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BACKGROUND: Whilst prevalence of youth smoking in middle and high income countries has decreased, inequality has prevailed. The introduction of legislation regulating tobacco use in public spaces varies across countries, impacting the tobacco control context. Thus reviewing our knowledge of how social networks may influence smoking differently within different contexts is required to facilitate the development of context-specific interventions. METHODS: The search, conducted on 31st May 2019, included the following smoking-related terms; schools, adolescents, peers and social networks. Inclusion and exclusion criteria were applied throughout the title and abstract screening and full text screening. Quality assessment and synthesis followed. Studies were narratively synthesised to identify changes according to legislative context. This synthesis was conducted separately for findings relating to three categories: socioeconomic status; social selection and influence; and network position. RESULTS: Thirty studies were included. Differences in the relationship between network characteristics and smoking according to socioeconomic status were measured in five out of fifteen studies in Europe. Results varied across studies, with differences in network characteristics and their association with smoking varying both between schools of a differing and those of a similar socioeconomic composition. For studies conducted both before and after the introduction of comprehensive smoking legislation, the evidence for selection processes was more consistent than influence, which varied according to reciprocity. Findings showed that isolates were more likely to smoke and in-degree and out-degree centrality were related to smoking both before and after the introduction of legislation. The relationship between popularity and smoking was contingent on school level smoking prevalence in studies conducted before the introduction of legislation, but not after. CONCLUSIONS: Overall, effects according to socioeconomic status were underreported in the included studies and no consistent evidence of change after the introduction of a comprehensive smoking ban was observed. Further network analyses are required using more recent data to obtain a comprehensive understanding of how network processes may influence smoking differently according to socioeconomic status, and how adaptation could be used to enhance intervention effectiveness. SYSTEMATIC REVIEW REGISTRATION: International Prospective Register of Systematic Reviews (PROSPERO) registration number: CRD42019137358 .
Assuntos
Nicotiana , Prevenção do Hábito de Fumar , Adolescente , Humanos , Fumar/epidemiologia , Classe Social , Uso de TabacoRESUMO
BACKGROUND: Friendships during adolescence play a significant role in the initiation and maintenance of tobacco use. Smoking behaviour among adolescent friends has not been explored among out of school youth (OSY) in South Africa. Out of school youth (OSY), described as those between 13 and 20â¯years old, have not completed their schooling and are not currently enrolled in school, are at greater risk for tobacco use. AIM: The main aim of this study is to examine whether the smoking behaviour of OSY is associated with that of their OSY friends. METHODS: Respondent driven sampling was used to recruit OSY and their OSY friends. A mixed effects logistic regression with a random intercept across school-province combinations was used to analyse survey data. Race and gender were also incorporated into the analyses as effect moderators (nâ¯=â¯391). RESULTS: Results of this study confirm that cigarette smoking was common among OSY and their OSY friends, with 53.5% of the respondents smoking in the past month (SDâ¯=â¯0.44). When OSY friends were either all non-smokers or half their friends were non-smokers, Coloured (mixed race) OSY were less likely to smoke compared to Black African and Other (mostly Asian descent) OSY. CONCLUSION: Cultural norms and values associated with the different race groups may play a role in the smoking behaviour of out of school youth friends. Understanding this relationship is useful for identifying those OSY that are vulnerable to the behaviours that place them at risk of tobacco related morbidity and mortality.
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The present study examined whether parenting and parental smoking can prevent children from selecting smoking friends during adolescence. 254 Adolescents of one Belgian secondary school participated. Self-administered questionnaires were distributed among 2nd-4th graders (mean ages = 14.2-16.2 years) during spring 2006. Follow-up was conducted 12 months later. Data was analyzed conducting longitudinal social network analyses. Results showed adolescents perceiving high parental psychological control had a significant higher tendency to select smoking friends. Perceived behavioral control and perceived parental support did not affect the selection of smoking friends. Furthermore, maternal smoking behavior affected the selection of smoking friends, although no effect of paternal smoking behavior on the selection of smoking friends was found. Adolescent smoking prevention efforts should focus on the influence of parents through their smoking behavior and their psychological control to decrease adolescents' tendency to select smoking friends resulting in fewer opportunities for negative peer influences to occur.
