RESUMEN
BACKGROUND: Evidence on the effectiveness of multidomain lifestyle interventions to prevent cognitive decline in older people without dementia is mixed. Embedded in the World-Wide FINGERS initiative, FINGER-NL aims to investigate the effectiveness of a 2-year multidomain lifestyle intervention on cognitive functioning in older Dutch at risk individuals. METHODS: Multi-center, randomized, controlled, multidomain lifestyle intervention trial with a duration of 24 months. 1210 adults between 60-79 years old with presence of ≥ 2 modifiable risk factors and ≥ 1 non-modifiable risk factor for cognitive decline were recruited between January 2022 and May 2023 via the Dutch Brain Research Registry and across five study sites in the Netherlands. Participants were randomized to either a high-intensity or a low-intensity intervention group. The multidomain intervention comprises a combination of 7 lifestyle components (physical activity, cognitive training, cardiovascular risk factor management, nutritional counseling, sleep counseling, stress management, and social activities) and 1 nutritional product (Souvenaid®) that could help maintain cognitive functioning. The high-intensity intervention group receives a personalized, supervised and hybrid intervention consisting of group meetings (on-site and online) and individual sessions guided by a trained lifestyle coach, and access to a digital intervention platform that provides custom-made training materials and selected lifestyle apps. The low-intensity intervention group receives bi-monthly online lifestyle-related health advice via the digital intervention platform. Primary outcome is 2-year change on a cognitive composite score covering processing speed, executive function, and memory. RESULTS: Within 17 months, participant recruitment has been successfully completed (N = 1210; mean age: 67.7 years (SD: 4.6); 64% female). Modifiable risk factors commonly present at baseline were physical inactivity (89%), low mental/cognitive activity (50%), low social engagement (39%), hypertension (39%) and high alcohol consumption (39%). The mean body mass index of participants was 28.3 (SD: 4.2) and the total serum cholesterol was 5.4 mmol/L (SD: 1.2). CONCLUSIONS: Baseline lifestyle and clinical measurements showed successful recruitment of participants with sufficient potential for prevention. Results of FINGER-NL will provide further insight into the efficacy of a multidomain lifestyle intervention to prevent cognitive decline in older adults. TRIAL REGISTRATION: ClinicalTrials.gov (ID: NCT05256199)/2022-01-11.
Asunto(s)
Disfunción Cognitiva , Estilo de Vida , Humanos , Anciano , Femenino , Masculino , Países Bajos , Persona de Mediana Edad , Disfunción Cognitiva/prevención & control , Cognición/fisiología , Ejercicio Físico/fisiología , Factores de Riesgo , Conducta de Reducción del RiesgoRESUMEN
INTRODUCTION: Hearing loss (HL) has been associated with cognitive decline and dementia. We examined the temporal association between prevalent and incident HL and cognitive change. METHODS: A total of 1823 participants (24-82 years) from the Maastricht Aging Study (MAAS) were assessed at baseline, 6 and 12 years, including pure-tone audiometry. Linear-mixed models were used to test the association between HL and cognition, adjusted for demographics and other dementia risk factors. RESULTS: Participants with prevalent and incident HL showed a faster decline in verbal memory, information processing speed, and executive function than participants without HL. Decline was steady from baseline to 6 and 12 years for prevalent HL, but time-delayed from 6 to 12 years for incident HL. Having a hearing aid did not change associations. DISCUSSION: Findings support the notion that HL is a risk factor for cognitive decline independent of other dementia risk factors. Onset of HL preceded onset of cognitive decline. HIGHLIGHTS: We examined cognitive change in prevalent and incident hearing loss. Prevalent and incident hearing loss were associated with faster cognitive decline. For prevalent hearing loss, decline was steady from baseline to 6 and 12 years. Onset of hearing loss preceded the onset of cognitive decline. Having a hearing aid did not change the observed associations.
