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1.
Water Sci Technol ; 70(5): 819-27, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25225928

RESUMEN

Rice straw was added to a sewage sludge digester and its effects on methane production, dewatering characteristics, and microbial communities in the digested sludge were examined by a continuous digestion experiment under mesophilic conditions (35 °C). Stable gas generation was monitored in all digestion experiments. Methane yield from raw sludge, chopped rice straw and softened rice straw were estimated to be 0.27, 0.18 and 0.26 NL/g total solids load, respectively. The capillary suction time of digested sludge was decreased by the addition of rice straw. Archaeal and bacterial communities in the sludge were elucidated by PCR-DGGE (polymerase chain reaction--denaturing gradient gel electrophoresis) targeting 16S rRNA genes. The Shannon index of DGGE profiles indicated that bacterial diversity increased with the addition of softened rice straw. DNA sequences of significant bands of the digested sludge were most closely related to Methanosaeta concilii (97.4% identity) and Methanoculleus bourgensis (100% identity). Meanwhile, those in the co-digested sludge with rice straw were most closely related to Methanosarcina barkeri (98.4% identity) and Methanoculleus bourgensis (99.3% identity). Although both Methanosaeta spp. and Methanosarcina spp. metabolize acetate to methane, Methanosarcina spp. have a competitive advantage at acetate concentrations of >70 mg/L. Results suggested that the quantity of acetate produced during rice straw degradation may change the archaeal community.


Asunto(s)
Reactores Biológicos/microbiología , Metano/metabolismo , Consorcios Microbianos , Oryza , ARN Ribosómico 16S/genética , Archaea/genética , Bacterias/genética , Biocombustibles , Electroforesis en Gel de Gradiente Desnaturalizante , Reacción en Cadena de la Polimerasa , Aguas del Alcantarillado/microbiología
4.
Acta Paediatr ; 99(3): 442-5, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20003102

RESUMEN

AIM: Minor recurrent aphthous stomatitis (MRAS) is a common, painful and inflammatory ailment of the oral cavity with juvenile onset and unknown aetiology. The purpose of this study was to evaluate the potential of ascorbate (vitamin C) to reduce the frequency of MRAS and severity of pain. PATIENTS AND METHODS: Sixteen MRAS patients (9 boys and 7 girls: mean age, 12.0 +/- 2.4 years old) were assigned to take an oral dosage of 2000 mg/m(2)/day ascorbate. SUBJECTS: Their baseline frequency of outbreaks and the level of pains were compared during the treatment; in addition, a crossover clinical trial was performed. Polymorphonuclear leucocytes play a role in the pathogenesis, and then superoxide anion production was evaluated in prior to ascorbate treatment. RESULTS: The data indicated a statistically significant 50% reduction in oral ulcer outbreaks and a decline of pain level. Neutrophils were primed for superoxide anion production in the patients with MRAS. CONCLUSION: Ascorbate may modulate the generation of reactive oxygen species and augment neutrophil apoptosis, which could prevent neutrophil-mediated inflammation. Ascorbate seems to be effective, but the findings of our study were preliminary and it should be re-evaluated with a larger randomized controlled clinical trials.


Asunto(s)
Antioxidantes/uso terapéutico , Ácido Ascórbico/uso terapéutico , Estomatitis Aftosa/prevención & control , Administración Oral , Adolescente , Niño , Estudios Cruzados , Esquema de Medicación , Femenino , Humanos , Masculino , Neutrófilos/metabolismo , Dolor/prevención & control , Prevención Secundaria , Índice de Severidad de la Enfermedad , Superóxidos/metabolismo , Resultado del Tratamiento
5.
Ultrasonics ; 42(1-9): 717-22, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15047373

RESUMEN

The authors have proposed a new type of ultrasonic microscopy for biological tissue characterization. The system is driven by a nanosecond pulse voltage, the generated acoustic wave being reflected at the front and rear side of the sliced tissue. In this report, a time-frequency analysis was applied to determine the sound speed thorough the tissue. Frequency dependence of sound speed was obtained with a myocardium of a rat sliced into 10 microm. As the reflected waveform had a significant amount of oscillating component, the waveform was once subjected to the deconvolution process. As the result, two reflections were clearly separated in time domain. Then these two reflections were separately analyzed by time-frequency analysis. Each reflection was extracted by using a proper window function. Phase angles of these reflections at the same frequency were compared. A sound speed micrograph at an arbitrary frequency in between 50 and 150 MHz was successfully obtained. A tendency was found that the sound speed slightly increases with frequency.


