RESUMEN
We succeeded in increasing the radial observation points of the microwave frequency comb Doppler reflectometer system from 8 to 20 (or especially up to 45) using the high sampling rate of 40 GS/s digital signal processing. For a new acquisition system, the estimation scheme of the Doppler shifted frequency is constructed and compared with the conventional technique. Also, the fine radial profile of perpendicular velocity is obtained, and it is found that the perpendicular velocity profile is consistent with the E × B drift velocity one.
RESUMEN
AIM: We investigated the association of habitual long-distance running with the thickness of skeletal muscles and subcutaneous fat in the body extremities and trunk in middle-aged men using ultrasonography. METHODS: Three groups of healthy middle-aged men [mean (SD), 62.1 (2.8) years] took part in this investigation: a high-level group of 11 master runners who had competed in a 42.195 km race and run 51.6 (21.7) km every week, an intermediary-level group of 10 master runners who had competed in a 5-20 km race and run 9.3 (4.9) km every week, and a low-level group of 7 untrained men who continued to do no systematic training. The muscle thickness at 8 sites and the subcutaneous fat thickness at 10 sites were measured by B-mode ultrasonography, and were compared among the 3 groups. RESULTS: The high-level group had 10.0(-1)5.2% higher values for muscle thickness at the erector spinae, hamstrings, tibialis anterior, and triceps surae, compared with the intermediary-level and the low-level groups (p<0.05-0.001). The thickness of the subcutaneous fat about the rectus abdominis and external oblique was lower in the high-level group than in the intermediary-level and the low-level groups (p<0.05). CONCLUSIONS: Middle-aged male master athletes who habitually run at a high level have more muscle thickness in the lower extremities and trunk, and less subcutaneous fat thickness in the central regions of the body than do middle-aged men who habitually run at an intermediary level or do not run at all.
Asunto(s)
Tejido Adiposo/fisiología , Composición Corporal/fisiología , Extremidades/fisiología , Músculo Esquelético/fisiología , Carrera/fisiología , Tejido Subcutáneo/fisiología , Músculos Abdominales/diagnóstico por imagen , Músculos Abdominales/fisiología , Tejido Adiposo/diagnóstico por imagen , Antropometría , Estudios de Casos y Controles , Extremidades/diagnóstico por imagen , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/diagnóstico por imagen , Tejido Subcutáneo/diagnóstico por imagen , UltrasonografíaRESUMEN
Although UDP-glucuronosyltransferases (UGTs) act as an important detoxification system for many endogenous and exogenous compounds, they are also involved in the metabolic activation of morphine to form morphine-6-glucuronide (M-6-G). The cDNAs encoding guinea pig liver UGT2B21 and UGT2B22, which are intimately involved in M-6-G formation, have been cloned and characterized. Although some evidence suggests that UGTs may function as oligomers, it is not known whether hetero-oligomer formation leads to differences in substrate specificity. In this work, evidence for a functional hetero-oligomer between UGT2B21 and UGT2B22 is provided by studies on the glucuronidation of morphine in transfected COS-7 cells. Cells transfected with UGT2B21 cDNA catalyzed mainly morphine-3-glucuronide formation although M-6-G was also formed to some extent. In contrast, cells transfected with UGT2B22 cDNA did not show any significant activity toward morphine. When UGT2B21 and UGT2B22 were expressed simultaneously in different ratios in COS-7 cells, extensive M-6-G formation was observed. This stimulation of M-6-G formation was not observed, however, when microsomes containing UGT2B21were mixed with those containing UGT2B22 in the presence of detergent. Furthermore, this effect was not very marked when human UGT1A1 and UGT2B21 were coexpressed in COS-7 cells. This is the first report suggesting that UGT hetero-oligomer formation leads to altered substrate specificity.
