RESUMO
The question of whether distribution of chromatids to daughter cells in mitosis is a random or nonrandom process was investigated by study of the distribution of labeled chromatin in anaphase pairs at M(1) and M(2) after a pulse of tritiated thymidine. Diploid and tetraploid rat and diploid human fibroblast-like cells in serial monlayer culture were synchronized by two different methods to "purify" M(1) and M(2) anaphases: metaphase shake, and FUdR block to DNA synthesis followed by exogenous thymidine. Exposed grains of NTB-2 emulsion were counted over M(1) and M(2) anaphase pairs. An analysis (by pair) of diploid M(2) anaphase grain counts showed two discrete populations of daughters with less and with more radioactivity. A similar analysis of diploid M(1) and tetrapolid M(2) anaphases showed a single grain-count distribution. These findings may support a nonrandom model of chromatid segregation for diploid mammalian cells but do not rule out random segregation until sound mathematical models are formulated for expected random grain distributions in M(2) anaphases of cells with differing numbers of chromosomes.
Assuntos
Cromossomos/análise , Técnicas de Cultura , Mitose , Ácidos Nucleicos/análise , Análise de Variância , Animais , Autorradiografia , Contagem de Células , Linhagem Celular , Cromátides/metabolismo , Cromossomos/metabolismo , Meios de Cultura , DNA/antagonistas & inibidores , Replicação do DNA , Diploide , Fibroblastos , Histocitoquímica , Humanos , Métodos , Modelos Biológicos , Ratos , Timidina/metabolismo , Trítio , Uridina/metabolismoRESUMO
Replication and the performance of a differentiated function have been considered antagonistic processes. When cells in culture are partially tially synchronized with 5-fluoro-2'-deoxyuridine (FUdR), the synthesis of the specialized protein (collagen) is not reduced during chromosomal replication (S period). Collagen synthesis varies with general protein synthesis through the S period.
RESUMO
Decreased function of human mitochondrial branched chain alpha-ketoacid dehydrogenase complex results in branched chain ketoacidemia or maple syrup urine disease. Activity of this multienzyme complex varies from 0 to approximately 15% of wild type branched chain alpha-ketoacid dehydrogenase complex activity within the population of homozygous affected individuals. We used the technique of Western Blotting with antibodies against purified bovine liver branched chain alpha-ketoacid dehydrogenase complex to screen mitochondrial proteins from cultured human fibroblasts for immunocrossreactive proteins. This method probes the physical structure of the proteins forming this multienzyme complex. One patient with branched chain ketoacidemia lacked an immunoreactive transacylase protein. This protein catalyzes the transfer of the branched chain acyl group from the decarboxylase to reduced coenzyme A. Kinetic analysis of the enzyme activity in cell lysates from this patient confirmed that the complex would not utilize coenzyme A. Thus, we have defined a structural basis for an impaired multienzyme complex of mitochondria in man.
Assuntos
Cetona Oxirredutases/deficiência , Doença da Urina de Xarope de Bordo/etiologia , Complexos Multienzimáticos/deficiência , 3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida) , Animais , Bovinos , Linhagem Celular , Fibroblastos/enzimologia , Humanos , Cetona Oxirredutases/isolamento & purificação , Fígado/enzimologia , Doença da Urina de Xarope de Bordo/enzimologia , Doença da Urina de Xarope de Bordo/genética , Mitocôndrias/enzimologia , Peso Molecular , Complexos Multienzimáticos/isolamento & purificaçãoRESUMO
A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.