Asunto(s)
Ecocardiografía/métodos , Animales , Diseño de Equipo , Ratas , Procesamiento de Señales Asistido por Computador , Factores de Tiempo
6.
Kyobu Geka ; 56(4): 294-7, 2003 Apr.
Artículo en Japonés | MEDLINE | ID: mdl-12701192

RESUMEN

To examine the mid term outcome of the lateral tunnel Fontan and the result is to be compared to extracardiac Fontan operation. Between March 1991 and May 2002, 72 lateral tunnel (LT) and 28 extracardiac conduit (EC) total cavopulmonary connection (TCPC) were performed. Right atrium was incised parallel to the sulcus terminalis and LT was created by using autologous right atrial wall. Lateral tunnel size was determined 1-2 mm larger than normal half pulmonary artery (PA) size according to the body weight. There were 1 early and 1 late death, both initial LT group. Supraventricular tachycardia was found in 1 patient with EC group and 4 in LT group (all heterotaxy syndrome). There were no differences in mortality and mobidity between LT and EC TCPC. Lateral tunnel TCPC is useful especially to small infants and children.


Asunto(s)
Procedimiento de Fontan , Puente Cardíaco Derecho/métodos , Cardiopatías Congénitas/cirugía , Adolescente , Adulto , Implantación de Prótesis Vascular , Niño , Preescolar , Femenino , Atrios Cardíacos/cirugía , Ventrículos Cardíacos/anomalías , Ventrículos Cardíacos/cirugía , Humanos , Lactante , Masculino , Persona de Mediana Edad , Politetrafluoroetileno , Resultado del Tratamiento
7.
Cell Struct Funct ; 26(4): 197-203, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11699636

RESUMEN

To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibited positive immunostaining for anti-GFAP antibody and A2B5 antibody, characteristics associated with type 2 astrocytes, and expressed glutamine synthetase. Acidic amino acid transporters, GLT-1 and system xc-, which consists of xCT and 4F2hc, were expressed in all TR-ASTs by RT-PCR. On the other hand, GLAST expression was found in TR-AST3 and 5. The characteristics of [3H]L-glutamic acid (L-Glu) uptake by TR-AST5 include an Na+-dependent and Na+-independent manner, concentration-dependence, and inhibition by L-aspartic acid (L-Asp) and D-aspartic acid (D-Asp). The corresponding Michaelis-Menten constants for the Na+-dependent and Na+-independent process were 36.3 microM and 155 microM, respectively. [3H]L-Asp and [3H]D-Asp uptake by TR-AST5 had an Na+-dependent and Na+-independent manner. This study demonstrated that GLT-1, system xc-, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.


Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Aminoácidos/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Línea Celular Transformada , Animales , Animales Modificados Genéticamente , Antígenos Transformadores de Poliomavirus/genética , Transporte Biológico Activo , Proteínas Portadoras/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Inmunohistoquímica , Cinética , Masculino , Ratas , Sodio/metabolismo , Temperatura
8.
J Cereb Blood Flow Metab ; 21(10): 1232-9, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11598501