Assuntos
Amigos , Relações Pais-Filho , Poder Familiar , Fumar/epidemiologia , Adolescente , Comportamento de Escolha , Feminino , Humanos , Masculino , Países Baixos/epidemiologia , Pais , Grupo Associado , Apoio Social , Inquéritos e QuestionáriosRESUMO
Preventing smoking initiation among adolescents of lower socio-economic groups is crucial for the reduction of socio-economic inequalities in health. The aim of the present study was to examine whether effective smoking prevention interventions in Europe are equally effective among adolescents of low- and high-socio-economic status (SES). As part of the European Union-funded TEENAGE project, three school-based smoking prevention intervention studies in Europe were selected for secondary analyses: (i) a Dutch class competition intervention, (ii) the European Smoking Prevention Framework (ESFA) study and (iii) the A Stop Smoking in Schools Trial (ASSIST) intervention. All three studies differed in effectiveness by SES. The Dutch class competition study only had a significant effect among higher SES adolescents. The results for the ESFA study and ASSIST study were mixed and depended on which SES indicator was used. The conclusion of the study is that stratified analyses provide important insights in differential intervention effects for higher and lower socio-economic groups. Although findings from the different studies were mixed, interventions that use a social network approach in which youngsters are allowed to deliver the intervention themselves may be a successful strategy in targeting adolescents from lower socio-economic groups.
Assuntos
Comportamento do Adolescente , Prevenção do Hábito de Fumar , Fumar/economia , Classe Social , Adolescente , Criança , Europa (Continente) , Feminino , Humanos , Masculino , Países Baixos , Ensaios Clínicos Controlados Aleatórios como Assunto , Instituições Acadêmicas , Resultado do TratamentoRESUMO
The aim was to study whether physical activity (PA) interventions in European teenagers are equally effective in adolescents of low versus high socio-economic status (SES). Based on a systematic review (Project TEENAGE), three school-based studies for secondary analyses were selected. SES stratified analyses were run in: (i) a Belgian multi-component intervention, (ii) a French multi-component intervention and (iii) a Belgian computer-tailored education trial. Results of the secondary analyses showed that no overall significant differences between low and high SES groups were found, but some interesting specific effects were revealed. Results from the first study showed an increase in objective PA in the low SES group (P = 0.015) compared with no significant effects in the high SES group. In the second study, larger effects were found in adolescents of high SES (increase of 11 min day(-1) P < 0.001), compared with adolescents of lower SES (increase of 7 min day(-1), P = 0.02) at the longer term. The third study showed a positive effect on school-related PA in adolescents of high SES (P < 0.05) and on leisure time transportation in adolescents of low SES (P < 0.05). To conclude, we were not able to show a significant widening or narrowing of inequalities in European adolescents.
Assuntos
Comportamento do Adolescente , Exercício Físico , Adolescente , Bélgica , Criança , Feminino , França , Humanos , Masculino , Fatores SocioeconômicosRESUMO
OBJECTIVE: This study examined the impact of future friends and the contribution of different social influence and selection processes in predicting adolescents' smoking behaviour by extending the theory of planned behaviour (TPB). We investigated the impact of previous smoking, direct pressure from friends, descriptive norms of present and future friends, smoking-based selection of future friends, and distinguished between reciprocal and desired friends. DESIGN: A longitudinal design with three measurements was used. METHODSL: The sample consisted of 1,475 Dutch high school students (mean age = 12.7 years) that participated as a control group in the European Smoking prevention Framework Approach study at three measurements. RESULTS: Structural equation modelling revealed that adolescent smoking was influenced by intention, previous smoking, descriptive norms of parents and siblings, and that desired as well as reciprocal friends were selected based on similar smoking behaviour. Future friends indirectly influenced adolescent smoking through intention, as did attitude, subjective norms of parents and siblings, previous smoking, and descriptive norms of reciprocal friends and siblings. CONCLUSIONS: The present results suggest that descriptive norms and selection of friends need to be considered as major factors explaining smoking behaviour among adolescents besides the TPB components. These insights contribute to the further refinement of smoking prevention strategies.
