breast cancer patients (age 35-85 years, mean 58.8) and 117 healthy controls (age 18-84 years, mean 51.2). One hundred and ninety-one compounds detected in at least 80% of the samples from the training set were used for further analysis. The predictive model that best-detected breast cancer at various clinical stages was constructed using a combination of two of the compounds, 2-propanol and 2-butanone. The sensitivity and specificity in the training set were 93.3% and 83.3%, respectively. Triplicated reproducibility tests were performed by randomly choosing ten samples from each group, and the results showed a matching rate of 100% for the breast cancer patient group and 90% for the healthy control group. Our prediction model using two VOCs is a useful complement to the current diagnostic tools. Further studies inclusive of benign tumors and non-breast malignancies are warranted.
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2-Propanol/orina , Biomarcadores de Tumor , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/orina , Butanonas/orina , Compuestos Orgánicos Volátiles/orina , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Biopsia Líquida , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Curva ROC , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
BACKGROUND: Early identification and control of pathogenic bacteria are important in the treatment of pneumonia. Currently, two rapid antigen detection kits for pneumococcal pneumonia are available: one uses urine samples and the other, named RAPIRUN® S. pneumoniae, uses sputum samples. RAPIRUN® has shown high sensitivity with nasopharyngeal swab samples from pediatric patients. In this study, we investigated the performance of RAPIRUN® with nasopharyngeal swabs from adult patients. METHODS: All adult patients diagnosed with pneumonia from November 2011 to April 2012 in St. Luke's International hospital were included in this cross-sectional study. Single sputum, nasopharyngeal swab, and urine samples obtained from patients were investigated using a rapid antigen detection kit. Sputum and blood cultures were also evaluated. We compared the characteristics of pneumococcal pneumonia patients diagnosed using RAPIRUN with a nasopharyngeal swab to those patients diagnosed using other methods. Sensitivity and specificity were also calculated. RESULTS: Seventeen out of 60 patients with pneumonia were diagnosed with pneumococcal pneumonia. In 4 out of the 17 cases, a positive test result was obtained using RAPIRUN with a nasopharyngeal swab. The sensitivity and specificity were 23.5 and 100 %, respectively. CONCLUSION: RAPIRUN performed with nasopharyngeal swabs from adult patients exhibited lower sensitivity for the diagnosis of pneumococcal pneumonia than the other compared methods. The causative pathogen of pneumonia should be identified using not only sputum cultures or rapid antigen detection kits but also clinical features or gram staining of sputum.
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We propose a novel Kretschmann-type surface plasmon resonance (SPR) sensor chip having a surface covered with electrodeposited gold nanostructures to enhance the sensitivity of SPR biosensing. The nanostructure is three-dimensional and has a larger surface area than a conventional flat surface chip, which increases the amount of protein binding and also induces a large change in the effective dielectric constant of the sensing area. The gold nanostructures were formed by electrodeposition under galvanostatic conditions, so their size could be controlled by manipulating the deposition time and current. The sensing characteristics, including the concentration dependence and noise level, indicated that the performance of the resulting chip (called a Au-black chip) was equivalent to that of a conventional sensor chip. We also determined the optimal electrodeposition conditions to obtain a sharp SPR curve for protein detection assay; the optimal thickness of the gold layer was 50-60 nm. Enhanced protein sensing was demonstrated by using a binding assay of anti-BSA antibody and BSA molecules. The protein binding signal was several times higher than that of the conventional assay. The insights into electrodeposition for SPR sensing presented here will enable improved sensitivity for detecting low-concentration and small proteins.
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Galvanoplastia/métodos , Oro/química , Nanoestructuras/química , Albúmina Sérica Bovina/metabolismo , Resonancia por Plasmón de Superficie/métodos , Animales , Bovinos , Unión ProteicaRESUMEN
Clostridium botulinum produces the botulinum neurotoxin, forming a large complex as progenitor toxins in association with non-toxic non-hemagglutinin and/or several different hemagglutinin (HA) subcomponents, HA33, HA17 and HA70, which bind to carbohydrate of glycoproteins from epithelial cells in the infection process. To elucidate the carbohydrate recognition mechanism of HA70, X-ray structures of HA70 from type C toxin (HA70/C) in complexes with sialylated oligosaccharides were determined, and a binding assay by the glycoconjugate microarray was performed. These results suggested that HA70/C can recognize both α2-3- and α2-6-sialylated oligosaccharides, and that it has a higher affinity for α2-3-sialylated oligosaccharides.