Asunto(s)
Glucuronosiltransferasa/metabolismo , Derivados de la Morfina/análisis , Morfina/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Clonación Molecular , ADN Complementario/análisis , Glucuronosiltransferasa/genética , Cobayas , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Using an anti-Tn monoclonal antibody, the Tn antigen was detected immunohistochemically in prenatal and early postnatal central nervous tissues. On embryonic day 9 (E9), the antigen was distributed throughout the single neuroepithelial layer in the neocortex and then became more prominent in the preplate than in the ventricular zone along with formation of the preplate. Following division of the preplate and concomitant formation of the cortical plate, distinct labeling of the neocortex occurred in the marginal, subplate and intermediate zones, whereas in the cortical plate and ventricular zone were virtually not immunostained. It is notable that thalamocortical afferent fibers were also immunostained specifically on E14. After birth, the localization of the antigen became less noticeable and by 3 weeks after birth, the antigen had substantially disappeared. In the developing cerebellum, prominent labeling was also observed in the molecular layer and outskirts of the cerebellar nuclei on early postnatal days. To characterize the glycoprotein bearing the Tn antigen biochemically, immunoblot analysis was performed. The glycoprotein, most of which was extracted with a salt solution, migrated as a broad smeared band corresponding to a molecular weight of about 250 kDa on SDS-PAGE. Among the various tissues examined, this glycoprotein was only detected in the brain and its amount increased until an early postnatal stage with a peak on postnatal day 3 (P3), and then decreased gradually with age. This spatially and developmentally regulated expression of the Tn antigen suggests that this antigen plays a significant role in brain development.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Sistema Nervioso Central/metabolismo , Glicoproteínas/metabolismo , Neuronas/metabolismo , Vías Aferentes/embriología , Vías Aferentes/crecimiento & desarrollo , Vías Aferentes/metabolismo , Envejecimiento/inmunología , Animales , Animales Recién Nacidos , Especificidad de Anticuerpos/fisiología , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Axones/metabolismo , Sistema Nervioso Central/embriología , Sistema Nervioso Central/crecimiento & desarrollo , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Feto , Glicoproteínas/inmunología , Immunoblotting , Inmunohistoquímica , Lectinas/inmunología , Ratones , Ratones Endogámicos ICR , Neuronas/citología , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Tálamo/embriología , Tálamo/crecimiento & desarrollo , Tálamo/metabolismoRESUMEN
The effects of a highly toxic coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl (PenCB), on triose phosphate metabolizing enzymes were studied. Male Wistar rats received 25 mg/kg PenCB, i.p. At this dose the compound provokes a wasting syndrome. The activity of triose phosphate metabolizing enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, glycerokinase, transaldolase and transketolase were significantly reduced by PenCB treatment to 50%, 60%, 50%, 70% and 40% of free-fed controls, respectively. An inhibition study with pyrazol, a specific inhibitor of alcohol dehydrogenase (ADH), showed that ADH makes about a 30% contribution to the formation of glycerol-3-phosphate from glyceraldehyde-3-phosphate. Our current study revealed that PenCB suppresses ADH at the protein expression level. The reduced formation of glycerol-3-phosphate from glyceraldehyde dehydrogenase by PenCB could be due to the suppression of ADH. The triose phosphate content in the liver cytosol of PenCB-treated rats was significantly lower than in free-fed controls. The suppression of triose phosphate metabolism could be a cause of the wasting syndrome provoked by highly toxic coplanar PCB.
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Hígado/metabolismo , Bifenilos Policlorados/farmacología , Fosfatos de Azúcar/metabolismo , Animales , Citosol/metabolismo , Glicerol Quinasa/metabolismo , Hígado/citología , Masculino , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Ratas Wistar , Transaldolasa/metabolismo , Transcetolasa/metabolismo , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
The present study was addressed on the effect of 3,3',4,4',5-pentachlorobiphenyl (PenCB) to the expression of molecular chaperon proteins, glucose regulated protein (GRP) 78, GRP94, calreticulin and calnexin in liver endoplasmic reticulum of rat by treatment with acute exposure. Male Wistar rats received PenCB in corn oil at once a dose of 10 mg/kg i.p., then at 5 days after treatment the microsomes were prepared. Free-fed and pair-fed control groups were given the vehicle. The microsomal proteins were separated on SDS-PAGE, transferred to membrane and blotted using antibody towards respective chaperone proteins. The protein levels of GRP78, GRP94, calreticulin and calnexin were significantly decreased with the acute exposure. In addition, albumin level in the microsomes was also significantly reduced by the PenCB treatment. The transferrin level was significantly higher than pair-fed but not from free-fed group. These chaperone proteins have important physiological functions against synthesized and/or denatured proteins, which include assembling, folding of proteins. PenCB-treatment may alter the extent of biosynthesis of secretory protein such as albumin through the decreasing levels of chaperone proteins in endoplasmic reticulum. Interestingly, reduced GRP78 protein level by PenCB was not due to decreased mRNA level. Our results suggested that a part of the toxicity of PenCB is associated to significant decrease of the chaperone proteins in the endoplasmic reticulum.
