RESUMO
OBJECTIVE: Because the number of elderly is increasing worldwide, cognitive dysfunction becomes important health care issue. This study investigated the association between cognitive dysfunction and mortality in the elderly. METHOD: Data were analyzed from a longitudinal mortality follow-up study of 2712 Korean elderly aged 60 and over, examined in 2002 with complete data followed an average 6.03 years. Measurements included socio-demographic and clinical factors and Mini-Mental State Examination (MMSE). MMSE was categorized into groups with no, mild, or moderate cognitive dysfunction, and the subscores of MMSE domains were categorized into no dysfunction or dysfunction. The Cox proportional hazards models were conducted to examine the association between MMSE score and mortality, after adjusting for age, gender, education and other socio-demographic factors. RESULTS: Death during follow-up occurred in 318 subjects. The mortality risk was significantly associated with the elderly with mild cognitive dysfunction [hazard ratio (HR) = 1.93] and with moderate cognitive dysfunction (HR = 2.66). 'Orientation-to-time' (HR = 1.39) and 'Attention' (HR = 1.48) domains of MMSE were independently associated with mortality. CONCLUSION: This study showed that cognitive dysfunction independently predicted mortality in the elderly. Cognitive dysfunction should be considered part of identifying the elderly at high risk for mortality.
Assuntos
Transtornos Cognitivos/mortalidade , Entrevista Psiquiátrica Padronizada/estatística & dados numéricos , Idoso , Transtornos Cognitivos/diagnóstico , Feminino , Seguimentos , Avaliação Geriátrica , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , República da CoreiaRESUMO
We have developed a simple method for preparing a tagged protein by PCR. With this method any protein sequence can be easily tagged. The techniques include three steps of DNA restriction, ligation and PCR. We could obtain a DNA construct containing SUMO-1 gene with His6 tag sequence with high efficiency by the next day.
Assuntos
Engenharia Genética/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismoRESUMO
Although mature mammalian retinal ganglion cells normally fail to regrow injured axons, exposure to the molecular environment of the peripheral nervous system stimulates regenerative growth. The present study used dissociated rat retinal ganglion cells purified by immunopanning to identify peripheral nervous system-derived factors that promote axonal outgrowth. Of the multiple growth factors investigated, only ciliary neurotrophic factor and the related cytokine, leukemia inhibitory factor, had striking neuritogenic activity, with half-maximal effects at 1-2 ng/ml. Brain-derived neurotrophic factor stimulated retinal ganglion cell survival nearly as well as ciliary neurotrophic factor, but had only minor effects on outgrowth. Thus, the neuritogenic effects of ciliary neurotrophic factor are not a simple consequence of increased survival. Ciliary neurotrophic factor-stimulated outgrowth was correlated with increased expression of the growth-associated membrane phosphoprotein, GAP-43, a hallmark of optic nerve regeneration in vivo. A high molecular weight fraction from media conditioned by rat optic or sciatic nerve mimicked the effect of ciliary neurotrophic factor in inducing axonal outgrowth. Ciliary neurotrophic factor was detected in the conditioned media on western blots, and the biological activity of the conditioned media was neutralized with an anti-ciliary neurotrophic factor antibody. These results indicate that ciliary neurotrophic factor has specific effects on axon outgrowth in retinal ganglion cells that are dissociable from its effects on cell survival, and that ciliary neurotrophic factor accounts for most of the axon-promoting activity for retinal ganglion cells present in either the sciatic or optic nerve.
Assuntos
Axônios/fisiologia , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Células Cultivadas , Fator Neurotrófico Ciliar , Meios de Cultura/farmacologia , Proteína GAP-43/metabolismo , Humanos , Fatores de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Óptico/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes , Células Ganglionares da Retina/efeitos dos fármacos , Nervo Isquiático/metabolismo , Fatores de TempoRESUMO
For promoter analyses of genes, it is usually necessary to amplify promoter DNA fragments by polymerase chain reaction (PCR) and clone them into a plasmid containing a reporter gene. In the present study we developed a novel plasmid, pGL2-X, which was constructed through a simple procedure of cloning an XcmI cassette from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene into the multicloning site of pGL2-Basic (Promega) pGL2-X was then converted by XcmI digestion into a T-vector which was named pGL2-T. Unfortunately, however, the firefly luciferase gene in pGL2-Basic contains one XcmI restriction site and therefore one base within the recognition site was silent-mutated. The cloning efficiency of the pGL2-T vector was approximately 63% when tested with a PCR product amplified from a promoter region (-501(-)+24) of the murine acetylcholine receptor delta subunit (AchR delta) gene. In C2C12 muscle cells transiently transfected with pGL2-T containing the AchR delta promoter, transcription of the silent-mutated luciferase gene increased 2.2-fold by neuregulin (EGF domain of heregulin beta 1; 100 ng/mL), a known stimulator of AchR delta expression. This result suggested that the pGL2-T vector was biologically functional. Thus, the present study provides an easy method to construct a variety of T-vectors containing different reporter genes.
