RESUMEN
Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a dual luciferase reporter assay in KISS1R-transfected HEK293T cells. In general terms the results reflect the outcomes of other assay formats and a number of potent agonists were identified among the analogues, including ß(2) -hTyr-modified and fluorescently labelled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Aminoácidos/química , Kisspeptinas/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Ligandos , Receptores Acoplados a Proteínas G/química , Receptores de Kisspeptina-1RESUMEN
UNLABELLED: Self-assessed masticatory ability has been shown to be significantly related to general health among elderly persons. OBJECTIVE: To identify oral factors associated with the self-assessed masticatory ability. BASIC RESEARCH DESIGN: Cross-sectional study. PARTICIPANTS: A total of 736 community-dwelling elderly persons. MAIN OUTCOME MEASURES: Data on background factors and the self-assessed masticatory ability were collected by questionnaire. An intraoral examination examined the pattern of posterior occluding pairs of natural teeth (POPs), the WHO Community Periodontal Index of Treatment Needs (CPI) and denture-related factors such as use of dentures, pain when using dentures and stability and retention of dentures. Chi-squared tests examined the relationships between the self-assessed masticatory ability and the background factors and oral conditions. Ordinal regression models were constructed with the self-assessed masticatory ability as the dependent variable and oral conditions as the principal independent variables, to adjust for the potential confounding variables. RESULTS: Self-assessed impairment of masticatory ability was associated with lost POPs (p < 0.001) and CPI (p = 0.012). In the participants with lost POPs, self-assessed impairment of masticatory ability was associated with not using dentures and pain when using dentures (p < 0.001). In the totally edentulous subjects, impairment of masticatory ability was not associated with stability and retention of dentures (p = 0.070). CONCLUSIONS: Factors affecting self-assessed masticatory ability include the pattern of POPs, periodontal status, denture use and pain when using dentures.
Asunto(s)
Vida Independiente , Masticación/fisiología , Anciano , Anciano de 80 o más Años , Diente Premolar/patología , Enfermedad Crónica , Estudios Transversales , Retención de Dentadura , Dentaduras , Escolaridad , Empleo , Composición Familiar , Femenino , Estado de Salud , Humanos , Arcada Edéntula/clasificación , Arcada Parcialmente Edéntula/clasificación , Masculino , Diente Molar/patología , Índice Periodontal , Autoevaluación (Psicología) , Participación Social , Encuestas y CuestionariosRESUMEN
Increasing need for long-term care in older adults is expected with the ageing of Japan's population. The aim of the present study was to show the relationship between perceived chewing ability and long-term care needs for over 5 years in elderly persons. The chewing ability of 812 elderly persons living independently was evaluated at baseline using self-assessed masticatory ability, and it was classified into one of three categories: ability to chew all foods (good masticatory ability), ability to chew only slightly hard food (fair masticatory ability), and ability to only chew soft or pureed food (poor masticatory ability). Participants' care needs were then followed through Japan's long-term care insurance system for over 5 years. The log-rank test and Cox proportional hazard model were used to examine statistical differences in the frequency of care-needs certification between participants with good and fair or poor masticatory ability. Among participants aged 65-79 years, the frequency of care-needs certification was significantly higher in those with fair or poor masticatory ability than in those with good masticatory ability, and the relative hazard ratio was significantly higher in those with fair or poor masticatory ability than in those with good masticatory ability, after adjusting for age, gender, current employment status, educational background, social interaction, chronic medical conditions and dentition status. These relationships were not found among those aged 80-93 years. Impairment in perceived chewing ability may be associated with higher incidence of certification in Japan's long-term care insurance system among elderly persons aged 65-79.
