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We propose and experimentally demonstrate a low-loss and low-crosstalk Mach-Zehnder mode/wavelength multi/demultiplexer for WDM/MDM transmission based on a Si-photonics platform. A broadband 3-dB mode divider, which is also newly devised here, makes it possible to compose a Mach-Zehnder filter for "mode" and "wavelength" simultaneously. Transmission characteristics of fabricated 3-dB mode dividers are in excellent agreement with theoretical results. Mach-Zehnder filters using the 3-dB mode divider with a free spectral range (FSR) of 20 and 1 nm are also fabricated and the modal crosstalk is less than -24 dB in the 40-nm wavelength range for the MZ filter with an FSR of 20 nm. The tuning of the peak wavelength position by the TiN heater is also demonstrated.
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BACKGROUND: Abdominal surgery results in neuronal mediator release and subsequent acute intestinal hypomotility. This phase is followed by a longer lasting inflammatory phase resulting in postoperative ileus (POI). Calcitonin gene-related peptide (CGRP) has been shown to induce motility disturbances and in addition may be a candidate mediator to elicit neurogenic inflammation. We hypothesized that CGRP contributes to intestinal inflammation and POI. METHODS: The effect of CGRP in POI was tested in mice treated with the highly specific CGRP receptor antagonist BIBN4096BS and in CGRP receptor-deficient (RAMP-1(-/-) ) mice. POI severity was analyzed by cytokine expression, muscular inflammation and gastrointestinal (GI) transit. Peritoneal and muscularis macrophages and mast cells were analyzed for CGRP receptor expression and functional response to CGRP stimulation. KEY RESULTS: Intestinal manipulation (IM) resulted in CGRP release from myenteric nerves, and a concurrent increased interleukin (IL)-6 and IL-1ß transcription and leukocyte infiltration in the muscularis externa and increased GI transit time. CGRP potentiates IM-induced cytokine transcription within the muscularis externa and peritoneal macrophages. BIBN4096BS reduced cytokine levels and leukocyte infiltration and normalized GI transit. RAMP1(-/-) mice showed a significantly reduced leukocyte influx. CGRP receptor was expressed in muscularis and peritoneal macrophages but not mast cells. CGRP mediated macrophage activation but failed to induce mast cell degranulation and cytokine expression. CONCLUSIONS & INFERENCES: CGRP is immediately released during abdominal surgery and induces a neurogenic inflammation via activation of abdominal macrophages. BIBN4096BS prevented IM-induced inflammation and restored GI motility. These findings suggest that CGRP receptor antagonism could be instrumental in the prevention of POI.
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Ileus/prevención & control , Inflamación/tratamiento farmacológico , Intestinos/efectos de los fármacos , Laparotomía/efectos adversos , Piperazinas/uso terapéutico , Quinazolinas/uso terapéutico , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Animales , Tránsito Gastrointestinal/efectos de los fármacos , Ileus/etiología , Ileus/metabolismo , Inflamación/metabolismo , Inflamación/patología , Intestinos/patología , Ratones , Ratones Noqueados , Músculo Liso/efectos de los fármacos , Piperazinas/farmacología , Periodo Posoperatorio , Quinazolinas/farmacologíaRESUMEN
Castrate resistant prostate cancer (CRPC) is a disease that is resistant to both hormone therapy and chemotherapy. At present, no curative therapy for CRPC has been established. Therefore, it is necessary to determine a novel molecular target for the development of therapeutic agents. We previously reported that AlkB homolog 3 (ALKBH3) is highly expressed in prostate cancer but not in benign prostatic hyperplasia or in normal prostate epithelium and that the expression levels of ALKBH3 protein are significantly correlated with the hormone-independent state of prostate cancer. Moreover, ALKBH3 regulates the invasion of prostate cancer cells via the regulation of matrix metalloproteinase 9. Here, we show that ALKBH3 gene silencing markedly induces apoptosis in hormone-independent prostate cancer cell line DU145 but not in the normal prostate epithelial cell line PNT2. Moreover, the in vivo tumorigenicity of DU145 cells was significantly inhibited by the administration of ALKBH3 siRNA. Furthermore, the anchorage-independent growth of DU145 cells was inhibited by ALKBH3 knockdown and promoted by ALKBH3 overexpression, significantly. ALKBH3 shRNA-expressing prostate cancer cells formed significantly smaller tumors than those of control shRNA transfectants in an in vivo xenograft model. These findings suggest that ALKBH3 is a promising target molecule for the development of CRPC therapeutic agents.
