RESUMEN

A novel alanine:glyoxylate aminotransferase (AGT) mutation involved in primary hyperoxaluria type 1 (PH1) was studied in Japanese patients. Two mutations in exon 7, c.751T>A and c.752G>A, lead to a W251K amino acid substitution. Proband 1 (patient 1) was homozygous for the W251K mutation allele (DDBJ Accession No. AB292648), and AGT-specific activity in the patient's liver was very low. To reveal the cause of the low enzymatic activity, the intracellular localization of AGT (W251K) was studied using immunohistochemistry and immunoelectron microscopy. The latter analysis showed that patient 2 had only one-fifth of the normal AGT expression per catalase, suggesting impairment of AGT (W251K) dependent transport into peroxisomes. Peroxisomal transport of human AGT is believed to be dependent on the presence of the type 1 peroxisomal targeting sequence. The C-terminal tripeptide of AGT, KKL is necessary for peroxisomal targeting. In cultured cells, EGFP-AGT (W251K) localized both in the peroxisome and cytosol. These results were consistent with the data obtained from liver analysis of patient 2. The subcellular distribution of AGT (W251K) and the results from a random mutagenesis study suggest that KKL is necessary for peroxisomal targeting of human AGT, but additional signal other than KKL may be necessary.

RESUMEN

BACKGROUND: Although the importance of monocyte trans-endothelial migration in early atherogenesis is well recognized, it is unclear whether and how one transmigration event affects endothelium to facilitate subsequent ones. In this study, we tested the hypothesis that monocyte transmigration alters endothelial junctional organization to facilitate subsequent transmigration. METHODS AND RESULTS: When human monocytes were added twice at intervals of ≈30 min to IL-1beta-prestimulated human umbilical vein endothelial cells in vitro, significant augmentation of transmigration was observed at the second addition (≈1.5-fold, analyzed from a total of 231 monocytes in 3 experiments). Endothelial surface expressions of two major junctional molecules, PECAM-1 and VE-cadherin, increased and decreased respectively, in response to monocyte addition, which could facilitate subsequent transmigration. To further investigate spatiotemporal dynamics of the increasing molecule, PECAM-1, we constructed a PECAM-1-GFP expression system and found that monocyte transmigration induced local accumulation of endothelial PECAM-1 around the transmigration spot, which was followed by transmigration of subsequent monocyte around the same location. Detailed analysis revealed that within the defined region around one transmigration event, 50% of later transmigrating monocytes used the same or similar location as the previous one (10 out of 20 transmigrating monocytes in 11 experiments). CONCLUSIONS: These findings show that monocyte trans-endothelial migration alters endothelial junctional organization to a more monocyte-permeable state (increased PECAM-1 and decreased VE-cadherin), resulting in the augmented transmigratory activity at a later stage. This positive feedback mechanism is partially associated with monocyte transmigration-induced local accumulation of endothelial PECAM-1, which promotes transmigration of following monocytes at the same location.

Asunto(s)

Antígenos CD/biosíntesis , Cadherinas/biosíntesis , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Uniones Intercelulares/metabolismo , Monocitos/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Migración Transendotelial y Transepitelial/fisiología , Cadherinas/antagonistas & inhibidores , Células Cultivadas , Regulación hacia Abajo/fisiología , Células Endoteliales/citología , Endotelio Vascular/citología , Humanos , Uniones Intercelulares/fisiología , Monocitos/citología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/fisiología , Regulación hacia Arriba/fisiología

RESUMEN

Proteins destined for the peroxisome matrix or membrane possess distinct targeting signals that engage signal sequence receptors to drive their transport to their final subcellular destination. Peroxisomal targeting signal 1 (PTS1) and 2 (PTS2) are well characterized as signaling the import from the cytosol into the peroxisomal matrix of soluble peroxisomal enzymes. The expression vectors of the enhanced green fluorescent protein (eGFP)-tagged alanine:glyoxylate aminotransferase (AGT) and deletion mutants were introduced into HeLa cells to identify the peroxisomal targeting signal of the AGT. Two sites of the targeting signals into peroxisome, an amino acid sequence containing 59-66 a.a. (8 amino acids) and 389-392 a.a. (4 amino acids, KKKL, but not KKL) on the AGT were clarified. In a three-dimensional structural model, their sites were separately located on the surface of the AGT protein.