Asunto(s)
Cuidados a Largo Plazo/estadística & datos numéricos , Masticación/fisiología , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Humanos , Seguro de Cuidados a Largo Plazo/estadística & datos numéricos , Masculino , Salud Rural , Autoinforme , Autoevaluación (Psicología)RESUMEN
Although division site positioning in rod-shaped bacteria is generally believed to occur through the combined effect of nucleoid occlusion and the Min system, several lines of evidence suggest the existence of additional mechanisms. Studies using outgrown spores of Bacillus subtilis have shown that inhibiting the early stages of DNA replication, leading up to assembly of the replisome at oriC, influences Z ring positioning. Here we examine whether Z ring formation at midcell under various conditions of DNA replication inhibition is solely the result of relief of nucleoid occlusion. We show that midcell Z rings form preferentially over unreplicated nucleoids that have a bilobed morphology (lowering DNA concentration at midcell), whereas acentral Z rings form beside a single-lobed nucleoid. Remarkably however, when the DnaB replication initiation protein is inactivated midcell Z rings never form over bilobed nucleoids. Relieving nucleoid occlusion by deleting noc increased midcell Z ring frequency for all situations of DNA replication inhibition, however not to the same extent, with the DnaB-inactivated strain having the lowest frequency of midcell Z rings. We propose an additional mechanism for Z ring positioning in which the division site becomes increasingly potentiated for Z ring formation as initiation of replication is progressively completed.
Asunto(s)
Bacillus subtilis/citología , Bacillus subtilis/genética , División Celular , Nucléolo Celular/genética , Replicación del ADN , Bacillus subtilis/metabolismo , Proteínas Bacterianas , Nucléolo Celular/metabolismo , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismoRESUMEN
The escalating medical costs are a social problem in many countries. Masticatory ability is thought to be related to the general health conditions. The purpose of this study was to show relationships between self-assessed masticatory ability and medical costs among the elderly living independently in community. Data on background factors and self-assessed masticatory ability were collected from 702 Japanese elderly persons by questionnaires. An intra-oral examination was performed to examine the number of remaining teeth. Self-assessed masticatory ability was classified into one of three categories: ability to chew all kinds of food (Good), ability to chew only slightly hard food (Fair) or ability to chew only soft or pureed food (Poor). Data on the annual medical excluding dental costs were obtained from the Japanese National Health Insurance system. The Kruskal-Wallis test was used to examine differences in outpatient costs and hospitalisation costs among the three groups of self-assessed masticatory ability. Univariate unconditional logistic regression models and multivariate logistic regression models were used with medical costs as the dependent variable and self-assessed masticatory ability as the principal independent variable. A significant difference (P=0·039) in hospitalisation costs but not outpatient costs was found among the three groups of self-assessed masticatory ability. The multivariate logistic regression analysis showed that severely impaired masticatory ability (Poor) was significantly related to higher costs of hospitalisation. Self-assessed impairment of masticatory ability may be a significant and independent indicator of higher costs of hospitalisation among community-dwelling elderly persons.
Asunto(s)
Costos de la Atención en Salud/estadística & datos numéricos , Costos de Hospital/estadística & datos numéricos , Hospitalización/economía , Masticación , Anciano , Anciano de 80 o más Años , Atención Ambulatoria/economía , Femenino , Humanos , Vida Independiente , Japón , Modelos Logísticos , Masculino , Programas Nacionales de Salud/economía , Autoevaluación (Psicología) , Estadísticas no Paramétricas , Encuestas y CuestionariosRESUMEN
The aim of the present study was to elucidate whether self-assessed masticatory ability (masticatory ability) is significantly related to muscle strength of the body evaluated as handgrip strength and skeletal muscle mass of the whole body (kg) (SMM) after adjusting for confounding variables, including, age, gender, height, weight, employment status, type of household, educational background, social interaction, chronic medical conditions, smoking habit, drinking habits and dentition status among the elderly. A total of 381 persons aged 67-74 years were enrolled. Masticatory ability was classified into one of three categories: ability to chew all kinds of food, ability to chew only slightly hard food or ability to chew only soft or pureed food. Handgrip strength was measured, and bioimpedance analysis was used to estimate SMM. One-way analysis of variance and Bonferroni methods were used to examine differences in handgrip strength and SMM among the three groups of masticatory ability. An ordinal regression model was conducted with masticatory ability as the dependent variable and handgrip strength as the principal independent variable. Handgrip strength was significantly lower in those individuals who could chew only soft or pureed food than in those individuals who could chew all kinds of food. No significant difference in SMM was found among the three groups of masticatory ability. Masticatory ability was significantly related to handgrip strength after adjusting for SMM, dentition status and background factors. Chewing ability may be related to muscle strength of the body evaluated as handgrip strength, but not evaluated as SMM.
