RESUMEN
A calcium-binding protein was detected in the cytosol of Morris hepatomas 7288, 5123tc (h), 5123tc, and 7795. This protein could not be detected in adult Buffalo liver, 24-hr regenerating liver, or 18- to 20-day-old fetal liver. The amount of this protein present was not clearly related to the growth rate of the tumors studied, although there was more in a faster growing tumor (5123tc) than in the slowest (7795). The protein from the four tumors was apparently identical. The calcium-binding protein was heat stable and was not the calcium-dependent regulator of cyclic nucleotide phosphodiesterase. It had an apparent molecular weight of less than 12,500.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/aislamiento & purificación , Neoplasias Hepáticas Experimentales/análisis , Proteínas de Neoplasias/aislamiento & purificación , Animales , Cromatografía en Gel , Feto/análisis , Hígado/análisis , Regeneración Hepática , Masculino , Peso Molecular , RatasRESUMEN
An antiserum has been developed against a M.W. 11,500 calcium-binding protein purified to homogeneity from Morris hepatoma 5123tc. The antiserum was specific and did not cross-react with an excess of two other calcium-binding proteins, calmodulin or parvalbumin. The amount of the tumor protein has been quantitated immunologically in several Morris hepatomas, early neoplastic liver nodules, and mouse sarcomas. This protein could not be detected in any normal rat tissue. The calcium-binding protein in mouse tumors appeared similar to that in rats by both crossed polyacrylamide gel electrophoresis-immunoelectrophoresis and crossed isoelectric focusing immunoelectrophoresis.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Animales , Proteínas de Unión al Calcio/inmunología , Citosol , Inmunoensayo , Peso Molecular , RatasRESUMEN
Oncomodulin, an apparently tumor-specific calcium-binding protein, has been detected in many chemically induced rat hepatomas. It is now possible to detect, by radioimmunoassay and immunofluorescence, the presence of oncomodulin in normal rat kidney cells virally transformed by avian sarcoma virus. By contrast, it was not detected in uninfected, nonneoplastic normal rat kidney cells. The protein was isolated and purified by a novel high-performance liquid chromatography procedure and shown to be identical to that isolated previously from rat hepatoma. The cellular levels of oncomodulin approached the levels of calmodulin in avian sarcoma virus-transformed normal rat kidney cells, suggesting that the total calcium-binding activity of the cell may play a role in expression of the transformed phenotype.
Asunto(s)
Virus del Sarcoma Aviar/genética , Proteínas de Unión al Calcio/genética , Transformación Celular Viral , Proteínas de Neoplasias/genética , Aminoácidos/análisis , Animales , Proteínas de Unión al Calcio/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Técnica del Anticuerpo Fluorescente , Neoplasias Renales/análisis , Neoplasias Hepáticas Experimentales/análisis , Proteínas de Neoplasias/aislamiento & purificación , Radioinmunoensayo , RatasRESUMEN
The HT-29 human adenocarcinoma cell line has been used extensively in the study of colonic cell differentiation and colon cancer. We report here that substitution of glucose with trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) depresses growth and promotes mucin-producing, goblet-like maturation of HT-29. An initial characterization of this process was made by analyzing several cDNA clones whose RNA templates were differentially expressed at elevated levels in cells grown in trehalose-containing medium. Seven of the 9 clones examined corresponded to 6 mitochondrial genes whose expression levels, relative to those from glucose-grown cells, ranged from approximately 3-fold for 16S rRNA to 8-23-fold for NADH dehydrogenase subunit 4. On the other hand, levels of mitochondrial DNA copy, measured by using NADH dehydrogenase subunit 4 cDNA as probe, were shown to be unaffected by trehalose treatment. Elevation of cellular NADH dehydrogenase subunit 4 RNA in HT-29 cultures grown in medium containing different components (sodium butyrate, galactose, no-sugar, glucose, cellobiose) generally correlated with depressed growth levels and specifically with increased numbers of mucin-producing cells present. Like butyrate, the sugar, trehalose, is an effective inducer of HT-29 differentiation, and may prove useful as a dietary therapeutic, and as a probe for elucidating mitochondrial involvement in colonic cell differentiation and transformation.
