[Linezolid for the treatment of newborns infected with vancomycin-resistant Enterococcus faecium]. / Uso de linezolid para el tratamiento de recién nacidos con infecciones por Enterococcus faecium resistentes a la vancomicina.

Five patients hospitalized in the neonatal unit between 2002 and 2007, infected with vancomycin-resistant Enterococcus faecium treated with linezolid are presented in this study. This antibiotic showed good clinical efficacy and safety, since no adverse events occured in this group of patients.

Aplicación de PCR a partir de cultivo en la identificación de subespecies del complejo Mycobacterium Tuberculosis / The use of culture-based PCR for the identification of subspecies of the mycobacterium tuberculosis complex

Los métodos diagnósticos clásicos de tuberculosis (TB) se basan en la utilización de baciloscopía y cultivo. La identificación del agente etiológico desde la positivización del cultivo requiere entre 10 y 15 días, mientras que el empleo de la reacción en cadena de la polimerasa (PCR) disminuye el tiempo a 24 h, lo que permite no solo identificar las subespecies del complejo Mycobacterium tuberculosis (CMTB) sino también diferenciarlas de otras especies ambientales clínicamente importantes (MOTT) facilitando el diagnóstico y tratamiento. El objetivo del presente trabajo fue determinar la utilidad de la PCR en la identificación temprana de las micobacterias pertenecientes al CMTB, a partir de cultivos positivos, de pacientes con sospecha de TB, atendidos en un hospital pediátrico de alta complejidad, durante un período de cuatro años. A cada muestra, se le realizó baciloscopía y cultivo en medio líquido. A los cultivos positivos, una inmunocromatografía lateral (TBIDR) y luego PCR. El 4,6% del total de muestras (510/11.162) pertenecientes a 198 pacientes presentó cultivos positivos. Cuatrocientos veintiseis (84%) correspondieron a muestras respiratorias. El rendimiento de la baciloscopía directa fue del 41% (194/470). Cuatrocientos treinta y ocho (86%) resultaron M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG y 44 (9%) MOTT. La utilización de medios de cultivos líquidos junto con el empleo de PCR favorecen una rápida orientación microbiológica y constituye una estrategia útil para optimizar el manejo clínico de estas infecciones, desde el punto de vista terapéutico y epidemiológico, especialmente en pediatría (AU)

Classical diagnostic methods for tuberculosis (TB) are based on the use of smear microscopy and culture. The identification of the etiological agent from positive culture requires 10 to 15 days, while the use of the polymerase chain reaction (PCR) reduces the time to 24 h, which allows not only to identify the subspecies of the Mycobacterium tuberculosis complex (MTC) but also to differentiate them from clinically important environmental mycobacteria other than tuberculosis (MOTT), facilitating diagnosis and treatment. The aim of this study was to determine the usefulness of PCR in the early identification of mycobacteria belonging to the MTC, from positive cultures of patients with suspected TB seen in a pediatric tertiary hospital over a 4-year period. For each sample, smear microscopy and culture in liquid medium was performed. Positive cultures were subjected to lateral immunochromatography (TBIDR) and then PCR. Of the total number of samples (510/11,162) belonging to 198 patients, 4.6% showed positive cultures; 426 (84%) were respiratory samples. The direct smear microscopy yield was 41% (194/470). Overall, 438 (86%) were found to be M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG, and 44 (9%) MOTT. The use of liquid culture media together with the use of PCR favors a rapid microbiological orientation and is a useful strategy to optimize the clinical management of these infections, from a therapeutic and epidemiological point of view, especially in children (AU)

Infecciones por Chlamydia trachomatis en una unidad de cuidados intensivos neonatales / Chlamydia trachomatis infections in a neonatal intensive care unit