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Toxinas Botulínicas/química , Carbohidratos/química , Clostridium botulinum/metabolismo , Ácido N-Acetilneuramínico/química , Oligosacáridos/química , Secuencia de Carbohidratos , Cristalografía por Rayos X/métodos , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Galactosa/química , Glucosa/química , Glicoproteínas/química , Hemaglutininas/química , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , TemperaturaRESUMEN
OBJECTIVE: Measurement of the serum level of theophylline is essential for its proper use; however, it is difficult to infer the metabolic ability of individual patients by only the serum theophylline level and to decide the appropriate medication. In this study, we simultaneously measured serum theophylline and metabolite levels in patients treated with theophylline, and investigated their usefulness. EXPERIMENTAL: The subjects were asthma patients who visited the outpatient clinic of Respiratory Medicine, St Luke's International Hospital, between April and October 2003, and were medicated with sustained-release theophylline tablets (Theodur®). The serum level of theophylline and its metabolites was measured by HPLC in patients who gave written consent. RESULTS: A strong correlation was noted between the serum theophylline (TP) and 1,3-dimethyluric acid (DMU) levels of 52 patients (r = 0.670), and DMU/TP was about 0.04. In a patient whose the DMU/TP was 0.216, it was recognized that metabolic ability was promoted due to a history of smoking. DISCUSSION: In this study, it was shown that simultaneous measurement by HPLC of the serum level of theophylline and its metabolites and DMU/TP was useful to assess the metabolic ability of individual patients.
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Broncodilatadores/administración & dosificación , Broncodilatadores/sangre , Teofilina/administración & dosificación , Teofilina/sangre , Asma/sangre , Asma/tratamiento farmacológico , Cafeína/sangre , Cromatografía Líquida de Alta Presión , Humanos , Fumar/sangre , Ácido Úrico/análogos & derivados , Ácido Úrico/sangreRESUMEN
Neural stem cells (NSCs) proliferate and generate new neurons in the adult brain. A carbohydrate-binding protein (lectin), Galectin-1, is expressed in the NSCs in the subependymal zone (SEZ) of the adult mouse brain. The infusion and knockout of Galectin-1 in the SEZ results in an increase and decrease, respectively, of NSCs and subsequently born progenitor cells. The molecular mechanism of this effect, however, has been unknown. Previous studies outside the brain suggest that Galectin-1 binds to a carbohydrate structure of beta1 Integrin and modulates cell adhesion. Here, we studied the functional interaction between Galectin-1 and beta1 Integrin in the adult mouse SEZ. Beta1 Integrin was purified from adult SEZ tissue by binding to a Galectin-1 affinity column, and this binding depended on Galectin-1's carbohydrate-binding activity. In adult brain sections, Galectin-1-binding activity was detected on beta1 Integrin-expressing cells in the SEZ. Furthermore, in the adult SEZ, the simultaneous infusion of a beta1 Integrin-neutralizing antibody with Galectin-1 protein reversed the increasing effect of Galectin-1 on the number of adult neural progenitor cells (NPCs). Finally, intact beta1 Integrin was required for Galectin-1's function in NPC adhesion in vitro. These results suggest that the interaction between beta1 Integrin and Galectin-1 plays an important role in regulating the number of adult NPCs through mechanisms including cell adhesion.
