RESUMEN
PURPOSE: To investigate the relationship between the change of daily step counts and low back pain (LBP) during pregnancy. Materials and METHODS: Pregnant women at less than eight weeks of gestation (WG) were recruited. Daily step counts were measured with a pedometer. To assess LBP, the Oswestry disability index (ODI) score was recorded. Thirty-six individuals were divided into the LBP and non-LBP groups. The effect of step counts on LBP between the two groups was analyzed. RESULTS: At 16-19 WG, step counts were not considerably changed in the non-LBP group but were significantly increased in the LBP group. At 24-27 and 32-35 WG, step counts were increased in the non-LBP group but were significantly decreased in the LBP group. CONCLUSIONS: Acute increase of daily step counts in early pregnancy is a risk for LBP, and gradual increases of step counts after mid-pregnancy is recommended for women.
Asunto(s)
Dolor de la Región Lumbar/epidemiología , Actividad Motora , Complicaciones del Embarazo/epidemiología , Caminata , Actigrafía , Adulto , Estudios de Cohortes , Femenino , Humanos , Embarazo , Estudios ProspectivosRESUMEN
INTRODUCTION AND OBJECTIVES: Although the complications of intravesical BCG treatment are well described, asymptomatic genitourinary granulomas after BCG therapy have rarely been reported and management strategy for these conditions remains controversial. The objective of this study is to evaluate the incidence rate of asymptomatic genitourinary granuloma formation mimicking bladder cancer recurrence after intravesical bacillus Calmette-Guérin (BCG) therapy and to identify the diagnostic and treatment strategies according to patient conditions. PATIENTS AND METHODS: A retrospective review was conducted on 162 patients who underwent intravesical BCG therapy. For patients who developed granulomas, we evaluated the time interval between BCG instillation and the development of granuloma, the presence of acid-fast bacteria on pathology specimens, culture/polymerase chain reaction results, management strategies for the lesions, and clinical outcomes. RESULTS: Asymptomatic genitourinary masses developed in 14 patients, of whom 5 underwent histological examinations and all were confirmed to have granulomatous inflammation. The affected organs included the kidney, bladder, prostate, and penis. While four of the five patients did not receive treatment for their granulomas, one patient was administered antituberculous medication to prevent worsening of the lesion during the perioperative period of the scheduled cystoprostatectomy. None of the patients experienced worsening or recurrence of granulomatous lesions. Patients who developed asymptomatic masses (nâ¯=â¯14) were significantly younger than those who did not (pâ¯=â¯0.0076) and multivariate analysis also showed that younger age was independently associated with the development of clinically suspicious lesions (pâ¯=â¯0.032); however, none of the parameters were associated with histologically confirmed granuloma formation. CONCLUSIONS: Genitourinary granulomas mimicking recurrence of carcinoma may develop in nearly 10% of patients after intravesical BCG therapy. Most patients can be managed without potentially toxic antituberculosis therapy.
Asunto(s)
Vacuna BCG , Neoplasias de la Vejiga Urinaria , Masculino , Humanos , Vacuna BCG/efectos adversos , Incidencia , Recurrencia Local de Neoplasia/epidemiología , Neoplasias de la Vejiga Urinaria/patología , Granuloma/etiología , Granuloma/microbiologíaRESUMEN
Our aims were to elucidate the factors that affected vancomycin (VCM) serum trough levels and to find the optimal initial dose based on creatinine clearance (CrCl) and body weight (BW) to minimize inadequate trough levels in a retrospective observational study among Japanese adults. One hundred and six inpatients, in whom VCM trough levels were measured after completing the third dosing, were consecutively recruited into our study in a tertiary hospital. We considered the frequency of <30% as low. In the generalized linear model, initial VCM total daily dose, CrCl, and BW were independent risk factors of VCM trough levels. In patients with CrCl ≥30 and <50 mL/min, 1 g/day yielded low frequencies of a trough level of ≥20 mcg/mL, regardless of BW. In patients with CrCl ≥50 mL/min, 2 g/day yielded low frequencies of a trough level of <10 mcg/mL in patients weighing <55 kg, but not in patients weighing ≥55 kg. Optimal VCM initial total daily dose may be 1 g/day in patients with CrCl ≥30 and <50 mL/min regardless of BW and 2 g/day in patients weighing <55 kg with CrCl ≥50 mL/min among Japanese adults.
