RESUMEN
Inter-subject variability has caused the majority of previous electrical impedance tomography (EIT) techniques to focus on the derivation of relative or difference measures of in vivo tissue resistivity. Implicit in these techniques is the requirement for a reference or previously defined data set. This study assesses the accuracy and optimum electrode placement strategy for a recently developed method which estimates an absolute value of organ resistivity without recourse to a reference data set. Since this measurement of tissue resistivity is absolute, in Ohm metres, it should be possible to use EIT measurements for the objective diagnosis of lung diseases such as pulmonary oedema and emphysema. However, the stability and reproducibility of the method have not yet been investigated fully. To investigate these problems, this study used a Sheffield Mk3.5 system which was configured to operate with eight measurement electrodes. As a result of this study, the absolute resistivity measurement was found to be insensitive to the electrode level between 4 and 5 cm above the xiphoid process. The level of the electrode plane was varied between 2 cm and 7 cm above the xiphoid process. Absolute lung resistivity in 18 normal subjects (age 22.6 +/- 4.9, height 169.1 +/- 5.7 cm, weight 60.6 +/- 4.5 kg, body mass index 21.2 +/- 1.6: mean +/- standard deviation) was measured during both normal and deep breathing for 1 min. Three sets of measurements were made over a period of several days on each of nine of the normal male subjects. No significant differences in absolute lung resistivity were found, either during normal tidal breathing between the electrode levels of 4 and 5 cm (9.3 +/- 2.4 Omega m, 9.6 +/- 1.9 Omega m at 4 and 5 cm, respectively: mean +/- standard deviation) or during deep breathing between the electrode levels of 4 and 5 cm (10.9 +/- 2.9 Omega m and 11.1 +/- 2.3 Omega m, respectively: mean +/- standard deviation). However, the differences in absolute lung resistivity between normal and deep tidal breathing at the same electrode level are significant. No significant difference was found in the coefficient of variation between the electrode levels of 4 and 5 cm (9.5 +/- 3.6%, 8.5 +/- 3.2% at 4 and 5 cm, respectively: mean +/- standard deviation in individual subjects). Therefore, the electrode levels of 4 and 5 cm above the xiphoid process showed reasonable reliability in the measurement of absolute lung resistivity both among individuals and over time.
Asunto(s)
Impedancia Eléctrica , Electrodos , Aumento de la Imagen/instrumentación , Interpretación de Imagen Asistida por Computador/métodos , Pulmón/fisiología , Pletismografía de Impedancia/instrumentación , Tomografía/instrumentación , Adulto , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Aumento de la Imagen/métodos , Pulmón/anatomía & histología , Masculino , Pletismografía de Impedancia/métodos , Control de Calidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tomografía/métodosRESUMEN
Clock genes, which mediate molecular circadian rhythms, are expressed in a circadian fashion in the suprachiasmatic nucleus and in various peripheral tissues. To establish a molecular basis for circadian regulation in the salivary glands, we examined expression profiles of clock-related genes and salivary gland-characteristic genes. Clock-related genes-including Per1, Per2, Cry1, Bmal1, Dec1, Dec2, Dbp, and Reverbalpha-showed robust circadian expression rhythms in the submandibular glands in 12:12-hour light-dark conditions. In addition, a robust circadian rhythm was observed in amylase 1 mRNA levels, whereas the expression of other salivary-gland-characteristic genes examined was not rhythmic. The Clock mutation resulted in increased or decreased mRNA levels of Per2, Bmal1, Dec1, Dec2, and Dbp, and in Cry1-/- background, Cry2 disruption also increased or decreased mRNA levels of these clock-related genes and the amylase 1 gene. These findings indicate that the Clock- and Cry-dependent molecular clock system is active in the salivary glands.