Assuntos
Vetores Genéticos , Luciferases/genética , Sequência de Bases , Primers do DNA , Reação em Cadeia da PolimeraseRESUMO
This article describes the clinical observation of a novel hemoglobin (Hb) variant found during the course of routine blood testing on a 61-year-old subject. The Hb variant was observed during HbA1c testing by ion-exchange high-performance liquid chromatography. Alkaline electrophoresis and DNA sequencing confirmed the presence of a new Hb variant, HBB:c.407C > A (p.Ala136Asp). This mutation has been reported to induce Hb Beckman variant in the Globin Gene Server. However, it was different from the only experimental report for Hb Beckman by Rahbar, Lee & Asmeron (p.Ala136Glu; Hb Beckman alpha2 beta2 135(H13) ala-to-glu: a new unstable variant and reduced oxygen affinity. Blood 78, 204a). And our case was asymptomatic with normal lab findings, while Rahbar et al.'s case showed the clinical manifestations of chronic anemia. This would be a report for a novel Hb variant suggesting new insight of Hb Beckman variant. This would be a report of a novel Hb variant suggesting new insights into Hb Beckman variant.
Assuntos
Hemoglobinas Anormais/genética , Adulto , Substituição de Aminoácidos , Povo Asiático/genética , Sequência de Bases , Feminino , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Epstein-Barr virus (EBV) is a well-known tumorigenic virus and is associated with lymphoproliferative disorders. Quantitations of EBV viral loads in plasma and peripheral blood mononuclear cells have been reported to be useful biomarkers for monitoring Hodgkin's lymphoma and EBV-associated non-Hodgkin lymphoma (NHL). METHODS: In the present study, whole blood specimens were used to determine quantitatively EBV viral loads, which were then compared with clinical data. Using real-time quantitative polymerase chain reaction (RQ-PCR), EBV-DNA was monitored in whole blood samples from patients with NHL (n = 61) at the time of diagnosis, relapse, and follow-up. RESULTS: A statistically significant correlation was observed between positive EBV viral load and extranodal involvement in diffuse large B-cell lymphoma at the time of diagnosis or relapse (n = 29, P = 0.009). In patients who were serially checked for EBV-DNA levels (n = 16), viral load was found to fall to undetectable levels during complete remission. On the contrary, progressive disease and relapse were found to be associated with sustained or elevated EBV-DNA levels. CONCLUSION: These results suggest that whole blood EBV-DNA quantitation might be of value as a convenient biomarker for therapeutic responsiveness of NHL.
Assuntos
DNA Viral/sangue , Herpesvirus Humano 4/genética , Linfoma não Hodgkin/virologia , Carga Viral/métodos , Biomarcadores/sangue , Infecções por Vírus Epstein-Barr/genética , Feminino , Humanos , Linfoma Difuso de Grandes Células B/virologia , Linfoma não Hodgkin/terapia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodosRESUMO
Nuclei in the synaptic region of multinucleated skeletal myofibers are transcriptionally distinct, since acetylcholine receptor genes are transcribed at a high rate by these nuclei, but not by nuclei elsewhere in the myofiber. Although this spatially restricted transcription pattern is presumably imposed by the motor nerve, the continuous presence of the nerve is not required, since synapse-specific transcription persists after denervation. These results suggest either that a transcriptional signal persists at synaptic sites after nerve terminals have degenerated, or that a transcriptional pattern in the myofiber, once established, is stable in the absence of a nerve-derived signal. To distinguish between these possibilities, we denervated muscle and damaged the myofibers and specialized cells located near synaptic sites, and then studied transcription of an acetylcholine receptor gene in myofibers that regenerated in their original basal lamina sheaths, but remained denervated. We show that synapse-specific transcription is re-induced in these regenerated myofibers, and we conclude that a signal for synapse-specific transcription is stably maintained in the synaptic basal lamina.
Assuntos
Músculos/fisiologia , Receptores Colinérgicos/genética , Regeneração/genética , Sinapses/fisiologia , Transcrição Gênica/fisiologia , Animais , Membrana Basal/fisiologia , Expressão Gênica , Hormônio do Crescimento/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Denervação Muscular/métodosRESUMO
Polymerase chain reaction (PCR) is one of the most powerful tools in cloning genes. For the direct cloning of PCR products, T-vectors, which contain complementary 3'-thymidine overhangs, are widely used. In the present study, we developed a plasmid, pNB-T, which was constructed by cloning an XcmI cassette with a sufficient length of DNA (over 500 bp long) between two XcmI restriction sites into pBluescript SK(+). An XcmI cassette was made by nonspecific PCR using a primer containing recognition sequences of XcmI so that pNB-T can easily be converted into a T-vector by restriction of the plasmid with XcmI. In addition, the recognition sequences for BamHI and NcoI were added at 5'-end of the primer in order to facilitate subcloning of the gene cloned in the T-vector. The cloning efficiency of a PCR product, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, was approximately 90%. Digestion of the recombinant plasmid containing the GAPDH gene with BamHI or NcoI liberated the DNA fragments with the expected size, demonstrating the usefulness of extra restriction sites. The method described in this report is quite simple and enables us to construct a variety of useful T-vectors.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Amplificação de Genes , Vetores GenéticosRESUMO
This article examines the role of innervation in regulating expression of acetylcholinesterase (AchE), butyrylcholinesterase (BuchE), and the muscarinic acetylcholine receptor (mAchR) in avian heart. Two distinct approaches are taken. The first approach examines the relation between the onsets of parasympathetic and sympathetic innervation and the appearance of AchE and BuchE. All molecular forms of AchE and BuchE are present in early embryonic chick heart well before the onset of parasympathetic and sympathetic innervation. These molecular forms are characterized by sedimentation coefficients of 4.5S, 11S, 15S, and 19S. With further development, the amounts of AchE fall; the reductions in AchE parallel the onset of functional parasympathetic innervation. The amounts of BuchE increase progressively throughout embryonic development, independent of autonomic innervation, and in mature chick heart predominate over the much less abundant amounts of AchE. The 15S and 19S forms of AchE in heart are lost during early embryogenesis but reappear in skeletal muscle during later embryogenesis. The second approach examines the influence of vagotomy and sympathetic denervation of 8-day-old chick myocardium on expression of the molecular forms of AchE, BuchE, mAchR, and beta-adrenergic receptors. The amounts of AchE and BuchE molecular forms in avian heart are not measurably influenced by bilateral vagotomy for a duration of 4 days, unilateral vagotomy for a duration of 25 days, or sympathetic denervation. A measurable upregulation is observed in muscarinic receptors (35-46%) after vagotomy but not sympathectomy and in beta-adrenergic receptors (29%) after sympathectomy but not vagotomy. In all cases, results in atria and ventricles are nearly identical. The present results indicate that expression of AchE in the myocardium is unique and different from that in skeletal muscle and not directly linked with autonomic innervation.
Assuntos
Acetilcolinesterase/metabolismo , Coração/crescimento & desenvolvimento , Coração/inervação , Miocárdio/enzimologia , Vagotomia , Fatores Etários , Animais , Butirilcolinesterase/metabolismo , Embrião de Galinha , Galinhas , Muscarina/análise , Sistema Nervoso Parassimpático/fisiologia , Receptores Colinérgicos/análise , Simpatectomia , Sistema Nervoso Simpático/fisiologiaRESUMO
Signaling between nerve and muscle is mediated by multiple mechanisms, including two transcriptional pathways. Signals provided by the nerve terminal activate transcription of acetylcholine receptor (AChR) genes in myofiber nuclei near the synaptic site, and signals associated with myofiber electrical activity inactivate AChR gene expression throughout the myofiber. These opposing effects of innervation are conferred by 1.8 kb of 5' flanking DNA from the AChR delta subunit gene. These results raise the possibility that synapse-specific and electrical activity-dependent gene expression are mediated by the same DNA sequence and that activation and repression are determined by differential regulation of the same DNA binding protein. We produced transgenic mice carrying AChR delta subunit-hGH gene fusions, and we show here that a binding site (E-box) for myogenic basic helix-loop-helix proteins is required for electrical activity-dependent but not for synapse-specific gene expression of the delta subunit gene. These results indicate that a change in the expression or activity of an E-box binding protein(s) mediates electrical activity-dependent gene regulation and that synapse-specific and electrical activity-dependent gene expression require different DNA sequences. Moreover, we show here that the cis-acting elements for both aspects of innervation-dependent gene regulation are contained in 181 bp of 5' flanking DNA from the AChR delta subunit gene.
