RESUMO
A biochemical and quantitative morphologic study of presynaptic endings during postnatal development was carried out in subcellular fractions from cerebral cortex of 1, 4, 8, 12, and 18 day old and adult rats. Crude mitochondrial fractions were subfractionated in Ficoll gradients and all resulting fractions were examined in the electron microscope. Presynaptic terminals and other intact processes were counted. Protein content and enzyme activities were assayed in the fractions and in total brain homogenate. In the first and fourth day of life, most of the presynaptic terminals were found in two "light" fractions, between supernatant and 7.5% Ficoll, where they accounted, respectively, for 6 and 22% of all the processes. Progressively with age, more presynaptic terminals were found in the traditional "synaptosomal" fractions between 7.5 and 13% Ficoll. In that region of the gradient, 40, 54, 75, and 89% of the processes were presynaptic endings at 8, 12, and 18 postnatal days and in the adult animal, respectively. A similar shift from the lighter to the heavier fractions was observed in the distribution of choline acetyltransferase and acetylcholinesterase between days 8 and 12. The rate of increase of the specific activity of these two enzymes paralleled that of the percentage of the presynaptic endings after day 8. This study indicates that subcellular fractions can be used to study formation and maturation of synapses during postnatal development.
Assuntos
Córtex Cerebral/citologia , Córtex Cerebral/enzimologia , Sinapses , Vesículas Sinápticas , Acetilcolina , Acetilcolinesterase/análise , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/enzimologia , Química Encefálica , Fracionamento Celular , Córtex Cerebral/análise , Córtex Cerebral/crescimento & desenvolvimento , Histocitoquímica , L-Lactato Desidrogenase/análise , Microscopia Eletrônica , Mitocôndrias , Morfogênese , Bainha de Mielina/análise , Proteínas/análise , Ratos , Ratos Endogâmicos , Ribossomos , Succinato Desidrogenase/análise , Sinapses/enzimologia , Vesículas Sinápticas/enzimologia , Extratos de Tecidos , Transferases/análiseRESUMO
The distribution of labeled RNA in the optic nerve of the rabbit was studied by quantitative ultrastructural autoradiography after the intraocular injection of [(3)H]uridine. The highest density of silver grains related to [(3)H]RNA (27-40 grains/100 microm(2)) was found in glial cell perikarya; a slightly lower density was present in the glial nuclei (19-20 grains/100 microm(2)). Axons (4-5 grains/100 microm(2)) and myelin (2-3 grains/100 microm(2)) had the lowest grain densities. 74-83% of all counted grains were located outside the axons. By comparing the grain density distribution over the axon with that expected in the case of an exclusive labeling of the surrounding myelin and glial cell processes, it was concluded that the axons contained a number of grains representing [(3)H]RNA significantly higher than that expected to scatter from myelin and glial processes. Most of these grains were concentrated at the periphery of the axon and were not related to axonal mitochondria.
Assuntos
Nervo Óptico/metabolismo , RNA/metabolismo , Uridina , Animais , Autorradiografia , Axônios/metabolismo , Olho , Injeções , Microscopia Eletrônica , Bainha de Mielina/metabolismo , Neuroglia/metabolismo , RNA/biossíntese , Coelhos , Trítio , Uridina/administração & dosagemRESUMO
A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30-50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75-77% of the total processes. 34-45% of the grains were related to processes containing ribosomes which accounted for only 4-7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.
Assuntos
Terminações Nervosas/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Vesículas Sinápticas/metabolismo , Aminoácidos/metabolismo , Animais , Autorradiografia , Córtex Cerebral/citologia , Cloranfenicol/farmacologia , Depressão Química , Leucina/metabolismo , Microscopia Eletrônica , Ratos , Sinaptossomos/metabolismo , TrítioRESUMO
The rate of endogenous carbon monoxide production ( Vco), determined by the closed rebreathing system technique, was elevated above the normal range in four of five patients studied with ineffective erythropoiesis (four patients with primary refractory anemia, one with thalassemia). The mean molar ratio of Vco to Vheme (rate of circulating heme catabolism, determined from (51)Cr red cell survival curves) was 3.0 +/- 0.6 (SE), indicating that most of the CO originated from sources other than circulating erythrocyte hemoglobin, in contrast to previous findings in patients with hemolytic anemia, where Vco paralleled Vheme closely.After administration of glycine-2-(14)C to these patients, endogenous CO was isolated by washout of body CO stores at high pO(2) or by reacting peripheral venous blood samples with ferricyanide. The CO was then oxidized to CO(2) by palladium chloride and trapped for counting in a liquid scintillation spectrometer. "Early labeled" peaks of (14)CO were demonstrated which paralleled "early labeled" peaks of stercobilin and preceded maximal labeling of circulating heme. Production of "early labeled" (14)CO in patients with ineffective erythropoiesis was greatly increased, up to 14 times that found in a normal subject. The increased Vco and "early (14)CO" production shown by these patients are presumably related mainly to heme catabolism in the marrow. The possibility exists that hepatic heme and porphyrin compounds may also contribute significantly to Vco, as suggested by the finding of a high Vco in an additional patient with porphyria cutanea tarda.
