RESUMEN
BACKGROUND: Intensive care unit (ICU) capacity is a scant and precious resource in hospitals. Therefore, an optimal occupancy rate as well as detailed occupation planning is of great importance. Most literature deals with admission to the ICU, while only few discuss discharge from the ICU. Specifically, a delay of transfer from the ICU can cause a shortness of beds, jeopardize urgent patient treatment and lead to a decrease in treatment quality as well as economic downsides. This study examined the incidence, costs and reasons for delayed discharge from the ICU and analyzed the influence of the department the patient was admitted to. METHODS: Over the course of 12 months, the discharges of all 1643 patients of two surgical intensive care units of a large academic medical center were analyzed. Delay in minutes and reasons were recorded and translated into financial figures. A univariate logistic regression model was developed to evaluate the impact of length of stay at the ICU, age, gender, subspecialty and specific ICU on the delay of transfer. In a next step, significant factors of the univariate logistic regression were incorporated into a multivariate regression model. RESULTS: In 326 out of 1312 patients ready for discharge (24.8%), the transfer to the floor was delayed. Time of delay for all patients added up to a total of 265,691 min in 1 year. The application of the internal cost allocation, in which 1 min corresponds to 0.75 Euro cents, led to costs of 199,268 Euros (~ $240,000) for the study period. In 91.7% of the cases, the reason for the delay was the lack of an available or appropriate bed on the regular ward. Multivariate regression analysis revealed that the type of department the patient is admitted to poses a significantly influencing factor for delayed discharge from the ICU. CONCLUSION: Delay in discharge from the ICU is a common problem of economic relevance. The main reason is a lack of appropriate floor beds. Patients from certain specific departments are at a higher risk to be discharged with delay. A solution to this problem lies in the focus on the downstream units. A proper use of the scarce resources is to be pursued because of ethical as well as economic reasons in an increasingly aging population.
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Unidades de Cuidados Intensivos/economía , Transferencia de Pacientes/economía , Humanos , Modelos Logísticos , Estudios ProspectivosRESUMEN
BACKGROUND: Laparoscopic liver resection belongs to the standard repertoire in hepatobiliary surgery. The advantages and disadvantages are still the subject of controversial discussion. OBJECTIVE: The aim of the study was to compare the perioperative and long-term outcomes of laparoscopic and open liver resections. MATERIAL AND METHODS: All patients who underwent liver resection in the Department of Surgery at the certified liver center of the municipal hospital Karlsruhe were analyzed. From a total of 268 hepatic resections 65 laparoscopic liver resections were identified and matched 1:1 with 65 open resections, based primarily on the extent of the resection and secondarily on diagnosis, age and gender of the patients. The demographic data, comorbidities, perioperative and long-term outcomes were compared. RESULTS: Both groups had comparable demographic parameters and comorbidities. Operation time, duration of intensive care stay and percentage of negative resection margins were comparable in both groups. The 30-day mortality was 0% and 90-day mortality 1.5% in both groups. The laparoscopic group showed lower intraoperative and postoperative transfusion rates (pâ¯< 0.001), shorter hospital stay (pâ¯< 0.001) and lower overall morbidity (pâ¯< 0.001). The 1-, 3- and 5-year overall and tumor-free survival of patients with colorectal liver metastases was comparable (pâ¯= 0.984; pâ¯= 0.947). The same applied for patients with hepatocellular carcinomas (pâ¯= 0.803; pâ¯= 0.935). CONCLUSION: Laparoscopic liver resections have identical long-term outcomes with lower overall morbidity. Laparoscopic liver resections offer advantages regarding transfusion rates, length of hospital stay and postoperative complications.
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Hepatectomía , Laparoscopía , Neoplasias Hepáticas , Hepatectomía/métodos , Humanos , Tiempo de Internación , Neoplasias Hepáticas/cirugía , Análisis por Apareamiento , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: Staple line and anastomotic leakages are life-threatening complications after bariatric surgery. Upper gastrointestinal (GI) tract X-ray examination with oral administration of a water-soluble contrast agent can be used to detect leaks. The aim of this study was to evaluate the impact of routine upper GI tract fluoroscopy after primary bariatric surgery. METHODS: Between January 2009 and December 2014 a total of 658 bariatric interventions were carried out of which 442 were primary bariatric operations. Included in this single center study were 307 sleeve gastrectomies and 135 Roux-en-Y gastric bypasses. Up to December 2012 upper GI tract fluoroscopy was performed routinely between the first and third postoperative days and the detection of leakages was evaluated. RESULTS: In the investigation period 8 leakages (2.6 %) after sleeve gastrectomy, 1 anastomotic leakage in gastrojejunostomy and 1 in jejunojejunostomy after Roux-en-Y gastric bypass occurred. All patients developed clinical symptoms, such as abdominal pain, tachycardia or fever. In one case the leakage was detected by upper GI fluoroscopy and in nine cases radiological findings were unremarkable. No leakages were detected in asymptomatic patients. CONCLUSION: Routine upper GI fluoroscopy is not recommended for uneventful postoperative courses after primary bariatric surgery.
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Cirugía Bariátrica , Fluoroscopía , Complicaciones Posoperatorias/diagnóstico por imagen , Adulto , Anastomosis en-Y de Roux , Fuga Anastomótica/diagnóstico , Femenino , Derivación Gástrica , Gastroplastia , Humanos , Laparoscopía , Masculino , Persona de Mediana Edad , Engrapadoras Quirúrgicas , Dehiscencia de la Herida Operatoria/diagnósticoRESUMEN
INTRODUCTION: Esophageal dilation (ED) has been described as a long-term complication following laparoscopic adjustable gastric banding (LAGB) with an incidence of 0.5-50%. The purpose of this study was to evaluate the effect of major ED on weight loss and find methods to diagnose ED and possible treatment strategies based on a classification. MATERIALS AND METHODS: We performed a retrospective analysis of all patients undergoing LAGB between 2004 and 2008 in three community-based hospitals. ED was classified in four stages of dilation using gastrografin swallow. We report body mass index (BMI), failure rates and reoperations among these patients, with a mean follow-up period of 6.7 years. RESULTS: Nineteen (18.4%) of 103 patients who underwent LAGB presented with esophageal dilation. Band deflation failed for all nine patients (8.7%) with major ED. The mean BMI at LAGB (BMI 1), revision (BMI 2), and 1 year after conversion (BMI 3) were 45.9±3.2, 42.8±4.9 and 30.3±5.5 kg/m2, respectively. No significant difference was found comparing BMI 1 and BMI 2 (p=0,065, EWL1: 14.2±21.7 kg/m2). In contrast, the weight loss after the revision surgery was significant (p=0.001, EWL2: 67.1±30 kg/m2). No significant difference was found concerning age, gender, ASA, preoperative (LAGB) weight, and mean interval between LAGB and revision comparing patients with major ED (IV) to patients with milder forms (ED I-III). CONCLUSION: ED is a serious long-term complication after LAGB and seems to prevent effective weight loss in stage IV. Furthermore, untreated dilation could cause long-term damage to the esophagus. Therefore, we suggest routine radiographic follow-up after LAGB even in asymptomatic patients and a treatment based on a classification with an early surgical revision for major ED.
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Cirugía Bariátrica/métodos , Enfermedades del Esófago/fisiopatología , Obesidad Mórbida/cirugía , Complicaciones Posoperatorias/fisiopatología , Adulto , Índice de Masa Corporal , Dilatación Patológica/epidemiología , Dilatación Patológica/fisiopatología , Dilatación Patológica/terapia , Enfermedades del Esófago/epidemiología , Enfermedades del Esófago/terapia , Esófago/cirugía , Femenino , Estudios de Seguimiento , Gastroplastia/métodos , Humanos , Incidencia , Laparoscopía/métodos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/terapia , Reoperación , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Pérdida de PesoRESUMEN
WHAT IS KNOWN AND OBJECTIVES: Incorrect drug preparation for patients with feeding tubes can result in harm for the patient and the preparing person. Combined intervention programs are effective tools to reduce such preparation errors. However, to date, intervention programs have been mostly tested in hospitals with computerized physician order entry (CPOE), unit-dose systems, or ward-based clinical pharmacists. Hence, the primary objective of this study was to develop and evaluate an intervention program tailored to hospitals without such preconditions. METHODS: We conducted a prospective pre-/post-intervention study on a gastroenterological intensive care unit (ICU) and a surgical ward for oral, dental and maxillofacial diseases (surgical ward). During the study periods, observers documented and evaluated drug preparation processes of all peroral drugs for patients with feeding tubes. The primary endpoint was the rate of inappropriately crushed and/or suspended solid peroral drugs in regards to all solid peroral drugs. RESULTS AND DISCUSSION: Altogether, we evaluated 775 drug preparation processes of solid peroral drugs on the ICU and 975 on the surgical ward. The intervention program significantly reduced incorrect crushing and/or suspending of solid peroral drugs for administration to patients with feeding tubes from 9·8% to 4·2% (P < 0·01) on the ICU and from 5·7% to 1·4% (P < 0·01) on the surgical ward. WHAT IS NEW AND CONCLUSION: The implementation of the newly developed intervention program significantly reduced the rate of inappropriately prepared solid peroral drugs, suggesting that it is an effective measure to enable safe drug administration for inpatients with feeding tubes.