Asunto(s)
Fuerza de la Mano/fisiología , Masticación/fisiología , Músculo Esquelético/fisiología , Anciano , Estudios Transversales , Femenino , Alimentos , Humanos , Japón , Masculino , Autoevaluación (Psicología)RESUMEN
CD44 is a highly glycosylated cell adhesion molecule that is involved in lymphocyte infiltration of inflamed tissues. We have demonstrated previously that sialic acid residues of CD44 negatively regulates its receptor function and CD44 plays an important role in the accumulation of T helper type 2 (Th2) cells in the airway of a murine model of acute asthma. Here we evaluated the role of sialidase in the hyaluronic acid (HA) receptor function of CD44 expressed on CD4+ T cells, as well as in the development of a mite antigen-induced murine model of acute asthma. Splenic CD4+ T cell binding of HA was examined with flow cytometry. Expression of sialidases (Neu1, Neu2, Neu3 and Neu4) in spleen cells was evaluated by quantitative real-time reverse transcription-polymerase chain reaction. Airway inflammation and airway hyperresponsiveness (AHR) were evaluated in the asthmatic Neu1-deficient mouse strain SM/J model. Splenic CD4+ T cells from asthmatic model mice displayed increased HA receptor activity of CD44 after culture with the antigen, along with characteristic parallel induction of sialidase (Neu1) expression. This induction of HA binding was suppressed significantly by a sialidase inhibitor and was not observed in SM/J mice. Th2 cytokine concentration and absolute number of Th2 cells in the bronchoalveolar lavage fluid, and AHR were decreased in SM/J mice. In conclusion, HA receptor activity of CD44 and acute asthmatic reactions, including Th2-mediated airway inflammation and AHR, are dependent upon Neu1 enzymatic activity. Our observation suggests that Neu1 may be a target molecule for the treatment of asthma.
Asunto(s)
Asma/inmunología , Asma/fisiopatología , Receptores de Hialuranos/metabolismo , Neuraminidasa/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Animales , Antígenos Dermatofagoides/inmunología , Asma/genética , Azidas/farmacología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/fisiopatología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Recuento de Células , Quimiocina CCL11/metabolismo , Inhibidores Enzimáticos/farmacología , Eosinófilos/citología , Eosinófilos/inmunología , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Expresión Génica/inmunología , Ácido Hialurónico/metabolismo , Inflamación/inmunología , Inflamación/patología , Interferón gamma/metabolismo , Interleucinas/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones Mutantes , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Neuraminidasa/farmacología , Aglutinina de Mani/metabolismo , Ácidos Siálicos/farmacología , Bazo/citología , Bazo/enzimología , Bazo/inmunología , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/citología , VacunaciónAsunto(s)
Proteínas del Citoesqueleto/genética , Neuropatía Axonal Gigante/genética , Neuropatía Axonal Gigante/patología , Disomía Uniparental/genética , Secuencia de Bases , Exoma/genética , Femenino , Homocigoto , Humanos , Imagen por Resonancia Magnética , Datos de Secuencia Molecular , Conducción Nerviosa/genética , Conducción Nerviosa/fisiología , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The three-dimensional double-echo steady-state (3D-DESS) sequence is being used to investigate sequence parameters for the assessment of knee cartilage by magnetic resonance imaging (MRI). With the flip angle (FA) values that are commonly used, contrast between cartilage and synovial fluid may be too low to delineate minor lesions of the cartilage surface. PURPOSE: To investigate the value of FA maximizing contrast between cartilage and synovial fluid using the 3D-DESS sequence. MATERIAL AND METHODS: In 10 healthy volunteers (five men, five women; age range 23-48 years), 3D-DESS imaging was performed to capture median sagittal sections of the knee by adjusting FA from 10 degrees to 90 degrees. Cartilage-synovial fluid contrast-to-noise ratio (CNR) was measured to determine the value of FA maximizing CNR. Water-excitation pulse was used to suppress fat signals. MRI was performed at 1.0 T using a circular polarization flex coil. RESULTS: Cartilage-synovial fluid CNR was highest with 90 degrees FA, yielding images with good contrast between cartilage and synovial fluid. CONCLUSION: Simply by increasing FA to 90 degrees in 3D-DESS imaging, the contrast between cartilage and synovial fluid increased substantially. Subtle cartilage lesions may thus be detectable using 3D-DESS sequences.