RESUMEN

In this study, the gamma-aminobutyric acid (GABA) transporter at the blood-brain barrier (BBB) was identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis, and the transport mechanism was characterized using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB) as an in vitro model of the BBB. gamma-Aminobutyric acid transport was studied by the cellular uptake of [ 3 H]GABA. [3H]GABA uptake by TM-BBB cells was Na (+)-, Cl(-)-, and concentration-dependent. The corresponding Michaelis-Menten constant was 679 +/- 80 micromol/L and the maximal uptake rate was 4,790 +/- 494 pmol/(mg protein x 5 minutes). [3H]GABA uptake by TM-BBB cells was significantly inhibited by betaine, beta-alanine, nipecotic acid, taurine, and quinidine, whereas probenecid, L-proline, creatine, and glycine had no effect. This type of inhibition is consistent with the predominant involvement of the GAT2/BGT-1 transporter in TM-BBB cells. RT-PCR analysis showed that GAT2/BGT-1 mRNA was expressed in TM-BBB cells, whereas Western blot analysis showed that TM-BBB cells and mouse brain capillaries express GAT2/BGT-1 protein. Moreover, confocal immunofluorescent microscopy of dual-labeled mouse brain sections demonstrated the colocalization of GAT2/BGT-1 and P-glycoprotein, a BBB-specific marker, on brain capillaries labeled with anti-GAT2/BGT-1 antibody and anti-P-glycoprotein antibody, respectively. These results are evidence that GAT2/BGT-1 is expressed at the BBB and is involved in GABA transport across the BBB.


Asunto(s)
Barrera Hematoencefálica/fisiología , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Ácido gamma-Aminobutírico/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/genética , Células Cultivadas , Proteínas Transportadoras de GABA en la Membrana Plasmática , Glucano Endo-1,3-beta-D-Glucosidasa/genética , Riñón/metabolismo , Cinética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos , Microscopía Confocal , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Urotelio/metabolismo
9.
Pharm Res ; 18(1): 16-22, 2001 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-11336348

RESUMEN

PURPOSE: To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). METHODS: Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degrees C during the first period of 3 days. Cells were subsequently cultured at 33 degrees C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. RESULTS: Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1 approximately 5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degrees C with a doubling-time of 35 approximately 40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. CONCLUSIONS: Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.


Asunto(s)
Aminoácidos/metabolismo , Barrera Hematoencefálica/fisiología , Línea Celular Transformada/metabolismo , Plexo Coroideo/citología , Plexo Coroideo/metabolismo , Células Epiteliales/metabolismo , Animales , Animales Modificados Genéticamente , Antígenos Transformadores de Poliomavirus/metabolismo , Transporte Biológico/fisiología , Ácido Glutámico/metabolismo , Masculino , Prealbúmina/metabolismo , Prolina/metabolismo , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo
10.
Exp Eye Res ; 72(2): 163-72, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11161732

RESUMEN

The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 approximately 9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33 degrees C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39 degrees C due to the reduced expression of large T-antigen, supporting temperature-dependent cell growth. TR-iBRBs expressed GLUT1 and exhibited 3- O -methyl- D -glucose (3-OMG) uptake activity. This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 +/- 0.51 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.


Asunto(s)
Barrera Hematorretinal , Endotelio Vascular/patología , Células Tumorales Cultivadas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Animales , Animales Modificados Genéticamente , Antígenos Virales de Tumores/análisis , Antígenos Virales de Tumores/genética , Western Blotting , Capilares , División Celular , Separación Celular , Genes MDR , Transportador de Glucosa de Tipo 1 , Calor , Modelos Animales , Proteínas de Transporte de Monosacáridos/análisis , Ratas , Proteínas Tirosina Quinasas Receptoras/análisis , Receptores de Factores de Crecimiento/análisis , Receptores de Factores de Crecimiento Endotelial Vascular , Vasos Retinianos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
11.
Pharm Res ; 18(12): 1669-76, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11785685