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Química Farmacéutica/estadística & datos numéricos , Capacitación en Servicio/métodos , Intubación Gastrointestinal , Errores de Medicación/estadística & datos numéricos , Suspensiones/química , Humanos , Personal de Enfermería en Hospital , Estudios ProspectivosRESUMEN
BACKGROUND: Bariatric surgery has been performed since 1983 at the Bad Cannstatt Hospital near Stuttgart, Germany. The aim of this study was to investigate the development of bariatric surgery during the past 25 years. METHODS: Data were collected retrospectively. The parameters were number of surgical procedures, hospital stay, and postoperative complications. RESULTS: In the 25-year period 1,041 primary bariatric operations were performed. Open horizontal bypass and open vertical banded gastroplasty were performed initially. Starting in 2003 there was a change to laparoscopic procedures (gastric banding and Roux-en-Y bypass). The mean hospital stays were 14.7+/-5.1 days for open procedures and 6.7+/-4.2 days for laparoscopic methods, with 30-day mortalities of 0.8% and 0.0% and short-term complications at 16.9% and 7.8%, respectively. CONCLUSIONS: Perioperative complications and hospital stay were reduced by performing laparoscopic bariatric surgery. Our study emphasizes the advantages of the laparoscopic procedures which are standard at our hospital and fit in with the remaining operations in a department of visceral surgery.
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Cirugía Bariátrica/estadística & datos numéricos , Obesidad Mórbida/cirugía , Adulto , Cirugía Bariátrica/efectos adversos , Cirugía Bariátrica/métodos , Cirugía Bariátrica/mortalidad , Índice de Masa Corporal , Femenino , Derivación Gástrica/métodos , Alemania , Hospitales Generales , Humanos , Laparoscopía , Laparotomía , Tiempo de Internación , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Reoperación , Estudios Retrospectivos , Factores de TiempoRESUMEN
BACKGROUND: The aim of this study was to compare reliability in handling and function of resterilized and single-use disposable ultrasonic scissors. METHODS: In a prospective randomized study, the surgeon blindly tested new and resterilized ultrasonographic scissors. The parameters were force of activation, cutting effect, coagulation effect, error messages, and disturbing generator noise. RESULTS: Fifty-one new and 49 resterilized instruments in 94 operations were evaluated. The differences in force of activation, cutting effect, and coagulation were not significant. Error messages and disturbing noises were rare in both groups. Six new instruments and two resterilized instruments had to be exchanged because of problems during surgery. CONCLUSION: This study demonstrates comparable reliability in function and handling of resterilized and new ultrasonic scissors. The use of resterilized instruments leads to distinctly reduced costs and could contribute to efficiency in laparoscopic surgery.
Asunto(s)
Equipo Reutilizado , Laparoscopía , Esterilización , Instrumentos Quirúrgicos , Terapia por Ultrasonido/instrumentación , Ahorro de Costo , Análisis de Falla de Equipo , Equipo Reutilizado/economía , Alemania , Humanos , Laparoscopía/economía , Estudios Prospectivos , Control de Calidad , Esterilización/economía , Instrumentos Quirúrgicos/economía , Terapia por Ultrasonido/economíaRESUMEN
BACKGROUND: The aim of this study was to compare reliability in handling and function of resterilised and single-use disposable ultrasonic scissors. METHODS: In a prospective randomized study, the surgeon blindly tested new and resterilised ultrasonic scissors. The parameters were force of activation, cutting effect, coagulation effect, error messages and disturbing generator noise. RESULTS: 51 new and 49 resterilised instruments in 94 operations were evaluated. The differences in force of activation, cutting effect and coagulation were not significant. Error messages and disturbing noises were rare in both groups. 6 new instruments and 2 resterilised instruments had to be exchanged because of problems during surgery. CONCLUSION: This study demonstrates comparable reliability in function and handling of resterilised and new ultrasonic scissors. The use of resterilised instruments leads to distinctly reduced costs and could contribute to efficiency in laparoscopic surgery.
RESUMEN
BACKGROUND AND OBJECTIVE: Surgery is an effective method to treat patients with morbid obesity. However health insurance companies frequently refuse to cover the costs for the procedure despite an existing DRG-code for this operation. Individual medical expertise are necessary to receive reimbursement. In the present study the acceptance of medical expertise to receive cost coverage was analysed in our patients of the years 2000-2003 eligible for obesity surgery. PATIENTS AND METHODS: 617 medical expertise of patients eligible for obesity surgery in our hospital were reviewed and the acceptance rate was evaluated. Parameters such as body mass index, personal medical history, diets, comorbidity and prognosis were included. Expertise were submitted to the health care insurance companies and in case of acceptance the operation was performed. RESULTS: The average age of our patients was 39.1 +/- 11.2 years, 72.1% were female, 27.9% male. The average BMI was 47.5 +/- 7.4 kg/m2. There was a high incidence of comorbidity in these patients (58.7% arterial hypertension, 38.6% diabetes mellitus, 95.8% dyspnoea, 96.1% arthropathy, 89.0% psychosocial disorders). The difference between accepted and non-accepted regarding these secondary complications was not significant. 209 patients (33.8%) were operated. 14 patients of these paid the costs themselves. Only in 195 cases (31.6%) the health care insurance company covered the costs for the operation. CONCLUSION: The high number of refusals of medical expertise is not justified in view of the strict criteria for indication, the high frequency of comorbidity and the good results after the operation.
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Cirugía Bariátrica/economía , Reembolso de Seguro de Salud/estadística & datos numéricos , Obesidad Mórbida/cirugía , Adulto , Comorbilidad , Diabetes Mellitus/epidemiología , Dislipidemias/epidemiología , Disnea/epidemiología , Hígado Graso/epidemiología , Femenino , Alemania/epidemiología , Humanos , Hipertensión/epidemiología , Artropatías/epidemiología , Masculino , Trastornos Mentales/epidemiología , Obesidad Mórbida/economía , Obesidad Mórbida/epidemiología , Guías de Práctica Clínica como Asunto , Pronóstico , Estudios RetrospectivosRESUMEN
Lipopolysaccharide (LPS), a potent activator of human monocytes, induced F-actin polymerization in a concentration- and time-dependent manner. To test whether cytoskeletal events participate in the control of the LPS-induced ROI and TNF-alpha production, three natural occurring actin-modulating substances, cytochalasin D (Cyt D), latrunculin B (Lat B), and jasplakinolide (JK), were used. Here we show that treatment of monocytes with Cyt D, Lat B, or JK led to a rearrangement of the actin cytoskeleton, which upon addition of LPS was further modified. Cyt D and Lat B induced generation of ROI in the absence of LPS and enhanced the LPS-triggered respiratory burst. JK also proved to be a potent activator of ROI-production but only in the presence of LPS. TNF-alpha production was hardly affected by the three substances. There was no correlation between a specific state of Cyt D-, Lat B-, or JK-modified actin polymerization and ROI-production. Inhibitors of ADP-ribosylation proved to be activators of F-actin polymerization. They were shown to prevent ROI- and TNF-alpha production and to reduce the capability of LPS to mediate maximal F-actin assembly. At concentrations at which inhibition was greatest, maximal blockage of ROI and TNF-alpha production was observed. These findings may argue for a role of ADP-ribosylation in the transduction pathways mediating the biological responses, with involvement in the assembly of actin-containing cytoskeletal microfilaments.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Depsipéptidos , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Citoesqueleto de Actina/efectos de los fármacos , Adenosina Difosfato Ribosa/metabolismo , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citocalasina D/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Monocitos/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Péptidos Cíclicos/farmacología , Fosforilación , Polímeros/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tiazoles/farmacología , TiazolidinasRESUMEN
Progesterone 5 beta-reductase, which catalyzes the reduction of progesterone to 5 beta-pregnane-3,20-dione, was purified 770-fold to homogeneity from the cytosolic fraction of shoot cultures of Digitalis purpurea. This purification involved DEAE-Sephacel, affinity chromatography (Blue-Sepharose CL-6B and adenosine 2',5'-bisphosphate-Sepharose 4B) and elution from a gel matrix after non-dissociating PAGE. The molecular mass determined by SDS/PAGE was 43 kDa and the molecular mass determined by gel-filtration chromatography on calibrated Sephadex G-200 was 280 kDa, thus indicating that the native protein is a polymer consisting of several subunits. The purified enzyme had a Km value of 6 microM for NADPH and 34 microM for progesterone. The enzyme had a strong substrate specificity for progesterone. The relative rates for other steroids such as testosterone, cortisone and cortisol were much lower. The trypsin digestion of the purified progesterone 5 beta-reductase resulted in 100 peptide fragments. The largest fragment after trypsin digestion and sequence analysis consisted of 13 amino acids.
