RESUMEN
Trichinellosis is an important zoonotic parasitic disease worldwide and is principally caused by ingesting animal meat containing Trichinella infective larvae. Aspartyl aminopeptidase is an intracytoplasmic metalloproteinase that specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids (aspartic acid and glutamate), and plays an important role in the metabolism, growth and development of organisms. In this study, a novel T. spiralis aspartyl aminopeptidase (TsAAP) was cloned and expressed, and its biological properties and roles in worm growth and development were investigated. The results revealed that TsAAP transcription and expression in diverse T. spiralis stages were detected by RT-PCR and Western blotting, and primarily localized at cuticle, stichosome and intrauterine embryos of this nematode by immunofluorescence test. rTsAAP has the enzymatic activity of native AAP to hydrolyze the substrate H-Glu-pNA. There was a specific binding between rTsAAP and murine erythrocyte, and the binding site was localized in erythrocyte membrane proteins. Silencing of TsAAP gene by specific dsRNA significantly reduced the TsAAP expression, enzymatic activity, intestinal worm burdens and female fecundity. The results demonstrated that TsAAP participates in the growth, development and fecundity of T. spiralis and it might be a potential target molecule for anti-Trichinella vaccines.
Asunto(s)
Glutamil Aminopeptidasa , Proteínas del Helminto/genética , Trichinella spiralis/enzimología , Animales , Clonación Molecular , Eritrocitos/parasitología , Femenino , Glutamil Aminopeptidasa/genética , Ratones , Ratones Endogámicos BALB C , Trichinella spiralis/genética , TriquinelosisRESUMEN
Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop vaccine to prevent T. spiralis infection in food animals. T. spiralis aspartic protease-2 (TsASP2) has been demonstrated to play a crucial role in larval invasion of intestinal epithelium cells (IECs). The purpose of this study was to assess the interaction between TsASP2 and IECs and to investigate the immune protection elicited by vaccination with rTsASP2. The results showed that the enzymatic activity of native aspartic protease was detected in crude proteins of all T. spiralis development stages other than NBL stage, the highest activity was observed in the IIL stage. The results of Western blot showed that TsASP2 protein was expressed at ML, IIL and AW but not NBL, and the TsASP2 expression level at IIL stage was significantly higher than those of other three worm stages (P < 0.05). The specific binding between rTsASP2 and IECs was observed by immunofluorescence test (IFT) and confocal microscopy, and the binding site was localized at the IEC membrane and this binding ability was inhibited by aspartic protease specific inhibitor pepstain A. The results of ELISA showed that the binding ability was protein dose-dependent. Vaccination with rTsASP2 triggered a mixed Th1/Th2 humoral and mucosal immune responses, as demonstrated by the elevation levels of Th1/Th2 cytokines (IFN-γ and IL-4) secreted by the spleen and mesenteric lymph nodes (MLNs) of immunized mice. The mice vaccinated with rTsASP2 exhibited a 54.17% reduction in enteral adult worms and a 54.58% reduction in muscle larvae after T. spiralis challenge. The results demonstrated that TsASP2 might be a potential molecular target for anti-Trichinella vaccines.
Asunto(s)
Proteasas de Ácido Aspártico/metabolismo , Enterocitos/parasitología , Proteínas del Helminto/metabolismo , Mucosa Intestinal/parasitología , Triquinelosis/parasitología , Animales , Femenino , Inmunidad Humoral , Inmunidad Mucosa , Ratones , Ratones Endogámicos BALB C , Trichinella spiralis/enzimología , Triquinelosis/inmunología , Vacunación , Vacunas/inmunologíaRESUMEN
A T. spiralis serine protease 1.2 (TsSP1.2) was identified in the muscle larvae (ML) and intestinal larvae surface/excretory-secretory (ES) proteins by immunoproteomics. The aim of this study was to determine the TsSP1.2 function in the process of T. spiralis intrusion, growth and reproduction by using RNA interference (RNAi). RNAi was used to silence the expression of TsSP1.2 mRNA and protein in the nematode. On 2 days after the ML were electroporated with 2 µM of TsSP1.2-specific siRNA 534, TsSP1.2 mRNA and protein expression declined in 56.44 and 84.48%, respectively, compared with untreated ML. Although TsSP1.2 silencing did not impair worm viability, larval intrusion of intestinal epithelium cells (IEC) was suppressed by 57.18% (P < 0.01) and the suppression was siRNA-dose dependent (r = 0.976). Infection of mice with siRNA 534 transfected ML produced a 57.16% reduction of enteral adult burden and 71.46% reduction of muscle larva burden (P < 0.05). Moreover, silencing of TsSP1.2 gene in ML resulted in worm development impediment and reduction of female fertility. The results showed that silencing of TsSP1.2 by RNAi inhibited larval intrusion and development, and reduced female fecundity. TsSP1.2 plays a crucial role for worm invasion and development in T. spiralis life cycle, and is a potential vaccine/drug target against Trichinella infection.
