RESUMEN
Leishmaniasis is a spectrum of conditions caused by infection with the protozoan Leishmania spp. parasites. Leishmaniasis is endemic in 98 countries around the world, and resistance to current anti-leishmanial drugs is rising. Our work has identified and characterised a previously unstudied galactokinase-like protein (GalK) in Leishmania donovani, which catalyses the MgATP-dependent phosphorylation of the C-1 hydroxyl group of d-galactose to galactose-1-phosphate. Here, we report the production of the catalytically active recombinant protein in E. coli, determination of its substrate specificity and kinetic constants, as well as analysis of its molecular envelope using in solution X-ray scattering. Our results reveal kinetic parameters in range with other galactokinases with an average apparent Km value of 76 µM for galactose, Vmax and apparent Kcat values with 4.46376 × 10-9 M/s and 0.021 s-1, respectively. Substantial substrate promiscuity was observed, with galactose being the preferred substrate, followed by mannose, fructose and GalNAc. LdGalK has a highly flexible protein structure suggestive of multiple conformational states in solution, which may be the key to its substrate promiscuity. Our data presents novel insights into the galactose salvaging pathway in Leishmania and positions this protein as a potential target for the development of pharmaceuticals seeking to interfere with parasite substrate metabolism.
Asunto(s)
Leishmania donovani , Proteínas Protozoarias , Proteínas Recombinantes , Leishmania donovani/enzimología , Leishmania donovani/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Especificidad por Sustrato , Galactoquinasa/metabolismo , Galactoquinasa/genética , Galactoquinasa/química , Cinética , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosa/metabolismoRESUMEN
To demonstrate the relevance of activity-based cost management (ABCM) for the occupational and environmental health community, the investigators used data generated by an ABCM model of a respiratory protection program (RPP) to develop options for solving a business problem. The RPP manager in this hypothetical but realistic business scenario is faced with a 25% budget cut and a 10% increase in demand for RPP services. The manager's dilemma is to maintain the integrity of the RPP while absorbing a significant budget cut. Various cost savings options are developed, and the assumptions under which these options operate are presented. It is emphasized that the RPP manager's primary responsibility is to assure worker health and safety by first understanding the technical issues, merits, and implications of any cost-cutting option that may be considered. It is argued that only then should the manager consider the financial merits of the possible solutions to this business problem. In this way worker health and safety, and environmental protection goals, can continue to be achieved in an economic climate of cost cutting and downsizing.
Asunto(s)
Contabilidad/métodos , Salud Ambiental/economía , Exposición por Inhalación/prevención & control , Servicios de Salud del Trabajador/economía , Dispositivos de Protección Respiratoria/economía , Presupuestos , Asignación de Costos/métodos , Control de Costos , Costos y Análisis de Costo , Humanos , Modelos OrganizacionalesRESUMEN
The antipseudomonal activities of ceftriaxone (CEF) or ceftazidime (CAZ), each combined with tobramycin (TOB) or netilmicin (NET), against 90 clinically significant Pseudomonas aeruginosa isolates were examined both by checkerboard and time-kill assays. As expected, susceptibility testing of single antibiotics by agar dilution demonstrated good activity for CAZ (89% susceptible), TOB (94%), and NET (58%), but poor activity for CEF (15%). Checkerboard studies revealed striking synergy (FIC indices < or = 0.5) for CEF, however, in combination with either TOB (72%) or NET (81%), compared with CAZ-TOB (44%) or CAZ-NET (60%) (P < 0.01, respectively). No antagonism (FIC indices > or = 4) was found in any of these combinations. The MIC90s of CEF, CAZ, or aminoglycosides in the combinations were reduced at least fourfold: CEF, from > 128 to 32 mg/liter; CAZ, from 16 to 4 mg/liter; TOB, from 4 to 0.5 mg/liter; and NET, from 32 to 4 mg/liter. With CEF and NET, the percentage of strains sensitive to < or = 8 mg/liter of both drugs alone and in combination increased from 9% to 69%. Results of the time-kill assay for CEF-NET agreed reasonably well with the checkerboard method (Spearman rank correlation coefficient, 0.40, P < or = 0.01), and generated a bactericidal outcome in 60% (24 of the 40 isolates), when tested with combinations at 1/4 MBC of either antibiotic alone. Importantly, concentrations of CEF and aminoglycoside combinations that demonstrated synergy by either checkerboard or time-kill assays were achievable in serum clinically. These data suggest a unique interaction of CEF-aminoglycoside combinations against P. aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ceftazidima/farmacología , Ceftriaxona/farmacología , Netilmicina/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Tobramicina/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/microbiologíaRESUMEN
The development and characterization of a mouse model of chronic Staphylococcus epidermidis foreign body infection was done with two clinical isolates that differed in degree of extracellular slime production. Segments of Silastic catheters bearing preformed S. epidermidis biofilms were implanted intraperitoneally, and mice were assessed after 3 and 6 months. Both test strains of S. epidermidis persisted at the implant site through the 6-month follow-up in 80% of the mice, regardless of the degree of slime production. There was no evidence of overt animal morbidity, and microbiologic assessment of other peritoneal sites did not reveal dissemination of bacteria from the infected focus. In comparison with control mice, animals harboring chronic foreign body infection presented marked peripheral neutrophilia and mild anemia.