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Antineoplásicos/farmacología , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/genética , Dioxigenasas/antagonistas & inhibidores , Dioxigenasas/genética , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Neoplasias Hormono-Dependientes/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Enzimas Reparadoras del ADN/metabolismo , Dioxigenasas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Humanos , Masculino , Terapia Molecular Dirigida/métodos , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND: We have demonstrated for the first time that a novel human AlkB homologue, ALKBH3, contributes to prostate cancer development, but its clinical and biological roles in lung cancer remain unclear. METHODS: Expression of both mRNA and protein of PCA-1 was examined by RT-PCR and western blotting. We also assessed association with senescence and in vivo ALKBH3 treatment on orthotopic tumour cell inoculation, and analysed it clinicopathologically. RESULTS: We have since found novel biological roles for ALKBH3 in human lung cancers, particularly in adenocarcinoma. Our immunohistochemical analysis of human adenocarcinomas and squamous cell carcinomas of the lung not only showed overexpression of ALKBH3 in these tumours but the percentage of cells positive for ALKBH3 also correlated statistically to recurrence-free survival in adenocarcinoma. Knockdown of ALKBH3 by siRNA transfection induced expression of p21(WAF1/Cip1) and p27(Kip1) in the human lung adenocarcinoma cell line A549, resulting in cell cycle arrest, senescence and strong suppression of cell growth in vitro. In vivo, peritoneal tumour growth and dissemination was inhibited in nude mice, previously inoculated with the A549 cell line, by intraperitoneal injection of ALKBH3 siRNA + atelocollagen, as demonstrated by the reduction in both number and diameter of tumours developing in the peritoneum. CONCLUSION: We suggest that ALKBH3 contributes significantly to cancer cell survival and may be a therapeutic target for human adenocarcinoma of the lung.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Enzimas Reparadoras del ADN/fisiología , Dioxigenasas/fisiología , Neoplasias Pulmonares/genética , Anciano , Anciano de 80 o más Años , Histona H2a Dioxigenasa, Homólogo 1 de AlkB , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Enzimas Reparadoras del ADN/genética , Dioxigenasas/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/fisiología , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , ARN Interferente Pequeño/farmacología , Homología de Secuencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Salvinorin A is the main active psychoactive ingredient in Salvia divinorum, a Mexican plant that has been widely available as a hallucinogen in recent years. The aims of this study were to investigate the stability of salvinorin A in rat plasma, esterases responsible for its degradation, and estimation of the degradation products. The apparent first-order rate constants of salvinorin A at 37 degrees C, 25 degrees C, and 4 degrees C were 3.8 x 10(-1), 1.1 x 10(-1), and < 6.0 x 10(-3) h(-1), respectively. Salvinorin A degradation was markedly inhibited by the addition of sodium fluoride, an esterase inhibitor. Moreover, phenylmethylsulfonyl fluoride (serine esterase inhibitor) and bis-p-nitrophenylphosphate (carboxylesterase inhibitor) also inhibited salvinorin A degradation. In contrast, little or no suppression of the degradation was seen with 5,5'-dithiobis-2-nitrobenzoic acid (arylesterase inhibitor),ethopropazine (butyrylcholinesterase inhibitor), and BW284c51 (acetylcholineseterase inhibitor). These findings indicated that carboxylesterase was mainly involved in the salvinorin A hydrolysis in rat plasma.4. The degradation products of salvinorin A estimated by liquid chromatography-mass spectrometry included the deacetylated form (salvinorin B) and the lactone-ring-open forms of salvinorin A and salvinorin B. This lactone-ring-opening reactions were involved in calcium-dependent lactonase.