Asunto(s)

Peroxisomas/metabolismo , Transaminasas/química , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Modelos Moleculares , Transaminasas/metabolismo

RESUMEN

During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cell-free system and time-lapse imaging. We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. This enabled us to define three stages of apoptotic chromatin condensation: stage 1 ring condensation; stage 2 necklace condensation; and stage 3 nuclear collapse/disassembly. Electron microscopy revealed that neither chromatin nor detectable subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectable DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolyzable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.

Asunto(s)

Apoptosis , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Tumoral , Sistema Libre de Células , Pollos , Cromatina/metabolismo , Desoxirribonucleasas/metabolismo , Humanos , Hidrólisis , Microscopía Electrónica , Modelos Biológicos , Factores de Tiempo

RESUMEN

We cloned the cDNA of a novel steroid receptor-binding protein, SRB-RGS, which suppressed the estrogen receptor (ER)alpha-mediated and other promoter-driven transcriptional activities. This study revealed the interaction between the full-length SRB-RGS and full-length ERalpha or ERbeta by a coimmunoprecipitation assay. The full-length SRB-RGS and full-length ERalpha interacted in COS-7 cell by a mammalian two-hybrid system. The interaction between intrinsic SRB-RGS and ERs in the nuclear ER extract from the rat uteri was observed by the gel-shift assay. These results strongly suggested that SRB-RGS interacts with ERs bound to DNA (estrogen response element) in the nuclei of the cells. SRB-RGS suppressed very efficiently the ERalpha-, ERbeta-, and ERalpha+ERbeta-mediated transcriptional activities. Green fluorescence of enhanced green fluorescence protein (EGFP)-tagged SRB-RGS was localized both in the nucleus and in the cytoplasm. Intrinsic SRB-RGS was immunostained in the nucleus and the cytoplasm of HeLa cells. The putative SRB-RGS deduced from cDNA sequence was identified by the immunostaining and Western blotting by using the anti-SRB-RGS antibody. Overexpression of SRB-RGS induced the cell death in the HeLa cells. The nucleotide sequence of SRB-RGS cDNA that we cloned previously is identical with that of the newly isolated RGS3 cDNA. SRB-RGS could interact with ERs bound DNA in the nuclei of the cells and suppressed the ERs-mediated transcriptional activities.

Asunto(s)

Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Esteroides/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Animales , Secuencia de Bases , Western Blotting , Células COS , Muerte Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa/análisis , Cloranfenicol O-Acetiltransferasa/metabolismo , Chlorocebus aethiops , Clonación Molecular , Citoplasma/metabolismo , ADN Complementario/química , ADN Complementario/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Directa , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunohistoquímica , Inmunoprecipitación , Microscopía Confocal , Unión Proteica , Proteínas RGS , Ratas , Ratas Sprague-Dawley , Receptores de Esteroides/química , Receptores de Esteroides/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Técnicas del Sistema de Dos Híbridos

RESUMEN

Indoleamine 2,3-dioxygenase (IDO; EC 1.13.11.42) is a rate-limiting enzyme involved in the catabolism of tryptophan, which is an essential amino acid. It is induced under pathological conditions, such as the presence of viral infections or tumour cells. This enzyme is induced by IFN-gamma in the mouse rectal carcinoma cell line CMT-93. It is known that both 1-methyl-L: -tryptophan (1-MT) and methylthiohydantoin-DL: -tryptophan (MTH-trp) are tryptophan analogues, and are authentic inhibitors of the enzymatic activity of IDO. In this study, we examined the effects of both 1-MT and MTH-trp on the IFN-gamma inducible IDO expression of CMT-93. As a result, the IFN-gamma inducible IDO mRNA and the protein levels in CMT-93 were suppressed by 1-MT and MTH-trp, independently. Moreover, tryptophan (Trp), as a substrate of IDO, also suppressed IDO induction by IFN-gamma at the transcriptional level. These results suggest that 1-MT and MTH-trp are as inhibitors of IDO enzymatic activity, and Trp suppresses IDO induction by IFN-gamma at the transcriptional level.