Asunto(s)
Ritmo Circadiano/genética , Glándula Submandibular/metabolismo , Transactivadores/análisis , Factores de Transcripción ARNTL , Amilasas/análisis , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Relojes Biológicos/genética , Proteínas CLOCK , Proteínas de Ciclo Celular , Criptocromos , Proteínas de Unión al ADN/análisis , Flavoproteínas/análisis , Secuencias Hélice-Asa-Hélice/genética , Proteínas de Homeodominio/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Proteínas Nucleares/análisis , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares , Proteínas Circadianas Period , Receptores Citoplasmáticos y Nucleares/análisis , Glándula Submandibular/enzimología , Factores de Transcripción/análisisRESUMEN
A phantom was constructed to simulate the electrical properties of the neck. A range of possible electrode configurations was then examined in order to improve the sensitivity of the impedance measurement method for the in vivo detection of air emboli. The neck phantom consisted of simulated skin, fat and muscle layers made of agar and a conductive rubber tube mimicking the common carotid artery. The ring-shaped electrodes with a guard electrode showed the highest sensitivity to emboli at short distances.
Asunto(s)
Enfermedades de las Arterias Carótidas/diagnóstico , Enfermedades de las Arterias Carótidas/fisiopatología , Impedancia Eléctrica , Electrodos , Embolia/diagnóstico , Embolia/fisiopatología , Pletismografía de Impedancia/instrumentación , Composición Corporal , Diagnóstico por Computador/instrumentación , Diagnóstico por Computador/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Fantasmas de Imagen , Pletismografía de Impedancia/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Non-invasive detection of air emboli in blood is investigated in vitro using a tetrapolar electrical impedance measurement. A cubic tank with a linear array of four electrodes, spaced approximately 1 cm apart down one side, was filled with 0.2 Sm(-1) saline. Bubbles were generated by carbon dioxide gas. Electrical transfer impedance was measured every 8.2 ms at 1.25 MHz. The movement of bubbles was recorded by a video camera, and their sizes and depths from the middle of the array were measured using captured video images. Changes in transfer impedance caused by passage of bubbles were clearly observed and almost identical with those calculated theoretically. Using lead field theory and experimental results, the fundamental limit on the detectable size of bubbles was estimated at the carotid artery, the great saphenous vein and the cephalic vein. The theoretical results showed that a 0.5 mm diameter bubble is detectable at a depth of 5.3 mm, similar to the depth of the great saphenous and the cephalic veins, and a 2.3 mm diameter bubble is detectable at a depth of 21 mm, similar to the depth of the common carotid artery.
Asunto(s)
Electrodiagnóstico/métodos , Embolia Aérea/diagnóstico , Impedancia Eléctrica , Embolia Aérea/patología , Humanos , Técnicas In Vitro , Modelos Cardiovasculares , Tomografía/métodosRESUMEN
Accurate electrical transfer impedance measurement at the high frequencies (> 1 MHz) required to characterise blood and intracellular structures is very difficult, owing to stray capacitances between lead wires. To solve this problem, an optically isolated measurement system has been developed using a phase-locked-loop technique for synchronisation between current injection (drive) and voltage measurement (receive) circuits. The synchronisation error between drive and receive circuits was less than 1 ns. The accuracy and reproducibility of the developed system was examined using a tissue equivalent Cole model consisting of two resistors and one capacitor. The absolute value Z and phase shift theta in impedance of the Cole model was measured at 1.25 MHz by both an LCR meter and the isolated measurement system. The difference between the values measured by the isolated measurement system and those measured by the LCR meter was less than 0.27omega (2.9%) in Z and 0.79 degree in theta. The standard deviation was less than 0.09 omega in Z and 0.60 degree in theta.
