RESUMEN
We evaluated the effect of pre-operative serratus anterior plane block on postoperative pain and opioid consumption after thoracoscopic surgery. We randomly allocated 89 participants to block with 30 ml ropivacaine 0.375% (n = 44), or no block without placebo or sham procedure (n = 45). We analysed results from 42 participants in each group. Serratus anterior plane block reduced mean (SD) remifentanil dose during surgery, 0.12 (0.06) mg.h-1 vs. 0.16 (0.06) mg.h-1 , p = 0.016, and reduced mean (SD) fentanyl consumption in the first 24 postoperative hours, 3.8 (1.9) µg.kg-1 vs. 5.7 (1.6) µg.kg-1 , p = 0.000004. Block also reduced the worst median (IQR [range]) pain scores reported in the first 24 postoperative hours: 6 (5-7 [3-10]) vs. 7 (6-7 [3-10]), p = 0.027. Block decreased dissatisfaction with pain management, categorised as 'highly unsatisfactory', 'unsatisfactory', 'neutral', 'satisfactory' or 'highly satisfactory': 1/2/21/18/0 vs. 1/14/15/11/1, p = 0.0038. There were no differences in the rates of nausea, vomiting, dizziness or length of hospital stay. Serratus anterior plane block may be used to reduce pain and opioid use after thoracoscopic lung surgery.
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Bloqueo Nervioso/métodos , Dolor Postoperatorio/prevención & control , Toracoscopía/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Analgésicos Opioides/administración & dosificación , Esquema de Medicación , Femenino , Fentanilo/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Dimensión del Dolor/métodos , Dolor Postoperatorio/etiología , Satisfacción del Paciente , Remifentanilo/administración & dosificación , Adulto JovenRESUMEN
Tetramethylpyrazine (TMP) is a biologically active ingredient, which is isolated from a popularChinese medicinal plant. It has been used effectively to treat ischemic heart problems, cerebrovascular and thrombotic vascular diseases. This study was designed to evaluate the effect of TMP on calciumsensing receptors in pulmonary artery smooth muscle in chickens. For this purpose forty day-old chicks were distributed into five groups: the control group, the hypoxia group (kept under low Oxygen treatment), and TMP groups (kept under low Oxygen treatment along with treatment of different concentrations of TMP). The pulmonary artery smooth muscle cells were also cultured on 6-well plates in high glucose culture medium and divided into the same five groups. We used in vivo and in vitro study models by applying immunohistochemistry, RT-qPCR assay and Western blotting analysis. Our results showed that pre-incubation with hypoxia markedly stimulated the activation of calcium-sensing receptor (CaSR) in pulmonary artery smooth muscle cells (PASMCs). The TMP decreased the mRNA and protein levels of CaSR. Treatment with TMP clearly inhibited the activation of all CaSR in a dose-dependent manner. Our data demonstrated that TMP can down-regulate the expression of CaSR. Therefore, these findings provide a new target to treat pulmonary arterial hypertension (PAH) under hypoxic conditions.