Asunto(s)
Proteínas del Helminto/metabolismo , Serina Proteasas/metabolismo , Trichinella spiralis/enzimología , Animales , Femenino , Proteínas del Helminto/genética , Mucosa Intestinal/parasitología , Larva , Masculino , Ratones , Ratones Endogámicos BALB C , Músculos/parasitología , Interferencia de ARN , ARN Interferente Pequeño , Serina Proteasas/genética , Trichinella spiralis/genéticaRESUMEN
A putative serine protease of T. spiralis (TsSP) was expressed in Escherichia coli and its potential as a diagnostic antigen was primarily assessed in this study. Anti-Trichinella IgG in serum samples from T. spiralis different animal hosts (mice, rats, pigs and rabbits) were detected on Western blot analysis with rTsSP. Anti-Trichinella antibodies were detected in 100% (30/30) of experimentally infected mice by rTsSP-ELISA. Cross-reactions of rTsSPELISA were not found with sera from mice infected with other parasites (S. erinaceieuropaei, S. japonicum, C. sinensis, A. cantonensis and T. gondii) and sera from normal mice. There was no statistical difference in antibody detection rate among mice infected with the encapsulated Trichinella species (T. spiralis, T. nativa, T. britovi, and T. nelsoni) (P>0.05). The results of rTsSP-ELISA showed that serum specific antibody IgG in mice infected with 100 or 500 T. spiralis muscle larvae (ML) were detectable early at 7-8 dpi, but not detected by ML ES antigen-ELISA prior to 10-12 dpi. Specific anti-Trichinella IgG was detected in 100% (18/18) of infected pigs by rTsSP-ELISA and ES-ELISA, but no specific antibodies was not detected in 20 conventionally raised normal pigs by two antigens. The results showed the rTsSP had the potential for early serodiagnosis of animal Trichinella infection, however it requires to be assayed with early infection sera of swine infected with Trichinella and other parasites.
Asunto(s)
Serina Proteasas/aislamiento & purificación , Trichinella spiralis/enzimología , Triquinelosis/diagnóstico , Animales , Anticuerpos Antihelmínticos/sangre , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Conejos , Ratas , Ratas Wistar , Pruebas Serológicas , Sus scrofaRESUMEN
Infection with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by the bacterial enteropathogen Citrobacter rodentium. To clarify the underlying mechanism, we analyzed fecal microbiota composition of control and helminth-infected mice and evaluated the functional role of compositional differences by microbiota transplantation experiments. Our results showed that infection of Balb/c mice with H. polygyrus resulted in significant changes in the composition of the gut microbiota, characterized by a marked increase in the abundance of Bacteroidetes and decreases in Firmicutes and Lactobacillales. Recipients of the gut microbiota from helminth-infected wide-type, but not STAT6-deficient, Balb/c donors had increased fecal pathogen shedding and significant worsening of Citrobacter-induced colitis compared to recipients of microbiota from control donors. Recipients of helminth-altered microbiota also displayed increased regulatory T cells and IL-10 expression. Depletion of CD4+CD25+ T cells and neutralization of IL-10 in recipients of helminth-altered microbiota led to reduced stool C. rodentium numbers and attenuated colitis. These results indicate that alteration of the gut microbiota is a significant contributor to the H. polygyrus-induced exacerbation of C. rodentium colitis. The helminth-induced alteration of the microbiota is Th2-dependent and acts by promoting regulatory T cells that suppress protective responses to bacterial enteropathogens.