RESUMEN

PURPOSE: The aim of this study was to characterize L-lactic acid transport using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as a model of in vitro inner blood-retinal barrier (iBRB) to obtain a better understanding of the transport mechanism at the iBRB. METHODS: TR-iBRB2 cells were cultured at 33 degrees C, and L-lactic acid uptake was monitored by measuring [14C]L-lactic acid at 37 degrees C. The expression and mRNA level of monocarboxylate transporter (MCT)1 and MCT2 were determined by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR with specific primers, respectively. RESULTS: The [14C]L-lactic acid uptake by TR-iBRB2 cells increased up to a pH of 5.0 and was inhibited in the presence of 10 mM L-lactic acid. The [14C]L-lactic acid uptake at pH 6.0 was both temperature- and concentration-dependent with a Michaelis-Menten constant of 1.7 mM and a maximum uptake rate of 15 nmol/(30 s mg of protein). This process was reduced by carbonylcyanide p-trifluoromethoxyphenylhydrazone (protonophore), alpha-cyano-4-hydroxycinnamate, and p-chloromercuribenzenesulfonate (typical inhibitors for H+-coupled monocarboxylic acid transport), suggesting that L-lactic acid uptake by TR-iBRB2 cells is a carrier-mediated transport process coupled with an H+ gradient. [14C]L-Lactic acid uptake was markedly inhibited by monocarboxylic acids but not dicarboxylic acids and amino acids. Moreover, salicylic and valproic acids competitively inhibited this process with an inhibition constant of 4.7 mM and 5.4 mM, respectively. Although MCT1 and MCT2 mRNA were found to be expressed in TR-iBRB2 cells, MCT1 mRNA was found to be present at a concentration 33-fold greater than that of MCT2 mRNA using quantitative real-time PCR. [14C]L-Lactic acid was significantly reduced by 5-(N,N-hexamethylene)-amiloride at pH 7.4 and Na+/H+ exchanger I mRNA was expressed in TR-iBRB2 cells. CONCLUSION: L-Lactic acid transport at the iBRB is an H-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.


Asunto(s)
Barrera Hematorretinal/fisiología , Ácido Láctico/farmacocinética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Endotelio Vascular , Concentración de Iones de Hidrógeno , Masculino , Modelos Biológicos , Compuestos Orgánicos/farmacología , ARN Mensajero/análisis , Ratas , Ratas Wistar , Vasos Retinianos/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
12.
Ann Thorac Surg ; 70(5): 1501-6, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11093477

RESUMEN

BACKGROUND: Since 1991 we have performed a multistage palliative approach to biventricular repair of pulmonary atresia or critical pulmonary stenosis with intact ventricular septum in infants with a detectable right ventricular infundibulum. METHODS: A total of 25 patients (19 pulmonary atresia and 6 critical pulmonary stenosis) underwent initial palliation consisting of a transarterial pulmonary valvotomy and a polytetrafluoroethylene shunt between the left subclavian artery and pulmonary trunk. Among the 23 survivors, 15 underwent balloon valvotomy. Six of these patients later required additional palliative surgery that consisted of repeat pulmonary valvotomy, adjustment of an atrial communication, and resection of the hypertrophied muscles in the right ventricle. RESULTS: Of the 25 patients, 23 (92%) survived. In all, 20 patients underwent definitive operations: 18 (90%) biventricular repair (12 pulmonary atresia, and 6 critical pulmonary stenosis), one bidirectional Glenn, and one Fontan procedure. The actuarial probability of achieving a biventricular repair at 36 months of age was 69%. In 18 patients right ventricular end-diastolic volume significantly increased but tricuspid valve diameter did not change. CONCLUSIONS: The multistage palliation procedure to promote right ventricular growth makes a definitive biventricular repair of pulmonary atresia or critical pulmonary stenosis with intact ventricular septum possible in the majority of infants with a patent infundibulum.


Asunto(s)
Atresia Pulmonar/cirugía , Estenosis de la Válvula Pulmonar/cirugía , Procedimientos Quirúrgicos Cardíacos/métodos , Cateterismo , Femenino , Procedimiento de Fontan , Tabiques Cardíacos , Ventrículos Cardíacos , Humanos , Lactante , Recién Nacido , Masculino , Cuidados Paliativos/métodos
13.
J Neurochem ; 75(5): 1907-16, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11032880

RESUMEN

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.