Assuntos
Comportamento do Adolescente/psicologia , Amigos/psicologia , Fumar/psicologia , Adolescente , Atitude Frente a Saúde , Criança , Comportamento de Escolha , Feminino , Humanos , Intenção , Relações Interpessoais , Estudos Longitudinais , Masculino , Países Baixos , Pais/psicologia , Grupo Associado , Irmãos/psicologia , Meio Social , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To examine whether smoking onset in young adolescents is predicted by peer or parental smoking. DESIGN: Longitudinal design with one pretest and one follow-up at 12 months. SETTING: Schools in Finland, Denmark, the Netherlands, the United Kingdom, Spain and Portugal. PARTICIPANTS: 7102 randomly selected adolescents from six countries. Mean age was 12.78 years. MAIN OUTCOME MEASURES: Smoking behaviour of adolescents, peers and parents. RESULTS: No support was found for peer smoking as an important predictor of smoking onset in most countries. Support was found for the selection paradigm, implying that adolescents choose friends with similar smoking behaviour. Support for the impact of parents on adolescent behaviour and the choice of friends was also found. CONCLUSIONS: Smoking uptake in this age cohort may be more strongly influenced by personal and parental influences than initially believed. Hence, social inoculation programmes teaching youngsters to resist the pressures to smoke may be less appropriate if youngsters have a positive attitude towards smoking, associate smoking with various advantages and look for peers with similar values. For this group attitudes towards smoking may thus guide future friend selection.
Assuntos
Comportamento do Adolescente/psicologia , Grupo Associado , Fumar/psicologia , Adolescente , Atitude Frente a Saúde , Criança , Europa (Continente)/epidemiologia , Feminino , Humanos , Relações Interpessoais , Estudos Longitudinais , Masculino , Modelos Estatísticos , Pais/psicologia , Fatores de Risco , Fumar/epidemiologiaRESUMO
Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Síndrome de Down/genética , Mutação da Fase de Leitura , Fragmentos de Peptídeos/metabolismo , Adulto , Precursor de Proteína beta-Amiloide/metabolismo , Linhagem Celular , Criança , Feminino , Expressão Gênica , Humanos , Recém-Nascido , Rim/citologia , Rim/metabolismo , Substâncias Macromoleculares , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , TransfecçãoRESUMO
During its physiopathological maturation, the beta-amyloid precursor protein undergoes several distinct proteolytic events by activities called secretases. In Alzheimer's disease, the main histological hallmark called senile plaque is clearly linked to the overproduction of the amyloid peptides Abeta40 and Abeta42, two highly aggregable betaAPP-derived fragments generated by combined cleavages by beta- and gamma-secretases. Recently, an alternative hydrolytic pathway was described, involving another category of proteolytic activities called caspases, responsible for the production of a 31 amino acids betaAPP C-terminal fragment called C31. C31 was reported to lower the viability of N2a cells but the exact mechanisms mediating C31-toxicity remained to be established. Here we show that the transient transfection of pSV2 vector encoding C31 lowers by about 80% TSM1 neuronal cells viability. Arguing against a C31-stimulated apoptotic response, we demonstrate by combined enzymatic and immunological approaches that C31 expression did not modulate basal or staurosporine-induced caspase 3-like activity and pro-caspase-3 activation. Furthermore, C31 did not modify Bax and p53 expressions, poly-(ADP-ribose)-polymerase cleavage and cytochrome c translocation into the cytosol. However, we established that C31 overexpression triggers selective increase of Abeta42 but not Abeta40 production by HEK293 cells expressing wild-type betaAPP751. Altogether, our data demonstrate that C31 induces a caspase-independent toxicity in TSM1 neurons and potentiates the pathogenic betaAPP maturation pathway by increasing selectively Abeta42 species in wild type-betaAPP-expressing human cells.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Caspases/metabolismo , Neurônios/enzimologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Caspases/toxicidade , Linhagem Celular , Grupo dos Citocromos c/metabolismo , Expressão Gênica/fisiologia , Humanos , Rim/citologia , Mamíferos , Neurônios/citologia , Estrutura Terciária de Proteína , TransfecçãoRESUMO