Asunto(s)
Peso Corporal , Creatinina/metabolismo , Cálculo de Dosificación de Drogas , Vancomicina/administración & dosificación , Vancomicina/farmacocinética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Pueblo Asiatico , Índice de Masa Corporal , Femenino , Humanos , Modelos Lineales , Masculino , Tasa de Depuración Metabólica , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Centros de Atención Terciaria , Vancomicina/sangre , Adulto JovenRESUMEN
N-cadherin is a homophilic cell adhesion molecule that stabilizes excitatory synapses, by connecting pre- and post-synaptic termini. Upon NMDA receptor (NMDAR) activation by glutamate, membrane-proximal domains of N-cadherin are cleaved serially by a-disintegrin-and-metalloprotease 10 (ADAM10) and then presenilin 1(PS1, catalytic subunit of the γ-secretase complex). To assess the physiological significance of the initial N-cadherin cleavage, we engineer the mouse genome to create a knock-in allele with tandem missense mutations in the mouse N-cadherin/Cadherin-2 gene (Cdh2 R714G, I715D, or GD) that confers resistance on proteolysis by ADAM10 (GD mice). GD mice showed a better performance in the radial maze test, with significantly less revisiting errors after intervals of 30 and 300 s than WT, and a tendency for enhanced freezing in fear conditioning. Interestingly, GD mice reveal higher complexity in the tufts of thorny excrescence in the CA3 region of the hippocampus. Fine morphometry with serial section transmission electron microscopy (ssTEM) and three-dimensional (3D) reconstruction reveals significantly higher synaptic density, significantly smaller PSD area, and normal dendritic spine volume in GD mice. This knock-in mouse has provided in vivo evidence that ADAM10-mediated cleavage is a critical step in N-cadherin shedding and degradation and involved in the structure and function of glutamatergic synapses, which affect the memory function.
Asunto(s)
Cadherinas/metabolismo , Hipocampo/metabolismo , Aprendizaje Espacial , Sinapsis/metabolismo , Análisis y Desempeño de Tareas , Proteína ADAM10/metabolismo , Alelos , Animales , Conducta Animal , Células CHO , Membrana Celular/metabolismo , Cricetulus , Miedo , Técnicas de Sustitución del Gen , Memoria , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Mutación/genética , Estabilidad Proteica , Células Piramidales/metabolismo , Sinapsis/patología , Sinapsis/ultraestructura , Transmisión Sináptica/fisiología , Sinaptosomas/metabolismo , Sinaptosomas/ultraestructuraRESUMEN
There are two microtubule systems in the posterior silk gland cells. One is a radial microtubule system in which the microtubules run radially from the basal to the apical cytoplasm and in which fibroin globules (secretory granules of fibroin) and mitochondria are arranged along these microtubules, thus composing a "canal system" which is assumed to be responsible for the intracellular transport of fibroin globules. The other is a circular microtubule system in the apical cytoplasm which is composed of bundles of microtubules and microfilaments running in a circular arrangement around the glandular lumen at an interval of approximately 4 mum at the end of the fifth instar. This system is presumably concerned with secretion and/or intraluminal transport of fibroin.
Asunto(s)
Bombyx/ultraestructura , Microtúbulos/ultraestructura , Animales , Glándulas Exocrinas/ultraestructura , Larva/ultraestructura , Metamorfosis Biológica , Microscopía Electrónica , Microscopía de Polarización , Mitocondrias/ultraestructuraRESUMEN
Electron microscope observations of thin sections of epoxy resin-embeded posterior silk gland cells at the later stage of the fifth instar revealed that the Golgi vacuoles and the secretory granules (fibroin globules) in the cytoplasm and the glandular lumen contain fine fibrous materials. In frozen thin sections these structures appear as electron-dense granules and electron-dense blocks, or a column, respectively. Immunoelectron microscopy has shown that ferritin particles or products of the peroxidase reaction are localized on these structures. It was concluded that the fine fibrous materials most probably represent native fibroin molecules or their aggregates.