Asunto(s)
Barrera Hematoencefálica/fisiología , Proteínas Portadoras/metabolismo , Sulfato de Deshidroepiandrosterona/metabolismo , Animales , Proteínas de Transporte de Anión , Aniones/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Proteínas Portadoras/genética , Células Cultivadas , Sulfato de Deshidroepiandrosterona/farmacocinética , Sulfato de Deshidroepiandrosterona/farmacología , Digoxina/farmacocinética , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Inulina/farmacocinética , Masculino , Ratones , Ratones Transgénicos , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
14.
Am J Gastroenterol ; 95(8): 2095-8, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10950064

RESUMEN

"Cap polyposis" is a rarely-encountered condition in which distinctive inflammatory polyps are located from the rectum to the distal descending colon. Microscopically, the polyps consist of elongated, tortuous, and distended crypts covered by a "cap" of inflammatory granulation tissue. Although the pathogenesis is unknown, mucosal prolapse has been postulated to be an important etiological factor, given certain clinical and histological similarities. We describe two cases of cap polyposis with protein-losing enteropathy. One was treated successfully by avoidance of straining at defecation. Another resolved after double-barreled transverse colostomy. Both successful treatments support a causal link of polyposis to prolapse.


Asunto(s)
Pólipos del Colon/terapia , Adulto , Colitis/complicaciones , Colon/diagnóstico por imagen , Colon/patología , Pólipos del Colon/complicaciones , Pólipos del Colon/etiología , Pólipos del Colon/cirugía , Colostomía , Defecación , Femenino , Humanos , Persona de Mediana Edad , Enteropatías Perdedoras de Proteínas/complicaciones , Radiografía
15.
AAPS PharmSci ; 2(3): E27, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11741243

RESUMEN

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37 degrees C but not at 39 degrees C. However, growth was restored when the temperature of the culture was lowered to 33 degrees C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33 degrees C, there was less of this complex at 37 degrees C and 39 degrees C. TM-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. The alkaline phosphatase and gamma-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 +/- 1.16 mmol/l. Moreover, P-glycoprotein (P-gp) with a molecular weight of -170 kDa was expressed in all TM-BBBs. Both mdr1a and mdr1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 micromol/l verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.


Asunto(s)
Antígenos Virales de Tumores/metabolismo , Encéfalo/irrigación sanguínea , Endotelio Vascular/citología , Virus 40 de los Simios/inmunología , 3-O-Metilglucosa/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Transporte Biológico , Barrera Hematoencefálica , Capilares , División Celular , Línea Celular , Endotelio Vascular/metabolismo , Transportador de Glucosa de Tipo 1 , Inulina/metabolismo , Manitol/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Transporte de Monosacáridos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temperatura , gamma-Glutamiltransferasa/metabolismo , Factor de von Willebrand/metabolismo , Miembro 4 de la Subfamilia B de Casete de Unión a ATP
16.
J Drug Target ; 8(6): 357-70, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11328662

RESUMEN

Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling-time of about 22-31 hr, but did not grow at 39 degrees C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the gamma-glutamyltranspeptidase activity in TR-BBBs was approximately 13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 +/- 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.


Asunto(s)
Barrera Hematoencefálica/fisiología , Línea Celular/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Endotelio Vascular/metabolismo , Guanosina/farmacocinética , ARN Mensajero/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Animales Modificados Genéticamente , Antígenos Virales de Tumores/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Línea Celular/citología , Claudina-5 , Endotelio Vascular/citología , Transportador de Glucosa de Tipo 1 , Guanosina/análogos & derivados , Moléculas de Adhesión de Unión , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Ocludina , Ratas , Ratas Wistar
17.
Genes Dev ; 12(21): 3325-30, 1998 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-9808619

RESUMEN

Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. A transgenic embryo assay was used to obtain evidence that the Drosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. The eve promoter contains an optimal TATA element and a GAGA sequence. The analysis of various chimeric promoters provides evidence that TATA is essential for promoter competition, whereas GAGA mediates enhancer blocking. The Trithorax-like (Trl) protein interacts with GAGA, and mutations in trl attenuate eve promoter insulator activity. We suggest that Trl-GAGA increases the stability of enhancer-promoter interactions by creating an open chromatin configuration at the core promoter.