FE65, a protein expressed in the nervous system, has the ability to bind the C-terminal domain of the amyloid precursor protein. This suggests a role for FE65 in the pathogenesis of Alzheimer's disease (AD). The present study was conducted to find out if the distribution of FE65 immunoreactivity was affected during the course of AD, and to determine the degree of co-localization of FE65 with other proteins known to be involved in AD. Single immunoperoxidase-labeling experiments, conducted on six sporadic AD patients and six nondemented age-matched controls, showed that the proportion of volume occupied by FE65 immunoreactivity was not modified in the isocortex of AD patients. However, in hippocampal area CA4, increased FE65 immunoreactivity seemed to be associated with the severity of the disease. Double-immunofluorescent labeling did not show any clear co-localization of FE65 with the amyloid precursor protein. FE65 immunoreactivity was also absent from focal and diffuse deposits of the beta-amyloid peptide. Unexpectedly double labeling experiments showed a co-localization of FE65 and tau proteins in intracellular tangles. Ultrastructural observations confirmed that FE65 was associated with paired helical filaments.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas do Tecido Nervoso/análise , Emaranhados Neurofibrilares/metabolismo , Proteínas Nucleares/análise , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/análise , Humanos , Imuno-Histoquímica , Emaranhados Neurofibrilares/patologia , Neurônios/química , Neurônios/patologiaRESUMO
In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Transporte Axonal/fisiologia , Histonas/metabolismo , Neurônios Motores/metabolismo , Degeneração Neural/metabolismo , Proteínas de Protozoários/metabolismo , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Cinesinas/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Mutação de Sentido Incorreto , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Regulação para CimaRESUMO
Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells.
Assuntos
Doença de Alzheimer/enzimologia , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana/fisiologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases , Baculoviridae/metabolismo , Western Blotting , Células CHO , Linhagem Celular , Cricetinae , Endocitose , Humanos , Presenilina-1 , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Mutations in presenilin 1 and presenilin 2 (PS1 and PS2, respectively) genes cause the large majority of familial forms of early-onset Alzheimer's disease. The physical interaction between presenilins and APP has been recently described using coimmunoprecipitation. With a similar technique, we confirmed this interaction and have mapped the interaction domains on both PS2 and APP. Using several carboxy-terminal truncated forms of PS2, we demonstrated that the hydrophilic amino terminus of PS2 (residues 1 to 87, PS2NT) was sufficient for interaction with APP. Interestingly, only a construct with a leader peptide for secretion (SecPS2NT) and not its cytosolic counterpart was shown to interact with APP. For APP, we could demonstrate interaction of PS2 with the last 100 but not the last 45 amino acids of APP, including therefore the A beta region. Accordingly, SecPS2NT is capable of binding to A beta-immunoreactive species in conditioned medium. In addition, a second region in the extracellular domain of APP also interacted with PS2. Comparable results with PS1 indicate that the two presenilins share similar determinants of binding to APP. Confirming these results, SecPS2NT is able to inhibit PS1/APP interaction. Such a competition makes it unlikely that the PS/APP interaction results from nonspecific aggregation of PS in transfected cells. The physical interaction of presenilins with a region encompassing the A beta sequence of APP could be causally related to the misprocessing of APP and the production of A beta1-42.