Asunto(s)
Bombyx/ultraestructura , Fibroínas/análisis , Animales , Reacciones Antígeno-Anticuerpo , Gránulos Citoplasmáticos/ultraestructura , Glándulas Exocrinas/ultraestructura , Ferritinas , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/ultraestructura , Larva , PeroxidasasRESUMEN
Ultracentrifugal analyses of the native silk proteins extracted from the various parts of the middle silk gland of the mature silkworm have revealed that there exist four components with S degrees (20,w) values of 10S, 9-10S, 9S, and 4S in the extract. It is suggested that the fastest 10S component is the native fibroin synthesized in the posterior silk gland and transferred to the middle silk gland to be stored there, while the slower three components probably correspond to inner, middle, and outer sericins which were synthesized in the posterior, middle, and anterior portion of the middle silk gland, respectively. Native fibroin solution was prepared from the most posterior part of the middle silk gland. Ultracentrifugal analyses have shown that the solution contains considerable amounts of aggregates in addition to the main 10S component. Treatment with lithium bromide (LiBr), urea, or guanidine hydrochloride solution up to 6 M all have failed to dissociate the 10S component. From the sedimentation equilibrium analyses and partial specific volume of 0.71(6), the molecular weight of the 10S component of the native fibroin solution was found to be between 3.2 - 4.2 x 10(5), with a tendency to lie fairly close to 3.7 x 10(5).
Asunto(s)
Bombyx , Fibroínas/análisis , Proteínas/análisis , Animales , Electroforesis , Microscopía Electrónica , Peso Molecular , UltracentrifugaciónRESUMEN
The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P-450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Transporte Biológico , Aparato de Golgi/ultraestructura , Hígado/ultraestructura , Masculino , Microscopía Electrónica , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/ultraestructura , Fenobarbital/farmacología , Ratas , Ratas EndogámicasRESUMEN
We have investigated the degradation in rat liver of two typical endoplasmic reticulum (ER) membrane proteins, phenobarbital (PB)-inducible cytochrome P-450 (P-450[PB]) and NADPH-cytochrome P-450 reductase (FP2). Autolysosomes, almost completely free from contamination by the other organelles such as ER, were prepared from leupeptin-treated rat livers according to the method of Furuno et al. (Furuno, K., T. Ishikawa, and K. Kato, 1982, J. Biochem., 91:1943-1950). Quantitative immunoblot analysis showed that these two proteins were found in large amounts in the autolysosomes regardless of PB treatment. The specific content of P-450 (PB) in the autolysosomes changed along with that in the microsomes during and after PB treatment, whereas hardly any P-450(PB) was detected in the cytosol fraction throughout the experiment. We also found a marked increase in the autolysosomal proteins 3 d after cessation of PB treatment when microsomal proteins are degraded most rapidly. Ferritin immunoelectron microscopy revealed directly that when the limiting membranes of the premature autolysosomes were partially broken the smooth vesicles segregated within the autolysosomes were heavily stained with ferritin anti-P-450(PB) conjugates. Thus, for the first time, we could present convincing evidence that P-450(PB) and FP2 are segregated to be degraded in the autolysosomes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Retículo Endoplásmico/metabolismo , Hígado/metabolismo , Lisosomas/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Inducción Enzimática , Cinética , Hígado/efectos de los fármacos , Lisosomas/ultraestructura , Masculino , Microscopía Electrónica , Fenobarbital/farmacología , Ratas , Ratas EndogámicasRESUMEN
Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauchi, K., R. Masaki, S. Taketani, A. Yamamoto, A. Akayama, and Y. Tashiro. 1991. J. Biol. Chem. 266:19536-19542). This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination. When expressed from cDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER. The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring. The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E. coli beta-galactosidase to the ER membrane. Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilic sequences on either side of the hydrophobic domain play an important role in ER targeting.