Las infecciones por Chlamydia trachomatis han aumentado su prevalencia, especialmente en jóvenes embarazadas. Esto adquiere relevancia en pediatría por el elevado riesgo de transmisión vertical al neonato y su potencial gravedad en el lactante. Estas infecciones requieren de un alto índice de sospecha, por cuadro clínico atípico y signos radiológicos inespecíficos. Los métodos diagnósticos convencionales presentan limitaciones para su detección. Las técnicas moleculares son las recomendadas por su elevada sensibilidad, especificidad y rapidez, lo cual permite una terapéutica adecuada y oportuna. En este estudio, desarrollado en una unidad de cuidados intensivos neonatales de un hospital de alta complejidad durante 12 años, se describieron las características de la población, su presentación clínica y evolución. La detección microbiológica se realizó por métodos moleculares. Se incluyeron 29 pacientes (p) con infección por C. trachomatis (3,9% del total de muestras enviadas),13 p con infección respiratoria y 16 p con compromiso ocular. La mediana de edad fue de 19 días al momento del diagnóstico y el 65% de las gestantes tenía <25 años. Veinticuatro p (83%) eran recién nacidos a término y 23 p (79%) previamente sanos. Nueve p (31%) presentaron fiebre al momento del ingreso y 12 (41%) eosinofilia. De los 13 p con enfermedad respiratoria, 9 (69%) consultaron por tos y 11 (85%) con hipoxemia, con requerimientos de oxígeno en 8 (61%), asistencia respiratoria mecánica en 3 (23%) y uno (16%) requirió ECMO. Los hallazgos radiológicos mostraron un patrón intersticial inespecífico. Nueve p (31%) presentaron coinfección y uno falleció asociado a influenza A (AU)

The prevalence of Chlamydia trachomatis infections has increased, especially among young pregnant women. This is of particular relevance in pediatrics due to the high risk of motherto-child transmission and the potential severity of the infection in infants. A high index of suspicion is required for these infections due to the atypical clinical features and non-specific radiological signs. The usefulness of conventional diagnostic methods is limited. Molecular techniques are recommended because of their high sensitivity, specificity, and speed, allowing for adequate and timely treatment. In this 12-year study conducted in a neonatal intensive care unit of a tertiary-care hospital, patient characteristics, clinical presentation, and outcome are described. Microbiological detection was performed using molecular methods. Twenty-nine patients with C. trachomatis infection (3.9% of the total samples submitted), of whom 13 had respiratory tract infection and 16 ocular involvement, were included. The median age at diagnosis was 19 days and 65% of the mothers were <25 years old. Twenty-four p (83%) were term newborns and 23 patients (79%) were previously healthy. On admission, 9 patients (31%) had fever and 12 (41%) had eosinophilia. Of the 13 patients with respiratory tract involvement, 9 (69%) consulted for cough and 11 (85%) had hypoxemia, requiring oxygen in 8 (61%), mechanical ventilation in 3 (23%), and ECMO in 1 (16%). Radiological findings showed a nonspecific interstitial pattern. Nine patients (31%) presented with coinfection, one of whom died due to an associated influenza A infection (AU)

Características clínicas y epidemiológicas asociadas con internación en niños con COVID-19 en un hospital pediátrico de alta complejidad: Estudio de cohorte enero-marzo 2022 / Clinical and epidemiologic characteristics associated with hospital admission in children with COVID19 in a tertiary-care pediatric hospital: A January-March 2022 cohort study