Asunto(s)
Aumento de la Imagen/métodos , Imagenología Tridimensional/métodos , Rodilla/anatomía & histología , Imagen por Resonancia Magnética/métodos , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Adulto JovenRESUMEN
Human plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is markedly upregulated in human cancers, leading to apoptosis suppression. To define molecular mechanisms and the possible target for NEU3, its encoding gene was silenced by small interference RNA (siRNA) or overexpressed in human cells. NEU3 siRNA-induced apoptosis with no special stimuli in HeLa cells, accompanied with decreased Bcl-xL and increased mda7 and GM3 synthase mRNA levels, whereas overexpression resulted in the opposite. Carcinoma HT-29 and MCF-7 cells appeared to be similarly affected, but normal cell lines demonstrated no significant changes. NEU3 siRNA was found to inhibit and NEU3 overexpression to stimulate Ras activation with consequent influence on extracellular signal-regulated kinases and Akt. Ras activation by NEU3 was abrogated by PP2 (src inhibitor) or AG1478 (epidermal growth factor receptor (EGFR) inhibitor), and NEU3 actually enhanced EGF-stimulated tyrosine-phosphorylation of EGFR, suggesting that the upstream targets might be tyrosine kinases including src and EGFR, and the subsequent stimulation of Ras cascade leads to the inhibition of cell apoptosis. Glycolipid changes observed seemed to be one of the causes of the cell effects. NEU3 may thus be an essential gene for cancer cell survival and siRNAs targeting this protein could have utility for gene-based therapy of human cancers.
Asunto(s)
Membrana Celular/enzimología , Membrana Celular/patología , Transformación Celular Neoplásica/patología , Neuraminidasa/fisiología , Apoptosis/genética , Línea Celular , Membrana Celular/genética , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Silenciador del Gen , Células HT29 , Células HeLa , Humanos , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Interferencia de ARN , ARN Interferente Pequeño/fisiología , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Cisplatin and ifosfamide are considered among the most active drugs in both neoadjuvant and salvage treatments for patients with cervical cancer. Nedaplatin is an analog of cisplatin and it exhibits lesser nephrotoxicity, neurotoxicity, and gastrointestinal toxicity than cisplatin. This study aimed to determine the recommended dosage of nedaplatin plus ifosfamide chemoradiotherapy for advanced squamous cell carcinoma (SCC) of the uterine cervix. Beginning with a dose of 65 mg/m(2), nedaplatin (day 1) combined with ifosfamide 1 g/m(2) (days 1-5) was designed to be administered for three cycles (minimum: two cycles); its dose was gradually escalated up to 80 mg/m(2). Dose-limiting toxicity (DLT) was defined as a more than 7-day delay in the planned radiation therapy and/or planned chemotherapy (prior to the completion of two cycles) due to toxicity. Chemotherapy was not interrupted prior to the completion of two cycles in any patients. Of the 12 patients, 11 received three cycles of chemotherapy. DLT did not occur in any patient. We confirmed a clinical complete response (CR) in ten and partial response (PR) in two patients. The median follow-up period was 39 months (range: 18-57 months). Ten patients (83%) were alive and disease free, one patient was alive with disease, and only one patient died due to the disease. Nedaplatin and ifosfamide combination chemotherapy is a feasible and active chemoradiation strategy for patients with advanced SCC of the uterine cervix. With the ifosfamide dose fixed to 1 g/m(2), the recommended nedaplatin dosage was determined to be 80 mg/m(2) to be administered for three cycles.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/radioterapia , Ifosfamida/uso terapéutico , Compuestos Organoplatinos/uso terapéutico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/radioterapia , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma de Células Escamosas/patología , Terapia Combinada/efectos adversos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ifosfamida/administración & dosificación , Persona de Mediana Edad , Estadificación de Neoplasias , Compuestos Organoplatinos/administración & dosificación , Neoplasias del Cuello Uterino/patologíaRESUMEN