Asunto(s)
Adenocarcinoma/patología , Neoplasias del Colon/patología , ARN Neoplásico/análisis , ARN/análisis , Trehalosa/farmacología , Adenocarcinoma/genética , Secuencia de Bases , Diferenciación Celular , División Celular/efectos de los fármacos , Neoplasias del Colon/genética , ADN Mitocondrial/análisis , Humanos , Datos de Secuencia Molecular , NADH Deshidrogenasa/genética , ARN Mitocondrial , Transcripción Genética , Células Tumorales CultivadasRESUMEN
A heat-stable Ca2+-binding protein was purified to homogeneity from Morris hepatoma 5123 tc. It had an apparent molecular weight of 11 000, and isoelectric point of 3.9, and bound two atoms of calcium per molecule of protein. The spectral and amino acid analysis indicated the tumour protein to be similar to the parvalbumins. This protein has been shown to be absent in liver.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Neoplasias Hepáticas Experimentales/análisis , Aminoácidos/análisis , Animales , Proteínas de Unión al Calcio/aislamiento & purificación , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Calor , Punto Isoeléctrico , Masculino , Peso Molecular , Ratas , Espectrometría de FluorescenciaRESUMEN
The crystal structure of oncomodulin, a 12,000 Mr protein isolated from rat tumours, has been determined by molecular replacement using the carp parvalbumin structure as a starting model. Refinement was performed by cycles of molecular fitting and restrained least-squares, using area-detector intensity data to 1.85 A resolution. For the 5770 reflections in the range 6.0 to 1.85 A, which were used in the refinement, the crystallographic R-factor is 0.166. The refined model includes residues 2 to 108, three Ca2+ and 87 water molecules per oncomodulin molecule. The oncomodulin backbone is closely related to that of parvalbumin; however, some differences are found after a least-squares fit of the two backbones, with root-mean-square (r.m.s.) deviations of 1 to 2 A in residues 2 to 6, 59 to 61 of the CD loop, 87, 90 and 108. The overall r.m.s. deviation of the backbone residues 5 to 108 is 0.62 A. Each of the two Ca2+ atoms that are bound to the CD and EF loops is co-ordinated to seven oxygen atoms, including one water molecule. The third Ca2+ is also seven-co-ordinated, to five oxygen atoms belonging to three different oncomodulin molecules and to two water molecules which form hydrogen bonds to a fourth oncomodulin; thus, this intermolecular Ca2+ and its equivalents interlink the molecules into zigzag layers normal to the b axis with a spacing of b/2 or 32.14 A. No such extensive molecular aggregation has been reported for any of the related Ca-binding regulatory proteins of the troponin-C family studied thus far. The Ca-O distances in all three polyhedra are in the range 2.07 A to 2.64 A, indicating tightly bound Ca polyhedra.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Gráficos por Computador , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Parvalbúminas/química , Parvalbúminas/genética , Conformación Proteica , Ratas , Homología de Secuencia de Ácido Nucleico , Difracción de Rayos XRESUMEN
DNA fragments of 50 and 10 kbp were found in ischemic brain in adult rats following two-vessel occlusion or in neonates following hypoxia-ischemia. These higher-order fragments were detected before any laddered oligonucleosomal DNA fragmentation characteristic of apoptosis. Both the 50- and 10-kbp fragments were also detected during necrosis produced by decapitation, but these led to smeared smaller fragments, not laddered patterns. End-group analysis showed the presence of both 3'-OH and 5'-OH ends in both the 50- and 10-kbp fragments but the predominance of 3'-OH ends in the laddered fragments. A higher proportion of 5'-OH to 3'-OH ends was found in the 10-kbp fragment compared to the larger 50-kbp fragment, suggesting a selective degradation of the 50-kbp DNA fragment to the laddered oligonucleosomal patterns. Overall, the mode of DNA fragmentation appeared different from that described in classic apoptosis of thymocytes.