Introducción: La proporción de casos reportados de niños y adolescentes con COVID-19 aumenta progresivamente. La hospitalización relacionada con COVID-19 en niños es infrecuente, pero causa morbilidad y sobrecarga al sistema de salud. Objetivos: Describir las características clínicas y evolutivas de los niños con diagnóstico de COVID-19 en un hospital pediátrico de alta complejidad. Comparar los pacientes que requirieron internación y los que no. Material y métodos: Cohorte prospectiva. Se incluyeron todos los pacientes con diagnóstico virológico de COVID-19 desde 1.1.2022 a 1.3.22 en un hospital pediátrico de alta complejidad. Se compararon los antecedentes, características clínicas y evolutivas de los pacientes según requirieran o no internación. Se utilizó STATA 16. Resultados: n: 1764 pacientes, de ellos 958 eran varones (54%). La mediana de edad fue 56 meses (RIC 17-116). Tenían enfermedad de base 789 pacientes (46%). Las más frecuentes fueron: enfermedad oncohematológica 215 (12%), neurológica 103 pacientes (6%) , enfermedad pulmonar crónica 68 (4%), cardiopatías congénitas 65 (4%) y síndrome genético 57 pacientes (3%). Eran inmunosuprimidos: 292 (17%). Presentaron síntomas relacionados con COVID-19 1319 pacientes (79%). Requirieron internación 591 (34%). Tuvieron coinfección con otros virus respiratorios 33 pacientes (2%). Ingresaron a Cuidados intensivos en relación a la COVID-19 22 pacientes (1.3%) y fallecieron en relación con la infección 8 (0.5%). En el análisis univariado, la presencia de comorbilidades, la coinfección viral y la inmunosupresión se asociaron estadísticamente con el requerimiento de internación. El antecedente de 2 o más dosis de vacuna para SARS-CoV-2 fue un factor protector para la internación en los mayores de 3 años. En el modelo multivariado, los pacientes menores de 3 años (OR 6.5, IC95% 1.2-36.8, p 0.03), con comorbilidades (OR 2.04, IC 95% 1.7- 3.3, p 0.00) y los huéspedes inmunocomprometidos (OR 2.89, IC95% 2.1-4.1, p 0.00) tuvieron más riesgo de internación. Ajustado por el resto de las variables, haber recibido dos o más dosis de vacuna fue un factor protector para la internación (OR 0.65, IC 95% 0.49-0.87, p<0.01). Conclusiones: En este estudio de cohorte prospectivo de niños con diagnóstico confirmado de COVID-19 predominó la enfermedad sintomática. Fueron admitidos en relación con el COVID-19, 34% de los pacientes. La vacunación con dos o más dosis fue un factor protector para la internación en el modelo multivariado. Además, se asociaron estadísticamente con la hospitalización, la edad menor de 3 años, las comorbilidades previas y la inmunosupresión (AU)

Introduction: The rate of reported cases of children and adolescents with COVID-19 is progressively increasing. COVID-19-related hospital admission in children is uncommon, but leads to morbidity and places a burden on the healthcare system. Objectives: To describe the clinical characteristics and outcome of children diagnosed with COVID-19 in a pediatric tertiary-care hospital and to compare patients who required hospital admission with those who did not. Material and methods: A prospective cohort study. All patients with a virological diagnosis of COVID-19 seen between 1.1.2022 and 1.3.22 in a tertiary-care pediatric hospital were included. We compared patient history, clinical characteristics, and outcome according to whether or not they required hospital admission. STATA 16 was used. Results: n: 1764 patients, 958 of whom were male (54%). The median age was 56 months (IQR, 17- 116). Overall, 789 patients had an underlying disease (46%), the most frequent of which were hematology-oncology disease in 215 patients (12%), neurological disease in 103 (6%), chronic lung disease in 68 (4%), congenital heart disease in 65 (4%), and a genetic syndrome in 57 (3%); 292 were immunosuppressed (17%). Overall, 1319 patients (79%) had COVID-19-related symptoms and 591 (34%) required hospital admission. A coinfection with other respiratory viruses was observed in 33 patients (2%). Intensive care admission due to COVID-19 was required in 22 patients (1.3%) and 8 (0.5%) died with COVID-19. In univariate analysis, the presence of comorbidities, viral coinfecton, and immunosuppression were statistically significantly associated with the need for hospitalization. A history of two or more doses of the SARSCoV2 vaccine was a protective factor against hospital admission in children older than 3 years. In the multivariate model, patients younger than 3 years (OR 6.5, 95% CI 1.2-36.8, p 0.03), with comorbidities (OR 2.04, 95%CI 1.7-3.3, p 0.00) and immunocompromised hosts (OR 2.89, 95% CI 2.1-4.1, p 0.00) had a higher risk of hospital admission. When adjusting for the remaining variables, having received two or more doses of the vaccine was found to be a protective factor against hospital admission (OR 0.65, 95% CI 0.49-0.87, p<0.01). Conclusions: In this prospective cohort study of children with a confirmed diagnosis of COVID-19, symptomatic disease predominated. Thirty-four percent of the patients were admitted for COVID-19. Vaccination with two or more doses was a protective factor against hospitalization in the multivariate model. In addition, age younger than 3 years, previous comorbidities, and immunosuppression were statistically associated with hospital admission (AU)

