RESUMEN
The standard breast MRI protocol includes T2 sequences (anatomy and signal analysis), T1 gradient-echo sequences which can detect markers placed after biopsy, and injected dynamic 3D sequences for performing volume and multiplanar reconstructions, which are particularly useful for locating lesions well. Good patient positioning is essential and is obtained by using foam wedges for small breasts, ensuring there are no folds, and the correct position of the nipples. These aspects limit movement artefacts which alter subtraction sequences, so that it must always be possible for reading these sequences to be assisted by comparing them with the native sequences. New functional imaging sequences are now appearing in an attempt to increase the specificity of MRI, which is one of its main limitations. Of these, magnetic resonance spectroscopy appears to be the most promising, highlighting an abnormal choline peak in malignant lesions. This molecular signature provides early information (24 hours after beginning neoadjuvant treatment) on the chemosensitivity of a breast tumour.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Imagen de Difusión por Resonancia Magnética/métodos , Interpretación de Imagen Asistida por Computador , Imagenología Tridimensional/métodos , Imagen por Resonancia Magnética/métodos , Mamografía/métodos , Artefactos , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Terapia Combinada , Femenino , Humanos , Espectroscopía de Resonancia Magnética/métodos , Terapia Neoadyuvante , Posicionamiento del Paciente , Pronóstico , Sensibilidad y EspecificidadAsunto(s)
Neoplasias de la Mama/diagnóstico , Carcinoma Ductal de Mama/diagnóstico por imagen , Mamografía/métodos , Enfermedad de Paget Extramamaria/diagnóstico por imagen , Neoplasias Cutáneas/diagnóstico por imagen , Axila , Femenino , Humanos , Persona de Mediana Edad , Enfermedad de Paget Extramamaria/patología , Neoplasias Cutáneas/patologíaRESUMEN
The validity and origin of category effects in the anomia demonstrated by individuals with dementia of the Alzheimer's type (DAT) remains controversial. Twenty DAT subjects were tested with picture naming and semantic association judgment tests. Picture and word stimuli were drawn from biological, nonbiological, and actions-verbs categories, all of equal difficulty and previously normed on elderly controls. DAT subjects made significantly more naming and semantic judgment errors in the biological category than in the nonbiological category. They were relatively more accurate in naming and making judgments for actions-verbs when presented as words or as 5-s animations. When line drawings of actions were shown for naming, performance deteriorated significantly. Converging results from these 2 tasks provide strong evidence for a semantic memory impairment preferentially affecting biological items to a greater extent than nonbiological items or action verbs in DAT.
Asunto(s)
Enfermedad de Alzheimer/complicaciones , Anomia/etiología , Trastornos del Conocimiento/etiología , Anciano , Anciano de 80 o más Años , Anomia/diagnóstico , Trastornos del Conocimiento/diagnóstico , Femenino , Humanos , Juicio , Masculino , Pruebas Neuropsicológicas , Semántica , Índice de Severidad de la EnfermedadRESUMEN
Mitomycin C (MMC) is an antineoplastic agent with an antiproliferative effect on fibroblasts. It is routinely used topically in ophthalmology. The goal of this research project is to evaluate if topically applied MMC at the site of a maxillary antrostomy will inhibit the healing process of the mucosa and reduce the risk of postoperative stenosis. Antrostomies were carried out in both maxillary sinuses of 10 rabbits. For each rabbit, MMC is applied at the site of the antrostomy on one side while the other side serves as control. Antrostomies are re-evaluated 3 weeks later and their areas measured precisely. Seven of 10 control antrostomies were closed compared to only 1 of the 10 treated with MMC (p = .014). Comparison of the areas of the control and treated sides also proved to be statistically significant (p = .0039). Topical application of MMC was not associated with any systemic side effects and did not cause any permanent microscopic changes since the mucosa re-epithelialized normally. These results suggest that MMC slows the postoperative healing process of the nasal mucosa of the rabbit without affecting re-epithelialization. This could allow antrostomies to heal with a wider diameter, decreasing the risk of postoperative stenosis. Our next step will be to reproduce these results following functional endoscopic surgery on humans.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Seno Maxilar/cirugía , Mitomicina/uso terapéutico , Complicaciones Posoperatorias/prevención & control , Cicatrización de Heridas/efectos de los fármacos , Administración Tópica , Animales , Antibióticos Antineoplásicos/administración & dosificación , Cicatriz/etiología , Cicatriz/prevención & control , Masculino , Seno Maxilar/patología , Mitomicina/administración & dosificación , Modelos Animales , Obstrucción Nasal/etiología , Obstrucción Nasal/prevención & control , Estomía/métodos , ConejosRESUMEN