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Proteínas Aviares/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Pirazinas/farmacología , Receptores Sensibles al Calcio/biosíntesis , Animales , Hipoxia de la Célula/efectos de los fármacos , Pollos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Arteria Pulmonar/patologíaRESUMEN
Objective: To investigate the relationship between the content of human telomerase reverse transcriptase (hTERT) and its clinical features in serum free DNA in patients with different degree of spinal cord injury. Methods: From December 2013 to December 2016, inpatients of the Central Hospital of Bazhong, Sichuan Province were enrolledand divided into the experimental group, the disease control group and the negative control group. For the experimental group: 46 patients with spinal cord injury were graded according to the criteria of the American Association of Spinal Cord Injury (ASIA), including 12 cases of grade A, 10 cases of grade B, 10 cases of grade C, 7 cases of grade D and 7 cases of grade E; for the disease control group: 15 patients with spinal fractures (without spinal cord injury) at the same period were included; and for the negative control group: 20 healthy adult volunteers aged 18-50 years were selected.Real-time fluorescence quantitative PCR and immunoblotting were performed to detect the content of hTERT in serum free DNA both in patients and healthy controls and to compare the difference between them. The results of the somatosensory evoked potential (SEP) of all patients were compared and analyzed.The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of hTERT content in serum free DNA in patients with spinal cord injury. Results: Comparison of serum free DNA hTERT content: in the experimental group, the serum free DNA hTERT content of grade A, B, C, D, E was (99.63±8.23), (76.24±4.37), (46.07±5.43), (16.30±0.95) and (15.74±1.12)µg/L, respectively.While it was (15.01±1.39)µg/L in the disease control group and (14.54±1.03)µg/L in the negative control group. The total difference was statistically significant between patients of each group and the control group (F=857.917, P<0.001). Comparison of the protein content of TERT: in the experimental group, the protein content of TERT of grade A, B, C, D, E was 0.736±0.214, 0.641±0.172, 0.606±0.184, 0.411±0.132 and 0.307±0.152, respectively.The protein content of TERT in the disease control group and the negative control group was about 0.312±0.098 and 0.322±0.177, the difference between patients of each group and the control group was statistically significant (F=62.461, P<0.001). Detection results of surface evoked potential (SEP) showed that in the experimental group, level A patients all had conduction block.Two cases of level B patients had conduction block and 8 cases had delayed conduction.Among level C patients, 1 case had conduction block, 9 cases had delayed conduction.Among level D patients and patients from the control group and the negative control group, SEP detection all had no conduction block. Conclusion: The detection of the hTERT content in serum free DNA in patients with spinal cord injury has a certain guiding significance for the diagnosis of spinal cord injury and the degree of injury.
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Traumatismos de la Médula Espinal , Adolescente , Adulto , ADN , Proteínas de Unión al ADN , Humanos , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa , Adulto JovenRESUMEN
PurposeTo report long-term outcome of new surgical technique for prolapsed subconjunctival orbital fat.Patients and methodsRetrospective study was conducted on 48 eyes of 37 patients who underwent excision of prolapsed subconjunctival orbital fat with conjunctival fixation to the sclera. Complications and recurrence were evaluated.ResultsThe mean follow-up period was 39 months (range, 8-101 months). Two eyes (4.4%) developed recurrence at 4 and 8 years after surgery. No long-term complication was found.ConclusionsThe new surgical technique to manage prolapsed subconjunctival orbital fat using conjunctival fixation to the sclera was very useful and effective, with few recurrence and no long-term complication.
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Tejido Adiposo , Conjuntiva/cirugía , Procedimientos Quirúrgicos Oftalmológicos/métodos , Enfermedades Orbitales/cirugía , Técnicas de Sutura/instrumentación , Suturas , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Orbitales/diagnóstico , Prolapso , Estudios Retrospectivos , Esclerótica/cirugía , Factores de TiempoRESUMEN
This study investigated the relationship between alterations in the hypothalamic-pituitary-adrenal (HPA) axis function and glucose and lipid metabolism in diabetic rats. To accomplish this a diabetes model was established by jointly administering a long-term high-fat diet plus Streptozotocin (STZ; 50 mg/kg ip). The rats were randomly divided into four groups: 1) a normal control group, 2) a model group, 3) astragalus polysaccharide (APS) group, and 4) a metformin group. APS and metformin hydrochloride were administered intragastrically (100 mgâkg(-1)d(-1)). Rat blood glucose and body weight were measured once per week, and urine was collected for 24 h after 30 days of administration of APS. The levels of blood lipids, insulin, and corticosterone (CORT), as well as hypothalamic CRH, pituitary ACTH, urine sugar and CORT were measured. Compared with the normal control group, the levels of blood sugar, urine sugar, TC, and TG significantly increased in the model group, and the levels of hepatic glycogen and HDL-C decreased. Administration of APS was shown to reverse these changes. Furthermore, as compared with the normal control group, the levels of insulin and hypothalamic CRH in the model group decreased significantly, while the levels of plasma ACTH and CORT, pituitary ACTH, and urine CORT were elevated. Again, APS administration improves these outcomes and returns their levels to normal. Thus, the glucose and lipid metabolic disorder in the high-fat diet and STZ-induced diabetes model may be related to increased HPA axis activity. The hypoglycemic effect of the traditional Chinese medicine, ASP, may improve HPA axis functioning and aid in the treatment of diabetes.