Asunto(s)
Catéteres de Permanencia , Cuerpos Extraños/complicaciones , Peritonitis/etiología , Infecciones Estafilocócicas/etiología , Staphylococcus epidermidis , Animales , Modelos Animales de Enfermedad , Femenino , Tejido de Granulación/patología , Humanos , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Diálisis Peritoneal Ambulatoria ContinuaRESUMEN
A retrospective study was conducted in 20 Canadian hospitals to assess peritonitis rates of CAPD patients utilizing the SCD 210 patient assist device during a 30 month study period. A total of 175 patients having a cumulative SCD experience of 1,494 patient months were included in the survey. Sixty-eight patients experienced 116 episodes of peritonitis, i.e., one episode every 12.9 patient months (pt mo). Patients that had used the SCD for their entire CAPD experience had a significantly lower peritonitis rate (1/15.2 pt mo) compared to patients who had used other CAPD systems (1/10.1 pt mo). Peritonitis rates for diabetic study patients or patients with impaired vision and/or dexterity were not significantly greater than non-diabetic or non-impaired study patients. Staphylococcus epidermidis and Staphylococcus aureus were the most frequent causative microorganisms, accounting for 27.7% and 16.0% of peritonitis episodes caused by single organisms, respectively. The proportion of peritonitis caused by skin commensals was consistent across all study patients, regardless of impairment of vision and/or dexterity. The study results demonstrate the successful application of this patient assist device in a CAPD population that consisted of a large proportion of high risk patients.
Asunto(s)
Diálisis Peritoneal Ambulatoria Continua/instrumentación , Peritonitis/prevención & control , Bacterias/aislamiento & purificación , Soluciones para Diálisis , Femenino , Humanos , Masculino , Diálisis Peritoneal Ambulatoria Continua/efectos adversos , Peritonitis/etiología , Peritonitis/microbiología , Estudios RetrospectivosRESUMEN
A mouse model of surgically induced renal failure harboring a peritoneal catheter implant was utilized to study the response to intracatheter (IC) peritoneal inoculation of 10(6) or 10(8) colony-forming units (CFU) Staphylococcus epidermidis. The kinetics of bacterial clearance from peritoneal structures, including the peritoneal catheter implant, was investigated in renal failure and sham-operated mice. All animals survived experimental challenge with 10(6) CFU; however, microbiological assessments conducted 1 week and 1 month after inoculation revealed that the catheter site of a small number of renal failure mice was persistently colonized. During 1 month following 10(8) CFU IC inoculation, significant animal mortality was observed in renal failure mice (29.4%), while sham-operated controls presented minimal lethality (5.9%). In surviving mice of both groups, the peritoneal catheter site proved to be a reservoir for persisting S. epidermidis, a finding confirmed by scanning electron microscopy. Throughout the study, local and systemic inflammatory responses were comparable in sham-operated and renal failure mice. Nevertheless, 3 and 4 weeks after 10(8) CFU inoculation, S. epidermidis recovery was significantly greater in sham-operated controls. Phenotypic colonial variation was observed in a majority of S. epidermidis isolates, and only in those specimens recovered 2 or more weeks after inoculation. Our results reveal that the peritoneal catheter implant provides a preferred site for persistent bacterial colonization up to 1 month after inoculation.