Características clínicas y evolutivas de niños con infección por SARS-CoV-2 en un hospital de alta complejidad: estudio de cohorte / Clinical and outcome characteristics of children with SARS CoV-2 infection in a tertiary-care hospital: a cohort study

Introducción: Las infecciones por SARS-CoV-2 representan un problema de salud pública a nivel mundial. En los niños se reporta menor incidencia y cuadros clínicos más leves. Se realizó el presente estudio con el objetivo de describir las características clínicas y evolutivas de los niños con diagnóstico de infección por SARS CoV-2 en el Hospital Juan P. Garrahan. Material y métodos: estudio de cohorte prospectivo. Se incluyeron todos los pacientes con diagnóstico confirmado por PCR de COVID-19 desde 20.4.20 hasta el 3.07.21 y con seguimiento en el hospital de Pediatría Juan P. Garrahan. Resultados: n: 1644. Eran varones 836 (51%). La mediana de edad fue 75 meses (RIC 22- 143). Tenían alguna enfermedad de base previa al diagnóstico de COVID-19: 884 pacientes (53,7%), la más frecuente fue la enfermedad oncohematológica. Estaban asintomáticos 423 pacientes (25,7%). De los pacientes sintomáticos, 1071 (65,1%) presentaron cuadro leve, 5 (0,3%) moderado, 69 (4,2%) grave y 76 (4,6%) crítico. La fiebre fue el hallazgo más frecuente n: 782; (47,5%). Se internaron 900 pacientes (54,7%), 33 en UCI (2%). Fallecieron 7 pacientes (0,4%), todos ellos con comorbilidades graves. Conclusiones: En este estudio de cohorte de niños con infección por SARS-CoV-2 confirmada, predominaron los pacientes con enfermedad de base y las formas leves de COVID-19. El ingreso a UCI fue menor al 2%. Fallecieron 7 pacientes (0.4%) todos ellos con comorbilidades y coinfecciones (AU)

Introduction: SARS-CoV-2 infections represent a worldwide public health problem. A lower incidence and milder clinical pictures are reported in children. The aim of this study was to describe clinical and outcome characteristics of children diagnosed with SARS-CoV-2 infection at Hospital de Pediatría Juan P. Garrahan. Methods: A prospective cohort study was conducted. All patients with a PCR-confirmed diagnosis of COVID-19 seen between 20.4.20 and 3.07.21 and followed-up at Hospital de Pediatría Juan P. Garrahan were included. Results: n: 1644; 836 males (51%) were male. Median age was 75 months (IQR, 22-143). Overall, 884 patients (53.7%) had an underlying disease prior to COVID-19 diagnosis, most frequently hematologic/ oncologic disease. 423 patients (25.7%) were asymptomatic. Of the symptomatic patients, 1071 (65.1%) had mild, 5 (0.3%) moderate, 69 (4.2%) severe, and 76 (4.6%) critical disease. Fever was the most frequent finding (n: 782; 47.5%). A total of 900 patients (54.7%) were admitted, 33 of whom to the ICU (2%). Seven patients (0.4%) died, all with severe comorbidities. Conclusions: In this cohort study of children with confirmed SARSCoV-2 infection, patients with underlying disease and mild forms of COVID-19 predominated. ICU admission occurred in less than 2%. Seven patients (0.4%) died, all of them with comorbidities and coinfections. (AU)

Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated.

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.

Phosphorylation of Na,K-ATPase by acetyl phosphate and inorganic phosphate. Sidedness of Na+, K+ and nucleotide interactions and related enzyme conformations.

