RESUMEN
Objective: To investigate the clinicopathological features of perivascular epithelioid cell tumor (PEComa) of the lung. Methods: Eight PEComa cases of the lung diagnosed at the First Affiliated Hospital of Soochow University, Suzhou, China from July 2008 to December 2021 were collected and subject to immunohistochemical staining, fluorescence in situ hybridization and next generation sequencing. The relevant literature was reviewed and the clinicopathological features were analyzed. Results: There were 5 males and 3 females, aged from 18 to 70 years (mean 39 years). There were 3 cases of the right upper lung, 3 cases of the left lower lung, 1 case of the left upper lung and 1 case of the right middle lung. Seven cases were solitary and 1 case was multifocal (4 lesions). Seven cases were benign while one was malignant. The tumors were all located in the peripheral part of the lung, with a maximum diameter of 0.2-4.0 cm. Grossly, they were oval and well circumscribed. Microscopically, the tumor cells were oval, short spindle-shaped, arranged in solid nests, acinar or hemangiopericytoma-like patterns, with clear or eosinophilic cytoplasm. The stroma was rich in blood vessels with hyalinization. Coagulated necrosis and high-grade nuclei were seen in the malignant case, and calcification was seen in 2 cases. Immunohistochemically, the tumor cells were positive for Melan A (8/8), HMB45 (7/8), CD34 (6/8), TFE3 (4/7), and SMA (3/8). All cases were negative for CKpan and S-100. TFE3 (Xp11.2) gene fusion was examined using the TFE3 break-apart fluorescence in situ hybridization in 5 cases, in which only the malignant case was positive. The next generation sequencing revealed the SFPQ-TFE3 [t(X;1)(p11.2;p34)] fusion. Follow-up of the patients ranged from 12 to 173 months while one patient was lost to the follow-up. The malignant case had tumor metastasis to the brain 4 years after the operation and then received radiotherapy. Other 6 cases had no recurrence and metastasis, and all the 7 patients survived. Conclusions: Most of the PEComas of the lung are benign. When there are malignant morphological features such as necrosis, high-grade nuclei or SFPQ-TFE3 gene fusion, close follow-up seems necessary.
Asunto(s)
Neoplasias de Células Epitelioides Perivasculares , Masculino , Femenino , Humanos , Hibridación Fluorescente in Situ , Neoplasias de Células Epitelioides Perivasculares/genética , Neoplasias de Células Epitelioides Perivasculares/cirugía , Neoplasias de Células Epitelioides Perivasculares/patología , Pulmón/patología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Necrosis , Biomarcadores de Tumor/análisisRESUMEN
A newly identified gene MK is transiently expressed in early stages of retinoic acid-induced differentiation of embryonal carcinoma cells (Kadomatsu, K., M. Tomomura, and T. Muramatsu, 1988. Biochem. Biophys. Res. Commun. 151:1312-1318). MK gene has been predicted to code a polypeptide that is rich in basic amino acids and cysteine and is not related to any other peptides so far reported. In the present study, we investigated MK expression during mouse embryogenesis by in situ hybridization. The MK transcript was detected all over the embryo proper of the 7-d embryo, while it was not detectable in the 5-d embryo. The ubiquitous expression continued in the 9-d embryo proper. On the 11th-13th d of gestation, the sites where MK gene was intensely expressed became progressively restricted; these sites were the brain ectoderm around the lens and brain ventricles, the anterior lobe of the pituitary gland, the upper and lower jaw, the caudal sclerotomic half of vertebral column, the limbs, the stomach, and the epithelial tissues of the lung, the pancreas, the small intestine, and the metanephros. These areas include the region where secondary embryonic induction is prominent. In the 15-d embryo, only the kidney expressed MK significantly. These data suggest that MK gene plays a fundamental role in the differentiation of a wide variety of cells; MK gene may also play some specific roles in generation of epithelial tissues, and remodeling of mesoderm.