Asunto(s)
Citrobacter rodentium/fisiología , Colitis/inmunología , Colon/patología , Infecciones por Enterobacteriaceae/inmunología , Microbiota/inmunología , Nematospiroides dubius/inmunología , Infecciones por Strongylida/inmunología , Linfocitos T Reguladores/inmunología , Células Th2/inmunología , Animales , Carga Bacteriana , Colitis/microbiología , Colitis/parasitología , Colon/microbiología , Colon/parasitología , Progresión de la Enfermedad , Heces/microbiología , Femenino , Inmunomodulación , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismoRESUMEN
Trichinella spiralis surface proteins are directly exposed to the host's immune system, making them the main target antigens which induce the immune responses and may play an important role in the larval invasion and development process. The analysis and characterization of T. spiralis surface proteins could provide useful information to elucidate the host-parasite interaction, identify the early diagnostic antigens and the targets for vaccine. The purpose of this study was to identify the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) Western-blot analysis and mass spectrometry. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66 kDa and isoelectric point (pI) from 4 to 7. Fourteen protein spots were recognized by sera of mice infected with T. spiralis at 42 dpi or at 18 dpi, and 12 spots were successfully identified by MALDI-TOF/TOF-MS, which represented 8 different proteins of T. spiralis. Out of the 8 T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity, which might be the invasion-related proteins and the targets for vaccine. The 4 proteins (deoxyribonuclease II family protein, serine protease, 53 kDa ES antigen and hypothetical protein Tsp_08444) recognized by infection sera at 18 dpi might be the early diagnostic antigens for trichinellosis.
Asunto(s)
Antígenos Helmínticos/análisis , Antígenos Helmínticos/inmunología , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Proteoma/análisis , Trichinella spiralis/química , Trichinella spiralis/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/química , Western Blotting , Electroforesis en Gel Bidimensional , Larva/química , Larva/inmunología , Masculino , Proteínas de la Membrana/química , Ratones Endogámicos BALB C , Peso Molecular , Músculos/parasitología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
UNLABELLED: Adherence to, and persistence with, treatments for osteoporosis are low. Adherence with teriparatide decreases over time. Higher copayments in the commercial/Medicare population were associated with worse persistence. Understanding factors such as prior screening, prior treatment history, and out of pocket costs that influence persistence with teriparatide may help clinicians make informed decisions. INTRODUCTION: The purpose of this study was to evaluate adherence and persistence with teriparatide. METHODS: Beneficiaries with at least one claim for teriparatide in 2003 or 2004 and continuous enrollment in the previous 12 months and subsequent 6 months were identified in a national commercial/Medicare and Medicaid administrative claims database (MarketScan®). Adherence was assessed through calculation of the medication possession ratio (MPR). Persistence was measured by time until discontinuation and time until first 60-day gap in treatment. Factors associated with persistence were assessed using Cox proportional hazards models. RESULTS: The average MPR at 6 months was 0.74 (N=2,218) and at 12 months, was 0.66 (N=1,303). At 6 months, 64.6% of patients remained on therapy and at 12 months, 56.7% remained. Bone mineral density screening and use of antiresorptive therapy within the 12 months pre-period, and lower patient copayments were associated with increased persistence. CONCLUSION: Patients appear to have good adherence with teriparatide over the first 6 months which declines over time. Prior screening and treatment of osteoporosis and out of pocket costs appear to impact persistence. To optimize patient outcomes, clinicians should consider clinical factors that impact persistence, while healthcare decision makers should consider the negative effect of higher patient copayments on persistence.
Asunto(s)
Conservadores de la Densidad Ósea/administración & dosificación , Medicaid/estadística & datos numéricos , Medicare/estadística & datos numéricos , Osteoporosis/tratamiento farmacológico , Fracturas Osteoporóticas/prevención & control , Cooperación del Paciente/estadística & datos numéricos , Teriparatido/administración & dosificación , Anciano , Anciano de 80 o más Años , Conservadores de la Densidad Ósea/economía , Seguro de Costos Compartidos , Femenino , Humanos , Seguro de Salud/economía , Masculino , Persona de Mediana Edad , Osteoporosis/economía , Estudios Retrospectivos , Factores de Riesgo , Teriparatido/economía , Estados UnidosRESUMEN