Asunto(s)
Aldehído Deshidrogenasa/química , Retículo Endoplásmico/enzimología , Microsomas/enzimología , Señales de Clasificación de Proteína/fisiología , Aldehído Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico/fisiología , Línea Celular , Retículo Endoplásmico/ultraestructura , Datos de Secuencia Molecular , Mutagénesis , Ratas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Growth of the posterior silk gland and biosynthesis of fibroin during the fifth larval instar of the silkworm, Bombyx mori, have been studied. In accordance with the exponential increase in the wet weight of the gland, the amounts of DNA, RNA, protein, and lipids per animal increased rapidly in the early stage of the fifth instar (0-96 hr). Biosynthesis of fibroin, on the contrary, mainly proceeds in the later stage of the fifth instar (120-192 hr). Electron microscopical observations have shown that, in the very early stage (0-12 hr), a number of free ribosomes exist in the cytoplasm. Rough endoplasmic reticulum (ER) with closely spaced cisternae was also observed. Then rough ER starts to proliferate rapidly, and at the same time lamellar ER is rapidly or gradually transformed into vesicular or tubular forms. In the later stage of the fifth instar (120-192 hr), the cytoplasm is mostly filled with tubular or vesicular ER. Golgi vacuoles, free vacuoles (fibroin globules), and mitochondria are also observed. It is concluded that in the early stage of the fifth instar the cellular structures necessary for the biosynthesis of fibroin are rapidly formed, while in the later stage the biosynthesis of fibroin proceeds at a maximum rate and utilizes these structures.
Asunto(s)
Bombyx/crecimiento & desarrollo , Fibroínas/biosíntesis , Animales , Bombyx/citología , ADN/análisis , Lípidos/análisis , Metamorfosis Biológica , Microscopía , Microscopía Electrónica , Tamaño de los Órganos , Proteínas/análisis , ARN/análisisRESUMEN
Cytolytic processes in posterior silk gland cells of the silkworm, Bombyx mori, during metamorphosis from larva to pupa have been studied. During this stage, the wet weight and the amounts of RNA and protein of the gland decrease rapidly and markedly, while the amount of DNA decreases slowly and slightly. The ultrastructural changes observed at the beginning of the prepupal stage consist of the appearance or the increase in the number of autophagosomes containing endoplasmic reticulum (ER), or "early autophagosomes" as we have called them, which seem to be gradually transformed to autolysosomes. A number of usual lysosomes, which frequently contain myelin figures, also appear in the cytoplasm. Sometimes they fuse with each other to form large conglomerates. In the middle of the prepupal stage, a number of smooth membrane-bounded vacuoles appear in cytoplasm. Towards the end of the prepupal stage the partition or sequestration of cytoplasm was observed. Thus large autophagosomes containing cytoplasmic organelles such as rough ER and/or mitochondria are formed. The nucleus is partitioned in a similar way by smooth membranes, and then autophagosomes containing condensed chromatin blocks are formed. These various kinds of autophagosomes, or "late autophagosomes" as we have generally called them, are continuously released into the hemolymph until the gland is completely disintegrated.
Asunto(s)
Bombyx/citología , Metamorfosis Biológica/fisiología , Animales , Bombyx/crecimiento & desarrollo , Citoplasma , ADN/análisis , Retículo Endoplásmico , Fibroínas/biosíntesis , Histocitoquímica , Lisosomas , Microscopía , Microscopía Electrónica , Mitocondrias , Proteínas/análisis , ARN/análisisRESUMEN
The development of the cells in the posterior silk gland of the silkworm, Bombyx mori, during the fourth larval instar has been studied. In the early stages of this instar, the wet weight of the gland and the amounts of RNA, DNA, and protein per animal increase logarithmically until they reach a stationary state at about 72 hr. At around 96 hr of the fourth instar, the larvae enter the molting state, which lasts for about 24 hr until the fourth ecdysis. Towards the end of the molt stage, the growth of the silk gland is resumed. Electron microscopical observation shows that in the early intermolt stage the cytoplasm is filled with free ribosomes and with rough endoplasmic reticulum (ER), first of the lamellar type (0-6 hr) and then of the vesicular or tubular type. The Golgi apparatus also is well developed. At the beginning of the molt stage (90-96 hr), however, most of the ER becomes lamellar in type, concentric lamellar structures being occasionally observed, and the Golgi vacuoles disappear. Autophagosomes and lysosomes increase markedly and the apical portion of the cytoplasm becomes extensively vacuolated; this suggests that the secretory activities are completely depressed, and pronounced degenerative changes appear during the molt stage. Towards the end of the molt stage, large lamellar ER elements are fragmented into smaller lamellae and there is a pronounced increase in the number of free ribosomes.