Asunto(s)
Proteínas Bacterianas , Proteínas de Drosophila , Drosophila/embriología , Elementos de Facilitación Genéticos/fisiología , Proteínas de Homeodominio/genética , Regiones Promotoras Genéticas/fisiología , Proteínas Represoras/genética , Factores de Transcripción , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Homeodominio/fisiología , Mutación/genética , Proteínas Represoras/fisiología , TATA Box/genética , TATA Box/fisiología
18.
Pathol Int ; 48(6): 486-90, 1998 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9702864

RESUMEN

A case of proliferative fasciitis arising in the left forearm of a 56-year-old man was examined. The lesion was preceded by blunt trauma, measured 1.5 x 1.3 x 1.0 cm, was poorly circumscribed and appeared white to light gray on the cut surface. Light microscopic examinations revealed that spindle cells and giant cells with one or two nuclei and abundant basophilic cytoplasm were arranged without any organized patterns in collagenous stroma. Ultrastructurally, well-developed rough endoplasmic reticulum separated by varying amounts of fine to course fibrillar materials was detected in the giant cells. Only vimentin immunoreactivity was detected in both spindle and giant cells. The Ki-67 labeling index of spindle cells was 35% but that of giant cells was less than 5%, and this reflects the quiescent or slow-growing features of these giant cells in proliferative fasciitis. DNA content of the cells, which was examined by image cytometry, demonstrated diploidy in both spindle (DNA index=1.01) and giant (DNA index=1.09) cells.


Asunto(s)
Fascitis/patología , Traumatismos del Antebrazo/patología , Ploidias , Heridas no Penetrantes/patología , División Celular , ADN/análisis , Retículo Endoplásmico Rugoso/ultraestructura , Fascitis/genética , Fascitis/metabolismo , Fascitis/cirugía , Traumatismos del Antebrazo/cirugía , Células Gigantes/ultraestructura , Humanos , Citometría de Imagen , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Vimentina/metabolismo , Heridas no Penetrantes/cirugía
19.
J Biochem ; 123(3): 540-5, 1998 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9538240

RESUMEN

A cDNA for mouse prolyl endopeptidase (PEP) was cloned and its nucleotide sequence determined. The overall amino acid sequence identity between mouse and other mammalian PEPs was about 96%. A specific inhibitor of PEP, N-benzyloxycarbonyl-thioprolyl-thioprolinal- dimethylacetal (ZTTA), inhibited DNA synthesis by Swiss 3T3 cells. Mouse PEP was shown to be localized partly in restricted nuclear regions. These results suggest that PEP participates in mammalian DNA synthesis.


Asunto(s)
Células 3T3/enzimología , ADN/biosíntesis , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Células 3T3/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , División Celular/efectos de los fármacos , Núcleo Celular/enzimología , Clonación Molecular , Citoplasma/enzimología , Técnica del Anticuerpo Fluorescente Indirecta , Immunoblotting , Ratones , Datos de Secuencia Molecular , Prolil Oligopeptidasas , Serina Endopeptidasas/inmunología , Tiazoles/farmacología
20.
Genes Dev ; 12(4): 547-56, 1998 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-9472023

RESUMEN

There are numerous examples of shared enhancers interacting with just a subset of target promoters. In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes. Here, we employ a transgenic embryo assay to obtain evidence that promoter selection is influenced by the TATA element. Both the AE1 enhancer from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer from the Bithorax complex (BX-C) preferentially activate TATA-containing promoters when challenged with linked TATA-less promoters. In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between these two classes of promoters. Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters. These results provide in vivo evidence that different core promoters possess distinct regulatory activities. We discuss the possibility that an invariant TFIID complex can adopt different conformations on the core promoter.


Asunto(s)
Proteínas Bacterianas , Proteínas de Drosophila , Drosophila/genética , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Proteína con Homeodominio Antennapedia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Unión al ADN , Drosophila/embriología , Factores de Transcripción Fushi Tarazu , Genes Reporteros , Proteínas de Homeodominio/genética , Modelos Genéticos , Proteínas Nucleares , Fosfoproteínas , Unión Proteica , Conformación Proteica , TATA Box , Factor de Transcripción TFIID , Factores de Transcripción TFII
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