This study was designed to evaluate, by means of a randomized controlled trial, the effects of a physical activity program incorporating aerobic, strength, and stretching exercises in individuals with osteoarthritis of the knee. We randomly assigned 137 volunteers ages >/=50 to an experimental group or a control group. The experimental group participated in three 1-hour sessions of supervised exercises per week over a 3-month period. The control participants were instructed to continue their usual daily activities, and they attended 1-hour education sessions twice a month. The effectiveness of the program was evaluated using repeated measurements of parameters related to self-reported health status, physical capacity, and joint tenderness.After 3 months, significantly greater improvements were observed in the experimental group than the control group in terms of: arthritis pain (p = 0.02), ability to walk and bend (p = 0.03), aerobic capacity (p < 0.0001), hamstring and low back flexibility (p = 0.003), quadriceps and hamstring strength (p <0.01), and the perception of changes relating to osteoarthritis of the knee and general condition (p < 0.0001). However, no significant differences were observed between the groups in isokinetic strength of the quadriceps (all p's >== 0.05), joint tenderness (p = 0.18), and health perception (p = 0.7). The overall results suggest that this program is effective for older persons with osteoarthritis of the knee and that it could contribute to maintaining their independence and improving their quality of life.
RESUMEN
Diffuse nasal polyposis remains a challenge despite recent improvements in endonasal surgery. The purpose of this study is to evaluate the results after a radical complete sphenoethmoidectomy with peroperative and postoperative frontal irrigation in cases of diffuse nasal polyposis. In this prospective study, we include 50 consecutive patients with diffuse nasal polyposis suffering from nasal obstruction, anosmia, and other symptoms of chronic sinusitis. All patients were refractory to medical therapy. In each patient an endoscopic complete sphenoethmoidectomy including total excision of all diseased ethmoid mucosa was performed. Preoperative and postoperative frontal irrigation was performed systematically. The patients were followed closely with serial endoscopic examination, and CT scanning was performed between 2 and 3 years after surgery. There were no complications. Thirty-nine of the 50 patients regained satisfactory olfaction. Partial nasal obstruction persisted in four of the 50 patients. Endoscopically, polyp recurrence was noted in 3% of posterior ethmoids, 23% of anterior ethmoids, and 50% of frontal recesses. We conclude that in cases of refractory and extensive nasal polyposis, a total sphenoethmoidectomy with perioperative frontal irrigation followed by long-term postoperative topical steroid therapy provides excellent improvement or cure with safety and reliability.
Asunto(s)
Pólipos Nasales/cirugía , Senos Paranasales/cirugía , Adulto , Anciano , Endoscopía , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Pólipos Nasales/fisiopatología , Procedimientos Quirúrgicos Otorrinolaringológicos , Estudios Prospectivos , Irrigación TerapéuticaRESUMEN
Mycetomas of paranasal sinuses are more frequently diagnosed with the widespread use of nasal endoscopy and computed tomography (CT). We present a series of 109 cases treated by functional endoscopic sinus surgery (FESS) with a mean follow-up of 29 months. All localizations were seen, and contrary to what was initially thought, seven cases presented in multiple sites. Several clinical presentations were found, from a pansinusal involvement to a simple mycetoma hanging in a superior meatus. A heterogeneous sinus opacity with microcalcifications on CT scan is very suggestive of the diagnosis, but a homogeneous opacity may be encountered even with bone lysis. FESS was performed in all cases to obtain a wide opening of the affected sinuses, permitting a careful extraction of all fungal material. In the postoperative period, no medical treatment is prescribed. With a mean follow-up of 29 months, only four recurrences were seen. This study reinforces the interest in FESS for cases of mycetoma of the paranasal sinuses.