RESUMEN

We previously identified perchloric acid-soluble protein (PSP) in the rat liver, kidney, brain and lung, and reported that it appeared to be related to repression of cell proliferation. In the present study, we clarified that PSP was expressed in the intestine, and found that the amino acid sequence of the intestinal PSP was consistent with those of other PSPs present in other tissues. An immunohistochemical study revealed that PSP was expressed in enterocytes and goblet cells, but not in other cell types among the lamina propria epithelial cells. A comparison of the expressions of PSP and proliferating cell nuclear antigen demonstrated that the proliferating cells did not express PSP. Intestinal PSP expression was induced by approximately 3-fold by oral administration of dietary fat. These findings indicate that the proliferation repression activity may be related to renewal of the intestinal epithelium, and that PSP is one of the fatty acid-inducible proteins.

Asunto(s)

Enterocitos/metabolismo , Células Caliciformes/metabolismo , Proteínas de Choque Térmico/metabolismo , Lípidos/farmacología , Ribonucleasas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Alimentación Animal , Animales , Secuencia de Bases , Bezafibrato/farmacología , ADN Complementario/genética , Proteínas de Choque Térmico/genética , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Ribonucleasas/genética , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico

RESUMEN

We examined the expression of perchloric acid-soluble protein (PSP) during liver regeneration after partial hepatectomy (PH) in rats. Liver regeneration was almost complete at 7-d after PH. Expression of PSP protein and mRNA decreased and then gradually increased during liver regeneration. An immunohistochemical study showed that PSP is distributed in cytosol and nuclei in normal liver, but localization in the nuclei was not be recognized in the regenerated liver.

Asunto(s)

Regulación de la Expresión Génica/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hepatectomía , Ribonucleasas/genética , Ribonucleasas/metabolismo , Animales , Inmunohistoquímica , Regeneración Hepática/genética , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

In the course of mammalian lens development, a transient capillary meshwork known as the pupillary membrane (PM) forms, which is located at the pupil area; the PM nourishes the anterior surface of the lens and then regresses to make the optical path clear. Although the involvement of apoptotic process has been reported in the PM regression, the initiating factor remains unknown. We initially found that regression of the PM coincided with the development of iris motility, and iris movement caused cessation and resumption of blood flow within the PM. Therefore, we investigated whether the development of the iris's ability to constrict and dilate functions as an essential signal that induces apoptosis in the PM. Continuous inhibition of iris movement with mydriatic agents from postnatal day 7 to day 12 suppressed apoptosis of the PM and migration of macrophage toward the PM, and resulted in the persistence of PM in rats. The distribution of apoptotic cells in the regressing PM was diffuse and showed no apparent localization. These results indicated that iris movement induced regression of the PM by changing the blood flow within it. This study suggests the importance of the physiological interactions between tissues-in this case, the iris and the PM-as a signal to advance vascular regression during organ development, and defines a novel function of the iris during ocular development in addition to the well-known function, that is, optimization of light transmission into the eye.

Asunto(s)

Apoptosis/fisiología , Movimientos Oculares/fisiología , Iris/irrigación sanguínea , Iris/fisiología , Microcirculación/fisiología , Animales , Animales Recién Nacidos , Pupila/fisiología , Ratas , Ratas Wistar

RESUMEN

Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.

Asunto(s)

Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Ribonucleasas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Brefeldino A/farmacología , Calcimicina/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cisplatino/farmacología , Aparato de Golgi/metabolismo , Proteínas de Choque Térmico/efectos de los fármacos , Membranas Intracelulares/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Ribonucleasas/efectos de los fármacos , Tapsigargina/farmacología , Tunicamicina/farmacología

RESUMEN

The herbicide paraquat (PQ) forms reactive oxygen species during enzymatic activation. We examined the effect of PQ on the relative levels of gene expression of antioxidant enzymes and glutathione (GSH) status in lungs of rats exposed to 20 mg/kg PQ. At 16 h after PQ intake, the mRNA expression level of glutathione reductase (GR) showed the greatest increase, and those of catalase (CAT) and manganese-superoxide dismutase (MnSOD) showed more modest increases. In contrast, PQ had little or no effect on the levels of mRNAs for copper/zinc-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPX). These findings indicate that CAT and MnSOD are coordinated and play a major role in removal of oxidants. On the other hand, PQ caused a significant increase in the GSH level in the lungs, but not in the liver. This increase in the lungs was, at least in part, caused by stimulation of the gamma-glutamylcysteine synthetase gene. However, the expression of GPX mRNA was not stimulated as described above. Because GSH is a substrate for GPX and serves as a scavenger of hydroxyl radicals, the increase in GSH as well as GR expression may be insignificant. This imbalance may be a result of oxidative stress due to PQ.