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Células COS , Mapeamento Cromossômico , DNA Complementar , Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese/fisiologia , Presenilina-1 , Presenilina-2 , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Recombinant adenoviruses were used for the expression of human amyloid precursor protein (APP) of Alzheimer's disease in primary cultures of rat cortical neurons and astrocytes. The catabolic pathways of human APP were studied 3 to 4 days after infection, when the equilibrium of APP production was reached. Although the expression of human wild type APP (WtAPP) by rat neurons induced the production of both extracellular and intraneuronal amyloid peptide (Abeta), Abeta was not detected in the culture medium of rat astrocytes producing human WtAPP. Because a low beta-secretase activity was previously reported in rodent astrocytes, we wondered whether modifications of the APP amino acid sequence at the beta-secretase clipping site would modify the astrocytic production of Abeta. Interestingly, rat astrocytes produced high amounts of Abeta after expression of human APP carrying a double amino acid substitution responsible for Alzheimer's disease in a large Swedish family (SwAPP). In both rat cortical neurons and astrocytes, the beta-secretase cleavage of the human SwAPP occurred very early in the secretion process in a cellular compartment in which a different sorting of SwAPP and WtAPP seems unlikely. These results suggest that human WtAPP and SwAPP could be processed by different beta-secretase activities.
Assuntos
Adenoviridae/genética , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/enzimologia , Córtex Cerebral/enzimologia , Endopeptidases/metabolismo , Neurônios/enzimologia , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases , Córtex Cerebral/citologia , Humanos , Ratos , Recombinação Genética , beta-Galactosidase/genéticaRESUMO
The evidence that betaA4 is central to the pathology of Alzheimer's disease (AD) came from the identification of several missense mutations in the amyloid precursor protein (APP) gene co-segregating with familial AD (FAD). In an attempt to study the proteolytical processing of mutated human APP in vivo, we have created transgenic mice expressing the human APP695 isoform with four FAD-linked mutations. Expression of the transgene was controlled by the promoter of the HMG-CR gene. Human APP is expressed in the brain of transgenic mice as shown by Western blot and immunohistology. The proteolytic processing of human APP in the transgenic mice leads to the generation of C-terminal APP fragments as well as to the release of betaA4. Despite substantial amounts of betaA4 detected in the brain of the transgenic mice, neither signs of Alzheimer's disease-related pathology nor related behavioural deficits could be demonstrated.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Mutação/genética , Regiões Promotoras Genéticas/genética , Proteínas/metabolismo , Animais , Imuno-Histoquímica , Camundongos , Camundongos TransgênicosRESUMO
We have developed mouse models for tumors affecting the epithelial cellular compartment of the thyroid which has been targeted using the bovine thyroglobulin (bTg) promoter. Transgenic mice expressing the human activated c-Ha-Ras gene developed papillary thyroid carcinomas demonstrating the oncogenic potential of activated Ras gene in the thyroid gland. Transgenic mice express the mutant form of the alpha subunit of the adenylate cyclase-coupled G alpha s with mutations at codon 201 (R201H). The expression of this mutant transgene is not by itself sufficient to produce benign tumors or even hyperplasia, but the transgenic mice have inherited a predisposition to develop thyroid adenomas.