Asunto(s)
Endoscopía , Micetoma/cirugía , Enfermedades de los Senos Paranasales/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Aspergilosis/cirugía , Aspergillus fumigatus , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades de los Senos Paranasales/diagnóstico por imagen , Senos Paranasales/diagnóstico por imagen , Recurrencia , Estudios Retrospectivos , Tomografía Computarizada por Rayos X , Resultado del TratamientoAsunto(s)
Angiofibroma , Neoplasias Nasofaríngeas , Complicaciones Neoplásicas del Embarazo , Adulto , Angiofibroma/diagnóstico por imagen , Angiofibroma/patología , Angiofibroma/cirugía , Femenino , Humanos , Neoplasias Nasofaríngeas/diagnóstico por imagen , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/cirugía , Embarazo , Complicaciones Neoplásicas del Embarazo/diagnóstico por imagen , Complicaciones Neoplásicas del Embarazo/patología , RadiografíaRESUMEN
We report our experience with 10 cases of sphenoidal aspergillomas treated by endoscopic sinus surgery (ESS). Chronic symptoms such as cough, post-nasal discharge, dysphonia and even facial pain can be encountered in the history. Computerised tomography and, occasionally, magnetic resonance imaging are of great help in the assessment of this disease, especially when extensive skull base involvement is present. The radiological presentation can vary from an heterogeneous to homogeneous opacity with or without bone lysis to a frank pseudotumoural appearance. Four diagnostic tools have been evaluated to confirm the diagnosis: histology, direct smear, fungal cultures, and serology for Aspergillus. ESS has been successfully carried out without morbidity in all cases. No recurrence of the disease is seen after a mean follow-up of 27 months.
Asunto(s)
Aspergilosis/cirugía , Aspergillus fumigatus , Seno Esfenoidal , Aspergilosis/diagnóstico , Endoscopía , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Enfermedades de los Senos Paranasales/diagnóstico , Enfermedades de los Senos Paranasales/microbiología , Enfermedades de los Senos Paranasales/cirugía , Factores de TiempoRESUMEN
OBJECTIVE: To describe the effect of a supervised physical activity program on the physical and psychological health of osteopenic women. DESIGN: A randomized controlled trial. SETTING: Sherbrooke, Quebec, Canada. PARTICIPANTS: A total of 124 community-living postmenopausal women, between 50 and 70 years of age, with low bone mass took part in the study. INTERVENTION: Subjects allocated to the experimental group performed weight-bearing exercises (walking, stepping up and down from benches), aerobic dancing, and flexibility exercises for 60 minutes, three times a week, over a period of 12 months. All subjects were invited to attend bi-monthly educational seminars covering topics related to osteoporosis. OUTCOME MEASURES: Spinal and femoral bone mineral density (BMD), functional fitness (flexibility, coordination, agility, strength/endurance, cardiorespiratory endurance), psychological well-being, back pain intensity, and self-perceived health. RESULTS: Spinal BMD stabilized in the exercisers while decreasing significantly in the controls (P = .031). No change in femoral BMD was observed in either group (P = .597). Four of the five parameters chosen to evaluate functional fitness, namely flexibility, agility, strength, and endurance, were affected positively by the exercise program (all P < .01). Adjusting for prescores by means of an analysis of covariance revealed a significant difference between the groups in psychological well-being, which favored the exercisers (P = .012). After 12 months, back pain reported by exercisers was lower than that reported by controls (P = .008). Finally, self-perceived health increased in the exercise group, whereas no difference was observed in the control group (P = .790). CONCLUSION: These results suggest that after 12 months, exercising can produce a significant increase above initial levels in the functional fitness, well-being, and self-perceived health of osteopenic women. Intensity of back pain can also be lowered by exercise. The exercise program succeeded in stabilizing spinal BMD but had no effect on femoral BMD.