Asunto(s)
Disfunción Cognitiva , Demencia , Pérdida Auditiva , Humanos , Pérdida Auditiva/epidemiología , Pérdida Auditiva/complicaciones , Envejecimiento/psicología , Disfunción Cognitiva/etiología , Cognición , Demencia/etiologíaRESUMEN
A 2013 systematic review and Delphi consensus study identified 12 modifiable risk and protective factors for dementia, which were subsequently merged into the "LIfestyle for BRAin health" (LIBRA) score. We systematically evaluated whether LIBRA requires revision based on new evidence. To identify modifiable risk and protective factors suitable for dementia risk reduction, we combined an umbrella review of systematic reviews and meta-analyses with a two-round Delphi consensus study. The review of 608 unique primary studies and opinions of 18 experts prioritized six modifiable factors: hearing impairment, social contact, sleep, life course inequalities, atrial fibrillation, and psychological stress. Based on expert ranking, hearing impairment, social contact, and sleep were considered the most suitable candidates for inclusion in updated dementia risk scores. As such, the current study shows that dementia risk scores need systematic updates based on emerging evidence. Future studies will validate the updated LIBRA score in different cohorts. HIGHLIGHTS: An umbrella review was combined with opinions of 18 dementia experts. Various candidate targets for dementia risk reduction were identified. Experts prioritized hearing impairment, social contact, and sleep. Re-assessment of dementia risk scores is encouraged. Future work should evaluate the predictive validity of updated risk scores.
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Técnica Delphi , Demencia , Conducta de Reducción del Riesgo , Humanos , Demencia/epidemiología , Demencia/prevención & control , Factores de Riesgo , Estilo de Vida , Pérdida Auditiva , Sueño/fisiologíaRESUMEN
BACKGROUND: Sleep disturbances have been linked with cognitive decline and a higher risk of dementia. However, there is a lack of studies with sufficient follow-up duration, a detailed neuropsychological assessment and adequate control of main confounders. OBJECTIVE: To investigate the relation between self-reported sleep quality and cognitive decline over 12 years in cognitively healthy individuals from the general population. METHODS: We used data from the Maastricht Aging Study (MAAS), a Dutch population-based prospective cohort study of 1,823 community-dwelling adults aged 24 to 82 years at baseline. Cognitive performance was measured at baseline, 6 and 12 years on verbal memory, executive functions, and information processing speed. Sleep quality was assessed at baseline using the sleep subscale score of the 90-item Symptom Checklist (SCL-90). Additional modifiable dementia risk factors were summarized in the LIfestyle for BRAin health (LIBRA) risk score. Weighted linear mixed models tested the association between continuous scores and tertiles of subjective sleep quality and change in cognitive performances over time. Models were adjusted for age, gender, educational level, LIBRA, and use of hypnotic (sleep) medication. RESULTS: Worse sleep quality was associated with faster decline in processing speed. At older age (≥65 years), it was also associated with faster decline in verbal memory. Association were independent of other modifiable dementia risk factors and use of hypnotic medication. Directionally similar but non-significant associations were found between worse sleep quality and executive functions. CONCLUSIONS: In this population-based study across the adult age range, poor self-reported sleep was associated with accelerated cognitive decline.
