RESUMO
When cosmic star formation history reaches a peak (at about redshift z ≈ 2), galaxies vigorously fed by cosmic reservoirs are dominated by gas and contain massive star-forming clumps, which are thought to form by violent gravitational instabilities in highly turbulent gas-rich disks. However, a clump formation event has not yet been observed, and it is debated whether clumps can survive energetic feedback from young stars, and afterwards migrate inwards to form galaxy bulges. Here we report the spatially resolved spectroscopy of a bright off-nuclear emission line region in a galaxy at z = 1.987. Although this region dominates star formation in the galaxy disk, its stellar continuum remains undetected in deep imaging, revealing an extremely young (less than ten million years old) massive clump, forming through the gravitational collapse of more than one billion solar masses of gas. Gas consumption in this young clump is more than tenfold faster than in the host galaxy, displaying high star-formation efficiency during this phase, in agreement with our hydrodynamic simulations. The frequency of older clumps with similar masses, coupled with our initial estimate of their formation rate (about 2.5 per billion years), supports long lifetimes (about 500 million years), favouring models in which clumps survive feedback and grow the bulges of present-day galaxies.
RESUMO
Comparative structural and functional results on the valine and tyrosine accepting tRNA-like molecules from turnip yellow mosaic virus (TYMV) and brome mosaic virus (BMV), and the corresponding cognate yeast tRNAs are presented. Novel experiments on TYMV RNA include design of variant genes of the tRNA-like domain and their transcription in vitro by T7 RNA polymerase, analysis of their valylation catalyzed by yeast valyl-tRNA synthetase, and structural mapping with dimethyl sulfate and carbodiimide combined with graphical modelling. Particular emphasis is given to conformational effects affecting the valylation capacity of the TYMV tRNA-like molecule (e.g., the effect of the U43----C43 mutation). The contacts of the TYMV and BMV RNAs with valyl- and tyrosyl-tRNA synthetases are compared with the positions in the molecules affecting their aminoacylation capacities. Finally, the involvement of the putative valine and tyrosine anticodons in the tRNA-like valylation and tyrosylation reactions is discussed. While an anticodon-like sequence participates in the valine identity of TYMV RNA, this seems not to be the case for the tyrosine identity of BMV RNA despite the fact that the tyrosine anticodon has been shown to be involved in the tyrosylation of canonical tRNA.
Assuntos
Vírus do Mosaico/genética , RNA de Transferência/metabolismo , RNA Viral/metabolismo , Saccharomyces cerevisiae/genética , Anticódon , Sequência de Bases , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Aminoacil-RNA de Transferência/metabolismoRESUMO
The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.
Assuntos
Aspartato-tRNA Ligase/metabolismo , Fenilalanina-tRNA Ligase/metabolismo , RNA de Transferência de Ácido Aspártico/ultraestrutura , RNA de Transferência de Fenilalanina/ultraestrutura , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Fúngico/metabolismo , RNA Fúngico/ultraestrutura , RNA de Transferência de Ácido Aspártico/metabolismo , RNA de Transferência de Fenilalanina/metabolismo , Saccharomyces cerevisiae , Relação Estrutura-Atividade , Especificidade por Substrato , Aminoacilação de RNA de TransferênciaRESUMO
A yeast aspartic acid tRNA with a 5' extension of 14 nucleotides was obtained by in vitro transcription with T7 DNA dependent RNA polymerase. This transcript, called extended tRNA(Asp) transcript, retains its aspartylation capacity with the same Km and only three times reduced kcat values as compared to those measured for canonical tRNA(Asp). This result indicates that the 5' extension of the amino acid acceptor stem of tRNA(Asp) does not interfere with recognition by aspartyl-tRNA synthetase. However, in contrast to the wild-type tRNA(Asp) transcript, the 5' extended molecule presents a reduced capacity to be mischarged by arginyl-tRNA synthetase, suggesting the existence of different structural requirements in aspartyl- and arginyl-tRNA synthetases for tRNA(Asp) recognition.
Assuntos
RNA Fúngico/metabolismo , RNA de Transferência de Ácido Aspártico/metabolismo , Saccharomyces cerevisiae/genética , Aminoacilação de RNA de Transferência/genética , Arginina-tRNA Ligase/metabolismo , Aspartato-tRNA Ligase/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , RNA Fúngico/genética , RNA de Transferência de Ácido Aspártico/genética , Relação Estrutura-Atividade , Fagos T/genéticaRESUMO
A synthetic gene of yeast aspartic acid tRNA with a promoter for phage T7 RNA polymerase was cloned in Escherichia coli. The in vitro transcribed tRNA(Asp) molecules are deprived of modified nucleotides and retain their aspartylation capacity. The solution conformation of these molecules was mapped with chemical structural probes and compared to that of fully modified molecules. Significant differences in reactivities were observed in Pb2+ cleavage of the RNAs and in modification of the bases with dimethyl sulphate. The most striking result concerns C56, which becomes reactive in unmodified tRNA(Asp), indicating the disruption of the C56-G19 base pair involved in the D- and T-loop interaction. The chemical data indicate that unmodified tRNA(Asp) transcripts possess a relaxed conformation compared to that of the native tRNA. This conclusion is confirmed by thermal melting experiments. Thus it can be proposed that post-transcriptional modifications of nucleotides in tRNA stabilize the biologically active conformations in these molecules.
Assuntos
Genes Sintéticos , Processamento Pós-Transcricional do RNA/fisiologia , RNA de Transferência de Ácido Aspártico/química , Saccharomyces cerevisiae/genética , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sondas RNA , RNA de Transferência de Ácido Aspártico/metabolismo , Temperatura , Transcrição GênicaRESUMO
This report presents the conceptual and methodological framework that presently underlies the experiments designed to decipher the structural features in tRNA important for its aminoacylation by aminoacyl-tRNA synthetases. It emphasizes the importance of conformational features in tRNA for an optimized aminoacylation. This is illustrated by selected examples on yeast tRNA(Asp). Using the phage T7 transcriptional system, a series of tRNA(Asp) variants were created in which conformational elements were modified. It is shown that aspartyl-tRNA synthetase tolerates conformational variability in tRNA(Asp) at the level of the D-loop and variable region, of the tertiary Levitt base-pair 15-48 which can be inverted and in the T-arm in which residue 49 can be excised. However, changing the anticodon region completely abolishes the aspartylation capacity of the variants. Transplanting the phenylalanine identity elements into a different tRNA(Asp) variant presenting conformational characteristics of tRNA(Phe) converts this molecule into a phenylalanine acceptor but is less efficient than wild-type tRNA(Phe). This engineered tRNA completely loses its aspartylation capacity, showing that some aspartic acid and phenylalanine identity determinants overlap. The fact that chimeric tRNA(Asp) molecules with altered anticodon regions lose their aspartylation capacity demonstrates that this region is part of the aspartic acid identity of tRNA(Asp).
Assuntos
RNA de Transferência de Ácido Aspártico/metabolismo , Saccharomyces cerevisiae/metabolismo , Aspartato-tRNA Ligase/metabolismo , Sequência de Bases , Sítios de Ligação , Engenharia Genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Transferência de Ácido Aspártico/química , RNA de Transferência de Ácido Aspártico/genética , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
Chemical modification studies have demonstrated that the ultra-violet difference spectrum of alpha-thrombin produced in the presence of sodium is due primarily to changes in the environment of tyrosine residues. This is based on the observation that the spectrum could be abolished by treatment of alpha-thrombin with tetranitromethane but not with dimethyl-(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Although lithium produces similar (UV) difference spectrum, circular dichroism studies indicate that sodium and lithium induce different conformational transitions. alpha-Thrombin tends to assume a more ordered structure in the presence of sodium whereas lithium has the reverse effect. This inverse behavior is consistent with the effects of these cations on the autolysis rate and thermal stability of the activities of alpha-thrombin.
Assuntos
Lítio , Sódio , Espectrofotometria Ultravioleta , Trombina/análise , Cálcio , Dicroísmo Circular , Humanos , Conformação MolecularRESUMO
It is known that storage at pH 6 stabilizes thrombin against inactivation. In order to determine whether structural changes accompany this stabilization, the conformation of human alpha-thrombin at pH 6.0 and 7.5 was investigated by chemical modification, difference spectroscopy, circular dichrosim, and thermal stability. It was shown that the CD spectra at the 230-200 nm peptide transition were indistinguishable at the two pH values, indicating no differences in the secondary structure as also indicated by the thermal stability of the enzyme at pH 6.0, 7.4 and 8.3. However, differences were observed in the 300-250 nm aromatic transition suggesting some changes in the microenvironment of the aromatic chromophores. Solvent perturbation in 20% ethylene glycol and 20% dimethylsulfoxide showed that at pH 7.5, 4.3 +/- 0.3 tryptophan and 8.6 +/- 0.4 tyrosine residues were exposed and accessible to the solvent whereas at pH 6.0 these values were 3.6 +/- 0.1 tryptophan and 7.8 +/- 0.4 tyrosine residues. At pH 7.5, 6.0 +/- 0.5 tryptophan residues were found reactive toward dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide while 2.5 +/- 0.3 were found reactive at pH 6.0. Accompanying these structural changes were ultraviolet absorption and CD spectral changes with transition midpoints at pH 6.45 characteristic of histidine ionization. These spectral changes were lost when alpha-thrombin was modified by diethylpyrocarbonate but not by N-alpha-tosyl-L-Lysinechloro-methyl ketone. It is concluded that a second histidine residue, not the active site His-43, is associated with the pH dependent conformational changes at pH 6.0. The ionization of this histidine residue and the accompanying conformational changes could explain the reduced catalytic efficiency and stability of alpha-thrombin at pH 6.
Assuntos
Trombina/análise , Dicroísmo Circular , Dietil Pirocarbonato , Histidina , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Conformação Proteica , Espectrofotometria Ultravioleta , Compostos de Sulfônio , Tosilina Clorometil CetonaRESUMO
A 30-year-old male died in Thailand after a scuffle. The corpse was embalmed and repatriated to France where an autopsy was performed. As usual in cases of embalmment, fluids such as blood and urine were unavailable and the toxicological analyses was performed on the bile and the liver. An overdose of heroin was determined as the cause of death. A review of the literature indicates that several drugs can be detected in fluids and tissues that contain formaldehyde. This case demonstrates that in embalmed corpses, toxicological assessment is still possible, e.g. after heroin fatalities.
Assuntos
Bile/química , Embalsamamento , Cabelo/química , Heroína/análise , Fígado/química , Entorpecentes/análise , Adulto , Overdose de Drogas , Medicina Legal/métodos , Cromatografia Gasosa-Espectrometria de Massas , Heroína/intoxicação , Humanos , Masculino , Entorpecentes/intoxicaçãoRESUMO
A case of fatal poisoning with cyamemazine is presented. The cyamemazine was identified in post-mortem blood using a specific gas chromatographic/mass spectrometry method. The autopsy blood concentration of cyamemazine was 1800 ng/ml. Chronic use of cyamemazine was demonstrated by the presence of the drug in hair. Two other drugs were also detected (bromazepam and trimeprazine). We think that this current blood concentration (1800 ng/ml) is a fatal blood concentration because of the negativity of the other parameters, but careful interpretation of analytical findings are important, the possibility that this death was a consequence of the toxicity of combined drugs could not be excluded. Not many therapeutics and toxic levels were previously reported in overdosage cases in which cyamemazine was involved. We consider that this concentration is only of guidance value for a fatal cyamemazine poisoning.
Assuntos
Antipsicóticos/intoxicação , Fenotiazinas/intoxicação , Ansiolíticos/análise , Antipruriginosos/análise , Antipsicóticos/análise , Bromazepam/análise , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Cabelo/química , Humanos , Pessoa de Meia-Idade , Fenotiazinas/análise , Trimeprazina/análiseRESUMO
The present study reexamines the accuracy of the supero-inferior femoral neck diameter for the determination of sex using a modern sample of French individuals. In 1998, Seidemann et al. used this univariate method for sex determination with the Hamann-Todd collection. Stojanowski and Seidemann in 1999 tested previous results on a modern sample taken from the University of New Mexico and concluded that the Caucasian male samples exhibited no significant differences between individuals born before and after 1900, but the Caucasian female subgroup did exhibit differences with an increase of the SID in the modern sample. The current study compares the previous results of the supero-inferior femoral neck diameter with a modern sample of elderly French individuals born after 1910. Both sides of the femur were measured. No statistical difference was found between the right and left side (p = 0.31). The results showed a significant difference between the pre-1900 and the modern sample, with an increase in femoral neck diameter in modern populations. The comparison of the SID values between the two modern samples (Mexico and Nice) showed no significant differences in the femoral neck diameter in the two male subgroups (p = 0.05), but the measurements of the SID in the female subgroup did exhibit significant differences with an increase of the neck femoral diameter (p < 0.01) in the modern French population. These results demonstrate an increase in the neck femoral morphology in the elderly European French females samples.
Assuntos
Antropometria/métodos , Colo do Fêmur/anatomia & histologia , Antropologia Forense/métodos , Caracteres Sexuais , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Análise Discriminante , Feminino , França , Humanos , Modelos Logísticos , MasculinoRESUMO
Forensic anthropologists are frequently asked to assess partial or badly damaged skeletal remains. One such request led us to compare the predictive accuracy of different mathematical methods using four non-standard measurements of the proximal femur (trochanter-diaphysis distance (TD), greater-lesser trochanter distance (TT), greater trochanter width (TW) and trochanter-head distance (TH)). These measurements were taken on 76 femurs (38 males and 38 females) of French individuals. Intra- and inter-observer trials did not reveal any significant statistical differences. The predictive accuracy of three models built using linear and non-linear modelling techniques was compared: discriminant analysis, logistic regression and neural network. The neural network outperformed discriminant analysis and, to a lesser extent, logistic regression. Indeed, the best results were obtained with a neural network that correctly classified 93.4% of femurs, with similar results in males (92.1%) and females (94.7%). Univariate functions were less accurate (68-88%). Discriminant analysis and logistic regression, both using all four variables, led to slightly better results (88.2% and 89.5%, respectively). In addition, all the models, save the neural network, led to unbalanced results between males and females. In conclusion, the artificial neural network is a powerful classification technique that may improve the accuracy rate of sex determination models for skeletal remains.
Assuntos
Análise Discriminante , Fêmur/anatomia & histologia , Modelos Logísticos , Redes Neurais de Computação , Determinação do Sexo pelo Esqueleto/métodos , Idoso de 80 Anos ou mais , Feminino , Antropologia Forense/métodos , França , Humanos , MasculinoRESUMO
Until now, determining the sex of a recently deceased individual using the measurement of the bicondylar breadth of the femur (also known as condylar width, epicondylar breadth and distal epiphyseal breadth) raised some concerns as to accuracy because no sample of contemporary French subjects was available. In this study, a sample of 88 female and male femurs taken from recently deceased elderly French people was studied. The bones were collected from subjects who had donated their bodies to the Medical School of Nice. The mean value of the male bicondylar breadth was found to be greater than that of females (84.3mm versus 74.8mm), confirming the sexual dimorphism of this parameter. Furthermore, the results showed a 95.4% accuracy rate for sexing individuals. To date, in the French population, as in some other samples, epicondylar breadth is the single most accurate measurement of sex determination, ahead even of head diameter. A discriminant function is presented to allow sex determination from remains of the distal femur. With regard to the data available in the literature, sexual dimorphism is probably the result of both genetic and environmental factors. The comparison of our results with those of other populations shows that there are inter-population variations of the bicondylar breadth, and also intra-population variations that account for the differences in the accuracy rate of this variable for the purposes of sex determination. These findings underscore the need to re-evaluate bone measurements within various contemporary populations.
Assuntos
Fêmur/anatomia & histologia , Antropologia Forense/métodos , Determinação do Sexo pelo Esqueleto/métodos , Idoso , Idoso de 80 Anos ou mais , Análise Discriminante , Feminino , França , Humanos , MasculinoRESUMO
[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.
Assuntos
Proteínas de Ligação a Tiroxina/metabolismo , Tiroxina/metabolismo , Aminoácidos/análise , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Humanos , Radioisótopos do Iodo , Cinética , Fragmentos de Peptídeos/análise , PronaseRESUMO
Viable single cell suspensions from rat colonic epithelium were obtained by using phosphate buffered saline containing 0-2 M mannitol. The method, which requires no prior enzyme treatment, provides undamaged cells in high yield within one hour. The procedure was also applied to neoplastic rat colonic tissue, which was induced by repeated intrarectal infusion of N-methyl-N-nitrosourea. Comparison between normal and neoplastic cells has shown that the latter have a higher nucleus: cytoplasm ratio and a higher metabolic activity.
Assuntos
Separação Celular/métodos , Neoplasias do Colo/patologia , Mucosa Intestinal/citologia , Animais , Células Epiteliais , Epitélio/patologia , RatosRESUMO
Peripheral nervous system myelin contains as the major structural protein a glycoprotein known as P0. Another glycoprotein present in smaller amounts, known as the 19K or X protein, has been previously identified as derived from P0 and identical with the main tryptic degradation product of P0 (TP0). Although both P0 and 19K protein incorporated fucose in vitro and stained on polyacrylamide gels with the periodic acid-Schiff stain for carbohydrate, only the P0 blotted to nitrocellulose paper showed immunoreactivity to an antibody to P0, whereas the 19K protein did not. Furthermore, when P0 was hydrolyzed with trypsin or elastase, the main degradation products reacted with P0 on immunoblots, whereas the 19K protein showed no immunoreactivity. From these studies and those of others, it may be concluded that the 19K protein shows some similarities to TP0, but probably has a different structure. P0 and 19K protein do not appear to be related as shown by lack of cross-immunoreactivity.
Assuntos
Proteínas da Mielina/análise , Nervo Isquiático/análise , Aminoácidos/metabolismo , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Fucose/metabolismo , Técnicas Imunológicas , Peso Molecular , Proteína P0 da Mielina , Proteínas da Mielina/imunologia , Proteínas da Mielina/metabolismo , Elastase Pancreática/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Endogâmicos , Tripsina/metabolismoRESUMO
Repeated incubation of rat colonic mucosa with phosphate-buffered mannitol yields morphologically intact single cell suspensions which are easily visualized microscopically with trypan blue. Cell dimensions are determined by means of a Nikon microcomparator.
Assuntos
Colo/citologia , Mucosa Intestinal/citologia , Animais , Adesão Celular , Células Cultivadas , Células Epiteliais , Feminino , Masculino , Ratos , Azul TripanoRESUMO
Cell growth and response to interferon (IFN) have been used to assess alteration in properties of cultured skin fibroblasts from patients with familial Alzheimer's Disease (AD). A comparison of the proliferative characteristics between "low" (7-11) and "high" (18-21) passage diploid fibroblasts from control and AD individuals shows that AD cells become severely restricted in their proliferative potential earlier than matched controls as a result of "in vitro aging". In addition, while control cells grow adequately in a serum-free, chemically-defined media (Nutridoma) (90-95% compared to FBS-supplemented media), AD cells grow poorly in Nutridoma (45-53% compared to FBS-supplemented media). An interferon (IFN)-inducible enzyme, 2'5'-oligoadenylate (2-5A) synthetase, is significantly reduced in IFN-treated AD cells compared to age-matched control cells in two cell passage-dependent manner. These data suggest that an intrinsic property of AD cells is the progressive reduced ability to interact with normal biologic signals provided by proteins such as interferons which in turn may contribute to the pathogenesis of AD.
Assuntos
2',5'-Oligoadenilato Sintetase/biossíntese , Doença de Alzheimer/genética , Fibroblastos/patologia , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , 2',5'-Oligoadenilato Sintetase/química , Adulto , Envelhecimento , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Divisão Celular , Linhagem Celular , Meios de Cultura , Indução Enzimática , Feminino , Fibroblastos/enzimologia , Humanos , Substâncias Macromoleculares , Proteínas RecombinantesRESUMO
Contacts between the tRNA-like domain in brome mosaic virus RNA and yeast tyrosyl-tRNA synthetase have been determined by footprinting with enzymatic probes. Regions in which the synthetase caused protections indicative of direct interaction coincide with loci identified by mutational studies as being important for efficient tyrosylation [Dreher, T. W. & Hall, T. C. (1988) J. Mol. Biol. 201, 41-55]. Additional extensive contacts were found upstream of the core of the tRNA-like structure. In parallel, the contacts of yeast tRNATyr with its cognate synthetase were determined by the same methodology and comparison of protected nucleotides in the two RNAs has permitted the assignment of structural analogies between domains in the viral tRNA-like structure and tRNATyr. Amino acid acceptor stems are similarly recognized by yeast tyrosyl-tRNA synthetase in the two RNAs, indicating that the pseudoknotted fold in the viral RNA does not perturb the interaction with the synthetase. A further important analogy appears between the anticodon/D arm of the L-conformation of tRNAs and a complex branched arm of the viral tRNA-like structure. However, no apparent anticodon triplet exists in the viral RNA. These results suggest that the major determinants for tyrosylation of yeast tRNATyr lie outside the anticodon stem and loop, possibly in the amino acid acceptor stem.
Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Vírus do Mosaico/genética , RNA Fúngico , RNA de Transferência Aminoácido-Específico , RNA de Transferência de Tirosina , RNA Viral , Saccharomyces cerevisiae/genética , Tirosina-tRNA Ligase/metabolismo , Acilação , Sequência de Bases , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Endorribonucleases , Células Eucarióticas/metabolismo , Cinética , Dados de Sequência Molecular , Vírus do Mosaico/metabolismo , Mutação , Conformação de Ácido Nucleico , RNA Fúngico/metabolismo , RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência de Tirosina/metabolismo , RNA Viral/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Tirosina/metabolismoRESUMO
General aspects of metabolic features of the most prominent CNS intermediate filament proteins, the 200,000 (200K), 150,000 (150K), and 70,000 (70K) dalton proteins of the neuron, and the glial fibrillary acidic protein (GFAP) have been explored using the incubated spinal cord slice from the rat. Measurement of short-term uptake of 3H-labeled amino acids into the individual proteins separated on polyacrylamide gels revealed that of the three neurofilament proteins, 200K was most metabolically active, 150K was less active, and 70K contained very little incorporated radioactivity. Glial fibrillary acidic protein based on Coomassie blue stain affinity showed less metabolic activity than any of the neurofilament proteins. Those relationships were constant at all ages, but the metabolic activity of all CNS intermediate filaments decreased with age. When Ca2+ was present in the medium of the incubated slices, the intermediate filaments were rapidly destroyed, but GFAP was more resistant to degradation than the neurofilament proteins. GFAP and probably the neurofilament proteins also were relatively resistant to Ca2+-activated degradative mechanisms in spinal cords of rats at younger ages (15 day) than in those of older animals (10-18 months). It is likely that the Ca2+ activated protease is less active in developing animals in which the nerve tracts are still elongating, than in adults. These results suggest that GFAP is less active metabolically and more resistant to degradation than the neurofilament proteins at all stages of maturation, but that metabolic activity of all CNS intermediate filaments decreases with age while the susceptibility to degradation increases.