Asunto(s)
Apoptosis/fisiología , Fragmentación del ADN , Ataque Isquémico Transitorio/genética , Ataque Isquémico Transitorio/patología , Nucleosomas/fisiología , Animales , Animales Recién Nacidos , Encéfalo/patología , Estado de Descerebración , Electroforesis en Gel de Agar , Electroforesis en Gel de Campo Pulsado , Hipoxia/complicaciones , Ataque Isquémico Transitorio/complicaciones , Masculino , Necrosis , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
The electrophoretic pattern of laddered DNA fragments which has been observed after cerebral ischemia is considered to indicate that neurons are dying by apoptosis. Herein the authors directly demonstrate using ligation-mediated polymerase chain reaction methods that 99% of the DNA fragments produced after either global or focal ischemia in adult rats, or produced after hypoxia-ischemia in neonatal rats, have staggered ends with a 3' recess of approximately 8 to 10 nucleotides. This is in contrast to archetypal apoptosis in which the DNA fragments are blunt ended as seen during developmental programmed cell death in dying cortical neurons, neuroblastoma, or thymic lymphocytes. It is not simply ischemia that results in staggered ends in DNA fragments because ischemic myocardium is similar to archetypal apoptosis with a vast majority of blunt-ended fragments. It is concluded that the endonucleases that produce this staggered fragmentation of the DNA backbone in ischemic brain must be different than those of classic or type I apoptosis.
Asunto(s)
Fragmentación del ADN , Hipertensión/genética , Isquemia Miocárdica/genética , Animales , Apoptosis/fisiología , Células Cultivadas , Hipertensión/patología , Masculino , Isquemia Miocárdica/patología , Neuronas/patología , Reacción en Cadena de la Polimerasa , Ratas , Ratas Endogámicas SHR , Ratas Sprague-DawleyRESUMEN
The time course of appearance of cells with DNA damage was studied in rats following transient severe forebrain ischemia. This DNA damage could be detected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day 1 in the striatum and later in the CA1 region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered apoptotic type of internucleosomal cleavage, which persisted without smearing for up to 7 days of reperfusion. In contrast, the DNA breaks following ischemia induced by decapitation were random and, after gel electrophoresis, consisted of smeared fragments of multiple sizes. There was some early regional cellular death, restricted to the dentate of the hippocampus, prior to the pannecrotic degeneration. It is concluded that transient forebrain ischemia leads to a type of neuronal destruction that is not random necrosis but that shares some component of the apoptotic cell death pathway.
Asunto(s)
Daño del ADN , Ataque Isquémico Transitorio/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Estado de Descerebración , Proteína Ácida Fibrilar de la Glía/metabolismo , Ataque Isquémico Transitorio/patología , Masculino , Necrosis , Prosencéfalo/irrigación sanguínea , Ratas , Factores de TiempoRESUMEN
The negative regulator of p53 transactivation, Mdm2, increased in the ischemic territory after 90 minutes of transient middle cerebral artery occlusion in spontaneously hypertensive rats compared to sham controls. Increased mdm2 mRNA was detected by semiquantitative reverse transcriptase polymerase chain reaction by 6 hours of reperfusion in the ipsilateral hemisphere. In situ hybridization histochemistry was used to localize increases in mdm2 mRNA which occurred in neurons of ischemic cortex and dorsolateral striatum. The number of labeled neurons increased by approximately 20-fold and the cells displayed five-fold increases of mdm2 mRNA in the cortex. Immunohistochemical staining for Mdm2 revealed that its mRNA was efficiently translated in the ischemic cortex, but not striatum, by 8 to 24 hours of reperfusion. Western blotting confirmed 30- to 40-fold increases in the full-length protein of 90 kd at these time points without evidence of alternative splicing. Because Mdm2 is a negative regulator of the apoptosis promoting activity of p53, increased expression of Mdm2 may be a component of a repair response in injured neurons, and supports Mdm2 being an indicator of DNA damage in the brain early after an ischemic insult in a similar way to Gadd45.
Asunto(s)
Encéfalo/metabolismo , Expresión Génica , Ataque Isquémico Transitorio/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas/genética , Animales , Western Blotting , Arterias Cerebrales , Corteza Cerebral/metabolismo , Constricción , Técnicas de Inmunoadsorción , Hibridación in Situ , Cinética , Masculino , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-mdm2 , ARN Mensajero , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , ReperfusiónRESUMEN
Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.