Asunto(s)
Impedancia Eléctrica , Electrónica Médica/instrumentación , Humanos , Modelos Biológicos , Reproducibilidad de los ResultadosRESUMEN
Immunohistochemical analysis showed that sterol 14-demethylase P450 (CYP51) is expressed in mature follicles and corpus lutea of rat ovaries. In follicles, CYP51 is expressed in granulosa and theca cells but not in oocytes. The ovarian CYP51 activity of hypophysectomized rats is very low and induced by pregnant mares' serum gonadotropin (PMSG) treatment together with ovarian growth. The expression of CYP51 first increases in growing follicles and then appears in the corpus lutea after luteinization. The former event may be due to the follicular-stimulating hormone action of PMSG, and the latter may be caused by the luteinizing hormone effect of PMSG. Sterol analysis indicated that the product of the CYP51-mediated lanosterol 14-demethylation, 4,4-dimethylcholesta-8,14,24-trienol, which has been identified as a meiosis-activating steroid (MAS) in mammals [Byskov et al. (1995) Nature 374, 559-562], accumulates (about 10 pmol/mg of ovary) in mature rat ovaries, and the content is enough to activate the resumption of meiosis. These lines of evidence suggest that the expression of ovarian CYP51 is dependent on gonadotropins, and ovarian CYP51 activity is enough for accumulating MAS. Serum insulin does not affect the ovarian CYP51 level, although it is essential for hepatic CYP51 expression. These findings indicate that expression of CYP51 is regulated differently among organs.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Gonadotropinas/farmacología , Ovario/efectos de los fármacos , Oxidorreductasas/biosíntesis , Esteroles/metabolismo , Animales , Antibacterianos/farmacología , Gonadotropina Coriónica/farmacología , Femenino , Gonadotropinas Equinas/farmacología , Hepatocitos/enzimología , Hipofisectomía/efectos adversos , Meiosis/fisiología , Ovario/enzimología , Ovario/metabolismo , Ratas , Ratas Sprague-Dawley , Maduración Sexual/fisiología , Esterol 14-Desmetilasa , Estreptozocina/farmacologíaRESUMEN
OBJECTIVE: The purpose of this study is to investigate stage- and hormone-dependent expression of chondrocyte-derived ezrin-like domain containing protein (CDEP), a putative guanine nucleotide exchange factor (GEF) for Rho in chondrocytes, and demonstrate the guanine nucleotide exchange activity of this protein in vitro, as well as the transforming activity in NIH3T3 cells. METHODS: The expression of CDEP mRNA in growth plate chondrocytes in vivo and in vitro was examined by RT-PCR Southern analysis. The guanine nucleotide exchange activity was determined using a recombinant CDEP peptide containing the DH and PH domains in Sf9 cell lysates. The transforming activity was examined using NIH3T3 cells transiently transfected with a truncated CDEP cDNA. RESULTS: CDEP mRNA was expressed at the highest level in the hypertrophic (terminal) stage of chondrocytes in vivoand in vitro. Parathyroid hormone (PTH) elicited a biphasic increase of CDEP mRNA in chondrocytes. The CDEP mRNA level increased within 1 h, then decreased nearly to the control level at 3 h. Thereafter the mRNA level started to increase at 6 h, reaching a plateau at 24 h. Dibutyryl cyclic AMP had a similar effect on CDEP expression in chondrocytes. The dissociation of [3H]GDP from RhoA was stimulated dose-dependently by Sf9 cell lysates containing the CDEP peptide. Furthermore, transfection of a truncated CDEP cDNA induced focus formation in NIH3T3 cultures. CONCLUSIONS: CDEP is a novel GEF for Rho family GTPases with the transforming activity. CDEP may play a role in mediating or modulating the action of cAMP-elevating hormones on maturing chondrocytes.