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Disfunción Cognitiva , Demencia , Humanos , Calidad del Sueño , Estudios Prospectivos , Disfunción Cognitiva/diagnóstico , Envejecimiento/psicología , Cognición , Demencia/epidemiología , Demencia/complicaciones , Hipnóticos y SedantesRESUMEN
Breast cancer is the leading cause of cancer-related death in women worldwide. Mutations of the PIK3CA gene are found in approximately 25% of breast carcinomas and are reported as activators of the PI3K/AKT/mTOR pathway. This study aims to compare three assays for the somatic mutation detection of PIK3CA gene in FFPE tissues of patients with breast cancer. We compared Cobas® PIK3CA Mutation Test (Roche Diagnostics, Meylan, France), PCR amplification-refractory mutation system Scorpions® (ARMS) and High-Resolution Melting PCR assay (HRM) for the detection of PIK3CA mutations. Discrepant samples were assessed using Next Generation Sequencing (NGS). 46 FFPE breast carcinomas samples of patients treated for breast cancer have been assessed for PIK3CA mutations using the three PCR assays. Among the 46 samples, 17 (37.8%), 13 (28.36%) and 19 (41.3%) had a PIK3CA mutation, with Cobas®, ARMS and HRM assays respectively. Three different mutations of PIK3CA have been detected for one sample. Calculated kappa were 0.95[0.86;1] between Cobas® and HRM, 0.75[0.55;0.95] between Cobas® and ARMS and 0.72[0.51;0.92] between HRM and ARMS. Five samples were found with discrepant results. Our study shows that the Cobas® assay is suitable for PIK3CA mutation assessment in patients with breast cancer. HRM assay is also suitable for PIK3CA mutation assessment but requires a mutation characterization with a specific assay.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Mutación , Adhesión en Parafina/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Neoplasias de la Mama/patología , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/estadística & datos numéricos , Estudios Retrospectivos , Análisis de Secuencia de ADN/métodosRESUMEN
Correction to: Cell Death Dis. 5, e1484 (2014); https://doi.org/10.1038/cddis.2014.408 ; published online 23 October 2014.
RESUMEN
Hyperproliferating cancer cells produce energy mainly from aerobic glycolysis, which results in elevated ROS levels. Thus aggressive tumors often possess enhanced anti-oxidant capacity that impedes many current anti-cancer therapies. Additionally, in ROS-compromised cancer cells ubiquitin proteasome system (UPS) is often deregulated for timely removal of oxidized proteins, thus enabling cell survival. Taken that UPS maintains the turnover of factors controlling cell cycle and apoptosis--such as p53 or p73, it represents a promising target for pharmaceutical intervention. Enhancing oxidative insult in already ROS-compromised cancer cells appears as an attractive anti-tumor scenario. TAp73 is a bona fide tumor suppressor that drives the chemosensitivity of some cancers to cisplatin or γ-radiation. It is an important drug target in tumors where p53 is lost or mutated. Here we discovered a novel synergistic mechanism leading to potent p73 activation and cancer cell death by oxidative stress and inhibition of 20S proteasomes. Using a small-molecule inhibitor of 20S proteasome and ROS-inducer--withaferin A (WA), we found that WA-induced ROS activates JNK kinase and stabilizes phase II anti-oxidant response effector NF-E2-related transcription factor (NRF2). This results in activation of Nrf2 target--NQO1 (NADPH quinone oxidoreductase), and TAp73 protein stabilization. The observed effect was ablated by the ROS scavenger--NAC. Concurrently, stress-activated JNK phosphorylates TAp73 at multiple serine and threonine residues, which is crucial to ablate TAp73/MDM2 complex and to promote TAp73 transcriptional function and induction of robust apoptosis. Taken together our data demonstrate that ROS insult in combination with the inhibition of 20S proteasome and TAp73 activation endows synthetic lethality in cancer cells. Thus, our results may enable the establishment of a novel pharmacological strategy to exploit the enhanced sensitivity of tumors to elevated ROS and proteasomal stress to kill advanced tumors by pharmacological activation of TAp73 using molecules like WA.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Neoplasias/patología , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estrés Fisiológico , Proteínas Supresoras de Tumor/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Eliminación de Gen , Humanos , Ratones , Modelos Biológicos , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/genética , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Witanólidos/farmacologíaRESUMEN
Enteroviruses (EV) are a common cause of aseptic meningitis in children. Virological diagnosis of EV meningitis is currently based on the detection of the viral genome in the cerebrospinal fluid (CSF). This study attempted to determine the correlation and the temporality of the polymerase chain reaction (PCR) assay in serum and CSF and to evaluate the possibility of diagnosing EV infection only on the serum PCR. The EV genome was sought by RT real-time PCR (Smart Cycler EV Primer and Probe Set(®), Cepheid) in CSF and serum, collected at the same time, for all children who underwent a lumbar puncture for suspected meningitis, between 1 June and 31 July 2010 at the Versailles Hospital. Forty-four patients were included in the study. EV infection was documented for 22 of them. In 10 patients, the EV genome was detected in CSF only; in 3 patients in serum only, and in 9 patients in both. Among patients with acute EV neurological infection, viremic children were significantly younger (1.6 months versus 5.8 years; P<0.001). Viremia was detected when the serum was sampled within 30 h after the beginning of symptoms. These results confirm previous reports of early and transient viremia in young children. This preliminary study shows the limits and added value of EV PCR in serum. It suggests that in some children and under certain conditions (age >3 months, clinical and biological compatibility with a viral infection, no previous antibiotic therapy, time from symptom onset to blood sampling <30 h, PCR in serum analyzed within 3h), PCR in serum, when positive, is a possible alternative. Therefore, it may be possible to diagnose EV infection without performing a lumbar puncture in a limited number of young children (11.4% of our suspected cases). This study needs to be reinforced by a multicenter study with a broader panel of patients.