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Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Metabolismo de los Lípidos , Sistema Hipófiso-Suprarrenal/metabolismo , Hormona Adrenocorticotrópica/sangre , Animales , Glucemia , Corticosterona/sangre , Hormona Liberadora de Corticotropina/sangre , Diabetes Mellitus Experimental/sangre , Femenino , Glucógeno/metabolismo , Insulina/sangre , Insulina/metabolismo , Lípidos/sangre , Hígado/metabolismo , RatasRESUMEN
We evaluated the effects of a prolonged inspiratory time on gas exchange in subjects undergoing one-lung ventilation for thoracic surgery. One hundred patients were randomly assigned to Group I:E = 1:2 or Group I:E = 1:1. Arterial blood gas analysis and respiratory mechanics measurements were performed 10 min after anaesthesia induction, 30 and 60 min after initiation of one-lung ventilation, and 15 min after restoration of conventional two-lung ventilation. The mean (SD) ratio of the partial pressure of arterial oxygen to fraction of inspired oxygen after 60 min of one-lung ventilation was significantly lower in Group I:E = 1:2 compared with Group I:E = 1:1 (27.7 (13.2) kPa vs 35.2 (22.1) kPa, respectively, p = 0.043). Mean (SD) physiological dead space-to-tidal volume ratio after 60 min of one-lung ventilation was significantly higher in Group I:E = 1:2 compared with Group I:E = 1:1 (0.46 (0.04) vs 0.43 (0.04), respectively, p = 0.008). Median (IQR [range]) peak inspiratory pressure was higher in Group I:E = 1:2 compared with Group I:E = 1:1 after 60 min of one-lung ventilation (23 (22-25 [18-29]) cmH2O vs 20 (18-21 [16-27]) cmH2O, respectively, p < 0.001) and median (IQR [range]) mean airway pressure was lower in Group I:E = 1:2 compared with Group I:E = 1:1 (10 (8-11 [5-15]) cmH2O vs 11 (10-13 [5-16]) cmH2O, respectively, p < 0.001). We conclude that, compared with an I:E ratio of 1:2, an I:E ratio of 1:1 resulted in a modest improvement in oxygenation and decreased shunt fraction during one-lung ventilation.
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Inhalación/fisiología , Ventilación Unipulmonar/métodos , Análisis de los Gases de la Sangre/métodos , Dióxido de Carbono/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxígeno/sangre , Intercambio Gaseoso Pulmonar , Mecánica Respiratoria/fisiología , Procedimientos Quirúrgicos Torácicos , Volumen de Ventilación Pulmonar , Factores de TiempoRESUMEN
To investigate different cells behaviors and genotoxicity, which were driven by specific microenvironments, three patterned surfaces (pillars, wide grooves and narrow grooves) and one smooth surface were prepared by template-based technique. Vinculin is a membrane-cytoskeletal protein in focal adhesion plaques and associates with cell-cell and cell-matrix junctions, which can promote cell adhesion and spreading. The immunofluorescence staining of vinculin revealed that the narrow grooves patterned substrate was favorable for L929 cell adhesion. For cell multiplication, the narrow grooves surface was fitted for the proliferation of L929, L02 and MSC cells, the pillars surface was only in favor of L929 cells to proliferate during 7 days of cell cultivation. Cell genetic toxicity was evaluated by cellular micronuclei test (MNT). The results indicated that topological surfaces were more suitable for L929 cells to proliferate and maintain the stability of genome. On the contrary, the narrow grooves surface induced higher micronuclei ratio of L02 and MSC cells than other surfaces. With the comprehensive results of cell multiplication and MNT, it was concluded that the wide grooves surface was best fitted for L02 cells to proliferate and have less DNA damages, and the smooth surface was optimum for the research of MSC cells in vitro.