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Células Madre Adultas/fisiología , Galectina 1/metabolismo , Integrina beta1/metabolismo , Neuronas/fisiología , Animales , Anticuerpos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/citología , Bromodesoxiuridina/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Cromatografía de Afinidad/métodos , Galectina 1/genética , Galectina 1/inmunología , Proteínas Fluorescentes Verdes/genética , Integrina beta1/genética , Lactosa/farmacología , Ratones , Neuronas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Edulcorantes/farmacología , Factores de TiempoRESUMEN
A 38 year-old female with no significant medical history was transferred to a medical centre in Hawaii after near-drowning at the beach. She was noted to have increasing shortness of breath. Subsequently she was placed on non-invasive ventilation and then intubated for respiratory support. She was thought to have early stage acute respiratory distress syndrome after sea water aspiration. By multidisciplinary treatment, she was able to be extubated successfully on hospital day 5, and then flew back to Japan. When visiting our hospital in Japan, further examinations were conducted for prolonged respiratory symptoms and pulmonary infiltrates by CT. A specimen obtained by transbronchial lung biopsy revealed organising pneumonia which was thought to be related to sea water aspiration. Methylprednisolone treatment resolved her respiratory symptoms and pulmonary infiltrates.
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BACKGROUND: Gemcitabine plus UFT combination chemotherapy are highly effective and less toxic in the first line setting in patients with non-small cell lung cancer (NSCLC). The purpose of the study is to confirm the feasibility of this regimen as second- or third-line therapy in NSCLC. METHODS: Fifteen patients with performance status of 0-1 were enrolled. UFT (tegafur 250 mg/m(2)/day) was administered orally twice a day from days 1-14, and gemcitabine of 900 mg/m(2) was administered intravenously on days 8 and 15 every three weeks on an outpatient setting. The treatment was repeated for at least 3 cycles and continued unless the disease progressed. RESULTS: The response rate and the disease control rate were 6.7% and 66.7%, respectively. Grade 3-4 toxicities included neutropenia in one patient and elevation of transaminases in one patient. The mean relative dose intensity of gemcitabine and UFT were 0.93 and 0.97, respectively. CONCLUSION: High disease control rate and less toxicity suggested the potential of gemcitabine and UFT combination chemotherapy as second- or third-line therapy in NSCLC.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pulmonares/tratamiento farmacológico , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Medicina Basada en la Evidencia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Terapia Recuperativa/métodos , Tegafur/administración & dosificación , Tegafur/efectos adversos , Uracilo/administración & dosificación , Uracilo/efectos adversos , GemcitabinaRESUMEN
The extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.
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Colorantes Fluorescentes/análisis , Glicoconjugados/análisis , Polisacáridos/análisis , Polisacáridos/metabolismo , Análisis por Matrices de Proteínas/métodos , Proteínas/análisis , Proteínas/metabolismo , Humanos , Unión Proteica , Especificidad por SustratoRESUMEN
Lectin microarray is an emerging technique, which will accelerate glycan profiling and discovery of glycan-related biomarkers. One of the most important stages in realizing the potential of the technique is to achieve sufficiently high sensitivity to detect even the low concentrations of some target glycoproteins which occur in sera or tissues. Previously, we developed a lectin microarray based on an evanescent-field fluorescence-assisted detection principle that allows rapid profiling of glycoproteins. Here, we report optimization of procedures for lectin spotting and immobilization to improve the sensitivity and reproducibility of the lectin microarray. The improved microarray allows high-sensitivity detection of even monovalent oligosaccharides that generally have a low affinity with lectins (K(d)>10(-6) M). The LOD observed for RCA120, a representative plant lectin, with asialofetuin, and an asialo-biantennary N-glycan probe were determined to be 100 pg/mL and 100 pM, respectively. With the improved lectin microarray system, closely related structural isomers, i.e., Le(a) and Le(x), were clearly differentiated by the difference in signal patterns on relevant multiple lectins, even though specific lectins to detect these glycan structures were not available. The result proved a previously proposed concept of lectin-based glycan profiling.