BACKGROUND: Systemic agents in cancer treatment were often associated with possible infusion reactions (IRs). This study estimated the incidence of IRs requiring medical intervention and assessed the clinical and economic impacts of IRs in patients with colorectal cancer (CRC) treated with cetuximab. PATIENTS AND METHODS: Details on patients with CRC receiving cetuximab in 2004-2006 were extracted from a large USA administrative claims database. IRs were identified based on the occurrence of outpatient treatment, emergency room (ER) visit, and/or hospitalization for hypersensitivity and allergic reactions. Multivariate regressions were used to examine potential risk factors and quantify the economic impact of IRs. RESULTS: A total of 1122 CRC patients receiving cetuximab were identified. The incidence of IRs requiring medical intervention was 8.4%. Sixty-eight percent of the patients had treatment disruptions and 34% discontinued cetuximab treatment. Mean adjusted costs were $13,863 for cetuximab administrations with an IR requiring ER visit or hospitalization and $6280 for those with an IR requiring outpatient treatment, compared with $4555 for those without an IR. CONCLUSIONS: The incidence rate of cetuximab-related IRs requiring medical intervention in clinical practice was found to be higher than rates reported in the product label and clinical trials. The clinical and economic impacts of these IRs are substantial.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/economía , Análisis Costo-Beneficio , Hipersensibilidad a las Drogas/economía , Infusiones Intravenosas/efectos adversos , Anticuerpos Monoclonales Humanizados , Cetuximab , Hipersensibilidad a las Drogas/diagnóstico , Hipersensibilidad a las Drogas/tratamiento farmacológico , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Años de Vida Ajustados por Calidad de Vida , Estudios Retrospectivos , Factores de Riesgo , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
UNLABELLED: The demographic and clinical characteristics of patients initiating teriparatide were compared with those of patients initiating bisphosphonates for the treatment of osteoporosis. In these samples of commercially insured, Medicare, and Medicaid patients, patients initiating teriparatide were older, in poorer health, and appeared to have more severe osteoporosis than patients initiating bisphosphonates. INTRODUCTION: The demographic and clinical characteristics of patients initiating teriparatide are compared with those of patients initiating bisphosphonates. METHODS: Beneficiaries (45 years and older) with at least one claim for teriparatide or a bisphosphonate from 2003 to 2005 and continuous enrollment in the previous 12 months and subsequent 6 months were identified from commercial, Medicare, and Medicaid administrative claims databases. Patients initiating teriparatide (commercial/Medicare (N = 2,218); Medicaid (N = 824)) were compared to patients initiating bisphosphonates (commercial/Medicare (N = 97,570); Medicaid (N = 77,526)) in terms of age, provider specialty, comorbidities, prior use of osteoporosis medications, fractures, BMD screening, health status, and resource utilization. RESULTS: Teriparatide patients were older and in poorer health than bisphosphonate patients. Approximately 38% of teriparatide patients in both groups had fractured in the pre-period compared to 16% of commercial/Medicare and 15% of Medicaid bisphosphonate patients. Teriparatide patients were more likely to have used osteoporosis medications in the pre-period (79.9% versus 32.1% (commercial/Medicare); 82.2% versus 19.6% (Medicaid)). CONCLUSIONS: In these samples of patients, those initiating teriparatide differed from those initiating bisphosphonates. Teriparatide patients were older, in poorer health, and appeared to have more severe osteoporosis than bisphosphonate patients. Comparisons of treatment outcomes should take these differences in patient characteristics into consideration.
Asunto(s)
Conservadores de la Densidad Ósea/uso terapéutico , Osteoporosis/tratamiento farmacológico , Teriparatido/uso terapéutico , Factores de Edad , Anciano , Difosfonatos/uso terapéutico , Prescripciones de Medicamentos/estadística & datos numéricos , Femenino , Humanos , Masculino , Medicaid , Medicare , Persona de Mediana Edad , Osteoporosis/etnología , Osteoporosis Posmenopáusica/tratamiento farmacológico , Osteoporosis Posmenopáusica/etnología , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Estados Unidos/epidemiologíaRESUMEN
Extracts of hawthorn (Crataegus oxycantha) have become popular herbal supplements for their well-recognized cardiotonic effects. Many commercial preparations have been used successfully in the treatment of congestive heart failure, although the active principles within these extracts have yet to be conclusively identified. Several hawthorn preparations were studied and found to have negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay using unpaced cells. As compared to conventional cardiac drugs (i.e., epinephrine, milrinone, ouabain, or propranolol), hawthorn extract has a unique activity profile. Hawthorn extract appears to be anti-arrhythmic and capable of inducing rhythmicity in quiescent cardiomyocytes. Hawthorn extract does not cause beta-adrenergic receptor blockade at concentrations which cause negative chronotropic effects. Commercial hawthorn preparations, extracts prepared from dried leaves and those made from dried berries have similar chronotropic activities. When crude extracts are separated using size-exclusion chromatography, several fractions retain multiple cardiac activities. Assays with chromatographic fractions reveal that multiple dissimilar cardioactive components may exist within the extract, making the identification of individual active constituents more challenging.