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Proteínas Portadoras/biosíntesis , Retículo Endoplásmico/metabolismo , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas de Choque Térmico , Hígado/metabolismo , Proteínas de la Membrana/biosíntesis , Chaperonas Moleculares/biosíntesis , Bifenilos Policlorados/farmacología , Ribonucleoproteínas/biosíntesis , Animales , Calnexina , Calreticulina , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Hígado/citología , Hígado/efectos de los fármacos , Masculino , RatasRESUMEN
We isolated glycoproteins carrying the Tn antigen, which was expressed spatiotemporally in the developing mouse brain. The Tn antigen was expressed on two molecular species with a molecular weight from 200 to 350 kDa and 110 to 160 kDa, as judged on SDS-PAGE. Although the two glycoproteins showed different susceptibilities to heparitinase I and solubilities in a salt solution, after treatment with V8 protease they showed the same mobility corresponding to a molecular weight of 90 kDa on SDS-PAGE, suggesting that these two molecules shared a common core protein. Partial N-terminal sequences of the glycoproteins were determined, i.e. AQRXRNENFERPV and ALAAPXAPAMLP, which were identified as the sequences of the N-terminal and central portions of syndecan-3, respectively. Both glycoproteins were reactive to anti-mouse syndecan-3 antibody. These results suggest that one is a soluble syndecan-3 cleaved between mucin-like domain and transmembrane domain, and the other is a membrane-bound syndecan-3 lacking N-terminal glycosaminoglycan attachment sites, and that both glycoproteins have a mucin-like domain characteristic of syndecan-3, in which the Tn antigen may be expressed.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/química , Química Encefálica , Glicoproteínas de Membrana/química , Proteoglicanos/química , Secuencia de Aminoácidos , Animales , Antígenos de Carbohidratos Asociados a Tumores/aislamiento & purificación , Sitios de Unión , Encéfalo/embriología , Carbohidratos/química , Carbohidratos/aislamiento & purificación , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Mucinas/química , Estructura Terciaria de Proteína , Sindecano-3RESUMEN
The Qdj:Sprague-Dawley (SD) rat is a mutant strain lacking in phenobarbital (PB)-mediated induction of CYP2B2. The presence of interindividual differences in the hepatic content of CYP2B proteins and testosterone 16beta-hydroxylase activity demonstrated that the breeding colony of Qdj:SD rats involves normal (+/+) and intermediate (+/-) phenotypes as well as mutant (-/-)-type rats. Although PB-treated Qdj:SD (-/-) rats expressed CYP2B1 normally, testosterone 16beta-hydroxylase activity in these rats was quite low. Analysis of regioselective metabolism of testosterone and 4-hydroxybiphenyl glucuronidation demonstrated normal catalytic activities associated with other forms of cytochrome P450s, including CYP2A, -2C, and -3A, as well as PB-inducible UDP-glucuronosyltransferase in Qdj:SD (-/-) rats. There were no serious mutations in the exons of the CYP2B1 gene in Qdj:SD (-/-) rats, demonstrating that this gene codes a functional CYP2B1. These observations suggest that CYP2B1 needs the interaction with CYP2B2 to exert the full function. The CYP2B2 gene in Qdj:SD (-/-) rats was the same as that in wild-type (+/+) rats in its length of the region containing all exon/introns and 5'-upstream up to -2.3 kilobase pairs. Malignant mutation such as stop codon formation was not observed in the exons, and no mutation was detected in the region containing the PB-responsive unit. These results strongly suggest that impaired induction of CYP2B2 in Qdj:SD (-/-) rats is attributable either to mutation at the region different from PB-responsive unit and exons or to absence or lowered expression of trans-acting factor(s) necessary for gene regulation.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP2B1/genética , Sistema Enzimático del Citocromo P-450/genética , Fenobarbital/farmacología , Esteroide Hidroxilasas/genética , Animales , Secuencia de Bases , Catálisis , Citocromo P-450 CYP2B1/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática/efectos de los fármacos , Masculino , Fenotipo , Ratas , Ratas Sprague-Dawley , Esteroide Hidroxilasas/biosíntesisRESUMEN