Asunto(s)
Catéteres de Permanencia , Fallo Renal Crónico/fisiopatología , Diálisis Peritoneal Ambulatoria Continua/instrumentación , Staphylococcus epidermidis/fisiología , Animales , Recuento de Colonia Microbiana , Femenino , Estudios de Seguimiento , Fallo Renal Crónico/terapia , Cinética , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Peritoneo/microbiología , Sepsis/microbiología , Infecciones Estafilocócicas/fisiopatología , Propiedades de Superficie , Tasa de SupervivenciaRESUMEN
The effect of repeated instillation of peritoneal dialysis (PD) solution on the peritoneal clearance of a Staphylococcus epidermidis challenge was investigated in a mouse model of surgically induced chronic renal failure. For periods of up to 2 weeks, mice bearing peritoneal catheter implants underwent daily (3 mL) or twice daily (1.5 mL) peritoneal instillation of PD solution (4.25% dextrose) by transcutaneous injection into the catheter lumen. Peritoneal instillation of PD solution did not have a significant influence on the microbiological status of peritoneal structures of renal failure or sham-operated mice following experimental intracatheter S epidermidis inoculation with 10(6) colony-forming units (CFU) (assessment 48 hours after inoculation) or 10(8) CFU (assessment 1 week after inoculation). Microbiological and scanning electron microscopy (SEM) assessments of recovered peritoneal catheters demonstrated that S epidermidis remained associated with the catheter site after other peritoneal structures had become culture negative. SEM of the parietal peritoneum revealed striking morphologic alterations of the mesothelial surface as a consequence of daily PD solution infusion. In the absence of S epidermidis inoculation, repeated instillation of PD solution caused a marked acute peritoneal inflammation without evidence of a concomitant systemic inflammatory response. Furthermore, peritoneal inflammatory response to S epidermidis challenge was augmented by the infusion procedure. Concurrent assessments of inflammatory response and microbiological status revealed that, in spite of heightened peritoneal inflammatory response with peritoneal infusion, bacterial clearance from the catheter site was not improved. Although the animal preparation was limited to peritoneal infusion without drainage, the influence of repeated peritoneal instillation of hyperosmolar, acidic PD solution on the response of mice to S epidermidis challenge was successfully addressed.
Asunto(s)
Soluciones para Diálisis , Fallo Renal Crónico/terapia , Peritonitis/microbiología , Animales , Catéteres de Permanencia , Femenino , Fallo Renal Crónico/complicaciones , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Diálisis Peritoneal Ambulatoria Continua/efectos adversos , Peritonitis/complicaciones , Infecciones Estafilocócicas/complicaciones , Staphylococcus epidermidis/crecimiento & desarrollo , Staphylococcus epidermidis/aislamiento & purificaciónRESUMEN
The influence of a permanent peritoneal catheter implant on the response of renal failure and control mice to peritoneal inoculation with 10(6) colony-forming units (CFU) Staphylococcus epidermidis was assessed 48 h after bacterial challenge. Two weeks after the surgical induction of renal failure or sham surgery, a segment of a peritoneal dialysis catheter was implanted entirely within the confines of the peritoneal cavity of mice. One month later peritoneal S. epidermidis inoculation was performed by transcutaneous injection through the abdominal wall either directly into the peritoneal cavity (i.p.) or via the catheter lumen (i.c.). Following i.p. inoculation, minimal bacterial growth was recovered from the peritoneal structures of all mice, including the peritoneal catheter. In contrast, following i.c. S. epidermidis challenge, the catheter site remained heavily colonized while peritoneal washings and parietal peritoneum again presented minimal bacterial recoveries. S. epidermidis recovery from the catheter site of renal failure mice was significantly greater than from the respective site of sham-operated controls. Scanning electron microscopy of catheter segments recovered from mice following i.c. inoculation revealed single cocci or microcolonies associated with the catheter surface and differential leukocyte counts of fluid aspirated from the catheter lumen revealed evidence of acute inflammation. Signs of inflammatory processes in peritoneal washings and peripheral blood, however, were not observed. These results are discussed in relation to S. epidermidis peritonitis and continuous ambulatory peritoneal dialysis.