Asunto(s)
Desarrollo Embrionario y Fetal , Expresión Génica/efectos de los fármacos , Teratoma/genética , Transcripción Genética , Tretinoina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Riñón/embriología , Ratones , Ratones Endogámicos ICR , Hibridación de Ácido Nucleico , Especificidad de Órganos , ARN/genética , ARN/aislamiento & purificación , Transcripción Genética/efectos de los fármacosRESUMEN
Exposure of mammalian cells to UV irradiation induces rapid and transient expression of early growth response-1 gene (Egr-1) encoding a transcription factor that plays a role in cell survival. These signals from the irradiated cell surface are likely to involve more than one pathway, and we show here that an essential pathway involves activation of several growth factor receptors by reactive oxygen intermediates (ROI). UVC irradiation causes the tyrosine phosphorylation of EGF receptor (EGFR) in mouse NIH 3T3 fibroblasts and HC11 mouse mammary cells. EGFR activation by irradiation of cells is abrogated by suramin, by antioxidants, and by the presence of a dominant negative EGFR. UV induces the formation of complexes between activated EGFR and SOS, Grb2, PLC gamma, and SHC that can be precipitated with antibodies to EGFR. The activation of EGFR by UV is mimicked by H2O2, suggesting that ROI may function upstream of EGFR activation. Our observations support the hypothesis that ROI and growth factor receptors operate in the early steps of the UV signal that lead to the enhanced expression and activity of Egr-1.
Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Proteínas Inmediatas-Precoces , Especies Reactivas de Oxígeno , Receptores de Factores de Crecimiento/fisiología , Transducción de Señal/efectos de la radiación , Rayos Ultravioleta , Células 3T3 , Animales , Antioxidantes/farmacología , Secuencia de Bases , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Proteína 1 de la Respuesta de Crecimiento Precoz , Epitelio , Receptores ErbB/análisis , Receptores ErbB/fisiología , Sustancias de Crecimiento/farmacología , Ratones , Datos de Secuencia Molecular , Fosforilación , Prolina/análogos & derivados , Prolina/farmacología , Proteínas/análisis , Suramina/farmacología , Tiocarbamatos/farmacología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Tirosina/metabolismoRESUMEN
BACKGROUND: The use of cultured pancreatic islet cells for diabetes treatment offers several advantages. In theory, cultured cells show greater purity and lower immunogenicity. However, cultured islet cells display a low survival rate in vitro. In the present study we grew islet cells on a polyglycolic acid (PGA) fibrous scaffold to promote cell adhesion, growth, and viability during prolonged culture. METHODS: Islets isolated from Wistar rat pancreata were digested with collagenase and purified by the Ficoll method. Cells were grown in culture with or without PGA scaffolds. Islet cell purity was determined using a dithizone stain; viability and survival rates were determined using an AO-PI stain. The insulin-secretion index was detected using radioimmunodetection and the growth on an adhesive scaffold analyzed using an inverted microscope and scanning electron microscope (SEM). RESULTS: In contrast to the scaffold-free control group, cells cultured on PGA scaffolds exhibited improved morphology, less cell death, and prolonged survival times. Cell viability and survival rates were significantly increased in scaffolded cells when compared to control cells (P < .05). Increased insulin secretion was observed in the culture solution of scaffolded cells following stimulation with low glucose (5.6 mmol/L) versus high glucose (16.7 mmol/L). The secretion indices of the two groups were significantly different (P < .05). Islet cell growth, as observed under SEM, was tightly circumvolute, adhesive, and three-dimensional. CONCLUSIONS: The present results demonstrated that islet cells can successfully grow and survive in culture on a PGA scaffold. These cells exhibited enhanced viability, survival, and insulin secretion.