Assuntos
Colágeno/síntese química , Oxigenases de Função Mista/metabolismo , Fragmentos de Peptídeos/síntese química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Células Cultivadas , Cromatografia/métodos , Cromatografia em Camada Fina , Colágeno/metabolismo , Síndrome de Ehlers-Danlos/enzimologia , Fibroblastos/enzimologia , Humanos , Marcação por Isótopo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pele/enzimologia , TrítioRESUMO
It has been proposed that benign nevi that fail to differentiate normally may undergo stepwise growth and morphologic changes resulting in progression toward dysplastic nevi, which in some cases progress into malignant melanoma. In this study, we sought to determine the relationship between production of endogenous growth factors and the appearance of chromosomal abnormalities in cultured nevi and melanomas. Newly established cultures from 8 nevi with benign histology and 6 malignant melanomas, and 2 malignant melanoma cell lines were studied. Assays for mitogenic growth factors were based on stimulation of [3H]thymidine incorporation into DNA in Hs0294 malignant melanoma cells, produced by serum-free conditioned medium from nevus or melanoma cultures. Karyotypes were examined in cultures of an equivalent passage. Three of the 8 nevus cultures were mitogen-negative and displayed normal karyotypes; one nevus culture was mitogen-positive and had a normal karyotype, although the biopsied tissue demonstrated histologic evidence of benign melanocytic proliferation; one was mitogen-negative initially, but had an extra chromosome 8 in 2 of 50 cells; 3 were mitogen-positive and chromosomally abnormal. Each of the cultures in this latter group exhibited reciprocal translocation (rcpt) as the only identifiable abnormality [rcpt(6;15), rcpt(10;15), rcpt(15;20)], or a constitutional rcpt(4;5). Thus, there was direct correlation between growth factor production and chromosome abnormality in 6 of 8 benign nevus cultures. In the newly established melanoma cultures there was also concordance between growth factor and chromosomal status; conditioned media from 4 of 6 were mitogen-positive by at least one assay, and all 4 of the mitogen-positive cultures had chromosomally abnormal cell populations. Of the 2 melanoma cultures negative for growth factors, one was also negative for chromosome abnormality; the other had chromosomal change consisting of increased polyploidy. Both melanoma cell lines had abnormal karyotypes and were mitogen-positive. Though numerous chromosome changes were noted in the karyotypically abnormal melanoma cells, 6 of the 8 cultures exhibited abnormalities in chromosomes 1, 6, and/or 7. These data suggest that steps in the progression from benign nevi toward dysplastic nevi or malignant melanoma include: proliferation resulting from altered production of endogenous mitogenic growth factors; and development of specific chromosomal abnormalities.
Assuntos
Aberrações Cromossômicas , Substâncias de Crescimento/biossíntese , Melanoma/genética , Nevo Pigmentado/genética , Neoplasias Cutâneas/genética , Células Cultivadas , Humanos , Melanoma/metabolismo , Melanoma/patologia , Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
A fetus with an open, noncystic myelomeningocele was detected at the 22nd week of gestation in a woman monitored for advanced maternal age. The lesion could not be demonstrated on amniography. In 13% of published reports amniography failed to detect significant spina bifida lesions. This false-negative rate seems related to the noncystic nature of some neural tube defects at midgestation.
Assuntos
Diagnóstico Pré-Natal , Espinha Bífida Oculta/diagnóstico , Líquido Amniótico/metabolismo , Diagnóstico Diferencial , Reações Falso-Negativas , Feminino , Fetoscopia , Humanos , Gravidez , alfa-Fetoproteínas/metabolismoRESUMO
A liveborn girl with 46,XX/47,XX+4 mosaicism is reported for the first time. The diagnosis of true mosaicism was established initially in the assay of cultured amniotic fluid cells, although fetal blood obtained by percutaneous umbilical blood sampling showed a 46,XX chromosome constitution. The liveborn infant had manifestations previously reported in dup(4p) and dup(4q) syndromes. Cells in cord and arterial blood samples also were 46,XX, but cultures of placenta and bilateral forearm skin biopsies showed 46,XX/47,XX,+4 mosaicism. This case illustrates the disadvantage of chromosome analysis from blood alone when tissue-specific mosaicism is present.