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Técnicas de Cultivo de Célula/instrumentación , Animales , Adhesión Celular , Línea Celular , Supervivencia Celular , Humanos , Ratones , Propiedades de Superficie , Vinculina/química , Vinculina/metabolismo , HumectabilidadRESUMEN
Tracheal intubations with the Airway Scope or the Clarus Video System, a new rigid fibrescope, were compared in 140 patients whose necks were immobilised by cervical collars. The time for intubation, success rate, number of attempts and number of optimisation manoeuvres were assessed. Mean (SD) intubation time was longer with the Airway Scope (30.4 (16.5) s) than with the Clarus Video System (18.9 (15.2) s; p = 0.003) and the median (IQR [range]) number of optimisation manoeuvres was also marginally different; 0 (0-1 [0-2]) with the Airway Scope, 0 (0-0 [0-2]) with the Clarus Video System; p = 0.004. The tracheas of 67 (95.7%) and 66 (94.3%) patients were successfully intubated with the Airway Scope and the Clarus Video System, respectively (p = 1.0). The number of attempts, vital signs and complications were not different between devices. The Clarus Video System was comparable to the Airway Scope in the success rate for tracheal intubation, but provided faster and easier intubations than the Airway Scope in patients with cervical collars.
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Tirantes , Intubación Intratraqueal/instrumentación , Laringoscopios , Adulto , Anciano , Vértebras Cervicales , Diseño de Equipo , Femenino , Tecnología de Fibra Óptica/instrumentación , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Grabación en Video/instrumentación , Adulto JovenRESUMEN
BACKGROUND: In general, there is a response time between actual arterial hypoxemia and its detection by pulse oximeters. We compared the desaturation and resaturation response times between two types of pulse oximeters, transmission and reflectance pulse oximeters, to find out which oximeter has a more rapid response time. METHODS: Thirty-three ASA 1 or 2 patients were enrolled in this study. A transmission pulse oximeter was placed on the index finger and a reflectance pulse oximeter was placed on the forehead and monitored simultaneously. After the induction of general anesthesia without pre-oxygenation, we waited until the oxygen saturation value of any of two pulse oximeters declined to 90%, and then mask ventilation was started with 100% oxygen. Oxygen saturation was recorded at an interval of 2 s during this time. RESULTS: The desaturation response time of SpO(2) to 95% after apnea was 82.0 s (interquartile range: 67.0-98.5 s) vs. 94.0 s (interquartile range: 84.0-106.5 s) (P<0.001) and SpO(2) to 90% was 94.0 s (interquartile range: 75.5-109.5 s) vs. 100.0 s (interquartile range: 84.5-114.5 s) (P<0.001) in the reflectance and transmission oximeters, respectively. The resaturation response time from mask ventilation to 100% SpO(2) was 23.2+/-5.6 vs. 28.9+/-7.6 s (P<0.001) in the reflectance and transmission oximeters, respectively. CONCLUSION: In clinical situations in which rapid changes in oxygen saturation are expected, we recommend the forehead reflectance pulse oximeter because it responds more quickly in detecting oxygen desaturation and resaturation compared with the transmission pulse oximeter.