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Infecciones por Enterovirus/diagnóstico , Meningitis Viral/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Edad , Niño , Preescolar , Enterovirus/genética , Infecciones por Enterovirus/sangre , Infecciones por Enterovirus/líquido cefalorraquídeo , Femenino , Genoma Viral/genética , Humanos , Lactante , Recién Nacido , Leucocitosis/sangre , Leucocitosis/líquido cefalorraquídeo , Leucocitosis/virología , Masculino , Meningitis Viral/sangre , Meningitis Viral/líquido cefalorraquídeo , Estudios Prospectivos , Punción Espinal , Factores de Tiempo , Viremia/sangreRESUMEN
Arachis hypogaea L., commonly known as the peanut or groundnut, is an important and widespread food legume. Because the crop has a narrow genetic base, genetic diversity in A. hypogaea is low and it lacks sources of resistance to many pests and diseases. In contrast, wild diploid Arachis species are genetically diverse and are rich sources of disease resistance genes. The majority of known plant disease resistance genes encode proteins with a nucleotide binding site domain (NBS). In this study, degenerate PCR primers designed to bind to DNA regions encoding conserved motifs within this domain were used to amplify NBS-encoding regions from Arachis spp. The Arachis spp. used were A. hypogaea var. Tatu and wild species that are known to be sources of disease resistance: A. cardenasii, A. duranensis, A. stenosperma and A. simpsonii. A total of 78 complete NBS-encoding regions were isolated, of which 63 had uninterrupted ORFs. Phylogenetic analysis of the Arachis NBS sequences derived in this study and other NBS sequences from Arabidopsis thaliana, Medicago trunculata, Glycine max, Lotus japonicus and Phaseolus vulgaris that are available in public databases This analysis indicates that most Arachis NBS sequences fall within legume-specific clades, some of which appear to have undergone extensive copy number expansions in the legumes. In addition, NBS motifs from A. thaliana and legumes were characterized. Differences in the TIR and non-TIR motifs were identified. The likely effect of these differences on the amplification of NBS-encoding sequences by PCR is discussed.
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Arachis/clasificación , Arachis/genética , Genes de Plantas , Filogenia , Arabidopsis/clasificación , Arabidopsis/genética , Secuencia de Bases , Cartilla de ADN , Inmunidad Innata , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Reacción en Cadena de la PolimerasaRESUMEN
By analysis of genomic clones, we have determined the complete nucleotide sequence of the gene encoding triosephosphate isomerase (TIM; EC 5.3.1.1) in the thermophilic bacterium, Bacillus stearothermophilus. The gene encodes a 253-amino-acid TIM which is 39% identical to that of the mesophile, Escherichia coli.
Asunto(s)
Geobacillus stearothermophilus/enzimología , Triosa-Fosfato Isomerasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano , Escherichia coli/genética , Geobacillus stearothermophilus/genética , Datos de Secuencia MolecularRESUMEN
We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues.