Asunto(s)
Adhesión Celular/fisiología , División Celular/fisiología , Islotes Pancreáticos/citología , Ácido Poliglicólico , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Diabetes Mellitus/cirugía , Femenino , Trasplante de Islotes Pancreáticos , Masculino , Ratas , Ratas Wistar , Andamios del TejidoRESUMEN
Human HT1080 fibrosarcoma cells, subclone H4, express little or no Egr-1 (Zif/268, Krox 24), an early growth response gene encoding a transcription factor. Phorbol ester (but not serum) treatment only can elicit a small increase in Egr-1 expression in H4, in contrast to the normally rapid, high transient expression of Egr-1 observed after the addition of a wide range of stimulating agents to normal or immortalized cell lines. Because several human tumor cell lines express little Egr-1, we tested the hypothesis that this loss was causal to transformation. We report here that the expression of exogenous mouse Egr-1 in H4 cells inhibits transformed growth in a dose-dependent manner and significantly suppresses tumorigenicity in athymic mice. By overexpression of the fragment in Egr-1 that is responsible for its DNA-binding activity, the zinc-finger domain, we show that this domain has a similar activity. Moreover, the expression of antisense mRNA encoding the DNA-binding domain increases the transformed character of the H4 cells. One possible conclusion is that endogenous Egr-1-like genes perform growth-regulatory functions. Other human tumor lines are also growth suppressed by Egr-1 overexpression including ZR-75-1 breast carcinoma, U251 glioblastoma, and to a lesser extent, SAOS-2 osteosarcoma cells. These results are surprising in light of the "early growth response" character of Egr-1 but extend our earlier report of suppression of growth in v-sis-transformed NIH3T3 cells.
Asunto(s)
ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibrosarcoma/patología , Proteínas Inmediatas-Precoces , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , División Celular/fisiología , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Fibrosarcoma/genética , Fibrosarcoma/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , ARN sin Sentido/genética , Factores de Transcripción/genética , Células Tumorales Cultivadas , Dedos de Zinc/genéticaRESUMEN
Connexins (cx), structural components of gap junction, are believed to play a role in the regulation of cell proliferation and suppression of the neoplastic phenotype. We used human brain glioblastoma tumor cells as a model system to test this hypothesis. Western blot and reverse transcription-PCR analysis indicate that the expression levels of the gap junction protein connexin 43 (cx43) are profoundly decreased in several human brain tumor cell lines examined. Transfection of human cx43 into human glioblastoma cell lines U251 and T98G profoundly reduces cell proliferation in monolayer culture, in soft agar, and in athymic nude mice. Surprisingly, these effects are not associated with the establishment of gap junction communication in cx43 transfected cells. We conclude that the loss of cx43 expression may play a role in the development of human gliomas and that cx43 acts as a tumor suppressor gene to human glioblastoma.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Conexina 43/metabolismo , Glioblastoma/metabolismo , Animales , Neoplasias Encefálicas/patología , Comunicación Celular , División Celular , Conexina 43/genética , Uniones Comunicantes , Genes Supresores de Tumor , Glioblastoma/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fenotipo , Transfección , Células Tumorales CultivadasRESUMEN
The response to ultra-violet (u.v.) irradiation varies among cells, but commonly involves the rapid increase in expression of one or more transcription factors. The specific roles of this increased expression are largely unknown. We show here that in mouse NIH3T3 cells, Egr-1 expression is increased two-fold 10 min after u.v. irradiation, rises to a maximum (eightfold induction) after about 2 h and then declines. The expression of p53 protein is also strongly induced but is maximal between 2 to 4 h before declining. In contrast, the expression of c-Fos, and C-Jun proteins are only slightly affected by u.v. The Egr-1 response is independent of the growth state of the cells but depends on tyrosine kinase and protein kinase C activities. c-Ha-Ras is also involved in the induction of Egr-1 in u.v. irradiated cells. Evidence presented suggests that the mechanism for the response involves oxidative stress rather than DNA damage. We show that Egr-1 functions in the protection of cells against u.v. damage since NIH3T3 cells that constitutively express antisense Egr-1 and consequently cannot produce an Egr-1 response to u.v., grow at a rate 26% less than similarly irradiated parental cells and 36% less than nonirradiated parental cells. This is the second protective role described for Egr-1.