Asunto(s)
Fragmentación del ADN , Hiperglucemia/patología , Ataque Isquémico Transitorio/patología , Animales , Apoptosis , Caspasa 3 , Caspasas/metabolismo , Cuerpo Estriado/patología , Grupo Citocromo c/metabolismo , Giro Dentado/patología , Activación Enzimática , Hipocampo/patología , Hiperglucemia/fisiopatología , Etiquetado Corte-Fin in Situ , Ataque Isquémico Transitorio/fisiopatología , Masculino , Microscopía Electrónica , Neocórtex/patología , Neuronas/patología , Ratas , Ratas Wistar , Tálamo/patologíaRESUMEN
The tryptophan analog, 5-hydroxytryptophan (5HW), has a significant absorbance between 310-320 nm, which allows it to act as an exclusive fluorescence probe in protein mixtures containing a large number of tryptophan residues. Here for the first time a method is reported for the biosynthetic incorporation of 5HW into an expressed protein, the Y57W mutant of the Ca2+ binding protein, oncomodulin. Fluorescence anisotropy and time-resolved fluorescence decay measurements of the interaction between anti-oncomodulin antibodies and the 5HW-incorporated oncomodulin conveniently provide evidence of complex formation and epitope identification that could not be obtained with the natural amino acid. This report demonstrates the significant potential for the use of 5HW as an intrinsic probe in the study of structure and dynamics of protein-protein interactions.
Asunto(s)
5-Hidroxitriptófano/metabolismo , Proteínas de Unión al Calcio/metabolismo , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Anisotropía , Proteínas de Unión al Calcio/inmunología , Epítopos , Sustancias Macromoleculares , Proteínas Recombinantes/biosíntesis , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Several synthetic peptides, modelled from a Ca(2+)-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb(3+)-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.
Asunto(s)
Proteínas de Unión al Calcio/química , Fragmentos de Péptidos/química , Terbio/metabolismo , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/síntesis química , Proteínas de Unión al Calcio/metabolismo , Yodoacetamida/análogos & derivados , Luminiscencia , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , SalicilatosRESUMEN
Using the rat hepatoma calcium-binding protein, oncomodulin, from Morris hepatoma 5123tc, an antiserum has been raised in rabbits useful for immunostaining of this tumor protein. The peroxidase-antiperoxidase (PAP) technique has been used to demonstrate oncomodulin in sections of Morris rat hepatomas 5123D, 5123tc, 7288Ctc, and 7777 fixed with Bouin's or Carnoy's fixatives, or using freeze substitution. Oncomodulin-specific staining was also shown by a hepatoma metastasis in lung. Optimal conditions for the indirect fluorescent antibody technique were established to demonstrate oncomodulin in virally transformed NRK cells (ASV-NRK). In both tumors and cultured neoplastic cells staining appeared which suggested that oncomodulin might occur in nucleus and cytoplasm. Normal untransformed tissues and uninfected cells did not show oncomodulin staining.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Neoplasias Hepáticas Experimentales/patología , Proteínas de Neoplasias/análisis , Animales , Línea Celular , Técnica del Anticuerpo Fluorescente , Técnicas para Inmunoenzimas , Neoplasias Hepáticas Experimentales/análisis , Radioinmunoensayo , Ratas , Ratas Endogámicas BUFRESUMEN
An antisense RNA probe complementary to rat oncomodulin mRNA has been prepared by run-off transcription. Blots of RNA isolated from tumors arising from both a chemically-induced hepatoma or virally transformed sarcomas, when probed with this antisense RNA, identified a common single hybridizable RNA of approx. 750 nucleotides, corresponding in size to that found in blots of RNA from rat placenta. Quantitative densitometry of dot blot autoradiographs allowed for relative measurements of oncomodulin mRNA in the various tumors. The increased sensitivity of detection afforded by the antisense probe permitted the measurement of oncomodulin mRNA in the developing placenta, which was not possible using DNA probes. In both tumors and placenta, a plot of mRNA versus protein revealed a direct relationship suggesting transcriptional control of oncomodulin abundance.