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Diterpenos de Tipo Clerodano/farmacocinética , Diterpenos/farmacocinética , Esterasas/metabolismo , Alucinógenos/farmacocinética , Animales , Diterpenos/sangre , Diterpenos de Tipo Clerodano/sangre , Estabilidad de Medicamentos , Inhibidores Enzimáticos/farmacología , Esterasas/antagonistas & inhibidores , Alucinógenos/sangre , Masculino , Ratas , Ratas Wistar , Salvia/químicaRESUMEN
1. The in vivo metabolism of alpha-methyltryptamine (AMT), a psychoactive tryptamine analogue, was studied in rats. 2. Male Wistar rats were administered 10 mg kg(-1) AMT orally and 24-h urine fractions were collected. After enzymatic hydrolysis of the urine sample, the metabolites were extracted by liquid-liquid extraction and analysed by gas chromatography/mass spectrometry (GC/MS). 3. 2-Oxo-AMT, 6-hydroxy-AMT, 7-hydroxy-AMT and 1'-hydroxy-AMT were detected as metabolites of AMT.
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Triptaminas/orina , Animales , Cromatografía de Gases y Espectrometría de Masas , Masculino , Ratas , Ratas Wistar , Triptaminas/administración & dosificación , Triptaminas/metabolismoRESUMEN
The in vivo metabolism of 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), a ring-substituted psychoactive phenethylamine, was studied in rat. Male Wistar rats were administered 10 mg/kg 2C-T-7 hydrochloride orally, and 24-h urine fractions were collected. After enzymatic hydrolysis of the urine sample, the metabolites were extracted by liquid-liquid extraction and analyzed by liquid chromatography/mass spectrometry. 2C-T-7-sulfoxide, N-acetyl-2C-T-7-sulfoxide, N-acetyl-2,5-dimethoxy-4-methylthiophenethylamine-sulfoxide, N-acetyl-2,5-dimethoxy-4-(2-hydroxypropylthio)phenethylamine-sulfoxide, and N-acetyl-2,5-dimethoxy-4-(2-hydroxypropylthio)phenethylamine-sulfone were detected as the primary metabolites of 2C-T-7. These findings suggest that sulfoxidation, sulfone formation, hydroxylation of the propyl side chain at the beta-position, and S-depropylation followed by methylation of thiol were the major metabolic pathways of 2C-T-7 in rat.
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Fenetilaminas/farmacocinética , Psicotrópicos/farmacocinética , Animales , Cromatografía Liquida , Masculino , Espectrometría de Masas , Fenetilaminas/química , Fenetilaminas/orina , Psicotrópicos/química , Psicotrópicos/orina , Ratas , Ratas WistarRESUMEN
The metabolism of methamphetamine (MA) and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in freshly isolated rat hepatocytes was investigated, and compared with in vivo results. A suspended hepatocyte culture, established from male Wistar rats using a collagenase perfusion technique, was incubated in the presence of MA or 2C-B. After enzymatic hydrolysis of the conjugated forms, the metabolites were extracted by liquid-liquid partition and analyzed by gas chromatography/mass spectrometry (GC/MS). Amphetamine, p-hydroxymethamphetamine and p-hydroxyamphetamine were detected in the culture fluids of the rat hepatocytes inoculated with MA. The alcohol derivative, carboxylic acid derivative, 2-O-desmethyl-2C-B, 2-O-desmethyl-N-acetyl-2C-B and 5-O-desmethyl-N-acetyl-2C-B were detected in the case of 2C-B. The major metabolite of MA in rat hepatocytes was p-hydroxymethamphetamine. This is in good agreement with the urinary excretion profile for rats that were fed MA. 2-O-Desmethyl-2C-B and the carboxylic acid derivative were the major recovered metabolites of 2C-B in the rat hepatocyte culture, a slight deviation from the in vivo findings, in which 5-O-desmethyl-N-acetyl-2C-B was found to be the main component. Metabolites with a hydroxy group were largely present in their conjugated forms in the culture fluids, except for 2-O-desmethyl-2C-B. Taking these results into consideration, a primary hepatocyte culture system has the potential to provide a quick and handy method for estimating the in vivo metabolic fate of abused drugs.