Assuntos
Músculos/fisiologia , Junção Neuromuscular/fisiologia , Receptores Colinérgicos/genética , Animais , Eletrofisiologia , Imunofluorescência , Expressão Gênica/fisiologia , Hormônio do Crescimento/genética , Camundongos , Camundongos TransgênicosRESUMO
Two different signalling pathways mediate the localization of acetylcholine receptors (AChRs) to synaptic sites in skeletal muscle. The signal for one pathway is agrin, a protein that triggers a redistribution of previously unlocalized cell surface AChRs to synaptic sites. The signal for the other pathway is not known, but this signal stimulates transcription of AChR genes in myofibre nuclei near the synaptic site. Neuregulins, identified originally as a potential ligand for erbB2 (Neu differentiation factor, NDF), stimulate proliferation of Schwann cells (glial growth factor, GGF), increase the rate of AChR synthesis in cultured muscle cells (AChR-inducing activity) and are expressed in motor neurons. These results raise the possibility that neuregulin is the signal that activates AChR genes in synaptic nuclei. Here we show that neuregulin activates AChR gene expression in C2 muscle cells and that the neuregulin response element in the AChR delta-subunit gene is contained in the same 181 base pairs that confer synapse-specific expression in transgenic mice. We use antibodies to show that neuregulins are concentrated at synaptic sites and that, like the extracellular signal that stimulates synapse-specific expression, neuregulins remain at synaptic sites in the absence of nerve and muscle. We show that C2 muscle cells contain erbB2 and erbB3 messenger RNA but little or no erbB4 mRNA, and that neuregulin stimulates tyrosine phosphorylation of erbB2 and erbB3, indicating that neuregulin signalling in skeletal muscle may be mediated by a complex of erbB2 and erbB3.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glicoproteínas/fisiologia , Músculo Esquelético/fisiologia , Junção Neuromuscular/fisiologia , Receptores Colinérgicos/genética , Animais , Anticorpos/imunologia , Linhagem Celular , Clonagem Molecular , Ativação Enzimática , Receptores ErbB/metabolismo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Técnicas Imunológicas , Camundongos , Camundongos Transgênicos , Neurregulinas , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3 , Receptor ErbB-4 , Sequências Reguladoras de Ácido Nucleico , Transdução de Sinais , Sinapses/fisiologia , Tirosina/metabolismoRESUMO
Although purinergic compounds are widely involved in the intra- and intercellular communication of the nervous system, little is known of their involvement in the growth and regeneration of neuronal connections. In dissociated cultures, the addition of adenosine or guanosine in the low micromolar range induced goldfish retinal ganglion cells to extend lengthy neurites and express the growth-associated protein GAP-43. These effects were highly specific and did not reflect conversion of the nucleosides to their nucleotide derivatives; pyrimidines, purine nucleotides, and membrane-permeable, nonhydrolyzable cyclic nucleotide analogs were all inactive. The activity of adenosine required its conversion to inosine, because inhibitors of adenosine deaminase rendered adenosine inactive. Exogenously applied inosine and guanosine act directly upon an intracellular target, which may coincide with a kinase described in PC12 cells. In support of this, the effects of the purine nucleosides were blocked with purine transport inhibitors and were inhibited competitively with the purine analog 6-thioguanine (6-TG). In PC12 cells, others have shown that 6-TG blocks nerve growth factor-induced neurite outgrowth and selectively inhibits the activity of protein kinase N, a partially characterized, nerve growth factor-inducible serine-threonine kinase. In both goldfish and rat retinal ganglion cells, 6-TG completely blocked outgrowth induced by other growth factors, and this inhibition was reversed with inosine. These results suggest that axon outgrowth in central nervous system neurons critically involves an intracellular purine-sensitive mechanism.