Asunto(s)
Bombyx/citología , Metamorfosis Biológica , Animales , Peso Corporal , ADN/análisis , Lípidos/análisis , Microscopía , Microscopía Electrónica , Proteínas/análisis , ARN/análisisRESUMEN
Direct ferritin immunoelectron microscopy was applied to visualize the distribution of the hepatocyte cell surface of the asialoglycoprotein receptor which is responsible for the rapid clearance of serum glycoproteins and lysosomal catabolism. For this purpose, rabbit antibody against the purified hepatic binding protein specific for asialoglycoproteins was prepared and coupled to ferritin by glutaraldehyde. The specific antibody conjugates were incubated with the hepatocytes, which were isolated from rat liver homogenate after fixation by glutaraldehyde perfusion. These cells preserved well the original polygonal shape and polarity, and it was easy to identify the sinusoidal, lateral, and bile canalicular faces. The surface density of the ferritin particles bound to the sinusoidal face was about four times higher than that of particles bound to the lateral face, while the bile canalicular face was hardly labeled and almost at the control level. Using the surface area of hepatocyte measured by morphometrical analyses, it was estimated that approximately 90% of bound ferritin particles were at the sinusoidal face, approximately 10% at the lateral face, and approximately 1% at the bile canalicular face. Nonhepatic cells such as endothelial and Kupffer cells had no receptor specific for asialoglycoproteins.
Asunto(s)
Hígado/ultraestructura , Receptores de Superficie Celular/análisis , Animales , Receptor de Asialoglicoproteína , Membrana Celular/análisis , Invaginaciones Cubiertas de la Membrana Celular/análisis , Endotelio/ultraestructura , Ferritinas , Macrófagos del Hígado/ultraestructura , Hígado/análisis , Microscopía Electrónica , Microvellosidades/análisis , Ratas , Receptores de Superficie Celular/inmunologíaRESUMEN
Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.
Asunto(s)
Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Membranas Intracelulares/análisis , Hígado/análisis , Microsomas Hepáticos/análisis , Animales , Reacciones Antígeno-Anticuerpo , Membrana Celular/análisis , Retículo Endoplásmico/análisis , Aparato de Golgi/análisis , Masculino , Microscopía Electrónica , Mitocondrias Hepáticas/análisis , Membrana Nuclear/análisis , Ratas , Fracciones SubcelularesRESUMEN
Rat livers were prefixed by perfusion with 0.6% glutaraldehyde and briefly homogenized with a Teflon-glass homogenizer. The prefixed cells isolated by low-speed centrifugation in high yield effectively preserved the original polygonal shape and polarity. These cells were incubated with ferritin-antibody conjugates monospecific for rat liver 5'-nucleotidase, and the localization of the enzymes on the surface of hepatocytes and endothelial cells was quantitatively investigated. It was revealed that the surface density of 5'-nucleotidase is much higher on the bile canalicular surface than on the sinusoidal surface and only a few ferritin particles were detected on the lateral surface. On the bile canalicular surface ferritin particles were almost exclusively found on the microvilli in larger clusters. Similar distribution was also observed on the sinusoidal surface but the size of cluster was much smaller. On both surfaces many fewer ferritin particles were found on the intermicrovillar region, including the coated pits region, than on the microvillar region. Ferritin particles were also found on the endothelial cell surface.