The effects of K+, Na+ and nucleotides (ATP or ADP) on the steady-state phosphorylation from [32P]Pi (0.5 and 1 mM) and acetyl [32P]phosphate (AcP) (5 mM) were studied in membrane fragments and in proteoliposomes with partially purified pig kidney Na,K-ATPase incorporated. The experiments were carried out at 20 degrees C and pH 7.0. In broken membranes, the Pi-induced phosphoenzyme levels were reduced to 40% by 10 mM K+ and to 20% by 10 mM K+ plus 1 mM ADP (or ATP); in the presence of 50 mM Na+, no E-P formation was detected. On the other hand, with AcP, the E-P formation was reduced by 10 mM K+ but was 30% increased by 50 mM Na+. In proteoliposomes E-P formation from Pi was (i) not influenced by 5-10 mM K+cyt or 100 mM Na+ext, (ii) about 50% reduced by 5, 10 or 100 mM K+ext and (iii) completely prevented by 50 mM Na+cyt. Enzyme phosphorylation from AcP was 30% increased by 10 mM K+cyt or 50 mM Na+cyt; these E-P were 50% reduced by 10-100 mM K+ext. However, E-P formed from AcP without K+cyt or Na+cyt was not affected by extracellular K+. Fluorescence changes of fluorescein isothiocyanate labelled membrane fragments, indicated that E-P from AcP corresponded to an E2 state in the presence of 10 mM Na+ or 2 mM K+ but to an E1 state in the absence of both cations. With pNPP, the data indicated an E1 state in the absence of Na+ and K+ and also in the presence of 20 mM Na+, and an E2 form in the presence of 5 mM K+. These results suggest that, although with some similarities, the reversible Pi phosphorylation and the phosphatase activity of the Na,K-ATPase do not share the whole reaction pathway.

Phosphatase activity of (Na+ + K+)-ATPase. Ligand interactions and related enzyme forms.

The prevailing conformations of partially purified pig kidney (Na+ + K+)-ATPase interacting with ligands related to its phosphatase activity were determined following time-dependent trypsin digestion and inactivation as well as the amounts of Rb+ or Ca2+ bound to the enzyme after passage through cation-exchange resin columns. In the presence of 150 mM choline chloride, alone or with 3 mM MgCl2, 3 mM MnCl2 or 1 mM CaCl2, the major enzyme conformation was E1. Similar forms were seen with 5 mM p-nitrophenyl phosphate with and without 3 mM MgCl2. KCl, at 0.5 mM or 150 mM, produced an E2 enzyme state; the effects of 0.5 mM KCl were completely counteracted by 5 mM p-nitrophenyl phosphate. Under optimal conditions for phosphatase activity (3 mM MgCL2/5 mM p-nitrophenyl phosphate/10 mM KCl) the (Na+ + K+)-ATPase was in the E2 state. At low ionic strength and 20 degrees C and under 85% of maximal RbCl-stimulated phosphatase turnover (1 mM RbCl/3 mM MgCl2/5 mM p-nitrophenyl phosphate) no Rb+ occlusion could be detected. Ca2+, at low ionic strength and in the presence of 3 mM MgCl2, stimulated an ouabain-sensitive phosphatase activity. The rates of hydrolysis obtained wit 1 mM CaCl2 were similar to those seen with 0.5 mM KCl; under both conditions, similar patterns of trypsin digestion and inactivation of the enzyme were obtained. On the other hand, Ca2+ could not mimic Rb+ in its ability to induce an E2-occluding state. These results suggest that during phosphatase activity of (Na+ + K+)-ATPase, the most abundant form is a non-occluding E2 and that at least one of the mechanisms of potassium stimulation of that activity it to take the enzyme into the E2 state.

The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump.

Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites.

Acetyl phosphate can act as a substrate for Na+ transport by (Na+ + K+)-ATPase.

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.

Phosphatase activity of Na+/K+-ATPase. Enzyme conformations from ligands interactions and Rb occlusion experiments.

The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 which has no K+ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na+ + K+-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.

Some total and partial reactions of Na+/K+-ATPase using ATP and acetyl phosphate as a substrate.