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Oximetría/instrumentación , Oxígeno/sangre , Adulto , Androstanoles/administración & dosificación , Anestésicos Intravenosos/administración & dosificación , Apnea/sangre , Mama/cirugía , Diseño de Equipo , Femenino , Fentanilo/administración & dosificación , Dedos/irrigación sanguínea , Frente/irrigación sanguínea , Humanos , Hipoxia/sangre , Persona de Mediana Edad , Fármacos Neuromusculares no Despolarizantes/administración & dosificación , Propofol/administración & dosificación , Respiración Artificial , Rocuronio , Tiroidectomía , Factores de Tiempo , Adulto JovenRESUMEN
Perinatal estrogens increase the number of vasopressin-expressing cells and the density of vasopressin-immunoreactive fibers observed in adult male rodents. The mechanism of action of estrogens on sexual differentiation of the extra-hypothalamic vasopressin system is unknown. We hypothesized that the sexually dimorphic expression of progestin receptors (PRs) during development would masculinize vasopressin expression in mice. We compared the number of vasopressin-expressing cells in the bed nucleus of the stria terminalis (BNST) and medial amygdala and the density of vasopressin-immunoreactive fibers in several brain regions of male and female wild type and PRKO mice using in situ hybridization and immunohistochemistry. As expected, sex differences in vasopressin cell number were observed in the BNST and medial amygdaloid nucleus. Vasopressin-immunoreactive fiber density was sexually dimorphic in the lateral septum, lateral habenular nucleus, medial amygdaloid nucleus, and mediodorsal thalamus. Sex differences were also observed in the principal nucleus of the BNST and medial preoptic area but not in the dorsomedial hypothalamus, which are thought to receive vasopressin innervation from the suprachiasmatic nucleus. Deletion of PRs did not alter the sex difference in vasopressin mRNA expression and vasopressin fiber immunoreactivity in any area examined. However, deletion of PRs increased the density of vasopressin fiber immunoreactivity in the lateral habenular nucleus. Our data suggest that PRs modulate vasopressin levels, but not sexual differentiation of vasopressin innervation in mice.
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Fibras Nerviosas/fisiología , Receptores de Progesterona/fisiología , Diferenciación Sexual/fisiología , Vasopresinas/fisiología , Animales , Recuento de Células , Implantes de Medicamentos , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Progesterona/genética , Diferenciación Sexual/efectos de los fármacos , Tinción con Nitrato de Plata , Esteroides/metabolismo , Testosterona/administración & dosificación , Testosterona/farmacología , Vasopresinas/biosíntesisRESUMEN
BACKGROUND: This study examined the effect of different levels of spinal anaesthesia, induced by solutions of different baricity but containing the same amount of local anaesthetic agent, on the requirement for sedation with propofol. METHODS: Thirty-six patients undergoing varicose vein surgery under spinal anaesthesia were randomly allocated to receive tetracaine 15 mg in 3 ml of either glucose 5% (hyperbaric) or CSF (isobaric). I.V. propofol was started 5 min after the intrathecal injection and was titrated to maintain a bispectral index (BIS) score of 65-75. The propofol requirements to maintain this range in the two groups were compared every 5 min. RESULTS: The propofol requirement was always lower in the hyperbaric group, with the differences becoming statistically significant 20 min after the intrathecal injection. Total consumption of propofol over the 55 min of the study was also less in the hyperbaric group. CONCLUSION: The known difference in level of spinal anaesthetic block induced by solutions of different baricity, but the same dose of local anaesthetic, was associated with different requirements for propofol sedation as determined by BIS assessment.