Asunto(s)
Ferritinas , Hígado/enzimología , Nucleotidasas/análisis , 5'-Nucleotidasa , Animales , Canalículos Biliares/citología , Endotelio/citología , Técnicas de Inmunoadsorción , Masculino , Microscopía Electrónica/métodos , Microvellosidades/enzimología , Ratas , Ratas Endogámicas , Propiedades de SuperficieRESUMEN
Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Microsomas Hepáticos/metabolismo , Proteínas Ribosómicas/biosíntesis , Animales , Sitios de Unión , Sistema Enzimático del Citocromo P-450/inmunología , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Membranas Intracelulares/metabolismo , Masculino , Microsomas Hepáticos/ultraestructura , Puromicina/farmacología , Ratas , Propiedades de SuperficieRESUMEN
Sucrose density gradient analyses of pH 5.5 and pH 7.4 extracts from rat liver nucleoli revealed the presence of two broad peaks of approximately 60S and 80S, and 60S and 80-100S, respectively. Ribonucleoprotein (RNP) particles containing precursor ribosomal RNA in these peaks have been characterized by electron microscopy and RNA analyses. Spherical particles only were found in the 60S peak of the pH 5.5 extract, from which 28S RNA and smaller RNA (23S and 18S RNA) exclusively were extracted. In the broad 80S peak of the pH 5.5 extract, about 60% of the particles were spherical while 30% were rodlike. In the RNA species present there were 28S plus smaller RNA (80%) and 35S RNA (20%). The 60Speak of the pH 7.4 extract contained mainly spherical particles (84%), and the RNA species present was mostly 28S plus smaller RNA (89%). In addition to spherical particles (43%), a number of rodlike (31%) and filamentous molecules (26%) were observed in the heavier side of the 80-100S peak of the pH 7.4 extract, from which 45S (14%), 35S (26%), and 28S and smaller RNA (60%) were extracted. Thus the precursor ribosomal particles containing 45S RNA and 35S RNA appear to be filamentous and rodlike molecules, respectively. Folding of loose ribonucleoprotein filaments into compact, spherical, large subparticles may be part of the maturation process of ribosomal large subparticles, in addition to the so-called sequential cleavage of RNA.
Asunto(s)
Nucléolo Celular/ultraestructura , Hígado/ultraestructura , Ribosomas/ultraestructura , Animales , Nucléolo Celular/análisis , Nucléolo Celular/metabolismo , Núcleo Celular/análisis , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Hígado/análisis , Hígado/metabolismo , Masculino , Microscopía Electrónica , Nucleoproteínas/análisis , ARN Ribosómico/análisis , ARN Ribosómico/metabolismo , Ratas , Ribosomas/análisis , Ribosomas/metabolismoRESUMEN
Induction of cytochrome P-450s by 3-methylcholanthrene (MC) and phenobarbital (PB) and distribution of P-450s in the rat liver nuclear envelope were investigated by biochemical analyses and ferritin immunoelectron microscopy using specific antibodies against the major molecular species of MC- and PB-induced cytochrome P-450. It was found, in agreement with Kasper (J. Biol. Chem., 1971, 246: 577-581), that the total amount of cytochrome P-450s determined by biochemical analysis was markedly increased by MC, but not by PB, treatment. Immunoelectron microscopic analysis, however, showed marked and slight increases in ferritin labeling by MC and PB treatment, respectively. The latter finding was interpreted as resulting from the induction of a particular molecular species of PB-induced cytochrome P-450s. Ferritin immunoelectron microscopic analysis of intact isolated nuclei, naked nuclei from which the outer membrane of the nuclear envelope was partially detached (mechanically), and isolated nuclear envelopes have shown that the ferritin particles are found exclusively on the cytoplasmic face of the outer nuclear envelopes. Neither the nucleoplasmic face of the inner membrane of the nuclear envelope nor the cisternal face of both membranes of the nuclear envelope showed any labeling with ferritin. This indicates that cytochrome P-450 is located only on the outer membrane of the nuclear envelope and does not diffuse laterally into the domain of the inner membrane of the nuclear envelope across the nuclear pores. Our results suggest that a marked heterogeneity exists in the enzyme distribution between the outer and inner membrane of the nuclear envelope and that microsomal marker enzymes such as cytochrome P-450 exist exclusively in the outer membrane. In addition, it appears that cytochrome P-450 is probably not a transmembrane protein but an intrinsic protein located on the cytoplasmic face of the outer membrane of the nuclear envelope.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Membrana Nuclear/enzimología , Animales , Inducción Enzimática/efectos de los fármacos , Ferritinas , Aparato de Golgi/enzimología , Hígado/ultraestructura , Masculino , Metilcolantreno/farmacología , Membrana Nuclear/ultraestructura , Fenobarbital/farmacología , Ratas , Ratas EndogámicasRESUMEN
Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus.