RESUMO
The present study evaluated the role of pecan nut (Carya illinoensis) shells aqueous extract (AE) against oxidative damage induced by cigarette smoke exposure (CSE) and behavioral parameters of smoking withdrawal. Mice were passively exposed to cigarette smoke for 3 weeks (6, 10, and 14 cigarettes/day) and orally treated with AE (25 g/L). CSE induced lipid peroxidation in brain and red blood cells (RBC), increased catalase (CAT) activity in RBC, and decreased plasma ascorbic acid levels. AE prevented oxidative damage and increased antioxidant defenses of mice exposed to cigarette smoke. In addition, AE reduced the locomotor activity and anxiety symptoms induced by smoking withdrawal, and these behavioral parameters showed a positive correlation with RBC lipid peroxidation. Our results showed the beneficial effects of this by-product of the pecan industry, indicating its usefulness in smoking cessation.
Assuntos
Antioxidantes/farmacologia , Ansiedade/induzido quimicamente , Carya , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Ácido Ascórbico/sangue , Catalase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , NozesRESUMO
Polyclonal antibodies were raised against a multiprotein 'holoenzyme' form of calf thymus DNA polymerase alpha-primase and used to probe a human cDNA-protein expression library constructed in the lambda gt11 vector. The probe identified a series of cDNA clones derived from a 3.2 kb mRNA which encodes a novel 105 kDa polypeptide, the P1 protein. In intact cells, the P1 protein was specifically associated with the nucleus, and in cell extracts, it was associated with complex forms of DNA polymerase alpha-primase. The synthesis of human P1-specific mRNA was stimulated upon addition of fresh serum to growth-arrested cells, and RNA blot analyses with the human P1-cDNA probe indicated that P1 is encoded by a strictly conserved mammalian gene. The amino acid sequence deduced from a 240-codon open reading frame resident in the largest human P1-cDNA (0.84 kb) displayed greater than 96% identity with that deduced from the equivalent segment of a 795-codon open reading frame of a larger mouse P1-cDNA (2.8 kb). Throughout its length, the primary structure of mammalian P1 displayed strong homology with that of Mcm3, a 125 kDa yeast protein thought to be involved in the initiation of DNA replication (Gibson et al. 1990. Mol. Cell. Biol. 10: 5707-5720). The P1-Mcm3 homology, the strong conservation of P1 among mammals, its nuclear localization, and its association with the replication-specific DNA polymerase alpha strongly suggest an important role of the P1 protein in the replication of mammalian DNA.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Proteínas Nucleares/genética , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Clonagem Molecular , DNA Primase , Proteínas Fúngicas/química , Humanos , Camundongos , Componente 3 do Complexo de Manutenção de Minicromossomo , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA Nucleotidiltransferases/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
We describe the nucleotide sequences of several overlapping cDNA clones specific for human glutaminyl-tRNA synthetase. The identified open reading frame indicates that the enzyme is composed of 1440 amino acids. A stretch of about 360 amino acids of the human enzyme is highly conserved in bacterial and yeast glutaminyl-tRNA synthetases. However, the human enzyme is three times larger than the bacterial and twice as large as the yeast enzyme suggesting that a considerable part of human glutaminyl-tRNA synthetase has evolved to perform functions other than the charging of tRNA. The sequence outside of the conserved core region includes three 57-amino acid repeats followed by a consecutive stretch of 11 charged amino acids. A computer assisted search of two protein data banks reveals that the human glutaminyl-tRNA synthetase shares small blocks of amino acid similarities with several other synthetases of different amino acid specificities. Interestingly, the enzyme also possesses some regions of similarities with eukaryotic translation elongation factor EF-1 but not with any other sequence stored in the protein data banks. The coding regions of human and mouse glutaminyl-tRNA synthetase cDNAs are identical at 94% of the codons. However, the 3'-noncoding regions of mouse and human mRNAs are more divergent (approximately 68%) but both possess the potential to form stable secondary structures of similar general architecture.
Assuntos
Aminoacil-tRNA Sintetases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Humanos , Ligação de Hidrogênio , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/metabolismo , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , SolubilidadeRESUMO
We have used a cDNA encoding the core region of the human glutaminyl-tRNA synthetase to determine the chromosomal localization of the corresponding gene. Southern blots of restricted DNA from a panel of rodent-human cell lines and in situ chromosome hybridization gave identical results showing that the human gene locus for glutaminyl-tRNA synthetase resides on the distal long arm of chromosome 1. There are now nine mapped aminoacyl-tRNA synthetase genes in the human genome.
Assuntos
Cromossomos Humanos Par 1 , Glutamato-tRNA Ligase/genética , Animais , Southern Blotting , Mapeamento Cromossômico , Sondas de DNA/genética , Eletroforese em Gel de Poliacrilamida , Ligação Genética , Humanos , Células Híbridas , Hibridização de Ácido Nucleico/genéticaRESUMO
We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.
Assuntos
Aminoacil-tRNA Sintetases/genética , Escherichia coli/genética , Glutamato-tRNA Ligase/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/enzimologia , Dados de Sequência Molecular , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Protein extracts were prepared at various times after serum stimulation of growth-arrested mouse 3T3 fibroblasts. The extracts were fractionated by sucrose gradient centrifugation and used to determine the activities of DNA polymerase alpha and DNA primase. We found that polymerase and primase appeared in close association in one homogeneous 8.2-S peak. Neither polymerase, free of associated primase, nor primase, free of polymerase, could be detected at any time after serum stimulation. The activities of both enzymes started to increase concomitantly at the beginning of the DNA replication phase of the cell cycle. We found five to six times more DNA primase activity in replicating than in resting 3T3 cells. Besides DNA primase, a second additional priming activity could be detected. This activity sedimented at 12.5 S and corresponded most probably to RNA polymerase I.