Assuntos
Cromossomos Humanos Par 4 , Mosaicismo/genética , Trissomia , Amniocentese , Feminino , Humanos , Lactente , Cariotipagem , GravidezRESUMO
We ascertained +i(12p) mosaicism during third trimester in a case of polyhydramnios and diaphragmatic hernia. Primary cultures of amniocytes had colonies with +i(12p), colonies without +i(12p), and mixed colonies with 46/47,+i(12p). The likely explanation was instability and loss of i(12p) during somatic divisions of amniocytes. Fetal blood in third trimester retained +i(12p) in 13% of cells. A review of mosaicism in published cases indicates that factors influencing the presence of +i(12p) include tissue type and in vitro and in vivo age. In blood, amniocyte, and probably bone marrow cultures, +i(12p) is less stable than in fibroblast-like cultures derived from skin and other tissues. Young cultures at early passage are more likely to have +i(12p) than old cultures. Cultures from young (especially fetal) donors are more likely to retain +i(12p) than cultures from adult donors. These rules will be important in determining appropriate tissues for diagnosis and interpretation of mosaicism in this disorder.
Assuntos
Aberrações Cromossômicas/genética , Cromossomos Humanos Par 12 , Linfócitos/patologia , Mosaicismo , Ultrassonografia Pré-Natal , Adulto , Líquido Amniótico , Células Cultivadas , Aberrações Cromossômicas/diagnóstico por imagem , Transtornos Cromossômicos , Feminino , Humanos , Linfócitos/metabolismo , Gravidez , Terceiro Trimestre da Gravidez , SíndromeRESUMO
In this study we used densitometry to evaluate DNA replication kinetics in a rearranged chromosome formed by the joining of two X chromosomes at region p22. No 45X mosaicism is present in peripheral blood or fibroblast cultures. The patient has primary amenorrhea, short stature, and gonadal dysgenesis. The sequence of replication in the majority of cells is p11, q11, q13, q22-24, q12, p22, q26, q28, q27, q25, and p21, q21. Thus p11 is the earliest region to replicate, and q21 is the last. In 66% of 127 cells analyzed, the replication pattern is asymmetric, and bands q12, q26, and q28 are most likely to be out of phase on the two sides of the breakpoint. We find that band p22 has a delay of replication compared to an abnormal X derived from two X chromosomes joined at the q23 region previously reported by us. Structural rearrangement may therefore delay replication in the region of the break.
Assuntos
Replicação do DNA , Aberrações dos Cromossomos Sexuais/genética , Cromossomos Sexuais/ultraestrutura , Cromossomo X/ultraestrutura , Adulto , Bandeamento Cromossômico , Densitometria , Mecanismo Genético de Compensação de Dose , Feminino , Disgenesia Gonadal/genética , Humanos , Cariotipagem , Cinética , Mosaicismo , Cromatina Sexual/análiseRESUMO
A new ELISA was used to measure H-Y antigen in cultured fibroblasts from a male with 46,X,dic(Y)(q11.2101) involving duplication of Yp. Aliquots of monoclonal H-Y antibody were absorbed with 1, 2, 4, or 8 X 10(6) cells from the 46,X,dic(Y) male, or with corresponding numbers of cells from a normal XY male and a normal XX female, and then were tested for residual activity against a soluble antigen source. Portions of the antibody absorbed with cells of the 46,X,dic(Y) male were found to be less reactive than portions absorbed with cells of the normal XY male, for all numbers of cells and both dilutions of plated antigen. The results, quantified in an electronic optical density reader, imply presence of excessive H-Y in cells of the 46,X,dic(Y) male, and suggest presence of a genetic determinant of H-Y on Yp or proximal Yq near the centromere.