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Moraxella/enzimología , Triosa-Fosfato Isomerasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Geobacillus stearothermophilus/enzimología , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Temperatura , Triosa-Fosfato Isomerasa/químicaRESUMEN
Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/l. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.
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Peces/metabolismo , Prolactina/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Riñón/metabolismo , Plásmidos , Prolactina/aislamiento & purificación , Prolactina/metabolismo , Unión Proteica , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The efficacy of captopril 25 mg/day as monotherapy or when necessary, in association with hydrochlorothiazide 25 mg/day, was studied during three months in 472 patients, average age 45 (17-59) years, 51% males with mild (73%) 95 less than PAD less than 104 mmHg, and moderate (27%) arterial hypertension 104 less than PAD less than 114 mmHg. Were included in the study hypertensive patients with previous antihypertensive therapy or when in the course of any previous antihypertensive treatment (52.4%) blood pressure control were not observed and side effects compromised patient's compliance. Captopril 25 mg/day was used once a day as single dose or subdivided in two daily doses (12.5 mg b.i.d.), during 30 days. If blood pressure was not normalized or dyastolic blood pressure drop was not equal or bigger than 10% after this period, it was added hydrochlorothiazide 25 mg/day. After three months under treatment, 411 (87%) patients normalized their dyastolic blood pressure DBP (less than 90 mmHg), from them, 273 (57.6%) had received only captopril and the others 138 (29.4%) with the addition of hydrochlorothiazide. The drop of mean arterial pressure, MAP = 2 DBP + 1 SBP was in average, 17.3 mmHg, in the 3 patients whose blood pressure normalized with captopril alone, and in average of 18.5 mmHg in those patients requiring addition of hydrochlorothiazide (difference without statistical significance). A small decrease of body weight, but with statistical significance (p less than 0.001) were observed during the use of captopril as monotherapy. Expressive reduction of side effects were observed during the period under captopril related to the period with previous antihypertensive therapy.
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Captopril/uso terapéutico , Hidroclorotiazida/uso terapéutico , Hipertensión/tratamiento farmacológico , Adolescente , Adulto , Presión Sanguínea/efectos de los fármacos , Ensayos Clínicos como Asunto , Quimioterapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Multicéntricos como AsuntoRESUMEN
We have isolated cDNA clones encoding tilapia prolactin (tiPRL) from a cDNA library prepared from tilapia (Oreochromis niloticus) anterior pituitary glands. A trout PRL cDNA fragment was used as hybridization probe to select the recombinant plasmids carrying the tiPRL coding sequence. Two types of PRL cDNA were isolated and their complete nucleotide sequence determined. The larger cDNA (tiPRL-I) codes for a polypeptide of 212 amino acids, including a putative signal sequence of 24 amino acids, and contains a 3' untranslated region of 787 bp. The second prolactin cDNA (tiPRL-II) encodes a polypeptide of 200 amino acids, including a presumptive signal peptide of 23 amino acids, and contains a noncoding region of 512 bp. tiPRL-I and tiPRL-II cDNA sequences are 81% similar, whereas the encoded proteins share 69% amino acid identity at optimal alignment. Mature tiPRL-I was efficiently expressed in Escherichia coli carrying a plasmid in which the tiPRL-I cDNA was under the control of the phi 10 promoter of T7 bacteriophage. The new recombinant protein representing about 45% of the total cellular proteins was found in inclusion bodies and cross-reacted with salmon PRL antiserum.
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Escherichia coli/genética , Prolactina/genética , Salmonidae/genética , Trucha/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Peces/genética , Humanos , Mamíferos/genética , Datos de Secuencia Molecular , Plásmidos , Señales de Clasificación de Proteína/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterior pituitaries. The recombinant clones carrying the cDNA sequence of tilapia growth hormone (tiGH) were selected using a fragment of the trout growth hormone (tGH) cDNA as hybridization probe. The nucleotide sequence of the full-length tiGH cDNA was determined. This cDNA encodes a protein of 204 amino acids, including the putative signal peptide of 17 amino acids. Mature tiGH cDNA was inserted in an Escherichia coli expression vector which led to the production of tiGH protein with a yield estimated to be 20% of the total bacterial proteins.