Asunto(s)
Condrocitos/fisiología , AMP Cíclico/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hormona Paratiroidea/fisiología , Piperazinas/metabolismo , Animales , Southern Blotting , Transformación Celular Neoplásica , Células Cultivadas , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , TransfecciónRESUMEN
Subthreshold electrical stimulation with an intensity less than the threshold for evoking M-waves is applied repetitively to the common peroneal nerve via surface electrodes. The stimulation intensity is varied by adjusting the pulse width from 0 to 240 micros, while the pulse interval (40 ms) and current amplitude are kept constant. Single magnetic stimuli are applied to the motor cortex using a circular coil. Motor evoked potentials are recorded from the anterior tibial muscle in six normal subjects for various subthreshold stimulation intensities. Signal processing (filtering in the time and frequency domains) removes the artifact caused by the subthreshold electrical stimulation from the motor evoked potential. Statistically significant motor evoked potential facilitation (p < 0.05) is observed for pulse widths ranging from 72 to 240 micros in all the tested subjects. A pulse width corresponding to 90% of the electrical threshold facilitated the motor evoked potential in five of the six subjects.
Asunto(s)
Terapia por Estimulación Eléctrica/métodos , Potenciales Evocados Motores , Músculo Esquelético/fisiología , Adulto , Electromiografía , Humanos , Procesamiento de Señales Asistido por ComputadorRESUMEN
DEC1 (BHLHB2)/Stra13/Sharp2, a basic helix-loop-helix (bHLH) transcription factor has been suggested to be involved in the control of proliferation and/or differentiation of several cells including nerve cells, fibroblasts and chondrocytes. In the present study, we examined the effect of parathyroid hormone (PTH), dibutyryl cAMP (Bt2cAMP) and forskolin on the expression of DEC1 in various cells. In rabbit chondrocyte cultures, PTH or Bt2cAMP increased the DEC1 mRNA level within 1 h. Thereafter, the DEC1 mRNA level rapidly decreased to the basal level at 3 h, and increased at 6-24 h. In cultures of a mouse embryo prechondrogenic cell line ATDC5, PTH or forskolin, an activator of adenylate cyclase, also increased the DEC1 mRNA level within 1 h. Furthermore, in all evaluated cell lines of human fibroblasts, canine epithelial cells, human carcinoma, human glioblastoma and human melanoma, Bt2cAMP increased the DEC1 mRNA level within 1-3 h. Studies with actinomycin D and cycloheximide indicated that the enhancement of DEC1 mRNA by cAMP was not due to mRNA stabilization and did not require new protein synthesis. These findings suggest that DEC1 is a novel direct target for cAMP in wide types of cells, and that the bHLH protein is involved in the control of gene expression in cAMP-activated cells.
Asunto(s)
AMP Cíclico/fisiología , Secuencias Hélice-Asa-Hélice/fisiología , Proteínas de Homeodominio/genética , Transducción de Señal/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Condrocitos/citología , Cicloheximida/farmacología , Dactinomicina/farmacología , Perros , Fibroblastos/citología , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Glioblastoma , Células HeLa , Humanos , Riñón/citología , Pulmón/citología , Melanoma , Ratones , Ratones Endogámicos BALB C , Hormona Paratiroidea/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/análisis , Conejos , TeratocarcinomaRESUMEN
Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.