Asunto(s)
Terapia por Ejercicio , Osteoporosis Posmenopáusica/terapia , Anciano , Densidad Ósea , Femenino , Estado de Salud , Humanos , Salud Mental , Persona de Mediana Edad , Osteoporosis Posmenopáusica/fisiopatología , Osteoporosis Posmenopáusica/psicología , Educación del Paciente como Asunto , Aptitud Física , Estadísticas no Paramétricas , Resultado del TratamientoRESUMEN
Sera from 20 cystic fibrosis patients, whose lungs were colonised by Pseudomonas aeruginosa, were examined in a 3-5-year prospective study for any relationship between IgG subclass antibody levels to P. aeruginosa a- and b-type flagellins and pulmonary function (FEV1 and radiological score). Patients were divided into two groups according to their pulmonary status: group 1 comprised 11 patients with poor pulmonary status; group 2 comprised nine patients with relatively good pulmonary status. High concentrations of IgG1, IgG2 and IgG3 antibodies to flagellins, particularly to the b-type, were found in most patients. IgG4 reactivity was observed in only a few patients. Comparison of the two groups of patients showed that those with poor pulmonary status (group 1) had a significantly higher concentration (p < 0.05) of IgG3 for two of the three periods studied and of IgG2 for the last period studied. Moreover, IgG3 and IgG1 reactivities to b-type flagellin and IgG3 to a-type flagellin were also increased significantly (p < 0.05) in group 1 patients between the first and the last period studied. These patients also showed a significant (p < 0.05) time-dependent increase in IgG3 and IgG1 antibody concentrations. These data demonstrate that cystic fibrosis patients with poorer pulmonary status have higher IgG3 levels to flagellin than other cystic fibrosis patients. High concentrations of strong opsonic IgG3 and, to a lesser degree, of IgG1 antibodies may increase pulmonary inflammation and induce heightened pulmonary deterioration.
Asunto(s)
Fibrosis Quística/complicaciones , Flagelina/inmunología , Inmunoglobulina G/biosíntesis , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Niño , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Volumen Espiratorio Forzado , Humanos , Immunoblotting , Pulmón/fisiopatología , Masculino , Estudios Prospectivos , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/fisiopatología , Tinción con Nitrato de PlataRESUMEN
To increase the possibilities of obtaining antibodies to surface-exposed epitopes of Pseudomonas aeruginosa protein F, we immunized mice with cloned and expressed oprF gene as a gIII-fusion protein displayed on the M13 phage surface. The fusion protein elicited mouse antibodies reacting with the purified protein F at a limit dilution of 1:10,000. Recombinant clones expressing antibody fragments were constructed from the genes of selected B cells of hyperimmunized mouse after a first round of panning against the protein F. Expression of single chain Fv (ScFv) antibody fragments to the protein of P. aeruginosa was detected by ELISA in 20 of 384 clones obtained after the first panning selection. The 20 positive clones recognizing different protein F epitopes as demonstrated by ELISA were assayed by flow cytometry to identify antibody fragments reacting only with surface-exposed epitopes of the protein F on whole bacteria; one of the 20 clones tested showed a level of reactivity compatible with surface-exposed epitope that can lead to ulterior developments in targeting studies.
Asunto(s)
Epítopos/inmunología , Fragmentos de Inmunoglobulinas/biosíntesis , Región Variable de Inmunoglobulina/biosíntesis , Porinas/inmunología , Pseudomonas aeruginosa/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Antígenos de Superficie/inmunología , Bacteriófago M13/inmunología , Secuencia de Bases , Clonación Molecular , Femenino , Citometría de Flujo , Fragmentos de Inmunoglobulinas/química , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Plásmidos , Porinas/análisis , Porinas/genéticaRESUMEN
The true incidence of endonasal dacryocystocele in the newborn is probably underestimated; the number of cases in the last few years has increased with better imaging techniques and diagnostic tools. Although infrequent, it must be considered in the differential diagnosis of neonatal respiratory distress and nasal obstruction. These children typically present an endonasal mass under the inferior turbinate, most often associated with inner canthal swelling. This series of four patients represents one of the largest to be reported since the first publication on the subject by Goralowna in 1979.