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Glicoproteínas/análisis , Lectinas/metabolismo , Oligosacáridos/metabolismo , Análisis por Matrices de Proteínas/métodos , Animales , Pollos , Fluorescencia , Glicoproteínas/metabolismo , Lectinas/química , Modelos Biológicos , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The Jacalin-related lectin (JRL) family comprises galactose-binding-type (gJRLs) and mannose-binding-type (mJRLs) lectins. Although the documented occurrence of gJRLs is confined to the family Moraceae, mJRLs are widespread in the plant kingdom. A detailed comparison of sugar-binding specificity was made by frontal affinity chromatography to corroborate the structure-function relationships of the extended mJRL subfamily. Eight mJRLs covering a broad taxonomic range were used: Artocarpin from Artocarpus integrifolia (jackfruit, Moraceae), BanLec from Musa acuminata (banana, Musaceae), Calsepa from Calystegia sepium (hedge bindweed, Convolvulaceae), CCA from Castanea crenata (Japanese chestnut, Fagaceae), Conarva from Convolvulus arvensis (bindweed, Convolvulaceae), CRLL from Cycas revoluta (King Sago palm tree, Cycadaceae), Heltuba from Helianthus tuberosus (Jerusalem artichoke, Asteraceae) and MornigaM from Morus nigra (black mulberry, Moraceae). The result using 103 pyridylaminated glycans clearly divided the mJRLs into two major groups, each of which was further divided into two subgroups based on the preference for high-mannose-type N-glycans. This criterion also applied to the binding preference for complex-type N-glycans. Notably, the result of cluster analysis of the amino acid sequences clearly corresponded to the above specificity classification. Thus, marked correlation between the sugar-binding specificity of mJRLs and their phylogeny should shed light on the functional significance of JRLs.
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Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/clasificación , Lectinas de Plantas/química , Lectinas de Plantas/clasificación , Polisacáridos/química , Secuencia de Aminoácidos , Cromatografía de Afinidad , Ligandos , Datos de Secuencia Molecular , FilogeniaRESUMEN
BACKGROUND: Many drugs and the combinations of drugs are recommended for each treatment step in bronchial asthma. However, there are few issues examined about the optimal drug and combination of drugs in a long term prognosis. In this study, we investigated the optimal drugs and combinations of drugs from a point of view of prognosis. METHODS: One hundred and ninety four patients who visited our hospital for treatment from November, 2003 to October, 2004 and were managed according to GINA guideline were surveyed retrospectively. We compared the rate of step up and the frequency of urgent visit and urgent hospitalization in one year between drug groups in each treatment step. RESULTS: The rate of step up was significantly higher in leukotriene receptor antagonist (LTRA) group than in inhalation corticosteroid (ICS) group and theophylline group in Step 2. The frequency of urgent visit and urgent hospitalization was significantly higher in ICS+LTRA group than in ICS+theophylline group and ICS+long-acting beta 2-agonist (LABA) group in Step 3. CONCLUSION: There is a possibility that the prognosis becomes bad when we use LTRA in the practical treatment according to GINA guideline.
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Corticoesteroides/administración & dosificación , Agonistas Adrenérgicos beta/administración & dosificación , Asma/tratamiento farmacológico , Antagonistas de Leucotrieno/administración & dosificación , Administración por Inhalación , Adolescente , Adulto , Anciano , Esquema de Medicación , Quimioterapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Guías de Práctica Clínica como Asunto , Pronóstico , Estudios RetrospectivosRESUMEN
The glycome represents the total set of glycans expressed in a cell. The glycome has been assumed to vary between cell types, stages of development and differentiation, and during malignant transformation. Analysis of the glycome provides a basis for understanding the functions of glycans in these cellular processes. Recently, a technique called lectin microarray was developed for rapid profiling of glycosylation, although its use was mainly restricted to glycoproteins of cell lysates, and thus unable to profile the intact cell surface glycans. Here we report a simple and sensitive procedure based on this technology for direct analysis of the live mammalian cell-surface glycome. Fluorescent-labeled live cells were applied in situ to the established lectin microarray consisting of 43 immobilized lectins with distinctive binding specificities. After washing, bound cells were directly detected by an evanescent-field fluorescence scanner in a liquid phase without fixing and permeabilization. The results obtained by differential profiling of CHO and its glycosylation-defective mutant cells, and splenocytes of wild-type and beta1-3-N-acetylglucosaminyltransferase II knockout mice performed as model experiments agreed well with their glycosylation phenotypes. We also compared cell surface glycans of K562 cells before and after differentiation and found a significant increase in the expression of O-glycans on differentiated cells. These results demonstrate that the technique provides a novel strategy for profiling global changes of the mammalian cell surface glycome.