Asunto(s)
Regulación hacia Abajo , Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Teriparatido/metabolismo , Transactivadores , Tretinoina/metabolismo , Regulación hacia Arriba , Animales , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Colágeno/genética , Cicloheximida/farmacología , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Placa de Crecimiento/citología , Proteínas Hedgehog , Humanos , Masculino , Proteínas de la Membrana/genética , Osteopontina , Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea , Receptores Patched , Receptor Patched-1 , Fragmentos de Péptidos/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/farmacología , ARN Mensajero , Conejos , Receptor de Hormona Paratiroídea Tipo 1 , Receptores de Superficie Celular , Receptores de Hormona Paratiroidea/genética , Sialoglicoproteínas/genética , Teriparatido/farmacología , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
Recently, we purified membrane-bound transferrin-like protein (MTf) from the plasma membrane of rabbit chondrocytes and showed that the expression levels of MTf protein and mRNA were much higher in cartilage than in other tissues [Kawamoto T, Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R., Nakamasu, K., Noshiro, M., Kawashima-Ohya, Y., Fujii, M., Shintani, H., Okada, Y. & Kato, Y. (1998) Eur. J. Biochem. 256, 503--509]. In this study, we isolated the MTf gene from a constructed mouse genomic library. The mouse MTf gene was encoded by a single-copy gene spanning approximately 26 kb and consisting of 16 exons. The transcription-initiation site was located 157 bp upstream from the translation-start codon, and a TATA box was not found in the 5' flanking region. The mouse MTf gene was mapped on the B3 band of chromosome 16 by fluorescence in situ hybridization. Using primary chondrocytes, SK-MEL-28 (melanoma cell line), ATDC5 (chondrogenic cell line) and NIH3T3 (fibroblast cell line) cells, we carried out transient expression studies on various lengths of the 5' flanking region of the MTf gene fused to the luciferase reporter gene. Luciferase activity in SK-MEL-28 cells was higher than in primary chondrocytes. Although no luciferase activity was detectable in NIH3T3 cells, it was higher in chondrocytes than in ATDC5 chondrogenic cells. These findings were consistent with the levels of expression of MTf mRNA in these cells cultured under similar conditions. The patterns of increase and decrease in the luciferase activity in chondrocytes transfected with various 5' deleted constructs of the MTf promoter were similar to that in ATDC5 cells, but differed from that in SK-MEL-28 cells. The findings obtained with primary chondrocytes suggest that the regions between -693 and -444 and between -1635 and -1213 contain positive and negative cis-acting elements, respectively. The chondrocyte-specific expression of the MTf gene could be regulated via these regulatory elements in the promoter region.
Asunto(s)
Proteínas de la Membrana/genética , Metaloproteínas/genética , Proteínas de Neoplasias , Regiones Promotoras Genéticas/genética , Transferrina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Células Cultivadas , Condrocitos/metabolismo , Clonación Molecular , Exones/genética , Proteínas Ligadas a GPI , Regulación de la Expresión Génica , Hibridación Fluorescente in Situ , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Mapeo Físico de Cromosoma , ARN Mensajero/análisis , ARN Mensajero/genética , Conejos , Eliminación de Secuencia/genética , Transfección , Transferrina/química , Transferrina/metabolismoRESUMEN
We studied the effects of bile acid sulfonate analogs, namely, 3alpha,7alpha,12alpha-trihydroxy-5beta-cholane-24-sulfonate (C-sul), 3alpha,7alpha-dihydroxy-5beta-cholane-24-sulfonate (CDC-sul), and 3alpha,7beta-dihydroxy-5beta-cholane-24-sulfonate (UDC-sul), on serum and liver cholesterol levels, cholesterol 7alpha-hydroxylase activity, and biliary bile acid composition in hamsters fed cholesterol. Of the three analogs studied, UDC-sul slightly but significantly decreased free, esterified, and total cholesterol concentrations in the serum. UDC-sul and CDC-sul reduced liver total cholesterol levels by 25% and 18%, respectively, particularly in the esterified cholesterol fraction. Analysis of biliary bile acids showed the presence of the administered analogs, indicating that sulfonate analogs efficiently participate in enterohepatic cycling. The proportion of cholic acid was increased in all groups fed sulfonate analogs, but the ratio of glycine to taurine conjugated bile acids (G/T) was elevated only in UDC-sul feeding hamsters. There was no significant change in cholesterol 7alpha-hydroxylase activity in hamsters fed C-sul or CDC-sul, while UDC-sul slightly stimulated the enzyme activity compared to the control. The UDC-sul induced decrease in serum and liver cholesterol concentrations may be secondary to enhanced bile acid synthesis. This is supported by the increased cholesterol 7alpha-hydroxylase activity and elevated G/T ratio in biliary bile acids observed following UDC-sul administration.