Assuntos
Anemia Sideroblástica/metabolismo , Anemia/metabolismo , Pigmentos Biliares/biossíntese , Monóxido de Carbono/biossíntese , Eritropoese , Heme/metabolismo , Porfirias/metabolismo , Talassemia/metabolismo , Adolescente , Adulto , Idoso , Bilirrubina/sangue , Bilirrubina/metabolismo , Isótopos de Carbono , Isótopos do Cromo , Fezes/análise , Feminino , Glicina , Hemoglobinometria , Humanos , Ferro/sangue , Isótopos de Ferro , Leucopenia/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
1. The purpose of this study was to investigate the effects of Bioplex Zn (a chelated zinc proteinate) and phytase supplementation in a maize-soybean meal diet on the performance and tissue zinc (Zn) content of broiler chicks. Treatment structure consisted of a 2 x 6 factorial arrangement with two inclusions of phytase (0 or 500 PU/kg) and 6 of Bioplex Zn providing 0, 2, 4, 8, 16 and 32 mg Zn/kg diet. A total of 864 chicks were randomly assigned to each of 12 dietary treatments with 6 replicate cages of 12 chicks. 2. Dietary inclusion of phytase increased feed intake, weight gain, plasma Zn content, tibia Zn content, tibia and ash weight. 3. Dietary supplementation of Bioplex Zn linearly increased feed intake, weight gain, gain to feed ratio, plasma Zn concentration, liver Zn concentration, tibia Zn content, tibia and ash weight. 4. An interactive effect of phytase and Bioplex Zn on feed intake, weight gain, tibia Zn concentration and tibia ash weight was found. 5. One slope, straight broken-line analysis of weight gain regressed on the supplemental Zn level provided as Bioplex Zn indicated that 12 mg/kg supplemental Zn without phytase and 7.4 mg/kg supplemental Zn with phytase were required for the optimal weight gain of chicks.
Assuntos
6-Fitase/farmacologia , Ração Animal/análise , Galinhas/crescimento & desenvolvimento , Glycine max , Zea mays , Zinco/análise , Zinco/farmacologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Galinhas/metabolismo , Dieta/veterinária , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Masculino , Aumento de Peso , Zinco/metabolismoRESUMO
Prostaglandin E1(PGE1) inhibits tyrosine phosphorylation induced by low thrombin concentration (0.05 U/ml), but this is overcome by a high thrombin (2.0 U/ml) concentration. Thromboxane A2 and ADP are endogenous platelet agonists released during platelet activation which potentiate platelet responses. We investigated how these endogenous agonists influenced the effects of PGE1 on thrombin (2.0 U/ml)-induced tyrosine phosphorylation by removing released ADP with apyrase (2.0 U/ml) and by inhibiting thromboxane A2 synthesis with indomethacin (1 microM). Adding PGE1 (1 microM) before thrombin in apyrase/indomethacin(A/I)-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of a 117 kDa protein while other substrates were not affected. This selective effect was evident only in the presence of apyrase and was not dependent on indomethacin. Addition of PGE1 to A/I-treated platelets after thrombin also caused selective tyrosine dephosphorylation of the 117 kDa protein. Conditions which prevented thrombin-induced 117 kDa protein tyrosine phosphorylation also decreased fibrinogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal antibody to FAK and by absence of its tyrosine phosphorylation in the presence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets.
Assuntos
Difosfato de Adenosina/fisiologia , Alprostadil/fisiologia , Plaquetas/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Trombina/farmacologia , Tromboxano A2/fisiologia , Tirosina/metabolismo , Plaquetas/metabolismo , Cálcio/metabolismo , AMP Cíclico/fisiologia , Citosol/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Fosforilação , Ativação Plaquetária/efeitos dos fármacosRESUMO
The product of the MAT alpha 2 gene is a DNA-binding protein that acts as a repressor of two different sets of cell type-specific genes. In alpha cells, the alpha 2 protein represses the transcription of several a-specific genes. In a/alpha cells, the alpha 2 protein acts together with the product of the MATa1 gene, the a1 protein, to repress several genes used by haploids in the mating process. In addition to the mat alpha 2 mutations that result in defects in both types of regulation, other mat alpha 2 alleles have been described that result in defects in the repression of a-specific genes but that do not affect the ability of the alpha 2 and a1 proteins to interact to repress the haploid-specific genes. We report here the isolation of a new class of mat alpha 2 mutations that do not affect the ability of the alpha 2 protein to repress a-specific genes, but that interfere with the ability of the alpha 2 protein to interact with the a1 protein to repress the haploid-specific genes and establish the a/alpha cell type. These mutations may help determine the means by which the a1 protein interacts with alpha 2 to expand the set of genes under its control.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Proteínas de Ligação a DNA/metabolismo , Genes , Genes Fúngicos , Dados de Sequência Molecular , Mutação , PlasmídeosRESUMO
Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for "omega" insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for the same cut plasmid. Recovery of ends-in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repair of chromosomal double-strand breaks.
Assuntos
Recombinação Genética , Saccharomyces cerevisiae/genética , Alelos , Cromossomos Fúngicos , Dano ao DNA , DNA Fúngico , Plasmídeos , Translocação GenéticaRESUMO
Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching. In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites. We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence. SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1. We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms.
Assuntos
DNA Ribossômico/genética , Histona Desacetilases/metabolismo , Meiose/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/metabolismo , Alelos , Diploide , Frequência do Gene , Haploidia , Histona Desacetilases/farmacologia , Hidroliases/genética , Recombinação Genética/efeitos dos fármacos , Retroelementos/genética , Sirtuína 2 , Sirtuínas , Transativadores/farmacologiaRESUMO
RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.
Assuntos
Reparo do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Recombinação Genética , Retroelementos , Saccharomyces cerevisiae/genética , DNA Ligases/metabolismo , DNA Complementar , Epistasia Genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , RNA Mensageiro/genética , Proteína Rad52 de Recombinação e Reparo de DNA , Proteínas de Saccharomyces cerevisiaeRESUMO
Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.
Assuntos
Reparo do DNA , DNA Fúngico/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Mutação da Fase de Leitura , Saccharomyces cerevisiae/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Fúngicos , Cruzamentos Genéticos , Troca Genética , Diploide , Genes Fúngicos , Genótipo , Haploidia , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , Mapeamento por Restrição , Saccharomyces cerevisiae/metabolismoRESUMO
We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.
Assuntos
Clonagem Molecular , Elementos de DNA Transponíveis , Mutação , Saccharomyces cerevisiae/genética , Cruzamentos Genéticos , Genes Fúngicos , Genótipo , Hibridização de Ácido Nucleico , PlasmídeosRESUMO
Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.
Assuntos
Elementos de DNA Transponíveis , Saccharomyces cerevisiae/genética , Sequência de Bases , Mapeamento Cromossômico , DNA Fúngico/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Choque Térmico/genética , Dados de Sequência Molecular , Mutagênese Insercional , RNA de Transferência de Arginina/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMO
Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Proteínas Fúngicas/genética , Mitose/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endonucleases , Haploidia , Mutação , FenótipoRESUMO
A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.
Assuntos
Elementos de DNA Transponíveis , Regulação Fúngica da Expressão Gênica , Peptídeos/genética , Receptores de Peptídeos , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição , Southern Blotting , Eletroforese em Gel de Campo Pulsado , Genes Fúngicos , Fator de Acasalamento , Mutagênese Insercional , Fenótipo , Receptores de Superfície Celular/genética , Receptores de Fator de Acasalamento , Saccharomyces cerevisiae/genéticaRESUMO
Optimal management of the diabetic patient includes normalization of plasma glucose concentration. Attainment of this goal is difficult because both food and stress result in acute elevations of blood glucose that cannot be matched with a single subcutaneous injection of NPH insulin. This paper examines the currently available methods for delivery of insulin to the diabetic subject and the degree of metabolic control attained. It suggests that optimal diabetic control will be achieved only when newer methods of insulin delivery are available to the clinician that match plasma insulin requirements to the simultaneous plasma glucose concentration.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus/tratamento farmacológico , Dieta para Diabéticos , Insulina/administração & dosagem , Estresse Fisiológico/complicações , Diabetes Mellitus/sangue , Feminino , Humanos , Hiperglicemia/etiologia , Infusões Parenterais , Injeções Intramusculares , Insulina/sangue , CinéticaRESUMO
We have cloned and characterized the FUN12 gene which is found on chromosome 1 of Saccharomyces cerevisiae. The complete nucleotide (nt) sequence of the cDNA and the genomic clones shows that FUN12 is expressed as a 3.7-kb message and should encode a 97 kDa-protein. Immunoprecipitations using antipeptide antibodies showed that the cells contain a Fun12p of this size. The databases contain no nt sequences that are homologous to FUN12 and no protein homologous to Fun12p. Gene disruption experiments showed that FUN12 is an essential gene.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Divisão Celular/genética , Proteínas Fúngicas/química , Genes Fúngicos/fisiologia , Dados de Sequência MolecularRESUMO
Peroxynitrite (ONOO-) induces nitration of tyrosine residues and inhibits tyrosine phosphorylation in cell free systems. We investigated the effect of peroxynitrite on protein tyrosine nitration and phosphorylation in resting or thrombin-activated platelets. Peroxynitrite (150 microM) rapidly induced tyrosine nitration of 187, 164, 113, 89, and 61 kDa proteins in gel-filtered platelets which persisted up to 4.5 h. Repeated exposure of platelets to peroxynitrite produced increasing levels of nitration. Peroxynitrite also rapidly increased tyrosine phosphorylation of 120, 117, 95, 80-85, and 70 kDa platelet proteins, but this decreased by 5 min. The same pattern of tyrosine phosphorylation, but with higher intensity, was induced by thrombin in control platelets. Pretreatment of platelets with peroxynitrite decreased thrombin-induced tyrosine phosphorylation at 0.05 and 1 U/ml thrombin but not at 2 U/ml thrombin. Platelet activation responses such as P-selectin expression, serotonin secretion, and aggregation were also decreased by peroxynitrite treatment at low thrombin concentrations. Peroxynitrite exposure and tyrosine nitration decreased platelet sensitivity to thrombin but did not absolutely prevent tyrosine phosphorylation and other platelet responses.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Nitratos/sangue , Nitratos/farmacologia , Fosfotirosina/sangue , Tirosina/sangue , Plaquetas/fisiologia , Proteínas Sanguíneas/metabolismo , Humanos , Selectina-P/sangue , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Serotonina/sangue , Trombina/farmacologiaRESUMO
PURPOSE: Several mechanisms have been proposed to explain the prolonged bleeding times and clinical bleeding in chronic renal failure. Recent evidence has implicated an abnormality in the structure or function of the von Willebrand factor or in its interaction with uremic platelets. We investigated this factor in 11 patients with chronic renal failure. PATIENTS AND METHODS: Blood samples for cell counts, chemistries, and coagulation studies were obtained from 11 patients with chronic renal failure and prolonged bleeding times. Concentrations of von Willebrand factor antigen and ristocetin cofactor activity were determined in plasma and platelets. Multimeric analysis of von Willebrand factor in plasma and platelets was conducted. In eight cases, the platelets of uremic patients were purified, and the thrombin- and ristocetin-induced binding of normal von Willebrand factor to these platelets was examined. RESULTS: The mean plasma von Willebrand factor antigen and activity (ristocetin cofactor assay) were elevated 2.77 mu/ml and 1.88 mu/ml, respectively (normal, 1.01 mu/ml and 1.07 mu/ml, respectively). The ratio of activity to antigen in uremic plasma was 0.67 (normal, 1.05). The mean platelet von Willebrand factor antigen and activity in the uremic patients was decreased (0.26 and 0.50 mu/10(9) platelets, respectively) compared with normal patients (0.46 and 0.93 mu/10(9) platelets, respectively). The oligomeric structure of the uremic plasma von Willebrand factor lacked the largest multimers. Collection of the blood for analysis in several protease inhibitors and/or EDTA did not change the multimeric structure. The von Willebrand factor multimeric structure of platelets from uremic patients was normal. The ristocetin-induced platelet aggregation of the uremic platelet-rich plasma was decreased compared with normal plasma samples. Thrombin and ristocetin-induced binding of normal von Willebrand factor to uremic patients' platelets was indistinguishable from the binding to normal platelets. CONCLUSION: These data suggest that the uremic platelet-binding sites for von Willebrand factor are intact and that the defect in ristocetin-induced platelet aggregation is most likely plasmatic in nature. At least one plasmatic defect was the observed reduction or absence of the largest plasma von Willebrand factor multimer in uremic patients. The platelet von Willebrand content was significantly decreased. These defects may play a role in the prolonged bleeding time and the clinical bleeding observed in patients with uremia.
Assuntos
Plaquetas/metabolismo , Uremia/sangue , Fator de von Willebrand/análise , Tempo de Sangramento , Cromatografia em Agarose , Humanos , Falência Renal Crônica/sangue , Peso Molecular , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Ligação Proteica/efeitos dos fármacos , Ristocetina/farmacologia , Fator de von Willebrand/metabolismoRESUMO
In this study, we demonstrate that platelets contain a small but significant amount of platelet-von Willebrand factor (vWf) not associated with alpha-granules. When platelets free of plasma proteins are exposed to micromolar concentrations of digitonin, plasma membrane permeabilization occurs without disruption of platelet granules. Employing this technique, we have found that upon exposure of a total platelet population to 8 microM digitonin, 5% of total platelet-vWf is released into the supernatant; this occurs without release of beta-TG from alpha-granules. When platelets of discrete buoyant density profiles are tested, this extragranular platelet-vWf increased with decreasing platelet density. These findings suggest that a redistribution of platelet-vWf from alpha-granule to non-granule sites occurs coincident with a decrease in platelet buoyant density.