Asunto(s)
Dacriocistitis/complicaciones , Dacriocistitis/diagnóstico , Dacriocistitis/patología , Hernia/complicaciones , Hernia/diagnóstico , Hernia/fisiopatología , Recién Nacido , Aparato Lagrimal/fisiopatología , Cavidad Nasal/patología , Diagnóstico Diferencial , Femenino , Lateralidad Funcional , Humanos , Aparato Lagrimal/cirugía , Masculino , Cavidad Nasal/cirugía , Obstrucción Nasal/etiología , Obstrucción Nasal/patología , Obstrucción Nasal/cirugíaRESUMEN
The conservation of oprF gene among 25 clinical Pseudomonas aeruginosa strains and a set of 17 serotype-specific representative strains of the international antigen typing scheme (IATS) was analysed by dot-blotting using five specific oligonucleotide probes. The oligo 1, 2, 3, 4, 5 correspond to five different regions of the oprF gene and hybridized strongly with respectively 88%, 88%, 76%, 94% and 71% of the IATS strains and 88%, 96%, 92%, 88% and 92% of the clinical strains. A parallel study performed with the whole oprF gene showed a lack of specificity of this probe: indeed, the probe hybridized not only with the 42 Pseudomonas aeruginosa strains but also with Escherichia coli and Salmonella minnesota. This study suggests that the gene sequence encoding the protein F is not totally conserved among Pseudomonas aeruginosa strains.
Asunto(s)
Sondas de Oligonucleótidos , Porinas/genética , Pseudomonas aeruginosa/genética , Secuencia de Bases , Secuencia Conservada , Genes Bacterianos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/síntesis química , Sondas de Oligonucleótidos/genética , Pseudomonas aeruginosa/clasificación , Homología de Secuencia de Ácido Nucleico , Serotipificación , Especificidad de la EspecieRESUMEN
Arthrobacter M5 was characterized genetically to determine if the catabolic pathway (controlled by the bph genes), responsible for polychlorinated biphenyl (PCB) biodegradation in this Gram-positive strain, was similar to the pathways characterized from various Gram-negative bacteria. Arthrobacter M5 was originally isolated as a contaminant from a culture of the PCB degrader, Acinetobacter sp. strain P6. A bph-specific oligodeoxyribonucleotide (oligo) gene probe (bphC2) was designed by aligning the published sequences of two bphC genes (encoding 2,3-dihydroxybiphenyl dioxygenase) and synthesizing a 29-nucleotide (nt) fragment from a conserved region of the gene. The bphC2 oligo was used as a probe to identify a 10-kb HindIII fragment of total DNA from Arthrobacter M5 and subsequently to isolate Escherichia coli clones possessing bphC. The PCB-degradation genes were expressed in E. coli, but expression was increased by subcloning in Pseudomonas aeruginosa. The nt and amino acid sequences of the region corresponding to the Arthrobacter M5 bphC gene showed a very high degree of homology with the published sequences of bphC genes from Gram-negative bacteria.
Asunto(s)
Arthrobacter/genética , Arthrobacter/metabolismo , Dioxigenasas , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/metabolismo , Oxigenasas/genética , Bifenilos Policlorados/metabolismo , Secuencia de Aminoácidos , Arthrobacter/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Biblioteca Genómica , Datos de Secuencia Molecular , Familia de Multigenes , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The authors reviewed retrospectively 1510 patients with breast cancer operated on between 1960 and 1980. They compared 1353 patients who had an isolated breast cancer (group 1) with 157 patients who also had breast cancer but had other cancers either previously or subsequently (group 2). The mean age of patients in group 2 was 2 years more than that of patients in group 1. Group 2 patients had fewer T3 tumours, more T1 tumours (TNM classification), a lower incidence of lymph-node involvement and clinically less advanced tumours than group 1 patients. Hormonal status, histologic type of tumour and surgical and adjuvant treatment were identical in both groups. The 10-year survival rate (considering death from breast cancer) was 54.6% in group 1 versus 78.1% in group 2. The overall survival rate (considering death from breast cancer or from the other cancer) was 54.1% in group 1 versus 64.5% in group 2. Survival was also better in group 2 for each clinical stage. The authors conclude that patients who have another cancer before or after the development of their breast cancer have a better survival rate than those who have isolated breast cancer with no previous or subsequent neoplasms.