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Anestesia Raquidea/métodos , Anestésicos Locales/química , Sedación Consciente/métodos , Hipnóticos y Sedantes/administración & dosificación , Propofol/administración & dosificación , Adolescente , Adulto , Anestésicos Locales/administración & dosificación , Esquema de Medicación , Electroencefalografía/efectos de los fármacos , Humanos , Persona de Mediana Edad , Gravedad Específica , Tetracaína/administración & dosificación , Tetracaína/química , Várices/cirugíaRESUMEN
This study was performed to evaluate the effects of cryoanalgesia combined with thoracic epidural analgesia on pain and respiratory complications in patients undergoing thoracotomy. Ninety patients were prospectively randomised to epidural analgesia alone (n = 45) or epidural analgesia and cryoanalgesia combined (n = 45). We monitored the use of rescue pain medication and changes in forced vital capacity and forced expired volume in 1 s, and recorded pain and opioid-related side-effects during the immediate postoperative period. The incidence of post-thoracotomy pain and numbness were also assessed up to the sixth month after surgery. Cryoanalgesia combined with thoracic epidural analgesia was associated with earlier recovery in pulmonary function, less pain during movement and a lower daily requirement for rescue analgesia one week after surgery. However, the combination of cryoanalgesia and epidural analgesia failed to decrease the incidence of long-term pain and numbness. In view of its associated long-term morbidity, cryoanalgesia combined with thoracic epidural analgesia is not recommended for patients undergoing thoracotomy.
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Analgesia Epidural/métodos , Criocirugía , Dolor Postoperatorio/prevención & control , Toracotomía , Adulto , Anciano , Analgésicos Opioides/administración & dosificación , Terapia Combinada , Esquema de Medicación , Femenino , Volumen Espiratorio Forzado , Humanos , Masculino , Persona de Mediana Edad , Morfina/administración & dosificación , Movimiento , Dimensión del Dolor/métodos , Complicaciones Posoperatorias/prevención & control , Capacidad VitalRESUMEN
Levels of l exA transcripts are markedly increased upon exposure of Xanthomonas axonopodis pathovar citri ( X. a. pv. citri) to the DNA-damaging agent mitomycin C. Preliminary electrophoretic mobility-shift data led us to propose that binding of LexA protein to the sequence upstream of the lexA coding region is responsible for low promoter activity in the uniduced state. We determined that the LexA protein binds to the region located between the transcription start site and the translation initiation codon of the lexA gene of X. a. pv. citri. Using a DNase I footprinting technique, we identified a 19-bp palindromic sequence, TTAGTAGTAATACTACTAA (TTAGN(11)CTAA), located in this region as the binding sequence for the LexA protein of X. a. pv. citri, and showed that the two halves of the palindrome have to be in the inverted repeat orientation to permit binding of LexA. We also showed that almost any mutation in this sequence, including changes in the length of the spacer region of the palindrome, destroyed its ability to bind LexA both in vitro and in vivo.
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Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Secuencias Repetitivas de Ácidos Nucleicos/genética , Respuesta SOS en Genética/genética , Serina Endopeptidasas/metabolismo , Xanthomonas/genética , Proteínas Bacterianas/genética , Sitios de Unión , Huella de ADN , Cartilla de ADN/química , Desoxirribonucleasa I/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Mitomicina/farmacología , Mutación/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Unión Proteica , Eliminación de Secuencia , Serina Endopeptidasas/genética , Xanthomonas/efectos de los fármacos , Xanthomonas/metabolismoRESUMEN
The role of the LexA protein and, specifically, its effect on recA expression were analyzed in Xanthomonas campestris pathovar citri (X.c. pv. citri). Overexpression of LexA from X.c. pv. citri, in the plant pathogen, as well as in Escherichia coli, results in increased sensitivity to the DNA-damaging agents mitomycin C and ultraviolet radiation, indicating that the recombinant X.c. pv. citri LexA protein is functional in a different bacterial species. Immunoblot analysis revealed that the overexpressed LexA protein functioned as a repressor of recA expression in X.c. pv. citri, and that the mitomycin C-induced increase in the abundance of RecA was accompanied by specific proteolysis of LexA that required RecA. Although the LexA protein from X.c. pv. citri also blocked the expression of recA in E. coli, the E. coli RecA protein was not able to support the autocatalytic cleavage of LexA from the plant pathogen. The transcription start site of the X.c. pv. citri lexA gene was identified, and the region upstream of this gene was shown to confer responsiveness to mitomycin C on a luciferase reporter gene construct. Electrophoretic mobility-shift assays demonstrated that X.c. pv. citri LexA interacts with the promoter region of X.c. pv. citri lexA, as well as with those of the recA genes of X.c. pv. citri and E. coli. These results indicate that LexA functions as a repressor of gene expression in X.c. pv. citri just as it does in E. coli.