Formation of conjugated metabolites from morphine at a very low level in brain was studied in vitro in rats. Incubation of a low concentration of 3H-morphine with brain homogenate followed by two successive high-performance liquid chromatographic analyses showed that endogenous morphine is converted by brain enzymes to its 3- and 6-glucuronides (M-3-G and M-6-G), and codeine glucuronide (Cod-G). However, the formation of morphine-6-sulfate was likely to be low if it was produced at all. All of the cerebral hemisphere, brain stem and cerebellum were capable of producing M-3-G, M-6-G and Cod-G, although there were differences in selectivity. The capacity of the brain for glucuronide formation was far less than that of the liver, but UDP-glucuronosyltransferase in brain was much more selective in forming M-6-G and Cod-G than liver enzymes.
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Encéfalo/enzimología , Glucurónidos/metabolismo , Glucuronosiltransferasa/metabolismo , Morfina/metabolismo , Animales , Tronco Encefálico/metabolismo , Cerebelo/metabolismo , Cromatografía Líquida de Alta Presión , Codeína/análogos & derivados , Codeína/metabolismo , Hígado/metabolismo , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Derivados de la Morfina/metabolismo , Ratas , Ratas Sprague-Dawley , Telencéfalo/metabolismoRESUMEN
The effects of 5alpha-androsten-3alpha-ol (ASE), and retinoic acids (RAs) and their precursors on the phenobarbital (PB)-mediated induction of CYP2B1 and 2B2 were examined in cultured rat hepatocytes. Two isomers of RA, 9-cis- and all-trans-RA, suppressed markedly the effect of PB on CYP2B1/2 expression, while ASE had no suppressive effect. The effect of 9-cis-RA appeared at a lower concentration than the all-trans-isomer, indicating the dominant action of the former isomer. Suppression with 9-cis-retinal was also observed, but all-trans-retinol and -retinal were without effect. These results suggest that: (1) ASE, an inverse agonist for the constitutive androstane receptor (CAR), does not play a major role in the suppression of the CYP2B; (2) 9-cis-RA suppresses CYP2B induction by reducing ligand-free retinoid X-receptors (RXR) available for dimerization with the CAR; and (3) enzymes responsible for RA formation play an important role in the mechanism governing CYP2B regulation.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP2B1/genética , Sistema Enzimático del Citocromo P-450/genética , Inducción Enzimática/efectos de los fármacos , Fenobarbital/farmacología , Esteroide Hidroxilasas/genética , Tretinoina/farmacología , Androstenoles/farmacología , Animales , Células Cultivadas , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Citocromo P-450 CYP2B1/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/metabolismo , Immunoblotting , Isomerismo , Ligandos , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Esteroide Hidroxilasas/antagonistas & inhibidores , Esteroide Hidroxilasas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVES: To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis. DESIGN AND METHODS: A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL). RESULTS: This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3-7.7%), accuracy (recovery: 90.6-103.8%), simplicity and rapidity (<4 h). Clinical performance of the assay was assessed by measurement of the Ox-LDL in the plasma of normal subjects (10.8 +/- 2.8 U/mL, mean +/- SD) and patients with coronary heart disease (CHD) (19.7 +/- 10.2). The present EIA had a sensitivity of 79% and a specificity of 75% for CHD. CONCLUSIONS: We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay.