Asunto(s)
Supervivencia Celular/efectos de la radiación , Proteínas de Unión al ADN/fisiología , Proteínas Inmediatas-Precoces , Factores de Transcripción/fisiología , Rayos Ultravioleta , Células 3T3 , Animales , División Celular/fisiología , División Celular/efectos de la radiación , Supervivencia Celular/fisiología , Daño del ADN/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica/efectos de la radiación , Ratones , Estrés Oxidativo/fisiología , Proteínas Quinasas/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/biosíntesis , Proteínas ras/fisiologíaRESUMEN
The transcription factor Egr-1, stimulates the activity of a number of genes and inhibits other genes, by binding to the sequence GCGGGGGCG in 5' enhancer regions. However, the functions of Egr-1 are obscure in spite of its rather ubiquitous expression. Egr-1 may play a role in proliferation in mitogen-stimulated cells but its expression is also correlated with the differentiated state in teratocarcinoma cells. The constitutive expression of Egr-1 appears to have little effect on the growth rate of normal immortalized cell-lines. We show that in NIH3T3 cells that are conditionally transformed by the expression of v-six, the presence of Egr-1 is inhibitory to the production of transformed colonies (foci) and to growth in soft agar. In addition, the first appearance of tumors in nu/nu mice is delayed in tumorigenicity tests with cells that over-express Egr-1 and tumor growth is suppressed compared to control cells. We used a series of fragments of Egr-1 cloned into expression vectors to show that not only full length, but also truncated Egr-1 fragments inhibit colony formation. Using deletion mutants, we observed that this inhibitory activity is dependent on the presence of the DNA-binding 'zinc-finger' region. Wilm's tumor protein, WT1, (known to be a tumor suppressor gene) that exhibits the same DNA binding activity is also inhibitory. In contrast, colony formation is stimulated by an Egr-1 antisense RNA-expressing plasmid, since colonies grow rapidly and the colony-forming frequency is higher than in the presence of v-sis alone. We conclude that proteins containing the Egr-1 'zinc-finger' domain can bind to the regulatory regions of one or more genes that are required for the transformation of fibroblasts by v-sis thus inhibiting transformation. One function for Egr-1 implied by these results is the restraint of transformed growth in mitogen-stimulated cells.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Unión al ADN/fisiología , Proteínas Inmediatas-Precoces , Proteínas Oncogénicas de Retroviridae/metabolismo , Factores de Transcripción/fisiología , Células 3T3/patología , Animales , Secuencia de Bases , Adhesión Celular , División Celular , Transformación Celular Neoplásica/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Mutación , Trasplante de Neoplasias , Proteínas Oncogénicas v-sis , ARN sin Sentido/metabolismo , ARN sin Sentido/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ensayo de Tumor de Célula Madre , Dedos de ZincRESUMEN
The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.
Asunto(s)
Apoptosis , Transformación Celular Neoplásica , Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de la radiación , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Pruebas de Carcinogenicidad , Caspasa 3 , Caspasas/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Ciclo Celular/efectos de la radiación , División Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Fragmentación del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Fibrosarcoma , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , MAP Quinasa Quinasa 4 , Proteína Quinasa 3 Activada por Mitógenos , Mutación , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Rayos UltravioletaRESUMEN
UV irradiation of normal or immortalized cells induces a rapid increase in the expression of several transcription factors and is thought to serve a protective function. The human fibrosarcoma cell line, HT1080 clone H4, expresses almost undetectable levels of Egr-1 and does not respond to UV-C irradiation by the induction of Egr-1. The H4 cells are hypersensitive to UV which induces apoptosis and reduces clonogenicity. The introduction of exogenous Egr-1 into H4 (H4E9 and H4E4 cell-lines) confers protection from UV damage as measured by a number of assays. In both NIH3T3 (with inducible Egr-1) and H4E9 (constitutive Egr-1) cells, UV irradiation gave enhanced transactivation of Egr-1 reporters that correlated with phosphorylated Egr-1. Studies using inhibitors indicated that protein kinase-C and tyrosine kinases are involved in the anti-apoptotic effects of Egr-1 after UV damage. This is the first description of a biological effect of phosphorylated Egr-1.
Asunto(s)
Apoptosis/fisiología , Apoptosis/efectos de la radiación , Proteínas de Unión al ADN/genética , Proteínas Inmediatas-Precoces , Factores de Transcripción/genética , Dedos de Zinc/fisiología , Células 3T3/química , Células 3T3/citología , Animales , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Colina O-Acetiltransferasa/genética , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Fibrosarcoma , Fase G2/fisiología , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Fase S/fisiología , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiología , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/enzimología , Rayos UltravioletaRESUMEN
A new antibody-based protein array assay is described. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL) and high-throughput of microspot. In this system, the capture proteins, either antibodies or antigens are spotted onto membranes in an array format. Biological samples are then incubated with membranes. After antigens or antibodies in the samples bind to their corresponding targets and unbound proteins are washed away, the membranes are exposed to Horseradish Peroxidase (HRP)-conjugated antibody(ies). The signals are finally visualized with ECL system. Experiments demonstrate that multiple cytokines and antibodies can be simultaneously detected using this new approach. The procedure is so simple that no sophisticated equipment is required. The concept should be able to be extended to develop a high-throughput protein array system. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics and discovery of new biomarkers.
Asunto(s)
Anticuerpos/análisis , Citocinas/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Sanguíneas/análisis , Medios de Cultivo Condicionados/análisis , Peroxidasa de Rábano Silvestre , Humanos , Membranas Artificiales , ProteomaRESUMEN
A cDNA clone isolated from a lambda gt11 expression library of teratocarcinoma OTT6050 specifies for a glycoprotein with a molecular weight of about 44,000. The new glycoprotein was termed heparin binding protein-44 (HBP-44), since it was absorbed to a heparin-agarose column and was eluted from it by a buffer containing 1.5 M NaCl. HBP-44 mRNA was intensely expressed in PYS-2 parietal endoderm cells and in the kidney, and the RNA level increased about 10-fold during differentiation of F9 embryonal carcinoma cells to parietal endoderm cells. From the cDNA sequence, HBP-44 was concluded to be rich in charged amino acids, and large segments of the protein appeared to form alpha-helixes. The protein was considered to be anchored to the membrane by a cluster of hydrophobic amino acids present in the N-terminal region. Indeed, the N-terminal sequence of HBP-44 was homologous to asialoglycoprotein receptor, which is anchored to the membrane by the N-terminal region. Furthermore, a portion of the N-terminal region of HBP-44 was homologous to the leucine zipper domain. Except for the N-terminal region, HBP-44 had over-all homology with structural proteins such as myosin heavy chain. We propose that HBP-44 is extruded from plasma membranes and interacts with heparin and related molecules and that it is involved in the interactions of plasma membranes with basement membranes.