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Clonación Molecular , Escherichia coli/genética , Hormona del Crecimiento/genética , Trucha/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular/métodos , ADN/biosíntesis , ADN/genética , Peces/genética , Hormona del Crecimiento/biosíntesis , Datos de Secuencia Molecular , Plásmidos , ARN Mensajero/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
We constructed a cDNA library using mRNA isolated from rainbow trout pituitaries. Two types of cDNA clones encoding growth hormone (GH) were isolated and their complete nucleotide sequences determined. Twenty seven nucleotide substitutions in the coding region and 108 in the noncoding region distinguish the cDNAs of trout GH-I and II. Both cDNAs encode polypeptides of 210 amino acids, including a putative signal peptide of 22 amino acids, which differ by 12 residues. In both trout and salmon, GH-I mRNA is predominant, which suggests that the variation in the amount of secreted GH originates from a transcriptional event. Moreover, comparison of rainbow trout and chum salmon GH reveals that, in both cases, the predominant GH-I has mutated less than its GH-II counterpart. Mature tGH-II was expressed in Escherichia coli using the pIN-III-ompA-Hind secretion vector.
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ADN , Hormona del Crecimiento/genética , Salmonidae/genética , Trucha/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/metabolismo , Hormona del Crecimiento/metabolismo , Datos de Secuencia Molecular , Salmón , Homología de Secuencia de Ácido NucleicoRESUMEN
We describe the isolation and characterization of a cDNA for trout prolactin (tPrl). An extensive analysis of tPrl recombinant clones by restriction analysis and sequencing revealed the presence of only one form of tPrl mRNA. The deduced protein sequence consists of 210 amino acids, including a signal peptide of 23 amino acids. The amino acid sequence of the mature protein is compared among teleosts and mammals, showing two domains of strong similarity that may be involved in biological activity.
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ADN , Genes , Prolactina/genética , ARN Mensajero/análisis , Salmonidae/genética , Trucha/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Prolactina/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
The functional R6K alpha origin is composed of two DNA elements, one of 580 bp carrying the alpha origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previously identified as also required for gamma and beta origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for alpha origin activity. The function of the alpha origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the alpha origin depends on the R6K replication initiation protein pi. Within the 580 bp of the alpha origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the beta replicon. Deletion of the 96 bp or 98 bp results in inactivation of the alpha and the beta origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the alpha and the beta origins of R6K. DNA homology analysis performed on alpha, beta and gamma origin sequences, also reveals 10-23 bp sequences in the alpha and the beta origins that are related to the family of 22 bp direct repeats in the gamma origin which were shown previously to be binding sites for the pi protein.
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Replicación del ADN , Escherichia coli/genética , Genes Reguladores , Factores R , Secuencia de Bases , Deleción Cromosómica , Genes Bacterianos , Conformación de Ácido Nucleico , Homología de Secuencia de Ácido NucleicoRESUMEN
We have studied the specificity of singlet oxygen (1O2) mutagenesis in single-stranded DNA phage by analysing 1O2-induced mutations in the lac insert of the M13 mp 19 hybrid phage. 107 lac mutants were analysed showing mainly single-base substitutions with a total of 93% and 7% of 40-50 base deletion mutations. Most of the substitutions are G----T and C----A transversions with respectively 27 and 54% of the mutations. The replicative form of the M13 mp 19 DNA (RFDNA) was used as substrate for the 1O2 reactions, there are then two types of progeny phages DNA's. As guanine residues are the targets of the oxidation, it appears that both types of transversions are provided by one type of lesion: the guanine oxidised by 1O2 is read like a thymine by E. coli DNA polymerase-I.