Asunto(s)
Apolipoproteínas B/sangre , Técnicas para Inmunoenzimas , Lipoproteínas LDL/sangre , Fosfatidilcolinas/sangre , Adulto , Anticuerpos Monoclonales/biosíntesis , Apolipoproteínas B/inmunología , Biomarcadores/sangre , Cromatografía en Gel , Ritmo Circadiano , Enfermedad Coronaria/sangre , Enfermedad Coronaria/diagnóstico , Electroforesis en Gel de Poliacrilamida , Humanos , Masculino , Análisis por Apareamiento , Persona de Mediana Edad , Fosfatidilcolinas/inmunología , Curva ROC , Valores de ReferenciaRESUMEN
A coplanar polychlorinated biphenyl, 3,3',4,4',5-pentachlorobiphenyl (PenCB), significantly suppresses the expression of rat liver carbonic anhydrase III (CAIII), an enzyme which has recently been suggested to prevent from H(2)O(2)-inducible apoptosis. Marked changes in the CAIII levels of liver cytosol were observed in rats following doses of PenCB ranging from 0.5 to 25 mg/kg body weight and maximum suppression was observed at a dose of 10 mg/kg. Northern analysis revealed that the level of CAIII mRNA in rat liver was dramatically reduced by PenCB treatment while only weak suppression was observed in pair-fed controls. Two AU-rich elements, considered as a destabilizing signal of mRNA, were found in the 3'-untranslated region of CAIII sequenced after reverse transcription-PCR and 3'-rapid amplification of the cDNA end. Dramatic decrease of CAIII in rat liver by PenCB could account for the suppression of the defense system for oxidative stress.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Hígado/enzimología , Bifenilos Policlorados/farmacología , Regiones no Traducidas 3' , Animales , Northern Blotting , Anhidrasas Carbónicas/genética , Clonación Molecular , Citosol/enzimología , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Immunoblotting , Masculino , Datos de Secuencia Molecular , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Protein-protein interactions between cytochrome P450 (P450) and other drug-metabolizing enzymes were studied by affinity chromatography using CYP1A1-, glycine-, and bovine serum albumin (BSA)-conjugated Sepharose 4B columns. Sodium cholate-solubilized microsomes from phenobarbital-treated rat liver were applied to the columns and the material eluted with buffer containing NaCl was analyzed by immunoblotting. Microsomal epoxide hydrolase (mEH) and UDP-glucuronosyltransferases (UGTs), as well as NADPH-P450 reductase, were efficiently trapped by the CYP1A1 column. Glycine and BSA columns exhibited no ability to retain these proteins. Protein disulfide isomerase and calnexin, non-drug-metabolizing enzymes expressed in the endoplasmic reticulum, were unable to associate with the CYP1A1 column. These results suggest that CYP1A1 interacts with mEH and UGT to facilitate a series of multistep drug metabolic conversions.
Asunto(s)
Citocromo P-450 CYP1A1/metabolismo , Epóxido Hidrolasas/metabolismo , Glucuronosiltransferasa/metabolismo , Animales , Western Blotting , Bovinos , Cromatografía de Afinidad , Masculino , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin alpha5beta1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925-930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)-GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)-GlcNS(6OS)](6) present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin alpha5beta1.