The nymphal locomotion ability (walking distance) of the stenophagous bean bug Riptortus pedestris (Fabricius) was studied in each instar. We measured the walking distance using two systems. The walking distance in photophase was measured for 6 h using a tracking system with a charge coupled device (CCD) camera and computer software. The daily activity of nymphs was measured by an actograph system counting the number of infrared beam intercepts. The actograph data were converted to distance using a linear regression against the data of the tracking system. The longevity of nymphs without food was also studied to estimate the potential walking distance. Using both the tracking and actograph systems, it was determined that first instars walked less than the other instars (only 10.7 m within 6 h). The second to fifth instars could move 20-25 m within 6 h, and this distance did not differ among instar. This indicates that first instars seldom move after hatching in the field. The walking distance for 24 h varied and was greatest for the third instars (80.8 m). The potential longevity of nymphs was found to increase with instar age. Potential locomotion ability (walking distance for 24 hxpotential longevity) was high in the third to fifth instars (approximately 340 m). The potential locomotion ability for the second instars was relatively low compared with the elder instars (approximately 180 m). From these results, nymphs of R. pedestris seem to adapt by identifying feeding site locations after hatching and elder instars may be able to find a novel feeding site after the degradation of previous habitat.
Asunto(s)
Heterópteros/fisiología , Locomoción/fisiología , Animales , Conducta Alimentaria , Longevidad , Actividad Motora/fisiología , Ninfa/fisiologíaRESUMEN
OBJECTIVES: To identify the relationship between diet-related indicators and overweight and obesity in older adults in rural Japan. DESIGN: Cross-sectional survey. SETTING: Obira, Hokkaido, Japan. PARTICIPANTS: Local residents aged between 65 and 74 years, except for those with poor health, were included. INTERVENTION: A health- and diet-related questionnaire was applied to participants house-to-house by trained health professionals. MEASUREMENTS: The following indicators were examined: number of meals, number of balanced meals, food diversity, food group diversity, chewing ability, alcohol intake habit, smoking habit, instrumental activities of daily living scores, age, and residing alone or with family. RESULTS: Of 550 residents, 317 residents completely responded to the questionnaire. Of these, 41 were had low body mass index (BMI≤20) and were excluded. This resulted in a sample of 307 subjects comprising 117 men and 190 women; 37.6% and 46.8% of men and women were classified as obese (BMI≥25), respectively. Women with a normal BMI (20Asunto(s)
Dieta
, Obesidad/epidemiología
, Sobrepeso/epidemiología
, Población Rural
, Actividades Cotidianas
, Anciano
, Índice de Masa Corporal
, Estudios Transversales
, Femenino
, Humanos
, Japón
, Estilo de Vida
, Modelos Logísticos
, Masculino
, Comidas
, Obesidad/prevención & control
, Sobrepeso/prevención & control
, Encuestas y Cuestionarios
RESUMEN
Human transitional cell carcinomas of the bladder frequently reveal chromosomal abnormalities that span a range between chromosome 9p12 and 9qter, even at early stages of bladder carcinogenesis. Because the gene that encodes an actin-regulatory protein, gelsolin, is localized in chromosome 9q33, we examined the expression of gelsolin in a number of human bladder cancer cell lines and tissues. In all 6 cell lines and in 14 of the 18 tumor tissues (77.8%), gelsolin expression was undetectable or extremely low in comparison with its expression in normal bladder epithelial cells. Furthermore, upon the introduction of the exogenous human or mouse authentic gelsolin cDNA into a human bladder cancer cell line, UMUC-2, gelsolin transfectants of UMUC-2 greatly reduced the colony-forming ability and the tumorigenicity in vivo. These results suggest that gelsolin plays a key role as a tumor suppressor in human urinary bladder carcinogenesis.