Assuntos
Carcinoma Papilar/genética , Camundongos Transgênicos , Neoplasias da Glândula Tireoide/genética , Adenilil Ciclases/genética , Animais , Bovinos , Proteínas de Ligação ao GTP/genética , Genes ras , Humanos , Camundongos , Mutação , Regiões Promotoras Genéticas , Tireoglobulina/genéticaRESUMO
The amyloid peptide (Abeta) of Alzheimer's disease (AD) is produced by proteolytic cleavage of a larger precursor, the amyloid peptide precursor or APP. The discovery of pathogenic mutations in the APP gene provides strong evidence for the hypothesis that APP metabolism is involved in the etiology of AD. To study the metabolism of the protein, human APP has been expressed in several mammalian cell types. Insect cells, infected by a recombinant baculovirus carrying the human APP sequence, also provide an interesting expression system because these cells do not produce endogenous APP. Baculovirus-infected cells synthesize very high amounts of extracellular soluble APP, after cleavage of the transmembrane protein, as described for mammalian cells. However, we demonstrate here that insect cells do not produce Abeta from APP. These results suggest that while the enzymatic activity needed for the production of soluble APP is conserved between insect and mammalian cells, the enzymes required for the production of Abeta from APP are only expressed in mammalian cells.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Spodoptera/metabolismo , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Baculoviridae/genética , Western Blotting , Células CHO/metabolismo , Cricetinae , Meios de Cultura , Endocitose , Vetores Genéticos , Humanos , Testes de Precipitina , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Spodoptera/genética , Spodoptera/virologiaRESUMO
Glutamic acid is the major excitatory amino acid of the central nervous system which interacts with two receptor families, the ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGluRs) are coupled to G proteins and can be divided into three subgroups based on their sequence homology, signal transduction pathway and pharmacology. In this study, we describe the cloning of the cDNA encoding the human metabotropic glutamate receptor type 3 (HmGluR3). It was obtained by reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to highly conserved sequences between rat mGluRs. The receptor shows 879 amino acids with 96% amino acid sequence identity with rat mGluR3. It is strongly expressed in fetal and adult whole brain, especially in caudate nucleus and corpus callosum. The gene was identified by fluorescence in situ hybridization on chromosome 7 band q22. Activation of the human mGluR3, permanently expressed in Baby Hamster Kidney (BHK) cells, by excitatory amino acid inhibits the forskolin-stimulated accumulation of intracellular cAMP. The rank order of potency is L-glutamic acid > or = (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R)-ACPD) >> ibotenic acid > quisqualic acid. (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG, 1 mM] is without effect on inhibition of forskolin-induced cAMP accumulation by L-glutamic acid.
Assuntos
AMP Cíclico/metabolismo , Ácido Glutâmico/farmacologia , Receptores de Glutamato Metabotrópico/genética , Sequência de Aminoácidos , Animais , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , RatosRESUMO
Production of soluble amyloid peptide precursor (APP) and amyloid peptide (A beta) was measured in CHO cells transfected by the wild-type APP 695 cDNA sequence or by the same sequence carrying missense mutations associated with familial Alzheimer's disease in Sweden. Deletion of the C-terminal domain of the protein corresponding to residues 654 to 695 of APP 695 not only inhibited very significantly the internalization of APP at 37 degrees C, but also led to the secretion of an uncleaved APP in the culture medium of CHO cells. This deletion did not affect A beta production from the Swedish APP but was able to inhibit the production of the wild-type APP. These results demonstrate that, in CHO cells, the internalization of the wild-type APP is needed for A beta production, while the production of the amyloid peptide from Swedish APP is independent of the internalization process.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Mutação , Deleção de Sequência , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sequência de Bases , Células CHO , Cricetinae , Primers do DNA , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Suécia , TransfecçãoRESUMO
An interaction between the apolipoprotein E (apoE) type 3 and the microtubule-associated protein tau has been demonstrated in vitro and suggested to modulate tau phosphorylation and/or formation of paired helical filaments. To evaluate in vivo the potential interaction between tau and apoE, we have investigated the expression and phosphorylation status of tau in the brain of apoE-deficient mice, using a panel of antibodies reacting with tau in a phosphorylation-dependent manner. The pattern and intensity of immunohistochemical labelling and the pattern of tau immunoreactivity on Western blots were similar in control and apoE-deficient mice. These results suggest that a lack of expression of apoE does not interfere with the expression, distribution and phosphorylation status of tau proteins.