Assuntos
Ensaio de Imunoadsorção Enzimática , Antígeno H-Y/análise , Aberrações dos Cromossomos Sexuais/genética , Cromossomo Y , Anticorpos Monoclonais , Bandeamento Cromossômico , Ensaio de Imunoadsorção Enzimática/métodos , Fibroblastos , Humanos , Lactente , MasculinoRESUMO
We report on a familial t(4;7)(q28;p22) with 2:2 adjacent-1 unbalanced segregation producing duplication of 4q28-->qter in multiple offspring. Within the large four-generation pedigree, a carrier had a reproductive outcome that was approximately equal for 1) the balanced translocation, 2) normal chromosomes, and 3) viable 4q trisomy or pregnancy loss. The three individuals with chromosomal confirmation of trisomy 4q28-->qter (comprising approximately 1.8% of the haploid autosomal length) had similar mental and developmental retardation, hypotonia, restricted speech, seizures, and facial anomalies but no cardiac, renal, or skeletal anomalies. It is suggested that these latter severe malformations, associated with the classic 4q2 to 3 group of anomalies, were from an imbalance outside 4q28-->qter and were not necessarily related to the relatively large size of the trisomic segment. Multiple different chromosomes are reported to be rearranged with 4q in the production of distal 4q trisomy. The incidence of 4q rearrangement remains unexplained, but once it is present in a family, viability of a large trisomy in 4q seems to explain the number of affected individuals reported.
Assuntos
Segregação de Cromossomos/genética , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 7/genética , Translocação Genética/genética , Anormalidades Múltiplas/genética , Adolescente , Adulto , Criança , Face/anormalidades , Feminino , Humanos , Lactente , Recém-Nascido , Cariotipagem , Masculino , Meiose , Linhagem , Fenótipo , Gravidez , Fatores de Risco , TrissomiaRESUMO
HLA typing was performed on 18 cultures of human amniotic fluid cells using cytotoxicity and absorption technics. Confirmation of antigen assignments was obtained in nine of ten instances, where HLA typing also was performed on cord blood. Three major problems were encountered in performing these studies: (1) complement cytotoxicity, (2) false-positive reactions, and (3) false-negative reactions. False-positive and false-negative reactions occurred more frequently with sera defining HLA-B locus specificities than with sera defining HLA-A locus specificities. Absorption studies were helpful in making antigen assignments when false reactions occurred. Preliminary studies suggest that the frequency of false-positive reactions can be decreased by absorbing HLA typing sera with antigen-negative amniotic fluid cultured cells, buffy coat, or platelets. Accurate antigen assignment is difficult when parental HLA types are unavailable.
Assuntos
Líquido Amniótico/citologia , Antígenos HLA/análise , Absorção , Líquido Amniótico/imunologia , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Reações Falso-Positivas , Feminino , Teste de Histocompatibilidade/métodos , Humanos , Masculino , GravidezRESUMO
We studied two unrelated individuals with Ehlers-Danlos syndrome type VI, which is characterized by congenital hypotonia, lax joints, severe kyphoscoliosis, friable skin, and hemorrhagic hypotrophic scars. The diagnosis was confirmed by decreased hydroxylysine residues in dermal collagen and decreased collagen lysyl hydroxylase activities in their cultured skin fibroblasts. Despite the diminished hydroxylysine residues in dermal collagen from the probands, we found no differences in hydroxylysyl residues of collagen synthesized by fibroblasts in culture. When patient 1 was given oral sodium ascorbate (5 g/d) for 3 weeks, ascorbate concentrations increased two-fold in plasma and 300-fold in urine. Urinary excretion of hydroxylysine and hydroxyproline increased during ascorbate administration. After a 1-year interval, bleeding time, wound healing, and muscle strength improved. Ascorbate supplementation (50 micrograms/mL) to confluent fibroblasts cultured from the two patients and controls increased hydroxyprolyl and hydroxylysyl residues of fibroblasts four to seven and three to four-fold respectively. Total protein associated with the cell layer increased 14% to 32% without concomitant change in cellular DNA. Total soluble collagenous material recovered from culture media increased 61% to 103% with ascorbate supplementation. These studies demonstrate that ascorbate improves the clinical status of patients with impaired collagen lysyl hydroxylase activity by enhancing lysyl and prolyl hydroxylation and total collagen production.