Asunto(s)
Gelsolina/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Vejiga Urinaria/metabolismo , Secuencia de Bases , Northern Blotting , Western Blotting , Gelsolina/fisiología , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Transfección , Células Tumorales Cultivadas , Ensayo de Tumor de Célula Madre , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Serotonin (5-HT) is a key regulator of mood and sexual behaviors. 5-HT reuptake inhibitors have been used as antidepressants. Really interesting new gene (RING) finger proteins have been associated with 5-HT regulation but their role remains largely unknown. Some RING finger proteins are involved in the serotonergic system, therefore, we speculate that the gene expression of RING finger protein38 (rnf38) is regulated by the serotonergic system. In the present study, we aimed to identify the full length sequence of medaka (Oryzias latipes) rnf38 mRNA and investigate its association with the serotonergic system using an antidepressant, citalopram (CIT). We identified the full length rnf38 cDNA, which consisted of 2726 nucleotides spanning 12 exons and the deduced protein sequence consisting of 518 amino acid residues including a RING finger domain, a KIT motif and a coiled-coil domain. Medaka exposed to 10(-7)M of CIT showed anxiety-like behavior. The expressions of 5-HT-related genes, pet1, solute carrier family 6, member 4A (slc6a4) and tryptophan hydroxylase (tph2) were significantly low (P<0.05) in the hindbrain. On the other hand, rnf38 gene was significantly high (P<0.05) in the telencephalon and the hypothalamus. This shows that 5-HT synthesis and transport in the hindbrain is suppressed by CIT, which induces rnf38 gene expression in the forebrain where 5-HT neurons project. Thus, the expression of rnf38 is negatively regulated by the serotonergic system.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Neuronas/metabolismo , Serotonina/metabolismo , Animales , Proteínas Portadoras/genética , Citalopram/farmacología , Clonación Molecular/métodos , Expresión Génica/fisiología , Masculino , Oryzias , ARN Mensajero , Triptófano Hidroxilasa/metabolismo , Dedos de ZincRESUMEN
We constructed oriC-deleted mutants of Bacillus subtilis by integrating the minimal replication region of plasmid pLS32 into the proA (115 degrees), spoIIIJ (360 degrees) and thrS (256 degrees) loci of the chromosome, respectively. All three mutants produced anucleate cells and the DNA/protein ratio was lower than that of the wild-type strain when grown in nutrient broth. However, when grown in minimal-glucose medium, the frequency of anucleate cells was reduced in all of them and the DNA/protein ratio was restored to normal. Especially, the oriC-deleted mutant in which the plasmid was integrated near oriC produced almost no anucleate cell. These results indicate that initiation frequency of chromosome replication from the integrated plasmid origin were reduced disproportionately to cell mass increase in rich medium, which in turn disrupted coordination between DNA replication cycle and cell division cycle. The locations of the plasmid origin relative to the natural oriC locus affected the production of anucleate cell remarkably, suggesting that partition mechanism of chromosome was also impaired by the translocation of its replication origin.