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Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica , Rec A Recombinasas/genética , Proteínas Represoras/fisiología , Serina Endopeptidasas/fisiología , Xanthomonas campestris/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Daño del ADN , ADN Bacteriano , Escherichia coli , Genes Bacterianos , Mitomicina/farmacología , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Regiones Promotoras Genéticas , Rec A Recombinasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Xanthomonas campestris/efectos de los fármacos , Xanthomonas campestris/efectos de la radiaciónRESUMEN
Two genes important in DNA repair, recA and lexA, were recently identified in Xanthomonas campestris pathovar citri (X.c. pv. citri). An open reading frame located immediately downstream of lexA and recA has now been isolated from this pathovar and characterized. This 486-bp open reading frame encodes a protein of 162 amino acids and shares substantial sequence similarity with recX of other bacterial species. The X.c. pv. citri RecX protein was overexpressed in Escherichia coli and purified; SDS-polyacrylamide gel electrophoresis revealed a molecular size of 18 kDa for the purified protein. Whereas Northern blot analysis failed to detect recX mRNA in X.c. pv. citri, recX transcripts were detected in this pathovar by reverse transcription and polymerase chain reaction analysis. The increased abundance of recX transcript in X.c. pv. citri revealed that the recX promoter was activated by exposure of cells to DNA-damaging agents. Southern blot and polymerase chain reaction analyses revealed the presence of a recX-related gene in all nine additional X. campestris pathovars tested. The genetic arrangement of lexA-recA-recX was apparent in X. campestris and each of the three genes transcribed from their own promoters.
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Proteínas Bacterianas/genética , Xanthomonas campestris/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Daño del ADN/fisiología , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Mediciones Luminiscentes , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Serina Endopeptidasas/genética , Proteínas no Estructurales Virales/genética , Xanthomonas campestris/metabolismoRESUMEN
Under normal cultivation conditions, a mixture of turbid and clear plaques is often apparent in cultures of bacterial cells infected with filamentous bacteriophages. Beginning with a culture of wild-type filamentous phage f1, which itself produces turbid plaques, a clear plaque strain (c1) was isolated. From c1, the turbid plaque strain t1 was isolated; from t1, the clear plaque strain c2 was isolated; and from c2, the turbid plaque strain t2 was isolated. Each of these strains was generated with a frequency of approximately 1 x 10(-4). Although filamentous phages have been thought not to induce host cell death, both turbid and clear plaque strains of f1 killed host bacteria. Plating of bacterial cells 1 h after infection revealed that colonies produced by cells infected with either wild-type f1 or strain c2 were smaller than those derived from uninfected cells, and that colony formation by infected cells was reduced by 15% and 38%, respectively. The time course of bacterial growth revealed that, at 4 h after infection, the number of CFU per milliliter of culture of cells infected with wild-type f1 or with strain c2 was reduced by 27% and 95%, respectively, compared with that for uninfected cells. Microculture analysis also revealed that the percentages of nondividing cells in f1 or c2 infected were 19% and 52%, respectively, 4 h after infection with wild-type f1 or with strain c2; no such cells were detected in cultures of uninfected cells. Negative staining and electron microscopy showed that 20% and 61% of cells infected with wild-type f1 or with strain c2 were dead 4 h postinfection. Finally, although the rates of DNA synthesis were similar for infected and uninfected cells, the rates of RNA and protein synthesis were markedly reduced in infected cells.
Asunto(s)
Colifagos/fisiología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/virología , Proteínas Bacterianas/metabolismo , División Celular , ADN Bacteriano/metabolismo , Escherichia coli/ultraestructura , ARN Bacteriano/metabolismo , Ensayo de Placa ViralRESUMEN
The lexA gene of Xanthomonas campestris pathovar citri (X.c. pv. citri) was cloned and sequenced. The 639-bp open reading frame encodes a protein of 213 amino acids that shares substantial sequence homology with the products of previously characterized lexA genes, sharing 46% identity with the LexA protein of Escherichia coli. Amino acids required for autocatalytic cleavage of LexA are conserved in the X.c. pv. citri protein, whereas domains thought to mediate DNA binding differ markedly from those of LexA proteins from E. coli and other bacteria. The X.c. pv. citri LexA protein was overexpressed in E. coli, and SDS-polyacrylamide gel electrophoresis revealed a molecular size of 23 kDa for the purified protein. A lexA mutant of X.c. pv. citri was constructed by gene replacement, and the basal level of recA expression in this mutant was shown to be similar to that for wild-type cells exposed to a DNA-damaging agent. These results indicate that LexA functions as a repressor of recA expression in X.c. pv. citri.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Serina Endopeptidasas/genética , Xanthomonas campestris/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN Bacteriano/análisis , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Mutación , Rec A Recombinasas/genética , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/farmacología , Xanthomonas campestris/químicaRESUMEN
The effects of the synthetic antioxidant germanium (Ge-132) were studied on liver oxidant damage induced by paraquat (PQ) in senescence-accelerated mice (SAM). PQ administered intravenously to SAM-P/8 (susceptible) or SAM-R/1 (resistant) mice increased liver DNA strand breakage and malondialdehyde (MDA) levels, indicators of oxidant damage. Ge-132 effectively blocked the PQ-induced effects on liver DNA strand breaks and MDA levels. In addition, Ge-132 significantly elevated the activities of hepatic superoxide dismutase (SOD) and catalase following PQ pretreatment. Histopathologically, Ge-132 inhibited PQ-induced hepatic mitochondrial injury in both strains, but more effectively in the susceptible strain. Data suggest that Ge-132 may be useful as an antioxidant in view of its ability to prevent PQ-induced hepatic oxidant injury.
Asunto(s)
Envejecimiento/genética , Antioxidantes/farmacología , Germanio/farmacología , Herbicidas/antagonistas & inhibidores , Hígado/metabolismo , Compuestos Organometálicos/farmacología , Estrés Oxidativo/efectos de los fármacos , Paraquat/antagonistas & inhibidores , Animales , Catalasa/metabolismo , Daño del ADN , Radicales Libres/metabolismo , Herbicidas/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/ultraestructura , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos , Microscopía Electrónica , Paraquat/toxicidad , Propionatos , Superóxido Dismutasa/metabolismoRESUMEN
Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf.
Asunto(s)
Proteínas Bacterianas/genética , Bacteriófagos/patogenicidad , Proteínas de Unión al ADN/genética , Proteínas Fimbrias , Genes Bacterianos , Xanthomonas campestris/genética , Xanthomonas campestris/virología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Proteínas de Unión al ADN/química , Datos de Secuencia Molecular , Mutagénesis Insercional , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The abundance of the RecA protein and of recA transcripts was markedly increased on exposure of Xanthomonas campestris pathovar citri to various DNA-damaging agents, including mitomycin C. The promoter sequence responsible for mediating the sensitivity of recA expression to DNA damage was investigated by subcloning a 426-bp restriction fragment of the 5' untranslated and coding region of the gene into a promoterless vector containing the luxAB genes of Vibrio fischeri. Xanthomonas campestris pv. citri cells transformed with this vector responded to DNA-damaging agents with a marked increase in luciferase activity. Deletion of nucleotides from the 5' end of the recA fragment inserted into the reporter plasmid revealed that the 58 bp upstream of the transcription initiation site are sufficient to mediate induction of recA expression by mitomycin C.