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Digitalis/enzimología , Oxidorreductasas/aislamiento & purificación , Plantas Medicinales , Plantas Tóxicas , Secuencia de Aminoácidos , Cardenólidos/metabolismo , Cinética , Datos de Secuencia Molecular , Peso Molecular , NADP/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Esteroides , Especificidad por SustratoRESUMEN
Catabolite repression (CR) of xylose utilization by Bacillus subtilis involves a 14-bp cis-acting element (CRE) located in the translated region of the gene encoding xylose isomerase (xylA). Mutations of CRE making it more similar to a previously proposed consensus element lead to increased CR exerted by glucose, fructose, and glycerol. Fusion of CRE to an unrelated, constitutive promoter confers CR to beta-galactosidase expression directed by that promoter. This result demonstrates that CRE can function independently of sequence context and suggests that it is indeed a generally active cis element for CR. In contrast to the other carbon sources studied here, glucose leads to an additional repression of xylA expression, which is independent of CRE and is not found when CRE is fused to the unrelated promoter. This repression requires a functional xylR encoding Xyl repressor and is dependent on the concentrations of glucose and the inducer xylose in the culture broth. Potential mechanisms for this glucose-specific repression are discussed.
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Isomerasas Aldosa-Cetosa , Bacillus subtilis/metabolismo , Carbohidrato Epimerasas/metabolismo , Genes Bacterianos/fisiología , Operón/fisiología , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Xilosa/metabolismo , Bacillus subtilis/genética , Secuencia de Bases , Carbohidrato Epimerasas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos/genética , Glucosa/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Mutación/fisiología , Operón/genética , Xilosa/genéticaAsunto(s)
Hígado/efectos de los fármacos , Venenos de Moluscos/toxicidad , Animales , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Hígado/metabolismo , Circulación Hepática/efectos de los fármacos , Venenos de Moluscos/aislamiento & purificación , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Perfusión , Ratas , Caracoles , EspectrofotometríaRESUMEN
We have selected a Bacillus subtilis 168-borne xylR Ser to Leu mutation at position 41 of the encoded amino acid sequence showing a constitutive expression phenotype for the xyl operon. When cloned on a multi-copy plasmid in a B. megaterium strain harbouring a single-copy xylA-lacZ fusion it leads to derepression of beta-galactosidase expression. Thus, it is trans dominant over the endogenous xylR, indicating that Xyl repressor functions as a multimer. This result also supports the assumption that the mutation is in a putative alpha-helix-turn-alpha-helix operator binding motif of Xyl repressor.
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Bacillus megaterium/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Regiones Operadoras Genéticas , Proteínas Represoras/genética , Secuencia de Aminoácidos , Expresión Génica , Genes Dominantes , Datos de Secuencia Molecular , Mutación , Plásmidos/genética , Homología de Secuencia de AminoácidoRESUMEN
A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 bp synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Operón , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Xilosa/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/metabolismo , Mapeo Restrictivo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The Bacillus subtilis xyl operon encoding enzymes for xylose utilization is repressed in the absence of xylose and in the presence of glucose. Transcriptional fusions of spoVG-lacZ to this operon show regulation of beta-galactosidase expression by glucose, indicating that glucose repression operates at the level of transcription. A similar result is obtained when glucose is replaced by glycerol, thus defining a general catabolite repression mechanism. A deletion of xylR, which encodes the xylose-sensitive repressor of the operon, does not affect glucose repression. The cis element mediating glucose repression was identified by Bal31 deletion analysis. It is confined to a 34 bp segment located at position +125 downstream of the xyl promoter in the coding sequence for xylose isomerase. Cloning of this segment in the opposite orientation leads to reduced catabolite repression. The homology of this element to various proposed consensus sequences for catabolite repression in B. subtilis is discussed.