Asunto(s)
Anticolesterolemiantes/farmacología , Ácidos y Sales Biliares/farmacología , Hipercolesterolemia/tratamiento farmacológico , Animales , Peso Corporal/efectos de los fármacos , Ácido Quenodesoxicólico/farmacología , Colesterol/sangre , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Ácidos Cólicos/farmacología , Cricetinae , Hipercolesterolemia/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Mesocricetus , Tamaño de los Órganos/efectos de los fármacos , Ácido Ursodesoxicólico/farmacologíaRESUMEN
DEC1 is a basic helix-loop-helix (bHLH) protein related to Drosophila Hairy, Enhancer of split and HES, and involved in the control of proliferation and/or differentiation of chondrocytes, neurons, etc. We report here the identification and characterization of human, mouse and rat DEC2, a novel member of the DEC subfamily. DEC2 had high (97%) and moderate (52%) similarities in the bHLH region and the Orange domain with DEC1, respectively. However, DEC2, but not DEC1, had alanine and glycine-rich regions in the C-terminal half. Unlike Hairy, Enhancer of split and HES, DEC2 lacked the WRPW motif for interaction with the corepressor Groucho. The DEC2 gene was mapped to human chromosome 12p11.23-p12.1, mouse chromosome 6 G2-G3 and rat chromosome 4q43 distal-q4, where the conserved linkage homology has been identified among these species. Unlike DEC1, which was broadly expressed in many tissues, DEC2 showed a more restricted pattern of mRNA expression. The DEC subfamily proteins may play an important role in tissue development.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 12 , Proteínas de Drosophila , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Clonación Molecular , Drosophila , Secuencias Hélice-Asa-Hélice , Humanos , Hibridación Fluorescente in Situ , Proteínas de Insectos/química , Cariotipificación , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Proteínas Recombinantes/química , Proteínas Represoras/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
DEC1/BHLHB2 is a novel cAMP-inducible basic helix-loop-helix (bHLH) transcriptional factor isolated from human chondrocyte cultures by the subtraction method [Shen et al. (1997) Biochem. Biophys. Res. Commun. 236, 294--298]. DEC1 seems to be involved in controlling the proliferation/differentiation of some cell lineages. We determined the structure of the human DEC1 gene and its chromosomal locus. Phylogenetic analysis and comparison of the gene structure showed that the DEC1 protein is a member of a new subgroup of the proline bHLH protein family that diverged earlier than other proline bHLH proteins including HES, hairy and E(spl). The human DEC1 gene spans approximately 5.7 kb and contains 5 exons. The putative promoter region contains multiple GC boxes but no TATA box. A primer extension study showed multiple transcriptional initiation sites. In the 5'-flanking region of the DEC1 gene, several transcriptional factor binding sites, including a cAMP-responsive element (CRE), were found using the transcription factor database. The DEC1 gene locates at Chromosome 3p25.3--26 by the FISH method. This is the first study to determine the genomic structure of the DEC1 gene subgroup.
Asunto(s)
Cromosomas Humanos Par 3/genética , Exones/genética , Secuencias Hélice-Asa-Hélice , Proteínas de Homeodominio/genética , Intrones/genética , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Southern Blotting , Clonación Molecular , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Filogenia , Mapeo Físico de Cromosoma , Sitios de Empalme de ARN/genéticaRESUMEN
BACKGROUND AND AIM: Although mucosal alpha- and beta-chemokines are considered to be involved in the pathogenesis of Helicobacter pylori-associated gastritis, little is known how these chemokines are related to the ulcerogenesis in peptic ulcer patients. We examined the levels of interleukin (IL)-8 and macrophage inflammatory protein-1alpha (MIP-1alpha) in organ cultures and the numbers of inflammatory cells infiltrating the lamina propria by using the mucosal tissues obtained from gastric ulcer (GU) patients with and without H. pylori infection. METHODS: Levels of IL-8 and MIP-1alpha secreted in organ cultures were measured by an enzyme-linked immunosorbent assay. Numbers of myeloperoxidase-positive neutrophils, CD68-positive macrophages, and mononuclear cells were determined in tissue sections. RESULTS: The mucosal tissues of both the gastric antrum and the ulcer site obtained from patients with H. pylori-positive GU showed significantly higher levels of IL-8 and MIP-1alpha and increased numbers of inflammatory cells compared with the corresponding mucosal tissues from those with H. pylori-negative GU or the antral mucosal tissues from H. pylori-negative controls. When the values were compared between the mucosal tissues from the gastric antrum and those from the ulcer site, the latter group of tissues showed significantly higher levels of IL-8 and MIP-1alpha and increased numbers of neutrophils and macrophages than the former group regardless of its healing process in patients with H. pylori-positive GU. CONCLUSION: Mucosal alpha- and beta-chemokines may be important to the ulcerogenesis in H. pylori-associated GU disease.