Asunto(s)
Actinas/fisiología , Fibronectinas/química , Heparina/metabolismo , Heparitina Sulfato/química , Glicoproteínas de Membrana/fisiología , Proteoglicanos/fisiología , Receptores de Fibronectina/fisiología , Animales , Sitios de Unión , Adhesión Celular , Citoesqueleto/fisiología , Fibronectinas/metabolismo , Fibronectinas/fisiología , Heparina/química , Humanos , Ratones , Oligonucleótidos Antisentido/farmacología , Oligosacáridos/química , Oligosacáridos/metabolismo , Oligosacáridos/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sindecano-2 , Células Tumorales CultivadasRESUMEN
The effect of 3,3',4,4',5-pentachlorobiphenyl (PenCB) on the synthesis of unsaturated fatty acids was studied in male Wistar rats. The arachidonic acid (20:4) content in the total lipids of liver homogenates was significantly reduced on day 5 of PenCB administration, while those of linoleic acid (18:2) and bishomo-gamma-linolenic acid (20:3) were increased. These changes in the total lipids of liver homogenates were observed following doses of PenCB ranging from 0.5 to 25 mg/kg of body weight. The same changes in these fatty acids were seen with four subtypes of microsomal glycerophospholipids in the liver. The marked reduction in the molar ratio of 20:4 to 18:2 in the lipids suggests alteration of the activity of the enzymes responsible for the synthesis of unsaturated fatty acids. The activity of Delta5 and Delta6 desaturases (arachidonic acid synthetase) in the liver microsomes was 17 and 13% of that of pair-fed control animals, whereas the activity of 1-acylglycerophosphorylcholine or 1-acylglycerophosphate acyltransferase, which transfers 20:4 or 18:2 to phospholipids, was not affected by the treatment. Thus, the reduction in the level of 20:4 that was observed can be explained by a reduction in desaturase activity. These results are evidence that the coplanar PenCB has a significant effect on the reduced synthesis of physiologically essential long-chain unsaturated fatty acids.
Asunto(s)
Ácido Araquidónico/biosíntesis , Ácido Graso Desaturasas/antagonistas & inhibidores , Microsomas Hepáticos/efectos de los fármacos , Bifenilos Policlorados/farmacología , Animales , delta-5 Desaturasa de Ácido Graso , Ácido Graso Desaturasas/metabolismo , Glicerofosfolípidos/metabolismo , Ácido Linoleico/metabolismo , Linoleoil-CoA Desaturasa , Masculino , Microsomas Hepáticos/enzimología , Bifenilos Policlorados/toxicidad , Ratas , Ratas WistarRESUMEN
The peripheral benzodiazepine receptor plays a role in the translocation of cholesterol into mitochondria where steroidogenesis occurs. Sterols have been suggested to be involved in the regulation of the cytochrome P-450 (CYP)2B subfamily as the endogenous suppressor of this CYP. To investigate the role of cholesterol metabolites on the expression of CYPs, the effect of PK11195, a specific ligand of the peripheral benzodiazepine receptor and a stimulator of cholesterol transportation, on CYP expression was examined in rats in vivo and in cultured hepatocytes. As judged by the change in testosterone metabolic activity catalyzed by liver microsomes, i.p. injection of PK11195 into rats increased the CYP2B subfamily significantly. A trend in the induction of the CYP2A1, 2C11, and 3A isozymes was also observed. When PK11195 was given to rats together with phenobarbital, an additive effect of these compounds on testosterone metabolic activity was observed. In cultured hepatocytes, PK11195 exhibited the same effect on CYP expression as seen in vivo, but the magnitude of the effect was much greater than that observed in vivo. The inductive effect of PK11195 toward the CYP2B and 3A subfamilies was 2.3- and 6.5-fold greater, respectively, than that with phenobarbital. The inductive effect of PK11195 was confirmed by immunoblotting with antibodies against CYP2A, 2B, 2C, and 3A proteins. These results indicate that PK11195 has an inductive effect on several subfamilies of CYPs by directly acting on liver cells and has no ability to suppress the expression of these CYPs. This observation suggests that, if certain sterols play a role in the suppressive control of the CYP2B subfamily, they are produced in organelles other than the mitochondria.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Isoquinolinas/metabolismo , Microsomas Hepáticos/enzimología , Receptores de GABA-A/metabolismo , Animales , Células Cultivadas , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar , Testosterona/metabolismoRESUMEN