Asunto(s)
Bacillus subtilis/genética , Ciclo Celular/genética , ADN Bacteriano/análisis , Mutagénesis Sitio-Dirigida , Origen de Réplica , Bacillus subtilis/citología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Ciclo Celular , Cromosomas Bacterianos/genética , ADN Helicasas/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , AdnB Helicasas , Microscopía Fluorescente , Plásmidos , Respuesta SOS en Genética , beta-Galactosidasa/análisisRESUMEN
Buchnera aphidicola is an intracellular prokaryote (endosymbiont) that lives in the body cavity of the aphid. Phylogenetic studies indicated that it is closely related to Escherichia coli and members of Enterobacteria. The gene order of the region containing the dnaA gene is well conserved in many bacteria. Seven genes of the endosymbiont of the aphid Schizaphis graminum, gyrB, dnaN, dnaA, rpmH, rnpA, yidD, and 60K. were found to be homologous in sequence and relative location to those of E. coli. We have further sequenced the region downstream of the 60K gene to elucidate the boundary of the conserved region, and found that one more gene, thdF, is conserved. The comparison of gene organizations of the dnaA region of the related bacteria supported the close phylogenetic relationship of B. aphidicola to E. coli. In addition, we have identified groES and groEL genes next to the thdF gene. GroEL protein was reported to be expressed at an elevated level in the endosymbionts of aphids, and is considered to play an important role in their association with the aphid host. Comparison of the structure of the groE operon with that of the endosymbiont of the aphid Acyrthosiphon pisum revealed the conservation of a sequence resembling the E. coli consensus heat shock promoter, and this sequence may be responsible for the high expression of the groEL gene in aphid endosymbionts.
Asunto(s)
Áfidos/microbiología , Proteínas Bacterianas/genética , Chaperonina 10/genética , Chaperonina 60/genética , ADN Bacteriano , Genes Bacterianos , Animales , Secuencia de Bases , Escherichia coli/genética , Evolución Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Operón , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , SimbiosisRESUMEN
Analysis of replication intermediates produced by in vitro replication of the Bacillus subtilis oriC plasmid revealed that replication initiated at an untranslatable DnaA box region downstream of the dnaA gene. In order to show that replication of the B. subtilis chromosome also starts at the same region in vivo, we have analyzed replication intermediates generated in vivo by the two-dimensional gel method. A bubble arc was detected when the downstream region was used as a probe. In contrast, only a simple Y arc was found when the upstream DnaA box region required for autonomous replication of the oriC plasmid was used as a probe. Furthermore, the bubble arc ranged from unit to almost double the size of a fragment in which the downstream region was located near the middle. These results indicate that replication of the B. subtilis chromosome initiates at the downstream DnaA box region of the dnaA gene and proceeds bidirectionally.
Asunto(s)
Bacillus subtilis/genética , Cromosomas Bacterianos , Electroforesis en Gel Bidimensional , Origen de Réplica , Proteínas Bacterianas/genética , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Familia de MultigenesRESUMEN
The phylogenetic position of the symbiotic oxymonad Dinenympha exilis, found in the hindgut of the lower termite Reticulitermes speratus, was determined by analysis of translation elongation factor 1 alpha (EF-1 alpha). cDNA corresponding to a major part of the amino acid coding region of EF-1 alpha mRNA was amplified by the reverse transcription polymerase chain reaction (RT-PCR) method from total mRNA of termite hindgut microorganisms without cultivation. The product was cloned into a plasmid vector, pGEM-T, and the clones were isolated and sequenced. One of the EF-1 alpha clones isolated was assigned to the protist D. exilis by whole-cell in-situ hybridization using a specific oligonucleotide probe with enzymatic signal amplification. The deduced amino acid sequence was aligned with those of other eukaryotic and archaeabacterial EF-1 alpha s, and the phylogenetic relationships among early branching eukaryotes were inferred by using the distance matrix method and the maximum parsimony method. The phylogenetic analysis indicated that the D. exilis offshoot occurred before mitochondria-containing organisms and D. exilis branched out after the diplomonads clade. These results indicate that the oxymonad D. exilis is one of the early branching organisms and suggest that the oxymonads form a lineage independent of other early branching organisms.