Asunto(s)
Infecciones por Helicobacter/inmunología , Helicobacter pylori , Interleucina-8/inmunología , Proteínas Inflamatorias de Macrófagos/inmunología , Úlcera Gástrica/microbiología , Adulto , Quimiocina CCL3 , Quimiocina CCL4 , Femenino , Infecciones por Helicobacter/patología , Humanos , Interleucina-8/análisis , Mucosa Intestinal/microbiología , Proteínas Inflamatorias de Macrófagos/análisis , Masculino , Persona de Mediana Edad , Úlcera Gástrica/inmunología , Úlcera Gástrica/patologíaRESUMEN
The role of serum insulin in regulating the expression level of hepatic sterol 14-demethylase P450 (CYP51) was examined. Administration of streptozotocin, which destroys pancreatic beta-cells, caused reduction of CYP51 mRNA level in rats in parallel with the loss of serum insulin. Streptozotocin treatment also reduced the CYP51 activity. The decreased mRNA level and activity of the streptozotocin-treated rats were restored to the normal level within 24 h by repeated administration of insulin. CYP51 level of normal rats was insensitive to the circadian variation of serum insulin and insulin administration, and no significant difference was observed between the hepatic CYP51 activities of Sprague-Dawley and Wistar lean rats, although the serum insulin concentration of the latter was higher than the former. These facts indicate that the expression of hepatic CYP51 is maintained by serum insulin, and its lowest physiological level is sufficient for supporting the expression of CYP51. The responses of CYP51 expression to streptozotocin and insulin treatments were closely similar to those of the sterol regulatory element binding protein (SREBP)-1c expression [Shimomura et al. (1999) Proc. Nat. Acad. Sci. USA 96, 13656-13661]. Based on this fact, the possible contribution of SREBP-1c to the insulin-dependent expression of hepatic CYP51 gene was also discussed.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Insulina/metabolismo , Hígado/enzimología , Oxidorreductasas/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Inyecciones , Insulina/sangre , Insulina/farmacología , Hígado/efectos de los fármacos , Masculino , Oxidorreductasas/efectos de los fármacos , Oxidorreductasas/genética , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Esterol 14-Desmetilasa , Estreptozocina/toxicidad , Testículo/efectos de los fármacos , Testículo/enzimologíaRESUMEN
Biodiversity is the most characteristic feature of cytochrome P450. Finding of CYP51 distributing widely in biological kingdoms provided breakthroughs for the discussion on the evolution and diversification of P450. Molecular phylogenetic analysis demonstrated that CYP51 appeared in the prokaryotic era and distributed into most kingdoms concomitant with phylogenetic divergence. This is the first evolutionary evidence indicating the prokaryotic origin of P450. Modification of substrate specificity of eukaryotic CYP51s occurred independently to adapt to the different sterol precursors existing in each kingdom. Formation of CYP51 variants through the mutation of active site and the selection of the advantageous ones from them were demonstrated by the emergence of azole-resistant CYP51s in Candida albicans under the environments rich in azole antifungal agents. These findings illustrate the most probable core process of P450 diversification consisting of modification of active site and selection of the resulting variants through interaction with endogenous and exogenous chemicals.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Evolución Molecular , Familia de Multigenes , Oxidorreductasas/genética , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Esterol 14-DesmetilasaRESUMEN