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Estimulantes del Sistema Nervioso Central/metabolismo , 2,5-Dimetoxi-4-Metilanfetamina/análogos & derivados , 2,5-Dimetoxi-4-Metilanfetamina/metabolismo , Alucinógenos/metabolismo , Hepatocitos/metabolismo , Metanfetamina/análogos & derivados , Metanfetamina/metabolismo , Anfetamina/metabolismo , Animales , Ácidos Carboxílicos/química , Células Cultivadas , Cromatografía de Gases y Espectrometría de Masas , Masculino , Modelos Animales , Estructura Molecular , Ratas , Ratas Wistar , Simpatomiméticos/metabolismo , p-Hidroxianfetamina/metabolismoRESUMEN
Protein tyrosine phosphatase (PTPase) dephosphorylation and protein tyrosine kinase (PTKs) phosphorylation of key signal transduction proteins may be regulated by extracellular signals, making PTPases important in the regulation of cell proliferation. Leucocyte common antigen (LAR), a receptor-like PTPase, consists of E-subunit, containing the cell adhesion molecule-like receptor region, and P-subunit specific for a short segment of the extracellular region, the transmembrane peptide, and two cytoplasmic PTPase domains. We produced a monoclonal antibody against the LAR P-subunit for immunohistochemical screening of LAR expression in normal and tumourous tissues. Gliomas and gastric, colorectal, lung, breast and prostate cancers showed weak and relatively infrequent expression. Intense and diffuse expression, however, was detected in 95% (227 out of 239) of thyroid carcinomas, but only 12% (22 out of 128) of adenomas and no cases of benign thyroid disease were immunopositive. In contrast to broad staining in carcinomas, LAR expression in thyroid adenomas was often found in small focal or locally invasive areas. Western blot analysis similarly detected LAR P-subunit protein in thyroid carcinomas, but not in normal tissues. We believe this to be the first demonstration of LAR overexpression in thyroid carcinoma and may help to elucidate the role of PTPases in the development of malignancy.
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Antígenos Comunes de Leucocito/genética , Neoplasias de la Tiroides/genética , Antígenos CD/genética , Carcinoma Papilar/genética , Carcinoma Papilar/inmunología , Carcinoma Papilar/patología , Humanos , Inmunohistoquímica , Subunidades de Proteína/genética , Glándula Tiroides/inmunología , Neoplasias de la Tiroides/inmunología , Neoplasias de la Tiroides/patologíaRESUMEN
The calcitonin gene-related peptide (CGRP) plays important roles as a neurotransmitter/neuromodulator in the central nervous system, and as a potent vasodilator when secreted from peripheral, perivascular nerves through its specific receptors. In this study, we cloned mouse cDNA counterparts of the human CGRP receptor composed of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1) and examined the signal transduction mechanism through the CGRP receptor. Mouse CRLR (mCRLR) is a 462-amino acid G protein-coupled heptahelical receptor, and mouse RAMP1 (mRAMP1) is a 148-amino acid single membrane-spanning protein with a short cytoplasmic portion. Specific binding of (125)I-CGRP was detected only when both mCRLR and mRAMP1 cDNAs were cotransfected to COS-7 cells, and the Kd value of the receptor was 2.2 x 10(-10) M. CGRP induced a marked elevation of the intracellular cAMP levels in COS-7 cells cotransfected with mCRLR and mRAMP1. CGRP signaling through the mCRLR/mRAMP1 receptor complex was found to increase the promoter activities of cyclic AMP responsive element and serum responsive element in the co-transfected HeLa cells. These results indicate that mCRLR and mRAMP1 constitute a functional mouse CGRP receptor for the transduction of CGRP signaling by PKA and extracellular signal-regulated kinase signal transduction pathways.
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Proteínas de la Membrana/genética , Ratones/genética , Receptores de Péptido Relacionado con el Gen de Calcitonina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS/metabolismo , Péptido Relacionado con Gen de Calcitonina/farmacología , Células Cultivadas/metabolismo , Chlorocebus aethiops , Clonación Molecular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , ADN Complementario/genética , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sustancias Macromoleculares , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína 1 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Péptido Relacionado con el Gen de Calcitonina/química , Receptores de Péptido Relacionado con el Gen de Calcitonina/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Linfocitos T/metabolismo , TransfecciónRESUMEN
We evaluated the usefulness of 99mTc-methoxy-isobutyl-isonitrile (MIBI) dual phase scintigraphy for detecting hyperfunctioning parathyroid adenoma. We retrospectively reviewed 18 hyperparathyroid patients who received MIBI prior to neck exploration and compared the radiological findings of MIBI with ultrasonography (US) and magnetic resonance imaging (MRI). Fifteen patients were studied with MRI, and 17 patients were examined with US. All patients were found to have a solitary parathyroid adenoma histopathologically. MIBI correctly revealed the location of 17 adenomas among 18 confirmed tumors. In our series, there was one false-positive case that was found to have thyroid adenoma. The diagnostic sensitivity of MIBI MRI and US was 94.4%, 80% and 52.5%, respectively. The positive predictive value (PPV) was 94.4% for MIBI, 81.8% for MRI and 92.3% for US. We conclude that MIBI is useful and accurate for the preoperative localization of adenoma in primary hyperparathyroidism.
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Adenoma/diagnóstico por imagen , Hiperparatiroidismo/diagnóstico por imagen , Glándulas Paratiroides/diagnóstico por imagen , Neoplasias de las Paratiroides/diagnóstico por imagen , Radiofármacos , Tecnecio Tc 99m Sestamibi , Adenoma/complicaciones , Adulto , Anciano , Femenino , Humanos , Hiperparatiroidismo/etiología , Masculino , Persona de Mediana Edad , Neoplasias de las Paratiroides/complicaciones , Cintigrafía , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
A 19-year-old male presented with dyspnea. Clinical examination revealed the left infant-head-sized testicular tumor, multiple lung metastases and retroperitoneal bulky lymph node metastasis with marked elevation of serum lactic dehydrogenase (LDH) and alpha-fetoprotein. Left radical orchiectomy followed by the chemotherapy with etoposide and cisplatin (EP) for 4 cycles was performed. The tumor weighed 1,700 g, and was pathologically diagnosed as mixed germ cell tumor consisting of embryonal carcinoma and yolk sac tumor. After the treatment, the tumor markers were normalized with partial response (PR) of lung metastases and complete response (CR) of retroperitoneal lymph node metastasis. Thereafter, biopsy of lung metastases through video-assisted thoracoscopic surgery (VATS) was performed, and pathologically no viable cells were detected. Five months after the treatment, he was seized with convulsion due to brain metastasis with hemorrhage. Therefore, a surgical resection of brain metastasis and 2nd line chemotherapy with etoposide, ifosfamide and cisplatin (VIP) chemotherapy for 3 cycles was performed. The patient has been free of recurrence for 21 months after the 2nd line chemotherapy.
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Carcinoma Embrionario/diagnóstico , Disnea/etiología , Tumor del Seno Endodérmico/diagnóstico , Neoplasias Pulmonares/secundario , Neoplasias Testiculares/diagnóstico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Embrionario/secundario , Carcinoma Embrionario/terapia , Cisplatino/administración & dosificación , Tumor del Seno Endodérmico/secundario , Tumor del Seno Endodérmico/terapia , Etopósido/administración & dosificación , Humanos , Neoplasias Pulmonares/complicaciones , Metástasis Linfática , Masculino , Neoplasias Primarias Múltiples , Orquiectomía , Neoplasias Testiculares/patología , Neoplasias Testiculares/terapiaRESUMEN
PURPOSE: To investigate the opacity pattern in corneas with an Arg124His (R124H) homozygous mutation of the BIG-H3 gene. METHODS: Slit-lamp examination was performed on eight patients with corneal dystrophy resulting from a genetically confirmed BIG-H3 R124H homozygous mutation. The birthplace of each patient also was determined. RESULTS: Slit-lamp examination disclosed two types of opacity patterns in corneas with the BIG-H3 R124H homozygous mutation. Type I (n = 4) is a spot-like opacity present in the anterior stroma in which the lesions are confluent. Type I is the same pattern that previous reports have shown to be caused by the BIG-H3 R124H homozygous mutation. The type II corneal opacity pattern (n = 4) is a reticular opacity in the anterior stroma with round translucent spaces. Type II opacity has not been reported previously in association with any corneal dystrophy. The patients with the type I opacity do not share a common birthplace; however, interestingly, the patients with the type II opacity traced their origin to Tottori prefecture in western Japan. CONCLUSION: The BIG-H3 homozygous R124H mutation induces the development of two distinct patterns of corneal opacity, the recognition of which can establish an accurate diagnosis of corneal dystrophy caused by the homozygous BIG-H3 R124H mutation independent of genetic analysis. In addition, genetic factors or circumstantial influences other than the gene responsible for the corneal dystrophy may influence the pattern of corneal opacity.
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Córnea/patología , Distrofias Hereditarias de la Córnea/patología , Opacidad de la Córnea/patología , Proteínas de la Matriz Extracelular , Proteínas de Neoplasias/genética , Mutación Puntual , Factor de Crecimiento Transformador beta/genética , Adulto , Distrofias Hereditarias de la Córnea/genética , Opacidad de la Córnea/genética , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agudeza VisualRESUMEN
PURPOSE: It has still not been determined whether the retinal mechanism causing form-deprivation myopia (FDM) is different from that causing lens-induced myopia (LIM). We previously reported that FDM was blocked by an intravitreal injection of the nitric oxide (NO) synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME). In this study, we investigated the effect of L-NAME on LIM in chicks. METHOD: The left eyes of 6-day-old chicks were injected with 30 microl of nontoxic concentrations of L-NAME (< or = 360 mM) or saline. The right eyes were injected with 30 microl of saline. A -16 dpt lens was placed in front of the left eye for 6 days. Another group of 6 chicks were injected with 180 mM L-NAME (left eye) and with saline (right eye) before placing -16 dpt lenses in front of both eyes. After removing the lens, the refraction and the axial length were measured. The effect of L-NAME (180 mM) on the retina of a separate group of chicks was examined by electroretinography 60 min after an intravitreal injection in non-LIM-treated eyes. RESULTS: The eyes of chicks that were injected with 180 or 360 mM L-NAME were less myopic and had significantly shorter axial lengths than control eyes. A significant decrease of the On response and an increase of the Off response were observed. CONCLUSION: The injection of L-NAME into developing chick eyes that were then covered with a -16 dpt lens resulted in a modifications of retinal function and an inhibition of the development of myopia. These results, combined with the earlier findings, suggest that NO modulates a common retinal pathway that leads to both LIM and FDM.
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Lentes de Contacto/efectos adversos , Inhibidores Enzimáticos/farmacología , Miopía/prevención & control , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Pollos , Electrorretinografía , Inyecciones , Miopía/etiología , Miopía/metabolismo , Óxido Nítrico/fisiología , Refracción Ocular , Retina/efectos de los fármacos , Retina/fisiologíaRESUMEN
BACKGROUND: In patients with branch retinal vein occlusion (BRVO), we investigated the presence of indocyanine green (ICG) and fluorescein hyperfluorescence at the site of occlusion. We also assessed the association of this feature with the clinical outcome of these patients. METHODS: Both indocyanine green (ICG) videoangiography and fluorescein angiography (FAG) were performed in 21 eyes with BRVO of less than 1 month duration. Deterioration of the disease was defined clinically as an increase in retinal hemorrhages or retinal edema. Capillary nonperfusion was quantified with computer image analysis from the FAG pictures. RESULTS: ICG videoangiography showed focal hyperfluorescence along the venous wall at the site of the affected A-V crossing in 9 of the 21 eyes, and FAG showed this feature in 10 eyes. The ICG hyperfluorescence was more prominently and focally detected than the hyperfluorescence on FAG, which was sometimes diffusely seen throughout the whole occluded area. Eight of the nine eyes showing ICG hyperfluorescence had clinical deterioration with an increase in retinal hemorrhage or edema. This deterioration occurred more frequently in eyes with hyperfluorescence and/or late leakage than in ones without these features. The mean nonperfused area was significantly larger in eyes with hyperfluorescence than in eyes without these features. CONCLUSION: The ICG hyperfluorescence at the site of A-V crossing is associated with disease deterioration in patients with fresh BRVO. The ICG hyperfluorescence was more easily detectable than the hyperfluorescence on FAG, although the difference in sensitivity between the two methods is not great.
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Angiografía con Fluoresceína , Verde de Indocianina , Oclusión de la Vena Retiniana/diagnóstico , Vena Retiniana/patología , Adulto , Anciano , Femenino , Fluorescencia , Humanos , Masculino , Microscopía por Video , Persona de Mediana Edad , Hemorragia Retiniana/diagnósticoRESUMEN
Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.
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Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/fisiología , Receptor de Insulina/metabolismo , Receptores de Superficie Celular , Animales , Sitios de Unión , Células COS , Cisteína , Eliminación de Gen , Immunoblotting , Técnicas de Inmunoadsorción , Insulina/farmacología , Mutagénesis , Fosforilación , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Serina , Relación Estructura-Actividad , Especificidad por Sustrato , TransfecciónRESUMEN
PURPOSE: To determine a method of rapid detection of M1S1 gene mutations in patients with gelatinous droplike corneal dystrophy. METHODS: Forty-one patients from 35 families with gelatinous drop-like corneal dystrophy were studied. The entire coding region of the M1S1 gene was screened using the protein truncation test (PYT), with a polymerase chain reaction fragment amplified from genomic DNA serving as a template of in vitro translation. RESULTS: Homozygous or compound heterozygous mutations were detected in all patients by a single reaction of the PTT. This result matched those obtained using the polymerase chain reaction-restriction fragment length polymorphism and direct sequence analyses. The Q118X mutation was present in 63 of the 70 alleles, accounting for 90% of the disease-associated chromosomes in Japanese patients. CONCLUSIONS: The PTT is useful for detecting mutations in the M1S1 gene. This technique showed that the Q118X mutation is a founder mutation in Japanese patients with gelatinous droplike corneal dystrophy, and it reflects the linkage disequilibrium reported previously.
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Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Distrofias Hereditarias de la Córnea/genética , Proteínas del Ojo/genética , Mutación , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Molécula de Adhesión Celular Epitelial , Prueba de Complementación Genética , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Activation of the cyclin-dependent kinase cdc2-cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine-14 and tyrosine-15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo-like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo-like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown. RESULTS: We show here that human Ste20-like kinase (SLK), which is a ubiquitously expressed mammalian protein related to xPlkk1, can phosphorylate and activate murine Plk1. During progression through the G2 phase of the mammalian cell cycle, the activity of endogenous SLK is increased. The amount of SLK protein is decreased in quiescent and differentiating cells. Treatment with okadaic acid induces a phosphorylation-dependent enhancement of SLK activity. CONCLUSIONS: We propose that SLK has a role in the regulation of Plk1 activity in actively dividing cells during the somatic cell cycle. SLK itself is suggested to be regulated by phosphorylation.
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Fase G2/fisiología , Mitosis/fisiología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Animales , Western Blotting , Células COS , Proteínas de Ciclo Celular , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fase G2/efectos de los fármacos , Células HeLa , Humanos , Ratones , Mitosis/efectos de los fármacos , Ácido Ocadaico/farmacología , Especificidad de Órganos , Fosforilación/efectos de los fármacos , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/farmacología , Proteínas Proto-Oncogénicas , Transfección , Xenopus , Proteínas de Xenopus , Quinasa Tipo Polo 1RESUMEN
CD45, a prototype of the receptor-like protein tyrosine phosphatase (PTPase) family, is one of the essential molecules in signal transduction through T cell receptors. Because at least 8 types of CD45 isoforms can potentially be produced by alternative mRNA splicing of exons 4, 5, and 6, the analyses at the transcription and protein levels of CD45 during the development and differentiation of T cells have been performed using RT-PCR and isoform-specific monoclonal antibodies, respectively. We report here that the ninth and smallest isoform of CD45, designated as CD45iota (CD45t), which is alternatively spiced from exons 4, 5, and 6 as well as exon 7, is present in the fetal thymus and splenic T cells of mice, and in murine Th1 clones, but not in Th2 clones. The expression of full-length CD45t mRNA as the functional CD45 PTPase was confirmed by RT-PCR analysis. Furthermore, the expression vector of CD45t was constructed, and its expression was detected in combination with anti-pan CD45 mAb and our newly established anti-LAR/CD45 PTPase domain mAb. These results suggested that CD45t might be an important isoform of CD45 for differentiation signaling of Th cells, and might be used as a marker to distinguish between Th1 and Th2 cells.
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Antígenos Comunes de Leucocito/biosíntesis , Bazo/inmunología , Células TH1/inmunología , Células Th2/inmunología , Timo/inmunología , Empalme Alternativo , Animales , Western Blotting , Células COS , Células Cultivadas , Feto , Vectores Genéticos , Antígenos Comunes de Leucocito/análisis , Antígenos Comunes de Leucocito/genética , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/biosíntesis , ARN Mensajero/análisis , Proteínas Recombinantes/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
The laminin alpha2-chain is a component of merosin, a member of the laminin family molecules, which is mainly expressed in the basement membranes of striated muscle. It is known that laminin alpha2 gene (lama2) null mutant mice (dy3k/dy3k) exhibit congenital muscular dystrophy (CMD). Because the laminin alpha2-chain is also expressed in the thymus, the role of merosin in the thymus was examined. In association with the onset of muscular dystrophy, CD4+ CD8+ double-positive (DP) thymocytes disappear by apoptotic cell death, while CD4+ CD8- or CD4- CD8+ thymocytes remain. In order to study the mechanisms leading to the selective death of DP cells in the absence of merosin, the role of the interaction between very late activation antigen-6 (VLA-6), a candidate merosin ligand in the thymus, and merosin was examined. The in vitro survival of thymocytes from normal mice was maintained by the addition of either anti-VLA-6 monoclonal antibodies (mAbs) or merosin. Furthermore, when the normal thymocytes were cultured on thymic epithelial cell lines, viable DP cell recoveries on wild-type epithelial cells were better than on cells from null mutant mice. The results suggest that DP cells are more sensitive to an uncharacterized apoptotic death signal, and that survival is supported by the interaction between VLA-6 and merosin.