Assuntos
Ácido Ascórbico/farmacologia , Colágeno/biossíntese , Síndrome de Ehlers-Danlos/metabolismo , Aminoácidos/metabolismo , Criança , Pré-Escolar , DNA/metabolismo , Síndrome de Ehlers-Danlos/classificação , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/deficiência , Pele/metabolismoRESUMO
Cultures from metastatic melanomas of 15 patients had detailed melanoma growth stimulatory activity (MGSA) and cytogenetic analysis. The presence of melanoma cells was confirmed by microscopic identification of melanin, tyrosinase activity, and electron microscopy characterization of melanosomes. The MGSA is found in cytoplasmic granules after immunocytochemical stain. Three of the cultures did not produce MGSA and showed no distinctive cytogenetic differences. Breakpoints in derivative chromosomes were concentrated in region 1p1, and among all cultures chromosome 1 was the most frequently rearranged. It also has a low copy number of normal homologs. Chromosomes 18, X, and Y were never derivative, and chromosomes 2 and 4 were rarely so. Thus the cytogenetic data indicate that 4q13-21, the hybridization site for MGSA cDNA, is spared from gross change, although it could be under the influence of another site on chromosome 1 that is lost or rearranged. The ratio of abnormal to normal autosomes (mean per cell) in no culture exceeded 0.5, and for no autosome exceeded 0.8, suggesting a limit to the rearrangement tolerated for cell survival. If the Y is retained, the X:Y ratio varies around a normal figure of 1. The ratio of autosomes to sex chromosomes varies around a normal figure of 22. These data suggest stability of the X chromosome in cells undergoing multiple rearrangements of the autosomes.
Assuntos
Aberrações Cromossômicas , Substâncias de Crescimento/genética , Melanoma/genética , Anticorpos Monoclonais , Bandeamento Cromossômico , Feminino , Humanos , Imuno-Histoquímica , Cariotipagem , Masculino , Melanoma/enzimologia , Melanoma/patologia , Monofenol Mono-Oxigenase/biossíntese , Ploidias , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/patologiaRESUMO
OBJECTIVE: To assess factors influencing uptake of amniocentesis after a positive Down's syndrome screening result. METHODS: Interviews of 53 Montana women with screening risks > or = 1 in 300 after delivery. RESULTS: Thirty had accepted amniocentesis ("yes" group) and 23 had declined ("no" group) (57% uptake). Age at delivery was significantly higher (p = 0.02) for the "no" than the "yes" group (mean 35.3 nu 31.7 years). The mean risk of Down's syndrome ascertained by screening was 1 in 190 for the "no" group and 1 in 115 for the "yes" group (p = 0.05). Statistically significant differences (p < or = 0.05) between opinions in the two groups included: (a) desire to know if the fetus had Down's syndrome; (b) perception of the burden of care for an affected child; (c) support of doctor, spouse, and relatives for choice about amniocentesis; (d) attitudes toward abortion; (e) importance of religion; and (f) concerns about the amniocentesis procedure. The most important factor for those choosing amniocentesis was knowing if the fetus had Down's syndrome, and for those not choosing amniocentesis, attitude about abortion. CONCLUSION: Our results show the need for prescreening education to enable pregnant women to make informed decisions about screening for Down's syndrome and diagnostic testing.
Assuntos
Amniocentese/estatística & dados numéricos , Síndrome de Down/diagnóstico , Doenças Fetais/diagnóstico , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Diagnóstico Pré-Natal/estatística & dados numéricos , Adulto , Atitude Frente a Saúde , Feminino , Educação em Saúde , Humanos , Montana , Gravidez , Medição de Risco , Inquéritos e QuestionáriosRESUMO
Monomer conjugation of an enzyme followed by copolymerization with free monomer is a useful method of enzyme immobilization. L-asparaginase was conjugated with N-succinimidyl acrylate. Analysis of the conjugated enzyme via isoelectric focusing showed that a molar ratio of 9.5 free monomers per enzyme was needed during the conjunction for each vinyl group bound. Only 3% of the enzyme activity was lost per vinyl group added, and conjugation of an average of four monomers per enzyme thermally destabilized the enzyme only at temperatures above 50 degrees C. Activity of the enzyme at physiological temperatures was relatively unaffected.