Asunto(s)
Proteínas de Unión al Calcio/genética , Neoplasias Experimentales/análisis , Placenta/análisis , ARN Mensajero/análisis , Animales , Femenino , Embarazo , RatasRESUMEN
Oncomodulin, first found in tumours, turns out to be a highly conserved oncodevelopmental protein in human and rodent placentas. The human and rat placental oncomodulins were visualised immunohistochemically. The placental oncomodulins are identical to each other, and to tumour oncomodulin with respect to amino acid composition, chromatographic elution and the pattern of the peptides released by trypsin action.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Placenta/análisis , Aminoácidos/análisis , Animales , Femenino , Humanos , Embarazo , Ratas , Ratas EndogámicasRESUMEN
Oncomodulin is a parvalbumin-like calcium binding protein of Mr 11,700 from rodent tumours. An antiserum to rat oncomodulin cross-reacts with extracts of several human solid tumour tissues. When purified, this human immunoreactive protein binds calcium, and has a molecular weight and amino acid composition similar to the rodent protein. This protein was not found in normal adult human tissue.
Asunto(s)
Proteínas de Unión al Calcio/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Neoplasias/análisis , Anciano , Animales , Humanos , Neoplasias Pulmonares/análisis , Masculino , Ratones , Persona de Mediana Edad , Peso MolecularRESUMEN
Oncomodulin was detected by immunocytochemical means in human placenta and found to occur in the cytotrophoblastic shell during implantation, in Langhans type villar cytotrophoblastic cells, and in extravillar cytotrophoblast or intermediate trophoblast. The presence of oncomodulin during first and early second trimester was confirmed by immunoradiometric assay (IRMA). In term placenta oncomodulin appeared in intermediate trophoblast cells, and was largely absent from the villi which lack cytotrophoblast. Furthermore, oncomodulin was abundant in the choriocarcinoma cell line BeWo, and detectable in JAR but not JEG-3. The function of this oncodevelopmental calcium-binding protein in normal development or in neoplasia is not known.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Coriocarcinoma/análisis , Placenta/análisis , Neoplasias Uterinas/análisis , Amnios/análisis , Línea Celular , Femenino , Edad Gestacional , Humanos , Inmunohistoquímica , EmbarazoRESUMEN
The mRNA for the oncodevelopmental calcium-binding protein oncomodulin (MW 11,700) has been detected in tissues of the rat conceptus by in situ hybridization using biotinylated RNA probes. Oncomodulin mRNA was detected in the basal zone and labyrinth of rat placenta, following a similar distribution to that shown for oncomodulin by immunohistochemistry. Oncomodulin mRNA was also detected in rat ectoplacental cone at ten days and in amnion and PYS, but not VYS from 11 days onward. Previously oncomodulin was not detected embryonically from day 14 to birth, but in the present study of oncomodulin mRNA and protein, both were detected in implantation stages from blastula through egg cylinder. Staining was also present on decidual tissue. The suggestion is made that the oncomodulin gene is initially active in all cell types, but later its activity is confined to extraembryonic tissues.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Implantación del Embrión , Proteínas de Neoplasias/análisis , Placentación , ARN Mensajero/análisis , Trofoblastos/análisis , Animales , Femenino , Embarazo , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
Removal of the thyroid in normocalcemic rats with functional parathyroid transplants was found to reduce the hepatocyte DNA synthetic activity which normally follows partial hepatectomy. This proliferative incapacitation of hepatocytes appeared to be due specifically to a calcitonin deficiency since it was overcome by a single injection of pure synthetic salmon calcitonin shortly after partial hepatectomy. Salmon calcitonin and bovine parathyroid hormone were equally able to reverse the similar proliferative incapacitation of hepatocytes in hypocalcemic rats which had both their parathyroid and thyroid glands removed one day before partial hepatectomy. However, these two hormones (individually or together) could not reverse the proliferative incapacity resulting from a more prolonged (3-day) exposure to the hypocalcemic conditions in thyroparathyroidectomized rats, but the proliferative incapacity could be reversed by simultaneous treatment with the vitamin D3 metabolite, 1 alpha,25-dihydroxycholecalciferol. We suggest that extracellular calcium ions are the actual regulators of this hormonally-controlled hepatocyte proliferative development and that parathyroid hormone and the vitamin D3 metabolite affect proliferation indirectly by determining the extracellular calcium concentration, while calcitonin directly, or indirectly, sensitizes hepatocytes to the action of calcium.