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Bacillus subtilis/genética , Operón , Transcripción Genética , Xilosa/metabolismo , Secuencia de Bases , Expresión Génica , Genes Bacterianos , Glucosa/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Biosíntesis de Proteínas , Homología de Secuencia de Ácido Nucleico , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Leaves of Digitalis purpurea contain an enzyme activity which catalyzes the conversion of progesterone to 5 beta-pregnane-3,20-dione. Since cardenolides without exception possess a 5 beta-configuration, 5 beta-pregnane-3,20-dione can serve as a precursor for this class of secondary metabolites. It is assumed that the enzyme is part of the putative biosynthetic pathway of cardenolides. This enzyme activity was spotted in the soluble fraction of a crude homogenate. Product formation was detected by gas chromatography and by gas chromatography/mass spectroscopy (g.c./m.s.). The enzyme had a pH optimum at 8.0 and an apparent Km value of 6 microM for progesterone. It required NADPH as a co-substrate with an apparent Km value of 22 microM. The optimum temperature in vitro was 30 degrees C. The activity was not dependent on monovalent and bivalent cations.
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Cardenólidos/metabolismo , Oxidorreductasas/análisis , Plantas/enzimología , Cationes/farmacología , Cromatografía Líquida de Alta Presión , Cinética , NAD/metabolismo , NADP/metabolismo , Oxidorreductasas/química , Plantas/efectos de los fármacos , Pregnanodionas/metabolismo , Progesterona/metabolismo , Solubilidad , Especificidad por SustratoRESUMEN
The xyl operator of Bacillus subtilis W23 was identified by deletion analysis of the xyl regulatory region as a 25-base-pair (bp) sequence located 10 bp downstream from the xyl promoter. The outer 10 bp of the xyl operator exhibit perfect palindromic symmetry, while 5 central bp are nonpalindromic. It was demonstrated that the penultimate base pair near the end of this sequence is important for repressor binding. The location of the xylR gene encoding the repressor was determined by its ability to mediate xylose-dependent repression of a xyl-cat fusion on a multicopy plasmid. The nucleotide sequence of 1,355 bp from this DNA was analyzed and contains an open reading frame with a coding capacity for 384 amino acids leading to a protein with a calculated molecular weight of 42,270. A mutant with a deletion in this reading frame showed no repression of the xyl-cat fusion. The coding sequence is preceded by a suitable ribosome-binding sequence and uses GTG as a start codon and TAA as a stop codon. The relationship of these results to corresponding data obtained from B. subtilis 168 is discussed.
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Bacillus subtilis/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Regiones Operadoras Genéticas , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Genes , Datos de Secuencia Molecular , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificaciónRESUMEN
Expression of xylose isomerase was repressed in Bacillus subtilis strains W23, 168, and BR151 and could be induced in the presence of xylose. The expression was also glucose repressed in strains 168 and BR151, although this effect was not observed with W23. A xyl-cat fusion gene was constructed on a multicopy plasmid, from which the xyl promoter located on a 366-base-pair (bp) DNA fragment derived from W23 directed the expression of chloramphenicol resistance. The regulation of expression was not very pronounced in this multicopy situation. The xyl promoter is a strong signal for transcription initiation. The 5' sequence of the xyl mRNA was identified by nuclease S1 mapping. The promoter consisted of the -10 sequence TAAGAT, the -35 sequence TTGAAA spaced by 17 bp, and an upstream poly(A) block with 14 As out of 17 bp. To study the regulation, a xyl-lacZ fusion gene was constructed and integrated as a single copy into the amygene of B. subtilis 168. This strain grows blue on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) indicator plates in the presence of xylose and white in the presence of glucose. Quantitatively, the induction of beta-galactosidase by xylose was 100-fold. In the presence of xylose plus glucose, the expression of the indicator gene was repressed to 30% of the fully induced level. About 25 to 60% of the maximal lacZ expression was obtained with this strain when the 366-bp xyl DNA fragment was provided in trans on a multicopy plasmid. This result indicates that repression in the absence of xylose is mediated in trans by a soluble factor which is expressed at a low level in B. subtilis 168. The xylose effect depended on negative regulation. The estimations of mRNA amounts by dot blot analysis showed unambiguously that the induction by xylose occurs at the level of transcription. The possible molecular mechanisms are discussed with respect to the nucleotide sequence of the 366-bp xyl regulatory DNA.