The present study was addressed on the effect of 3,3',4,4',5-pentachlorobiphenyl (PenCB) to the expression of glucose regulated protein (GRP) 78 and GRP94 in liver endoplasmic reticulum of rat by treatment with the schedule after acute or subacute exposure. In the acute exposure, male Wistar rats received PenCB in corn oil at once a dose of 25 mg/kg i.p., then at 5 days after treatment the microsomes were prepared. Free- and pair-fed control groups were given the vehicle. The microsomal proteins were separated on SDS-PAGE, transferred to membrane and blotted using anti-sera to the GRPs. The reduction of GRP78 and GRP94 was associated significantly with the acute exposure. In subacute exposure, the rats received PenCB in corn oil at once a dose of 0.1 or 1.0 mg/kg i.p. At 4 weeks after treatment, liver microsomes were obtained. The expression level of GRP78 and GRP94 are also decreased at 1.0 mg PenCB/kg treatment as similar as the acute exposure. But the reduction was not notable at 0.1 mg PenCB/kg treatment. GRP78 and GRP94 are a member of GRPs and the expression is regulated by glucose in cells as stress proteins. GRP78 and GRP94 have also the function for chaperone protein. Chaperone proteins have important physiological functions against synthesized and/or denatured proteins, which include assembling, folding of proteins. Our results suggested that a part of the toxicity of PenCB is associated to significant decrease of the chaperone proteins in the endoplasmic reticulum.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Bifenilos Policlorados/toxicidad , Animales , Citosol/metabolismo , Técnicas In Vitro , Hígado/citología , Hígado/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
We report here that a highly toxic coplanar polychlorinated biphenyl (PCB), 3,3',4,4',5-pentachlorobiphenyl (PenCB) induces molecular chaperones, HSP70 and HSP90 in liver cytosol of rats. Male Wistar rats received PenCB in corn oil once at a dose of 25 mg/kg i.p. Pair-fed control groups were treated with the vehicle and given the amount of chow matched with that taken by the PenCB-treated animals, and free-fed controls were given the vehicle. The liver cytosolic HSP70 level in rats treated with PenCB was 5-fold higher than those in free-fed controls, though that for pair-fed controls was approximately 2-fold higher than that in free-fed controls. The liver cytosolic HSP90 alpha and HSP90 beta levels were also higher in PenCB-treated rats than in both control groups, but the induction extent was lesser than that for HSP70. Inductive effect on the chaperones was examined with a single different dose of PenCB 0, 0.5, 1.0, 5.0, 10 and 25 mg/kg. Marked induction of the HSP70 level was observed with a minimum dose of PenCB 0.5 mg/kg. The HSP90 alpha level was induced with PenCB-dose dependent manner although the HSP90 beta induction was greatest with a dose of PenCB 5.0 mg/kg. HSP70 and HSP90 are essential for cells under normal conditions and act as molecular chaperones. HSP90 is well known to modulate the function of sex steroid hormone or aromatic hydrocarbon receptors while HSP70 is required for receptor-HSP90 heterocomplex assembly. The role of molecular chaperones may be involved in the endocrine disrupting properties of coplanar PCB and dioxins.
Asunto(s)
Citosol/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Hígado/metabolismo , Chaperonas Moleculares/metabolismo , Bifenilos Policlorados/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Hígado/citología , Masculino , Ratas , Ratas WistarRESUMEN
Our previous study indicated that picrotoxin competes with phenobarbital (PB) for the binding to liver microsomes, although the target of binding and the physiological role were not determined (unpublished observation). To seek information on the target site of PB, reflecting the induction of hepatic enzymes, we examined here the effect of picrotoxin on PB-mediated induction of hepatic cytochrome P450 and UDP-glucuronosyltransferase in vivo in rats. The induction of the CYP2B1/2 was estimated by immunoblot analysis and by measuring the activity of testosterone 16beta-hydroxylation. Intraperitoneal injection of picrotoxin alone slightly increased CYP2B1/2 protein. An experiment on co-treatment of picrotoxin and PB showed that picrotoxin enhanced rather than antagonized the inducing effect of PB. The results suggest two possibilities: that 1) picrotoxin increases the CYP2B subfamily by binding to the same site as PB; or 2) the site in microsomes for the competition between PB and picrotoxin does not reflect the induction of P450.