Acetyl phosphate, as a substrate of (Na+ + K+)-ATPase, was further characterized by comparing its effects with those of ATP on some total and partial reactions carried out by the enzyme. In the absence of Mg2+ acetyl phosphate could not induce disocclusion (release) of Rb+ from E2(Rb); nor did it affect the acceleration of Rb+ release by non-limiting concentrations of ADP. In K+-free solutions and at pH 7.4 sodium ions were essential for ATP hydrolysis by (Na+ + K+)-ATPase; when acetyl phosphate was the substrate a hydrolysis (inhibited by ouabain) was observed in the presence and absence of Na+. In liposomes with (Na+ + K+)-ATPase incorporated and exposed to extravesicular (intracellular) Na+, acetyl phosphate could sustain a ouabain-sensitive Rb+ efflux; the levels of that flux were similar to those obtained with micromolar concentrations of ATP. When the liposomes were incubated in the absence of extravesicular Na+ a ouabain-sensitive Rb+ efflux could not be detected with either substrate. Native (Na+ + K+)-ATPase was phosphorylated at 0 degrees C in the presence of NaCl (50 mM for ATP and 10 mM for acetyl phosphate); after phosphorylation had been stopped by simultaneous addition of excess trans-1,2-diaminocyclohexane-N,N,N',N' tetraacetic acid and 1 M NaCl net synthesis of ATP by addition of ADP was obtained with both phosphoenzymes. The present results show that acetyl phosphate can fuel the overall cycle of cation translocation by (Na+ + K+)-ATPase acting only at the catalytic substrate site; this takes place via the formation of phosphorylated intermediates which can lead to ATP synthesis in a way which is indistinguishable from that obtained with ATP.

ATP activation of plasma membrane yeast H(+)-ATPase shows complex kinetics independently of the degree of purification.

ATP stimulation of plasma membrane H(+)-ATPase activity from a wild baker's yeast (Saccharomyces cerevisiae) was followed under conditions of progressive degrees of purification. A particular emphasis was put to cover a wide range of concentrations which went from 2 microM up to 3000 microM ATP. The preparations used were (i) crude membrane fraction, (ii) untreated plasma membrane fraction obtained by differential centrifugation, (iii) residual plasma membrane treated with Triton X-100, (iv) enzyme solubilized with either Zwittergent 3-14 alone or after Triton X-100 treatment. Under all conditions the fitting of the dose-response curves required an equation composed by the sum of two Michaelian terms. Depending on the treatment, the Km values and Vmax values varied. The fitted curves displayed a high affinity-low Vmax (Km values of 7-60 microM and Vmax values of 0.03-0.50 mumol P(i)/mg per min) and a low affinity-high Vmax component (Km values of 408-1960 microM and Vmax values of 0.26-5.82 mumol P(i)/mg per min). The complex ATP activation curve of the yeast plasma membrane H(+)-ATPase is in line with similar behavior found for the H(+)-ATPase of higher plants and all known animal cation transport ATPases.

Detection of a highly ouabain sensitive isoform of rat brainstem Na,K-ATPase.

The present work provides evidence for the existence in rat brainstem of a form of Na,K-ATPase catalytic subunit that displays a high affinity for ouabain (Kd about 10(-9) M). Its kinetic identification was made out from studies on dose response curves of ouabain inhibition of Na,K-ATPase activity, ouabain inhibition of Na(+)-dependent phosphorylation from ATP and ouabain stabilized phosphoenzyme formation from inorganic phosphate (Pi). In all these studies this isoform comprises around 11 percent of the total Na,K-ATPase enzyme. The PAGE electrophoretic mobility of its phosphoprotein obtained from Pi in the presence of ouabain is lower than that of the alpha-1 form but it cannot be distinguished from that of alpha-2. Whether this highly ouabain sensitive form corresponds to the alpha-3 isoenzyme or represents the translational product of one of the additional genes described for the large catalytic subunit remains at the moment an open question.

Some properties of the H-ATPase activity present in root plasmalemma of Avena sativa L. Two different enzymes or one enzyme with two ATP sites?