Asunto(s)

Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Herbicidas/farmacología , Sistema Inmunológico/efectos de los fármacos , Paraquat/farmacología , Animales , Sistema Inmunológico/enzimología , Pulmón/efectos de los fármacos , Oxidantes/metabolismo , Estrés Oxidativo/fisiología , Ratas

RESUMEN

Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.

Asunto(s)

Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN , Genoma Viral , Hepacivirus/genética , Hígado/virología , Proteínas Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Western Blotting , Caseína Quinasas , Ciclo Celular , División Celular , Línea Celular , Separación Celular , Clonación Molecular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , ADN Complementario/metabolismo , Factores de Transcripción E2F , Citometría de Flujo , Regulación Viral de la Expresión Génica , Genes Reporteros , Hepatocitos/metabolismo , Humanos , Immunoblotting , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Factores de Tiempo , Transfección

RESUMEN

To analyze the root components of compounds causing drug addiction, we have developed our own GC/MS libraries(PESTICID.LIB consisting of 20 pesticides and Herbicides, and DRUG.LIB with 57 drugs). We have usually utilized standardized agents, but gastric contents, gastric specimens, serum and urine samples from patients were also used for the analyses. We were able to add libraries of various difficult to purchase psychotropic drugs and legally restricted agents by extracting them from the patient samples. Comments about the retention time, the base peak of the mass spectrum and 5 typical ion chromatograms in the libraries have been useful for laboratory analysis, and consequently have improved the accuracy of detection and identification. They were also found to be a useful guideline for discrimination of the unchanged materials and the metabolites. We are attempting to improve the accuracy of the library to avoid the effects of GC column conditions such as the column size, column temperature and different inserts by using a retention time index.

Asunto(s)

Cromatografía de Gases y Espectrometría de Masas/métodos , Plaguicidas/análisis , Preparaciones Farmacéuticas/análisis , Cromatografía de Gases y Espectrometría de Masas/instrumentación , Humanos , Plaguicidas/envenenamiento , Intoxicación/diagnóstico , Sensibilidad y Especificidad

RESUMEN

Tryptophan pyrrole ring cleavage enzymes were assayed in pregnant uterus of mouse. The highest activity was observed at 6.5 days post-coitus (dpc) with a small activity shoulder at 9.5 to 12.5 dpc concepti and placenta. The highest peak at early concepti of 6.5 dpc was coincided with gene expression of tryptophan 2,3-dioxygenase (TDO). And the shoulder from 9.5 dpc was coincided with the expression of indoleamine 2,3-dioxygenase (IDO). These enzymes showed a different inhibitory behavior to a proper IDO inhibitor, 1-methyl tryptophan. In situ hybridization analysis revealed that TDO was expressed in the decidua of the early concepti. This is the first report of the extra hepatic TDO.

Asunto(s)

Placenta/enzimología , Triptófano Oxigenasa/metabolismo , Triptófano/análogos & derivados , Animales , Secuencia de Bases , Femenino , Hibridación in Situ , Indolamina-Pirrol 2,3,-Dioxigenasa , Ratones , Ratones Endogámicos ICR , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triptófano/metabolismo , Triptófano/farmacología , Triptófano Oxigenasa/genética

RESUMEN

Primer extension of human liver poly(A)(+) RNA revealed that the main transcription start site of the human alanine:glyoxylate aminotransferase gene (AGXT) is situated near 45 bp upstream from the translation start site. Deletion analysis using the 1203 bp 5'-flanking region of the AGXT gene and a luciferase reporter suggested that the promoter sequence is most likely located 2-325 bp upstream from the translation start site, possibly with enhancer elements 440-700 bp upstream. It was also suggested that the region -2 to -64 is important for the expression of the AGXT gene. The region -2 to -325 has two TATA boxes and some initiator elements.

Asunto(s)

Transaminasas/genética , Transcripción Genética/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , ADN Complementario/química , Genes Reporteros , Humanos , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas