RESUMEN
The activity of hexokinase was studied in several normal and malignant human tissues. The enzyme activity in tumors was significantly higher. Isoenzyme studies on normal gastric mucosa and stomach cancer extracts showed that malignancy is accompanied by a "simplification" of the hexokinase isoenzyme pattern due to "deletion" of the slowest isoenzyme. Preparative polyacrylamide gel electrophoreis was used to isolate hexokinase isoenzymes from normal and malignant tissues. Tumor hexokinase isoenzymes displayed an increased affinity to glucose when compared to the corresponding normal prototypes (Km/glucose, 10(-6) M and 10(-5) M, respectively; Km = Michaelis constant). The molecular weights, subunit composition, and peptide patterns were identical for corresponding isoenzyme pairs from normal and tumor tissues.
Asunto(s)
Mucosa Gástrica/enzimología , Hexoquinasa/metabolismo , Isoenzimas/metabolismo , Neoplasias Gástricas/enzimología , Femenino , Glucosa/metabolismo , Hexoquinasa/aislamiento & purificación , Humanos , Isoenzimas/aislamiento & purificación , Cinética , Masculino , Peso Molecular , Péptidos/aislamiento & purificación , Conformación ProteicaRESUMEN
The distribution of ceruloplasmin-coding sequences among the fragments of rat nuclear DNA obtained after the complete digestion with seven restriction endonucleases (EcoRI, BamHI, BspI, HindIII, KpnI, BglII and XhoI) was studied using highly specific cDNA probes. Although only a single copy of this gene per rat haploid genome was detected in DNA-cDNA hybridization in solution, the number of restriction fragments carrying the sequences of ceruloplasmin (CP) gene varied from two to five, depending upon the enzyme used, and their total length was several times higher than the minimal length of CP-coding gene, as deduced from the size of mRNA (2.3 Md for double-stranded DNA). The partial double stranded DNA transcript of ceruloplasmin mRNA coding for about 70% of its length (from 3'-end) does not contain recognition sites for some restriction endonucleases generating multiple fragments of CP gene in cellular DNA. These data are consistent with a split pattern of CP gene which seems to consist of several exons and introns. The partial protection from S1 nuclease of discrete fragments of full-length cDNA after annealing with high molecular weight nuclear RNA is consistent with this assumption and seems to be an indication that exons and introns are joined into a functional unit coding for high mol wt. CP pre-mRNA.
Asunto(s)
Ceruloplasmina/genética , ADN/genética , Genes , Animales , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , ARN Mensajero/genética , Ratas , Transcripción GenéticaRESUMEN
Highly purified ceruloplasmin (CP) was isolated from human bile using affinity chromatography. Biliary CP is represented by two molecular species. One of those is identical to oxidase CP from normal human serum while the other is analogous to oxidase-lacking CP specific for the serum of the carriers of Wilson's mutation with respect to immunological specificity, electrophoretical mobility and molecular mass of the large fragments from spontaneous proteolysis.
Asunto(s)
Bilis/química , Ceruloplasmina/metabolismo , Ceruloplasmina/química , Ceruloplasmina/aislamiento & purificación , Cromatografía de Afinidad , Cobre/metabolismo , Electroforesis , Degeneración Hepatolenticular/sangre , Humanos , ImmunoblottingRESUMEN
Effects of fructoso-1,6-diphosphate (FDP) and cyclic 3',5'-adenosine monophosphate (cAMP) on various steps of protein biosynthesis in isolated rat liver mitochondria were investigated. It was shown that FDP repressed and cAMP depressed the incorporation of both 14C-amino acid and [3H]uridine into mitochondrial polysomes. Cyclic 2',3'-adenosine monophosphate, a physiologically inactive analog of cAMP, had no depressing effect on the polysomes formation in mitochondria. Effects of FDP and cAMP on the synthesis of mitochondrial RNA at different periods of incubation (5, 10, 30 min) were studied. It was found that FDP repressed the high molecular weight mitochondrial RNA biosynthesis and prevented the mRNA formation. cAMP derepressed the FDP effect. Rifampicin prevented the derepressing action of cAMP. The rate of protein synthesis in the translation system isolated from mitochondria was affected neither by FDP nor by cAMP. Authors concluded that in the mammalian mitochondria the repression of protein synthesis by a glycolytic metabolite (FDP) and its derepression by cAMP represented regulatory mechanism acting at the transcription level like catabolite repression-derepression in microorganisms.
Asunto(s)
AMP Cíclico/farmacología , Fructosafosfatos/farmacología , Mitocondrias Hepáticas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Animales , Hexosadifosfatos/farmacología , Cinética , Mitocondrias Hepáticas/efectos de los fármacos , Peso Molecular , Polirribosomas/efectos de los fármacos , Polirribosomas/metabolismo , ARN/biosíntesis , RatasRESUMEN
Highly purified preparations of mRNA coding for ceruloplasmin (CP) ere isolated from rat liver polyribosomes using indirect immunoprecipitation of CP polysomes and poly(U)-sepharose chromatography of polysomal RNA. The homogeneity of CP mRNA was as high as 86--90%. The molecular weight of CP mRNA is 1.3 . 10(6) daltons which is in excess when compared to the minimal size of mRNA necessary to code for CP precursor (about 700 amino acid residues). The base composition of CP mRNA is of AU-type. The experiments on end-labeling with [3H]borohydride after periodate oxidation whowed that CP mRNA contains 3'-terminal poly(A). Poly(U)-sepharose chromatography with stepwise temperature elution revealed length heterogeneity of poly(A) consisting of particular, different thermal subfractions of CP mRNA contain poly(A) consisting of 38, 90 and 165 adenylate residue. 5'-end of CP mRNA is block with inverted 7-methylguanosine (m7G) which is reducible with [3H]borohydride after periodate oxidation. This m7G residue is a component of RNAse- and alkali-resistant oligonucleotide, which structure according to net charge value and its shifts after various enzymatic treatments, is m7G5'ppp5'XmpAp.
Asunto(s)
Ceruloplasmina/biosíntesis , Hígado/metabolismo , ARN Mensajero , Animales , Composición de Base , Cinética , Peso Molecular , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Ratas , Ribonucleasas , Ribonucleótidos/análisisRESUMEN
Mapping of ceruloplasmin gene in human and mouse chromosomes was carried out using the cloned fragments of rat chromosomal ceruloplasmin gene and of ceruloplasmin cDNA as specific hybridization probes. DNA probes were nick-translated with 125I-dCTP up to the high specific capacity. The number of silver grains as well as their distribution along the differentially stained chromosomes were analyzed in 120 metaphase plates from bone marrow cells of laboratory mice and in 181 plates from human lymphocyte cultures. The most probable localization of human ceruloplasmin gene is centromeric region q11-13 of chromosome 15(14?). In laboratory mice ceruloplasmin gene is assigned to the euchromatic part of D-disc of chromosome 9. The possible causes for gene synteny in laboratory mouse and in man as well as its evolutionary implication are discussed.
Asunto(s)
Ceruloplasmina/genética , Mapeo Cromosómico , Cromosomas Humanos 13-15/ultraestructura , Cromosomas/ultraestructura , ADN/genética , Hibridación de Ácido Nucleico , Animales , Autorradiografía , Células Cultivadas , Bandeo Cromosómico , Técnicas Genéticas , Humanos , Metafase , Ratones , Ratas , Translocación GenéticaRESUMEN
Specific DNA-probes representing the fragments of chromosomal ceruplasmin gene (lambda RCp-1, lambda RCp-2, lambda RCp-3) and its cDNA copy of the corresponding mRNA were heavily labelled with 125J dCTP (the specific activity of the probes varying from 1.5 X 10(7) to 3.4 X 10(8) dpm). These probes were used for in situ hybridization on metaphase chromosomes. The total number of silver grains and their distribution along differentially stained chromosomes were determined in 653 metaphase plates from bone marrow cells of laboratory rats. The results of in situ hybridization were very similar for all four specific DNA-probes tested and allow to assign ceruplasmin gene to the q13 region of chromosome 7. The local increase of silver grain count over chromosome 15 was also registered and discussed.
Asunto(s)
Ceruloplasmina/genética , Mapeo Cromosómico , ADN/genética , Genes , Hibridación de Ácido Nucleico , Animales , Médula Ósea/ultraestructura , Bandeo Cromosómico , Clonación Molecular , Vectores Genéticos , Masculino , Metafase , Plásmidos , RatasRESUMEN
The relationship between differences in the clinical polymorphism of hepatolenticular degeneration (Wilson's disease) and characteristics of CP (ceruloplasmin) structural changes were investigated. The comparative study of Wilson's disease patients revealed two forms of clinical development of this disease which differ from each other by the expression of the visceral symptoms preceding the establishment of the typical neurological picture. The peptide map analysis of tryptic hydrolysates of the CP from individual patients has demonstrated the altered peptide patterns in five cases. Clinical and genetic heterogeneity of Wilson's disease is discussed.
Asunto(s)
Ceruloplasmina/genética , Degeneración Hepatolenticular/genética , Adolescente , Adulto , Catálisis , Fenómenos Químicos , Química , Niño , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Masculino , Dispersión Óptica Rotatoria , Péptidos , Polimorfismo Genético , Conformación Proteica , TripsinaRESUMEN
A specific novel molecular form of ceruloplasmin (CP) was detected in the sera of Wilson's disease patients and their closest relatives using two-dimensional cross-immunoelectrophoresis. This protein shares some antigenic properties with normal CP but is not completely identical to the latter. Besides, anomalous CP has no oxidase activity of normal CP and differs from it in electrophoretic mobility in agarose and polyacrylamide gels. Anomalous CP was purified to homogeneity and monospecific antibodies to this protein were obtained. The quantitative analysis showed that the ratios of normal CP to anomalous CP in homo- and heterozygous carriers of the "Wilsonian" gene are reproducibly different and can be used as a diagnostic test allowing the differentiation between these groups.
Asunto(s)
Ceruloplasmina/análisis , Tamización de Portadores Genéticos , Degeneración Hepatolenticular/genética , Adolescente , Adulto , Anciano , Ceruloplasmina/inmunología , Niño , Femenino , Degeneración Hepatolenticular/sangre , Degeneración Hepatolenticular/diagnóstico , Humanos , Inmunoelectroforesis Bidimensional , Masculino , Persona de Mediana Edad , Estructura MolecularRESUMEN
Hexokinase of the endometrium and gastric mucosa is represented by 5 isoenzymes. The "simplification" of HK isoenzyme spectrum is characteristic of cancer tissue. So, in gastric cancer there is a disappearance of the "slowest" isoenzyme, while in malignant endometrium the "fastest" one was absent. Hexokinase isoenzymes of the serum were identical to those in the tumors in question, that indicates the tumor origin of the body fluid hexokinase. The latter was not observed in normal body fluids. The isoenzymic composition of hexokinase in uterine fibromyoma did not differ from that in normal tissues. If hexokinase appeared in the serum of these patients, its isoenzymic composition was similar to that in the normal uterus. The study on the hexokinase isoenzyme composition may be a valuable adjunct in establishing the differential diagnosis between benign and malignant tumors.
Asunto(s)
Líquidos Corporales/enzimología , Hexoquinasa/metabolismo , Neoplasias/diagnóstico , Pruebas Enzimáticas Clínicas , Diagnóstico Diferencial , Activación Enzimática , Femenino , Humanos , Isoenzimas/metabolismo , Leiomioma/diagnóstico , Neoplasias Gástricas/diagnóstico , Neoplasias Uterinas/diagnósticoAsunto(s)
Núcleo Celular/metabolismo , Mitocondrias Hepáticas/metabolismo , Biosíntesis de Proteínas , ARN/metabolismo , Aerobiosis , Animales , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Cicloheximida/farmacología , Dactinomicina/farmacología , Técnicas In Vitro , Membranas/metabolismo , Ratas , Ribonucleasas/metabolismo , Moldes GenéticosAsunto(s)
Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Animales , Sitios de Unión , Sitios de Unión de Anticuerpos , Centrifugación por Gradiente de Densidad , Citoplasma/metabolismo , Digitonina , Inmunoglobulina G , Radioisótopos de Yodo , Hígado/citología , Membranas/metabolismo , Unión Proteica , Conejos/inmunología , Ratas , Receptores de Droga , Solubilidad , Espectrofotometría Ultravioleta , Fracciones Subcelulares/metabolismo , TensoactivosAsunto(s)
Cromosomas , Mitocondrias Hepáticas/efectos de los fármacos , Biosíntesis de Proteínas , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , AMP Cíclico/farmacología , Fructosafosfatos/farmacología , Glucosa/farmacología , Técnicas In Vitro , Oligomicinas/farmacología , Fosforilación Oxidativa/efectos de los fármacos , RatasAsunto(s)
Ceruloplasmina/metabolismo , Cobre/metabolismo , Degeneración Hepatolenticular/metabolismo , Adolescente , Adulto , Niño , Cobre/sangre , Electroencefalografía , Femenino , Degeneración Hepatolenticular/sangre , Degeneración Hepatolenticular/genética , Heterocigoto , Humanos , Hígado/metabolismo , Masculino , RadioisótoposAsunto(s)
Líquido Ascítico/enzimología , Jugo Gástrico/enzimología , Enfermedades Hematológicas/enzimología , Hexoquinasa/análisis , Neoplasias/enzimología , Adulto , Niño , Pruebas Enzimáticas Clínicas , Femenino , Neoplasias Gastrointestinales/enzimología , Humanos , Neoplasias Gástricas/enzimología , Neoplasias Urogenitales/enzimologíaRESUMEN
It is well known that mitochondria are only partly an autonomous system since they are subjected to nuclear control. For this reason, in studying mitochondrial genes one has to consider constantly the integration of mitochondrial and nuclear genetic systems. This fact makes experimental approaches still more sophisticated, especially, when one turns from individual genetic structures to mitochondrial heredity on the level of cells and multicellular organisms. Here we shall discuss some theoretical aspects of mitochondrial heredity that have been comparatively rarely dealt with in the literature.
Asunto(s)
ADN Mitocondrial/fisiología , Genes , Animales , Mitocondrias/fisiología , Mutación , Especificidad de la Especie , Replicación ViralRESUMEN
The review deals with the analysis of the molecular basis of cytoplasmic genetic determinants and their contribution to inherited disease, as well as with the primary genetic defects underlying some single gene human disorders, such as hepatolenticular degeneration, alpha 1-antitrypsin deficiency, familial hypercholesterolemia, and cystic fibrosis. The results of molecular genetic studies of inherited diseases have been applied to the antenatal and preclinical diagnosis at the levels of mutant genes and anomalous proteins, gene products.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Fibrosis Quística/diagnóstico , Fibrosis Quística/genética , Femenino , Expresión Génica , Genes Recesivos , Tamización de Portadores Genéticos , Degeneración Hepatolenticular/diagnóstico , Degeneración Hepatolenticular/genética , Humanos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Masculino , Biología Molecular , Embarazo , Diagnóstico Prenatal , Investigación , Deficiencia de alfa 1-AntitripsinaRESUMEN
A mutation for multiple resistance to tetracycline, cycloheximide and oligomycin appears to be followed by reconstruction of the mitochondrial genome resulting in the formation of independent nucleotide sequences that determine different resistant phenotypes. Heterozygotes for the cross resistance factor lack locus T responsible for relation tetracycline which comes from the alpha-parent. The nuclear recessive gene-suppresor i induces deletion of the whole determinant for multiple resistance. The loss of mt-DNA on ethidium bromide treatment does not lead to the loss of this determinant which remains in the cells either in an active or in a passive state.