Asunto(s)
Cateterismo , Enfermedades Renales/inmunología , Peritoneo , Peritonitis/inmunología , Infecciones Estafilocócicas/inmunología , Animales , Cateterismo/efectos adversos , Femenino , Enfermedades Renales/patología , Ratones , Ratones Endogámicos C57BL , Peritonitis/etiología , Infecciones Estafilocócicas/etiologíaRESUMEN
A mouse model of renal failure, which is induced by the sequential electrocoagulation of the right renal cortex and left nephrectomy, was examined for the capacity to reproduce the characteristics of chronic uremia. Assessment was conducted six weeks after the second surgical procedure in 13 week old female C57BL/6 inbred mice with renal failure and in normal and sham-operated controls. The surgery, which was well tolerated, was free of local and systemic signs of inflammation or infection. Growth was significantly delayed in all animals post surgery however renal failure mice presented the most severe growth retardation. Biochemical analysis of plasma revealed multiple abnormalities with commensurate elevations of urea and creatinine. In addition to the expected hyperphosphatemia, hyperkalemia and acidosis, a significant increase in cholesterol was present. Furthermore, in contrast to controls, renal failure mice produced large volumes of urine which contained significant levels of protein. Renal failure mice presented profound hematological changes in the red cell series in which anemia was evident. Changes in plasma biochemistry and in bone histology revealed the presence of severe secondary hyperparathyroidism. It was therefore concluded that the described mouse model of chronic renal failure presented characteristics consistent with those observed clinically in end-stage renal disease.
Asunto(s)
Modelos Animales de Enfermedad , Uremia/fisiopatología , Animales , Presión Sanguínea , Nitrógeno de la Urea Sanguínea , Huesos/patología , Fallo Renal Crónico/fisiopatología , Ratones , Ratones Endogámicos C57BL , Recuento de PlaquetasRESUMEN
The role of renal failure in the pathogenesis of the Staphylococcus epidermidis (S. epidermidis) peritonitis presented by end-stage renal disease patients treated with continuous ambulatory peritoneal dialysis was investigated in a mouse model of surgically-induced renal failure. Six weeks after the surgery, an i.p. inoculum of 10(6) colony forming units S. epidermidis was administered to renal failure mice and their sham-operated and normal controls, and assessment of bacterial clearance and inflammatory response was conducted over the next 72 hours. Peritoneal clearance of S. epidermidis was complete in most animals; however, the process was significantly delayed in renal failure mice compared to sham-operated controls. Viable bacteria invariably remained associated with the peritoneum after peritoneal washings had become culture negative. Peritoneal inflammatory response was markedly diminished in renal failure mice, the early polymorphonuclear cell response being particularly affected. Peripheral response consisted of a prompt and short-lived polymorph increase which was similar in renal failure and sham-operated mice. The factors responsible for the observed impairment of local inflammatory response in association with delayed bacterial clearance in renal failure mice following i.p. challenge with S. epidermidis remain to be defined.
Asunto(s)
Fallo Renal Crónico/inmunología , Diálisis Peritoneal Ambulatoria Continua , Peritonitis/inmunología , Infecciones Estafilocócicas/inmunología , Animales , Femenino , Tolerancia Inmunológica , Inmunidad Celular , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Staphylococcus epidermidis/inmunologíaRESUMEN
A gradual loss of cell viability was observed during in vitro incubation of peritoneal cells from chronically uremic mice in commercial peritoneal dialysis solutions. The magnitude of this cytotoxicity toward peritoneal cells harvested from uremic mice and controls was comparable. Resident peritoneal cells were always found to be more susceptible than thioglycolate-elicited peritoneal populations of either macrophages or polymorphonuclear cells. In order to elucidate the factors contributing to this phenomenon, resident peritoneal cells recovered from normal mice were incubated in vitro for 1 h in various solutions of known pH and osmolarity consisting of buffered and unbuffered commercial peritoneal dialysis solutions. The results clearly show that the major part of the cytotoxicity is attributable to the low pH of the solutions. Once pH was corrected, the hyperosmolarity of these solutions had no effect on cell viability; however, a small but significant cytotoxicity remained. Factors other than those addressed in this study probably account for the observed residual cytotoxicity.