The effects of Mg2+, K+ and ATP on a H-ATPase activity from a native plasmalemma fraction of oat roots were explored at 20 degrees C and pH 6.5. In the presence of 3 mM ATP and no K+, H-ATPase activity vs. [Mg2+] approached a monotonic activation but it became biphasic, with a decline above 3 mM Mg2+, in the presence of 20 mM K+. Mg2+ inhibition occurred also in K-free solutions when [ATP] was lowered to 0.05 mM. Also, an apparent monotonic H-ATPase activation by [K+] at 3.0 mM ATP was transformed in biphasic (inhibition by high [K+]) when [ATP] was reduced to 0.05 mM. The best fits of the ATP stimulation curves of hydrolysis satisfied the sum of two Michaelian functions where that with higher affinity had lower Vmx. Taking into consideration all conditions of activity assay, the high-affinity component (1) had a Km about 11-16 microM and a Vmx around 0.14-0.28 mumol Pi/mg per min whereas that with lower affinity (2) had a Km of 220-540 microM and a Vmx of 0.5-1.0 mumol Pi/mg per min. Km2 was markedly affected by the [K+] and [Mg2+]; at optimal concentrations of these cations (1 mM Mg2+ and 10 mM K+) it had a value of 235 +/- 24 microM which was increased to 540 +/- 35 microM at 20 mM [Mg2+] and 60 mM [K+]. In addition, Vmx1 was reduced to about a half when the concentrations of Mg2+ and K+ were increased to inhibitory levels. These results could be explained by the existence of two different enzymes or one enzyme with two ATP sites. In the second case, we could not tell at this stage if both are catalytic or one is regulatory.

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase. Their relevance to phosphatase activity.

The K+-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na+ + K+)-ATPase from pig kidney shows substrate inhibition (Ki about 9.5 mM at 2.1 mM Mg2+). Potassium antagonizes and sodium favours this inhibition. In addition , K+ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K+ activation. In the absence of Mg2+, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb+ from the Rb+ occluded unphosphorylated enzyme. With no Mg2+ and with 0.5 mM KCl, trypsin inactivation of (Na+ + K+)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl2, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E1 enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E1 state is the basis for substrate inhibition of the p-nitrophenylphosphatase activity of(Na+ + K+)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.

Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants.

We investigated the nature of the complex ATP activation kinetics of plant H+-ATPases. To this aim we analyzed that activation in three isolated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis thaliana. The isoforms were obtained by heterologous expression in endoplasmic reticulum of yeast. ATP stimulation was always with low affinity (K0.5 between 500 and 1800 [mu]M). In addition, the curves were not Michaelian and displayed positive cooperativity. Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine exhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kinetics with negative cooperativity, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule. Under this condition the ATP activation kinetics was Michaelian or had a slight negative cooperativity and the K0.5ATP was reduced 3-fold. These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativity and kinetics competence of the E(ATP) and E(ATP)2 complexes. Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of the enzyme molecule.

A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na/Ca exchange in squid nerve fibers.

The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long-standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na-gradient-dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de-regulated dialyzed squid axons, completely restores the MgATP stimulation of Na(o)-dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.

Comparative analysis of outcome and clinical features in children with pleural empyema caused by penicillin-nonsusceptible and penicillin-susceptible Streptococcus pneumoniae.

OBJECTIVE: To describe the clinical characteristics of pleural empyema in children caused by Streptococcus pneumoniae nonsusceptible to penicillin and compare their clinical outcome with pleural empyemas caused by penicillin-susceptible organisms. METHODS: Records of 109 children with complicated pleural effusions between January 1996 and December 1998 were retrospectively reviewed. RESULTS: Pathogens were recovered in 45 of these cases. Streptococcus pneumoniae represented 71% of the isolates, with 20 (62%) and 12 (38%) of the strains proving susceptible and nonsusceptible to penicillin, respectively. The average age for children with penicillin-nonsusceptible infections was 18.3 months compared with an average age of 40.9 months for those with penicillin-susceptible infections (P = 0.03). Previous antibiotic treatment was more frequent in children with penicillin-nonsusceptible organisms (P = 0.05). No significant differences were found between the two groups for gender, underlying diseases, duration of fever and tachypnea, need of surgical treatment, bacteremia incidence, mean duration of therapy, or length of hospital stay. CONCLUSIONS: Pleural empyemas caused by penicillin-nonsusceptible S. pneumoniae appear to be associated with younger age and previous antibiotic treatment. By contrast, the outcome was not influenced by the penicillin susceptibility of the pneumococcus strains.