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Lectinas , Polisacáridos/química , Polisacáridos/metabolismo , Análisis por Matrices de Proteínas , Animales , Células CHO , Secuencia de Carbohidratos , Diferenciación Celular/fisiología , Cricetinae , Cricetulus , Glucólisis , Glicosilación , Humanos , Células K562 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/fisiología , Fenotipo , Polisacáridos/análisis , Sensibilidad y Especificidad , Bazo/citología , Bazo/metabolismoRESUMEN
Frontal affinity chromatography (FAC) is a quantitative method that enables sensitive and reproducible measurements of interactions between lectins and oligosaccharides. The method is suitable even for the measurement of low-affinity interactions and is based on a simple procedure and a clear principle. To achieve high-throughput and efficient analysis, an automated FAC system was developed. The system designated FAC-1 consists of two isocratic pumps, an autosampler, and a couple of miniature columns (bed volume, 31.4 microl) connected in parallel to either a fluorescence or an ultraviolet detector. By use of this parallel-column system, the time required for each analysis was reduced substantially. Under the established conditions, fewer than 10 hrs are required for 100 interaction analyses, consuming as little as 1 pmol pyridylaminated oligosaccharide for each analysis. This strategy for FAC should contribute to the construction of a lectin-oligosaccharide interaction database essential for future glycomics. Overall features and practical protocols for interaction analyses using FAC-1 are described.
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Cromatografía de Afinidad , Lectinas , Oligosacáridos , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , Lectinas/química , Lectinas/metabolismo , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismoRESUMEN
To investigate protein-carbohydrate interactions in a comprehensive and high-throughput manner, carbohydrate biosensors including microarrays have recently attracted increased attention. In this context, carbohydrate and lectin microarrays are emerging as techniques to meet such requisites. However, most of these methods adopt a conventional immuno-detection system, which requires repetitive washing steps before detection. Since lectin-carbohydrate interactions are relatively weak compared with those between antigens and antibodies, a more precise analytical method, which does not require any washing step, is desirable. We describe here a novel platform for lectin microarray that enables direct observation of lectin-carbohydrate interactions under equilibrium conditions, on the basis of an evanescent-field fluorescence-assisted detection principle. This method allows the analysis of a panel of glycoproteins (glycopeptides) in an extremely sensitive manner. The system also allows real-time observation of lectin-glycoprotein interactions in an aqueous phase. No washing procedures are required, thus relatively weak interactions are detectable. The described lectin microarray is expected to be useful for various fields of glycomics requiring high-throughput analysis of not only purified glycoproteins but also of crude samples.