Asunto(s)
Proteínas Portadoras/biosíntesis , Células Madre Neoplásicas/metabolismo , Lectinas de Plantas , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Células Tumorales Cultivadas/metabolismo , Secuencia de Aminoácidos , Asialoglicoproteínas/análisis , Secuencia de Bases , Northern Blotting , Proteínas Portadoras/química , Diferenciación Celular , Clonación Molecular , Células Madre de Carcinoma Embrionario , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/análisis , Humanos , Inmunoquímica , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Lectinas/farmacología , Datos de Secuencia Molecular , Células Madre Neoplásicas/patología , Glicoproteínas de Membrana Plaquetaria/química , ARN Neoplásico/análisis , Mapeo Restrictivo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patologíaRESUMEN
The binding of the transcription factor early growth response-1 (Egr-1) to its specific DNA-binding sequence GCGGGGGCG occurs through the interaction of three zinc finger motifs. DNA binding by Egr-1 can be modified by alteration of reduction-oxidation (redox) state. Using gel retardation assays, we show that binding of Egr-1 protein is specific and is dependent on the presence of reducing agents in a dose-dependent manner. The zinc finger region is the domain subject to conformation changes by redox. Oxidized or metal-free Egr-1 does not bind. Nuclear extracts of several cell types contain a heat-sensitive factor(s) that induces the ability of Egr-1 protein to bind to DNA in otherwise suboptimal conditions containing insufficient reducing agent. This inducing activity may be replaced by Ref-1, a protein identified and characterized by Curran and co-workers (Xanthoudakis and Curran, 1992). The possibility arises that the transcription-regulating activity of Egr-1 may be regulated by the redox state in the cell via factors such as Ref-1 that modulate its DNA-binding activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Proteínas de Saccharomyces cerevisiae , Transactivadores , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Núcleo Celular/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/fisiología , Oxidación-Reducción , Proteínas RecombinantesRESUMEN
Histologic studies have demonstrated both a decrease in size and loss in number of type II muscle fibers with increasing age. Although these age-related histologic changes are believed to result in decreased strength and functional capacity, age-related changes in muscle force dynamics have not been clearly elucidated. Using vibromyographic (VMG) techniques, we recorded muscle activity of the soleus in 40 healthy adult volunteers spanning the age range of 20-82 years to test whether changes in postural muscle dynamics, in the frequency range of 0.1-50 Hz, were also associated with age. Although muscle dynamics below 15 Hz do not change with aging, the 30-50 Hz frequency components of the VMG were found to change significantly with advancing age (r = -.619, p = .0001). This was observed in both sexes independently. The observed age-related changes in muscle force dynamics demonstrate distinct physiologic alterations in muscle fiber activity. Further research will be required to fully elucidate the relationship between age-related changes in muscle fiber activity and other age-related conditions such as postural instability and osteoporosis.
Asunto(s)
Envejecimiento/fisiología , Músculo Esquelético/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Electromiografía/métodos , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Postura , VibraciónRESUMEN
Pancreatic islet transplantation is a promising therapeutic treatment for type 1 diabetes mellitus. In the present study, we cocultured islets with or without a polyglycolic acid (PGA) fibrous scaffold for 5 days and transplanted the PGA-islet grafts into the leg muscles of Wistar rats with streptozotocin-induced diabetes; controls were injected with saline. The results showed that the blood glucose concentrations of the group given islets embedded with the PGA scaffold were lower than those without the scaffold or controls. On the other hand, the insulin content of the PGA-islet group was higher at all 5 time points compared with the insulin contents of the other 2 groups. After transplantation, many islets in the PGA-islet grafts showed normal morphology (as seen under the scanning electron microscope) and were surrounded by red blood cells. A fibrous extracellular matrix was visible around the PGA-islet grafts. These results demonstrated that PGA-islet grafts improved blood glucose and insulin concentrations in rats with induced diabetes.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/cirugía , Insulina/sangre , Trasplante de Islotes Pancreáticos/métodos , Animales , Células Cultivadas , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Islotes Pancreáticos/ultraestructura , Trasplante de Islotes Pancreáticos/patología , Microscopía Electrónica de Rastreo , Ratas , Ratas WistarRESUMEN
Protein arrays hold a promise in basic and clinical applications. As the first step to develop such array system, I used an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL) to demonstrate the feasibility of simultaneous detection of multiple proteins. In the direct ELISA system, different known immunoglobulin Gs (IgGs) were immobilized onto polyvinylidine difloride (PDVF) membrane through 96-well format Bio-Dot unit. The antigens were then individually and collectively detected by incubation of membranes with different antibodies coupled with ECL. In the sandwich ELISA system, the cytokine capture antibodies were immobilized onto PDVF membranes. The membranes were then incubated with single cytokine or a combination of different cytokines. The captured cytokines were detected by biotin-conjugated antibodies coupled with ECL system. Experiments demonstrated that multiple IgGs and cytokines could be simultaneously detected using this approach with high specificity and sensitivity. More importantly, cytokines from biological samples were detected using this approach, which can be used in any general laboratory setting without any sophisticated equipment. This concept could be extended to develop a protein-based array system.