Asunto(s)
Neoplasias de la Mama/mortalidad , Neoplasias Primarias Múltiples , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Terapia Combinada , Femenino , Humanos , Metástasis Linfática , Persona de Mediana Edad , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
A new species of Mermithidae was found parasitizing the larvae of Culex tritaeniorhynchus and Culex fatigans in Henan, China and then named Romanomermis yunanensis. Thirty-one species of Mosquitoes involving six genera have been tested for susceptibility to R. yunanensis and Culicinae mosquito have consistently been highly susceptible. At a 1:5 ratio of mosquito larvae to nematode juveniles, the parasitisms of Aedes aegypti, Ae. albopictus, Culex fatigans, Culex tritaeniorhynchus, Anopheles sinensts, Psorophora columbiae and Culesta inornata were 98.2%, 98.5%, 98.6%, 97.1%, 0%, 98.8%, and 99.0% respectively. R. yunanensis presented a phenomenon of retarted development in Anopheles maculatus, which remained at the parasitic stage both in the larvae and pupa of An. maculatus. The parasitism of An. dirus was 89.7%, but most (88.8%) parasitizing mermithids underwent melanization and died. The comparative susceptibility of some North American mosquitoes to R. yunanesis and R. culicivorax at a 1:5 infective ratio showed that both percentage infection and intensity of infection statistically supported the generalization that, under laboratory conditions, R. yunanesis is a more vigorous and aggressive parasite of Culicinae.
Asunto(s)
Culex/parasitología , Culicidae/parasitología , Mermithoidea , Aedes/parasitología , Animales , Anopheles/parasitología , Interacciones Huésped-Parásitos , Especificidad de la EspecieRESUMEN
A retrospective study was carried out of 1520 consecutive patients with breast cancer operated on at the Hôpital Notre-Dame in Montreal between 1960 and 1980. Age and hormonal status of the patient, and duration, size, location, histologic type and stage of the tumour were studied. The authors grouped the patients according to four types of surgical treatment: radical (487 patients), modified radical (497 patients) and simple (220 patients) mastectomies and conservative procedures (316 patients). Adjuvant treatments included radiotherapy in 60%, hormone therapy in 4.7% and chemotherapy in 6.7% of patients. Overall survival at 5, 10 and 15 years was 71.3%, 58.7% and 51.1%; in patients with stage I disease, survival rates were, respectively, 86%, 78% and 72%; rates for patients with stage II disease were 74%, 62% and 53%. The population characteristics and survival rates were similar to those reported by others. The four types of treatment did not produce significantly different survival rates in patients with stage I lesions. However, this was not the case in patients with stage II and III lesions in whom simple mastectomy (McWhirter procedure) was associated with significantly worse results. Finally, this study confirmed the prognostic importance of stage, size of the tumour and degree of axillary lymph-node involvement.
Asunto(s)
Neoplasias de la Mama/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Metástasis Linfática , Persona de Mediana Edad , Estadificación de Neoplasias , Quebec , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.
Asunto(s)
Bacillus thuringiensis/genética , Toxinas Bacterianas/genética , Precursores de Proteínas/genética , Animales , Toxinas Bacterianas/análisis , Toxinas Bacterianas/farmacología , Células Cultivadas , Clonación Molecular , Cristalización , Bromuro de Cianógeno , Enzimas de Restricción del ADN , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Insecticidas , Lepidópteros/efectos de los fármacos , Fragmentos de Péptidos/aislamiento & purificación , Precursores de Proteínas/análisis , Precursores de Proteínas/farmacología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/farmacologíaRESUMEN
The hemA gene of Escherichia coli K12 was cloned by complementation of a hemA mutant of this organism. Subcloning of the initial 6.0 kb HindIII fragment allowed the isolation of a 1.5 kb NheI-AvaI fragment which retained the ability to complement the hemA mutant. DNA sequencing by the dideoxy chain terminator method of Sanger showed the presence of an open reading frame (ORF) of 1254 nucleotides, which ends 6 nucleotides beyond the AvaI site. Primer extension experiments showed the existence of a putative transcription initiation site for the hemA gene, located at position 130. A possible promoter sequence was identified upstream from this transcription initiation site, and its functional activity was confirmed by the use of the pK01 promoter-probe vector. Protein synthesis in an in vitro coupled transcription-translation system showed a 46 kDa protein, which corresponds to the mol. wt. of the hemA protein, as deduced from the nucleotide sequence of the gene. No homology was found between the amino acid sequence of the hemA protein of E. coli K12 and known sequences of other delta-aminolevulinic acid synthases (delta-ALAS), suggesting that this protein is different from other delta-ALAS enzymes.