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Carbohidratos/química , Lectinas/química , Análisis por Micromatrices/métodos , Animales , Glicoproteínas/análisis , Análisis por Micromatrices/instrumentaciónRESUMEN
The mucin-type sialoglycoprotein, podoplanin (aggrus), is a platelet-aggregating factor on cancer cells. We previously described up-regulated expression of podoplanin in malignant astrocytic tumors including glioblastoma. Its expression was associated with tumor malignancy. In the present study, we investigated podoplanin expression and platelet-aggregating activities of glioblastoma cell lines. First, we established a highly reactive anti-podoplanin antibody, NZ-1, which inhibits podoplanin-induced platelet aggregation completely. Of 15 glioblastoma cell lines, LN319 highly expressed podoplanin and induced platelet aggregation. Glycan profiling using a lectin microarray showed that podoplanin on LN319 possesses sialic acid, which is important in podoplanin-induced platelet aggregation. Interestingly, NZ-1 neutralized platelet aggregation by LN319. These results suggest that podoplanin is a main reason for platelet aggregation induced by LN319. We infer that NZ-1 is useful to determine whether platelet aggregation is podoplanin-specific or not. Furthermore, podoplanin might become a therapeutic target of glioblastoma for antibody-based therapy.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Glioblastoma/inmunología , Glicoproteínas de Membrana/inmunología , Agregación Plaquetaria/inmunología , Animales , Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Línea Celular Tumoral , Femenino , Glioblastoma/patología , Glicoproteínas de Membrana/química , Agregación Plaquetaria/efectos de los fármacos , Estructura Terciaria de Proteína , RatasRESUMEN
Two bacterial strains isolated from different hospitals in Japan were subjected to a polyphasic analysis. Strains IFM 0803(T) and IFM 10383 were found to have morphological, biochemical, physiological and chemotaxonomic properties consistent with their classification in the genus Nocardia. Strains IFM 0803(T) and IFM 10383 clustered with the type strain of Nocardia xishanensis, showing 16S rRNA gene sequence similarities of 98.6-98.9 % with this species. The novel strains could be distinguished from N. xishanensis by a range of phenotypic properties. Based on their phenotypic and phylogenetic characteristics, the two isolates are proposed as members of a novel species of the genus Nocardia, Nocardia exalbida sp. nov., with the type strain IFM 0803(T) (=NBRC 100660(T) = JCM 12667(T) = DSM 44883(T)).
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Nocardiosis/diagnóstico , Nocardia/aislamiento & purificación , Humanos , Japón , Datos de Secuencia Molecular , Nocardia/clasificación , Nocardia/genética , Nocardia/crecimiento & desarrollo , Nocardia/metabolismo , FilogeniaRESUMEN
We recently developed a novel system for lectin microarray based on the evanescent-field fluorescence-detection principle, by which even weak lectin-oligosaccharide interactions are detectable without a washing procedure. For its practical application, cell glycan analysis was performed for Chinese hamster ovary (CHO) cells and their glycan profile was compared with those of their glycosylation-defective Lec mutants. Each of the cell surface extracts gave a significantly different profile from that of the parental CHO cells in a manner reflecting denoted biosynthetic features. Hence, the developed lectin microarray system is considered to be fully applicable for differential glycan profiling of crude samples.
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Lectinas/genética , Polisacáridos/química , Análisis por Matrices de Proteínas , Animales , Células CHO , Cricetinae , Cricetulus , Glicosilación , Lectinas/química , Lectinas/metabolismo , Mutación , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.
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Lectinas/química , Análisis por Micromatrices/métodos , Polisacáridos/análisis , Polisacáridos/química , Animales , Fluorescencia , Ratones , Análisis por Micromatrices/normas , Reproducibilidad de los ResultadosRESUMEN
A new method for the quantitative evaluation of molecular interactions that are observed in electrophoresis is described. One component taking part in the interaction is labeled with a fluorescent dye and is subjected to capillary zone electrophoresis with fluorescence detection in the presence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant time intervals, and the electropherograms are converted to represent the fluorescence signal against mobility. After baseline subtraction, the first statistical moment of fluorescence signals on the mobility axis is calculated. This moment represents the average mobility of a labeled component. The change in the mobility moment in the presence and absence of the unlabeled component is used to evaluate the degree of saturation of the binding site of a labeled molecule with an unlabeled molecule. Mixtures of fluorescence-labeled protein (Fab' fragment of antibody or concanavalin A) and its unlabeled interacting partner (alpha(1)-antitrypsin or succinylated ovalbumin, respectively) at various concentrations were injected into a bare-silica capillary, and zone electrophoresis was carried out. The change in the mobility moment of the fluorescence-labeled molecules was used to determine the dissociation constants of the complexes. The determined constants are comparable to those obtained by a well-established method, that is, an analysis based on the peak height of the complex. Since the mobility moment analysis is not affected by the total intensity of the signals, it should be advantageous in analyses in which multiple capillaries are used, in which the injection volume and the sensitivity of detection might be more difficult to control at constant values. The mobility moment analysis also has advantages for the analysis of heterogeneous samples, since the identification of peaks is not necessarily required.