Asunto(s)
Antiarrítmicos/farmacología , Crataegus/química , Miocitos Cardíacos/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Antiarrítmicos/análisis , Fármacos Cardiovasculares/farmacología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Ratones , Extractos Vegetales/químicaRESUMEN
Nod factor is a critical signalling molecule in the establishment of the legume/rhizobial symbiosis. The Nod factor of Sinorhizobium meliloti carries O-sulphate, O-acetate and C16:2 N-acyl attachments that define its activity and host specificity. Here we assess the relative importance of these modifications for the induction of calcium spiking in Medicago truncatula. We find that Nod factor structures lacking the O-sulphate, structures lacking the O-acetate and N-acyl groups, and structures lacking the O-acetate combined with a C18:1 N-acyl group all show calcium spiking when applied at high concentrations. These calcium responses are blocked in dmi1 and dmi2 mutants, suggesting that they function through the Nod factor signal transduction pathway. The dmi3 mutant, which is proposed to function in the Nod factor signal transduction pathway downstream of calcium spiking, shows increased sensitivity to Nod factor. This increased sensitivity is only active with wild-type Nod factor and was not present when the plants were treated with mutant Nod factor structures. We propose that the Nod factor signal transduction pathway is under negative feedback regulation that is activated at or downstream of DMI3 and requires structural components of the Nod factor molecule for activity.
Asunto(s)
Lipopolisacáridos/farmacología , Medicago/fisiología , Rhizobiaceae/fisiología , Transducción de Señal/fisiología , Secuencia de Carbohidratos , Retroalimentación , Cinética , Lipopolisacáridos/química , Medicago/efectos de los fármacos , Medicago/crecimiento & desarrollo , Datos de Secuencia Molecular , Oligosacáridos/químicaRESUMEN
The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.
Asunto(s)
Plásmidos/genética , Sinorhizobium meliloti/genética , Agrobacterium tumefaciens/genética , Aminoácidos/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas Portadoras/genética , ADN Bacteriano/genética , Células Eucariotas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Biblioteca de Genes , Genes Bacterianos , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Fenotipo , Replicón/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Transcripción Genética/genéticaRESUMEN
Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.
Asunto(s)
Etilenos/farmacología , Lipopolisacáridos/metabolismo , Medicago sativa/metabolismo , Transducción de Señal/efectos de los fármacos , Calcio/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Medicago sativa/genéticaRESUMEN
The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.
Asunto(s)
Genoma Bacteriano , Análisis de Secuencia de ADN , Sinorhizobium meliloti/genética , Simbiosis/genética , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Cromosomas Bacterianos/genética , Biología Computacional , Elementos Transponibles de ADN , Metabolismo Energético/genética , Evolución Molecular , Duplicación de Gen , Genes Bacterianos , Genes Esenciales , Genes Reguladores , Medicago sativa/microbiología , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Plásmidos , Polisacáridos Bacterianos/genética , Replicón , Rhizobiaceae/genética , Sinorhizobium meliloti/fisiologíaRESUMEN
During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica , Sinorhizobium meliloti/crecimiento & desarrollo , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Ciclo Celular/fisiología , Secuencia Conservada/genética , Genes Esenciales , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/genéticaRESUMEN
Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2- microm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 microm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.
Asunto(s)
Medicago sativa/citología , Raíces de Plantas/citología , Polaridad Celular , Pared Celular/fisiología , Pared Celular/ultraestructura , Procesamiento de Imagen Asistido por Computador , Medicago sativa/crecimiento & desarrollo , Medicago sativa/ultraestructura , Microscopía Fluorescente , Microesferas , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/ultraestructuraRESUMEN
The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium-host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod- and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , Genes de Plantas , Medicago sativa/genética , Medicago sativa/microbiología , Sinorhizobium meliloti/fisiología , Mapeo Cromosómico , Prueba de Complementación Genética , Cinética , Medicago sativa/fisiología , Mutagénesis , Fijación del Nitrógeno , Oscilometría , Raíces de Plantas/microbiología , Raíces de Plantas/fisiología , SimbiosisAsunto(s)
Cinamatos , Farmacorresistencia Microbiana/genética , Genética Microbiana/métodos , Higromicina B/análogos & derivados , Nucleotidiltransferasas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Rhizobiaceae/genética , Marcadores Genéticos , Proteínas Fluorescentes Verdes , Higromicina B/farmacología , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Neomicina/farmacología , Plásmidos/genética , Proteínas Recombinantes de Fusión/análisis , Replicón/genética , Rhizobiaceae/efectos de los fármacos , Sinorhizobium meliloti/efectos de los fármacos , Sinorhizobium meliloti/genética , Especificidad de la Especie , Espectinomicina/farmacologíaRESUMEN
To facilitate sequencing of the Sinorhizobium meliloti 1021 pSyma megaplasmid, a high-resolution map was constructed by ordering 113 overlapping bacterial artificial chromosome clones with 192 markers. The 157 anonymous sequence tagged site markers (81,072 bases) reveal hypothetical functions encoded by the replicon.