Asunto(s)
Quimiocina CCL2/análisis , Citocinas/análisis , Inmunoglobulinas/análisis , Proteínas/análisis , Animales , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulinas/química , Mediciones Luminiscentes , Polivinilos/análisis , Polivinilos/química , Sensibilidad y EspecificidadRESUMEN
The transcription factor, Early growth response-1, Egr-1, occurs in several forms differing in length due to three alternate translation start sites (1). Egr-1 has also been reported to be phosphorylated although a role for this modification is unknown. Using dephosphorylation and hyperphosphorylation studies, we show here that phosphorylated Egr-1 is much more efficiently bound to its DNA binding element (GCE) and binds in a dose-dependent fashion. Egr-1 plays complex roles in growth regulation and we suggest that alternate forms of phosphorylated Egr-1 may account for this.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Animales , Baculoviridae , Sitios de Unión , ADN/metabolismo , Mariposas Nocturnas , Fosfoproteínas/metabolismo , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Relación Estructura-Actividad , Dedos de ZincRESUMEN
Using the lambda gt11 expression library of murine teratocarcinoma cells, we isolated cDNA clones encoding a core protein carrying a developmentally regulated carbohydrate marker, namely the binding site for Dolichos biflorus agglutinin. The deduced amino acid sequence of the polypeptide (molecular weight, 37,102) revealed a leader sequence, nine potential asparagine glycosylation sites, and a transmembrane region. Sequence homology to variable domain of immunoglobulin kappa chain has been detected in a domain; homologous amino acids near cysteine residues are those conserved in many members of the immunoglobulin gene superfamily. In contrast to other members of the superfamily, the core protein gene was significantly expressed in embryonal carcinoma cells, which are similar to undifferentiated cells of early embryos.
Asunto(s)
Genes de Inmunoglobulinas , Glicoproteínas/inmunología , Teratoma/inmunología , Secuencia de Aminoácidos , Animales , Bacteriófago lambda/genética , Secuencia de Bases , ADN/genética , Glicoproteínas/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Receptores Mitogénicos/genética , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
Embigin is a member of the immunoglobulin superfamily found in embryonal carcinoma cells. The present study deals with embigin gene expression in adult and embryonic cells. By RNA blot analyses, high levels of embigin mRNA were detected in embryonal carcinoma cells with different differentiation potentials, namely F9 and PCC4 cells. Similar or slightly higher levels of the RNA were detected in the embryonic and extraembryonic portions of 9-day mouse embryos. When F9 cells were induced to differentiate by treatment with retinoic acid and dibutyryl cyclic AMP for 2-5 days, the RNA level increased significantly. On the other hand, embigin mRNA dramatically dropped to almost background levels in embryos, from 11 days postcoitus (p.c.). Many organs of adult mice expressed only low levels of embigin mRNA, but considerable amounts of the transcript were found in the ovary and also in the uterus of pregnant mice (4 days p.c. and 12 days p.c.). In situ hybridization experiments revealed large amounts of embigin RNA in the embryo from 7 to 9 days p.c. The visceral endoderm of 7-day embryos, and the brain, visceral yolk sac and presumptive foregut of 9-day embryos were the sites where intense signals of embigin RNA were localized.