BACKGROUND: Although increased levels of interleukin (IL)-8 are known to be associated with infiltration of neutrophils in the gastric mucosa with Helicobacter pylori infection, no study has investigated the relationship between local IL-8 levels and neutrophil infiltration in the duodenal mucosa of patients with duodenal ulcer (DU). METHODS: Duodenal mucosal biopsy specimens with and without gastric metaplasia (GM) were obtained from patients with DU and controls with an endoscopic methylene blue (MB) staining method. Levels of IL-8 secreted in the organ cultures of biopsy specimens were measured with an enzyme-linked immunosorbent assay. The number of myeloperoxidase-positive neutrophils infiltrating the lamina propria was determined in immunohistochemically stained tissue sections. RESULTS: Histologic assessment showed that there was a strong correlation between the absence of endoscopic MB staining and the extent of GM. The levels of IL-8 in both duodenal and antral mucosal tissues were significantly higher in patients with H. pylori infection than in those without infection. In patients with DU the duodenal mucosal tissues with GM (MB-unstained mucosa) showed significantly higher levels of IL-8 than those without GM (MB-stained mucosa) or the antral mucosa. The number of neutrophils showed similar variations among DU and control patients with a positive correlation with IL-8 activity. The levels of IL-8 and the number of neutrophils decreased after H. pylori eradication in both duodenal and antral mucosal tissues, and these changes were more remarkable in the duodenal mucosal tissues with GM. CONCLUSIONS: Increased IL-8 activity in the duodenal mucosa with GM may be important for ulcerogenesis in H. pylori-positive DU patients.
Asunto(s)
Úlcera Duodenal/etiología , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/aislamiento & purificación , Interleucina-8/metabolismo , Mucosa Intestinal/inmunología , Estómago/patología , Adulto , Anciano , Biopsia , Úlcera Duodenal/inmunología , Úlcera Duodenal/patología , Endoscopía del Sistema Digestivo , Ensayo de Inmunoadsorción Enzimática , Femenino , Infecciones por Helicobacter/tratamiento farmacológico , Humanos , Mucosa Intestinal/patología , Recuento de Leucocitos , Masculino , Metaplasia , Persona de Mediana Edad , Neutrófilos , Estómago/inmunologíaRESUMEN
BACKGROUND: Enhanced gastric mucosal chemokine activity has been demonstrated in patients with Helicobacter pylori infection. However, little is known about the mechanisms involved in this phenomenon. AIM: To examine whether in vivo chemokine activity is similar to in vitro response of gastric epithelial cells infected by H. pylori. PATIENTS AND METHODS: Antral biopsy specimens were obtained from patients with H. pylori infection for organ culture, isolation of H. pylori and histological examination. RESULTS: In organ cultures of mucosal tissues, the levels of interleukin-8 and growth-related gene product a were elevated in patients with peptic ulcer disease compared with those with erosive gastritis or endoscopically normal mucosa. However, there were no significant differences in in vitro cultures of MKN45 or KATO III cells that were infected with H. pylori isolated from these same patients. These in vivo and in vitro alpha-chemokine levels showed no significant association with the presence of cagA gene and CagA protein, ureB genotype, or binding capacity to MKN45 or KATO III cells in individual H. pylori isolates. In contrast, in vivo mucosal alpha-chemokine activity correlated with H. pylori colonization density. CONCLUSION: Mucosal chemokine profiles and inflammatory responses in H. pylori infection may be associated more closely with host factors, including those determining bacterial adhesiveness, than with differences in H. pylori strains.
Asunto(s)
Quimiocinas/biosíntesis , Infecciones por Helicobacter/metabolismo , Helicobacter pylori , Antígenos Bacterianos/genética , Línea Celular , Células Epiteliales/fisiología , Mucosa Gástrica/metabolismo , Gastritis/metabolismo , Gastritis/microbiología , HumanosRESUMEN
Sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocin-induced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.