Experimental study of an asymmetric valveless pump to elucidate insights into strategies for pediatric extravascular flow augmentation.

Asymmetric pumping is a sub-category of valveless pumping in which a flexible tube is rhythmically compressed in the transverse symmetry plane. Due to the resulting asymmetry between the suction and discharge pipes, a net pumping head is achieved. Asymmetric pumping is regarded as one of the main mechanisms responsible for the Liebau effect in addition to impedance pumping. However, there remains a paucity of research surrounding the governing parameters of asymmetric pumping. Here, we conducted an experimental study of the performance of an asymmetric pump, with an aim to assess its potential for extravascular flow augmentation. A custom flexible latex tube and experimental platform were developed for this purpose. We tested various tube thicknesses and pinching frequencies. Our results demonstrate that the performance is within the range of physiological requirements for pediatric circulatory devices (~ 1 L/min and < 30 mmHg). We conclude that due to the absence of reverse flow and its mechanical simplicity, pure asymmetric pumping is promising for selected cardiovascular applications with less complexity than other valveless techniques.

Oesophageal motility disorders in infected immigrants with Chagas disease in a non-endemic European area.

BACKGROUND: Immigration-related new diseases pose a growing challenge for healthcare services in receptor countries. Following Latin American migration, Chagas disease has inevitably appeared in Europe. AIM: To determine the prevalence and characteristics of oesophageal motility disorders in immigrants infected with Trypanosoma cruzi, using high resolution oesophageal manometry (HREM). METHODS: In all newly-diagnosed cases with chronic Chagas infection referring upper digestive symptoms, a protocolized clinical evaluation and complementary tests including barium oesophagogram and HREM were carried out. As control group, 14 healthy subjects from the same endemic areas were studied with HREM. RESULTS: We included 61 patients (46 female, 15 male; age range 26-63 years). Only seven patients (11%) had a minor alteration on barium oesophagogram. By contrast, 23 (37%) patients showed an alteration in oesophageal manometry, mainly minor motility disorders (34%). Only one healthy control (7%) had a minor motility disorder at HREM (p = 0.029 vs. patients). CONCLUSIONS: Oesophageal motor disorders in infected immigrants with Chagas disease are common, and mainly characterized by a minor motility disorder that is not detected by barium oesophagogram. Hence, as well as barium oesophagogram examination, HREM should be considered, to assess oesophageal damage in this specific group of patients.

Utilidad de la serie blanca en el diagnóstico diferencial de la mononucleosis infecciosa / Utility of the white series in the differential diagnosis of infectious mononucleosis

Introducción: la mononucleosis infecciosa (MI) es una enfermedad frecuente en la infancia. Nos planteamos comparar la serie blanca de niños con sospecha de MI, en función de la serología positiva/negativa para virus Epstein-Barr (VEB), citomegalovirus (CMV) y Paul-Bunnell. Material y métodos: estudio descriptivo transversal. Se revisaron niños atendidos en Urgencias en 2010-2011, con diagnóstico de síndrome mononucleósico y serología positiva para VEB o CMV e igual número de niños con serologías negativas como grupo de control. Se compararon variables epidemiológicas, clínicas y serológicas. Resultados: se obtuvieron 50 niños con serologías positivas y 50 niños con serologías negativas (edad media de 5,81 años). Tuvieron serología positiva para VEB 44 niños, 2 para CMV y 4 para ambos. De los 48 niños con serología positiva para VEB, 26 eran Paul-Bunnell negativos y 22 positivos, siendo estos 22 niños el total de Paul-Bunnell positivos. La media de linfocitos, monocitos y basófilos fue mayor en niños con serología positiva para VEB y los neutrófilos fueron más bajos. En los dos casos con CMV positivo encontramos cifras de neutrófilos totales mayores. Ninguna edad se asoció con mayor probabilidad de VEB y Paul-Bunnell positivos. Conclusiones: existe predominio de linfocitos, monocitos y basófilos en niños con MI por VEB. El descenso de neutrófilos es la única variación analítica en los niños con MI por CMV. Estos valores analíticos pueden orientarnos en el diagnóstico de MI. Todos los niños con Paul-Bunnell positivo tenían positividad para el VEB sin relación con la edad (AU)

Introduction: the infectious mononucleosis (IM) is a common disease in childhood. We propose to compare the white series of children with suspected IM, based on serology positive/negative for Epstein -Barr Virus (EBV), Cytomegalovirus (CMV) and Paul- Bunnell. Material and methods: descriptive study. Children were reviewed, taken to hospital in 2010-2011, diagnosed with mononucleosis syndrome and positive serology for EBV or CMV, and equal number of children who were seronegative control group. Epidemiology, clinical and serological variables were compared. Results: there were 50 children with positive serology and 50 negative children (mean age 5.81 years ). EBV serology were 44 children, 2 and 4 both CMV. Of the 48 children with positive serology for EBV, 26 were negative Paul- Bunnell and 22 positive, and these 22 children total positive Paul-Bunnell. The average number of lymphocytes, monocytes and basophils was higher in children with positive serology for EBV and neutrophils were lower. Children were CMV negative but elevated neutrophils. No age was associated with increased likelihood of EBV and Paul- Bunnell positive. Conclusions: there is a predominance of lymphocytes, monocytes and basophils in children with EBV IM. The increase in neutrophils is the only analytical variation in children with CMV IM. These analytical values can guide the diagnosis of IM. All children with positive Paul- Bunnell positive for EBV had no relation with age (AU)

Singlet molecular oxygen quenching by the antioxidant dimethylmethoxy chromanol in solution and in ex vivo porcine skin.

Singlet-oxygen is a non-radical reactive oxygen species believed to play a major role in many photooxidation processes in connection with diverse photo-biological processes such as skin ageing or photocarcinogenesis. Dimethylmethoxy chromanol (3,4-dihydro-6-hydroxy-2,2-dimethyl-7-methoxy-1(2H)-benzopyran) is a potent antioxidant used in cosmetic and pharmaceutical formulations. We have assessed the singlet oxygen quenching ability of dimethylmethoxy chromanol, by monitoring the near-IR phosphorescence of singlet-oxygen in solution and in ex vivo porcine skin samples. Dimethylmethoxy chromanol quenches singlet oxygen with a rate constant of (1.3 ± 0.1) × 108 M⁻¹ s⁻¹ in solution. Consistent with this, a clear reduction in the singlet oxygen lifetime and emission intensity was observed when ex vivo porcine skin samples were treated with dimethylmethoxy chromanol.

Claridad de las recomendaciones al paciente en el informe de alta hospitalaria / Clarity of recommendations for patients in the hospital discharge report

Fundamento. Las recomendaciones al paciente (RP) son una de las partes fundamentales del informe de alta hospitalaria. El objetivo del estudio fue conocer la frecuencia de RP en los informes de distintas especialidades y la proporción de ellas consideradas de fácil comprensión. Material y método. Estudio observacional transversal. Ámbito: hospital general de agudos. Se revisó una muestra de informes de alta con análisis de la frecuencia y tipos de RP según su contenido y claridad. Resultados. Se analizaron 840 informes y 2.097 RP con un promedio de 2,5 RP por informe. Las RP más habituales se referían al seguimiento del paciente (46% del total), seguidas de las recomendaciones específicas para los días inmediatos al alta (37%), solo el 16% se referían a estilos de vida. Los informes de especialidades quirúrgicas contenían 3,2 RP por alta, frente a 2,3 de las especialidades médicas. Un 90,3% de las RP se consideraron de comprensión clara para un paciente medio. Conclusiones. Los informes de alta analizados contenían pocas RP, en especial las referidas a estilos de vida. Una proporción apreciable de las RP no estaban expresadas con suficiente claridad(AU)

Background. Recommendations for patients (RP) are one of the key parts of the hospital discharge report (HDR). The objective was to study the frequency of RP in the HDR of different specialties and the proportion of them considered to be easy to understand. Material and method. Observational and cross-sectional study. Setting. General acute care hospital. Interventions. Review of a sample of HDR, analysis of the frequency and types of PR based on their content and clarity. Results. A total of 840 HDR and 2,097 PR were analysed with an average of 2.5 RP per report. The most common RP referred to the patient follow-up (46% of total), followed by specific recommendations for the days immediately following discharge (37%), with only 16% related to lifestyle. Reports by surgical specialties contained 3.2 RP compared to 2.3 in medical specialties. The large majority (90.3%) of the RP were considered clear to understand for a standard patient. Conclusions. The HDR analysed contained few RP, in particular those related to lifestyles. A substantial proportion of the RP were not expressed with enough clarity(AU)

[Ultrasound-guided infraclavicular axillary vein cannulation]. / Canalización de la vena axilar infraclavicular guiada por ecografía.

BACKGROUND AND OBJECTIVE: Percutaneous central venous cannulation is a challenging procedure. Traditionally, an external landmark technique has been used to identify puncture site. We wanted to evaluate an ultrasound-guided technique for the axillary vein cannulation, looking specifically at the ease of use, success rate and decreased complications. METHODS: Sixty consecutive surgical patients scheduled for central venous catheter placement were registered. An ultrasound scanner made for guiding an in plane puncture of axillary vein was used. After locating the vessels, an echo-guided sterile procedure was performed to cannulate the vein. RESULTS: Cannulation was successful in all patients, and there were no complications during insertion of the catheters. Both axillary veins were cannulated, and the vein was punctured successfully at first attempt in 95% of the patients. The median time from the start of the first puncture (of the skin) until the aspiration of blood was 15 (7- 135) seconds. CONCLUSION: This ultrasound-guided technique for inserting central venous catheters in axillary vein was easy to apply. This procedure could increase precision and safety in patients undergoing axillary vein cannulation.

Canalización de la vena axilar infraclavicular guiada por ecografía / Ultrasound-guided infraclavicular axillary vein cannulation

Introducción y objetivos. La canalización venosa central percutánea es un procedimiento que puede ser difícil. Tradicionalmente se han utilizado técnicas basadas en referencias anatómicas externas para identificar los sitios de punción. Hemos evaluado una técnica guiada por ultrasonidos para la canalización de la vena axilar infraclavicular. Método. Se incluyeron pacientes quirúrgicos programados para la colocación de un catéter venoso central. Utilizamos una sonda lineal de ultrasonidos, 12MHz, para guiar la punción en plano de la vena axilar infraclavicular. El miembro superior ipsilateral fue colocado en abducción a 90° del eje cráneo-caudal. Se analizó el número de intentos, el tiempo empleado en la técnica (desde punción de la piel hasta aspiración de sangre) y la incidencia de complicaciones. Resultados. Fueron incluidos 60 pacientes. Fueron escaneadas ambas venas axilares infraclaviculares y el catéter se pudo colocar en un primer intento en el 95% de los pacientes. No hubo fallos de canalización. La mediana de tiempo de la técnica fue de 15 (rango 7-135) segundos. No hubo complicaciones mayores durante el procedimiento de inserción del catéter. Conclusión. La visualización por ultrasonidos de la vena axilar infraclavicular permitió su canalización con un mínimo cambio de posición del paciente, incluso durante la cirugía, en un tiempo clínicamente adecuado y sin complicaciones inmediatas(AU)

Background and objective. Percutaneous central venous cannulation is a challenging procedure. Traditionally, an external landmark technique has been used to identify puncture site. We wanted to evaluate an ultrasound-guided technique for the axillary vein cannulation, looking specifically at the ease of use, success rate and decreased complications. Methods. Sixty consecutive surgical patients scheduled for central venous catheter placement were registered. An ultrasound scanner made for guiding an in plane puncture of axillary vein was used. After locating the vessels, an echo-guided sterile procedure was performed to cannulate the vein. Results. Cannulation was successful in all patients, and there were no complications during insertion of the catheters. Both axillary veins were cannulated, and the vein was punctured successfully at first attempt in 95% of the patients. The median time from the start of the first puncture (of the skin) until the aspiration of blood was 15 (7 - 135) seconds. Conclusion. This ultrasound-guided technique for inserting central venous catheters in axillary vein was easy to apply. This procedure could increase precision and safety in patients undergoing axillary vein cannulation(AU)

[Clarity of recommendations for patients in the hospital discharge report]. / Claridad de las recomendaciones al paciente en el informe de alta hospitalaria.

BACKGROUND: Recommendations for patients (RP) are one of the key parts of the hospital discharge report (HDR). The objective was to study the frequency of RP in the HDR of different specialties and the proportion of them considered to be easy to understand. MATERIAL AND METHOD: Observational and cross-sectional study. SETTING: General acute care hospital. INTERVENTIONS: Review of a sample of HDR, analysis of the frequency and types of PR based on their content and clarity. RESULTS: A total of 840 HDR and 2,097 PR were analysed with an average of 2.5 RP per report. The most common RP referred to the patient follow-up (46% of total), followed by specific recommendations for the days immediately following discharge (37%), with only 16% related to lifestyle. Reports by surgical specialties contained 3.2 RP compared to 2.3 in medical specialties. The large majority (90.3%) of the RP were considered clear to understand for a standard patient. CONCLUSIONS: The HDR analysed contained few RP, in particular those related to lifestyles. A substantial proportion of the RP were not expressed with enough clarity.

Drenaje torácico / Thoracic drainage

La patología pleural supone un problema clínico frecuente. En algunos casos, eltratamiento incluye drenaje de la cavidad que puede efectuarse mediante toracocentesisevacuadoras, pero en ocasiones exige mantener un drenaje permanentede la cavidad pleural. El drenaje torácico consiste en la colocación de una sondaen el espacio pleural para evacuar la presencia de aire, líquido o sangre, lo cualproduce un colapso pulmonar de grado variable con repercusión clínica en funciónde la reserva ventilatoria previa de paciente y el grado de colapso. Existenvarios modelos de tubos torácicos, así como sistemas de drenaje pleural, y su lugarde inserción dependerá del tipo de patología ante la que nos encontremos.Enfermería es fundamental en todo el proceso, tanto en la preparación del paciente,inserción, y mantenimiento adecuado para el éxito del tratamiento, comodurante la extracción y posteriores cuidados. Resulta fundamental que dichos profesionalesconozcan el material utilizado así como su mantenimiento. Una buenatécnica de cura del punto-orificio de inserción prevendrá numerosas complicacionesque podrían resultar fatales para el paciente. Se crea un protocolo de actuaciónen los cuidados de enfermería de los pacientes portadores de drenaje torácicotanto en los cambios de apósito como en cura del drenaje para servir dereferente, de guía de actuación sistemática y homogénea [1-24](AU)

Pleural pathology is a frequent clinical problem. In some cases, treatment includesdraining the cavity which can be carried out by thoracentesis evacuators, but onoccasions treatment requires maintaining a drainage permanently inside the pleuralcavity. Pleural drainage consists in inserting a catheter in the pleural sack todrain the presence of air, liquid or blood which causes a variable degree of lungcollapse having a clinical consequence in function of the reserve breathing capacitythe patient previously had and the degree of collapse. There are variousmodels of thoracic tubes as well as systems to drain the pleural cavity and theirspot for insertion depends on the type of pathology being dealt with for thepatient under treatment. Nursing is fundamental in this entire process, includingin the preparation of the patient for this treatment, the insertion of the catheterand the adequate maintenance so that this procedure succeeds as well as duringthe removal of the catheter and the subsequent care required. It is fundamentalthat the nursing professionals know the materials used as well as their maintenance.A good technique to cure the punt/orifice where a catheter is inserted willprevent numerous complications which could be deadly for the patient. Theauthors create a procedural protocol for nurses to use when treating patients whohave thoracic drains; this protocol deals with changing the catheters as well as theentire process related to how to treat patients with a pleural drain. This protocolshould serve as reference material and as a guide to a systematic and homogenousworking procedure [1-24](AU)

Hot water dipping of olives (Olea europaea) for virgin oil debittering.

Olives (Olea europaea L.) of the Manzanilla, Picual, and Verdial varieties harvested at the green mature stage of ripening were dipped in hot water at a range of temperatures between 60 and 72 degrees C for 3 min. Immediately after treatment, oils were physically extracted from the olives. Olive heating promotes a reduction of oil bitterness in direct relationship to the temperature used. Fruit heating at > or =60 degrees C for 3 min did not cause significant changes in acidity, UV absorption, peroxide index, and panel test score of the oils obtained but decreased its oxidative stability. Oils extracted from heated fruit showed higher concentrations of chlorophylls and carotenes and lower total phenol content.

[Evolution and characteristics of primary care patients with painful shoulder]. / Evolución y características de los pacientes con hombro doloroso en atención primaria.

OBJECTIVES: To describe the population attending teaching health centres in Area V, Asturias, to consult for painful shoulder. To describe the examination, diagnosis, treatment and clinical response. DESIGN: Longitudinal, descriptive study. SETTING: Teaching health centres in Area V, Asturias. PARTICIPANTS: Patients consulting because of pain in their shoulders in October and November 2002. MAIN MEASUREMENTS: Two measurements, the first of which in December 2002 appraised age, sex, job, time off work, arc of pain, flection, internal-external rotation, and diagnostic impression. The second, in May 2003, assessed evolution, x-rays, echography, medication, infiltration, physiotherapy, exercises at home, referral to specialist, and final diagnosis. Bivariant and kappa index descriptive analyses were run. RESULTS: 110 cases, 58.2% women and 41.8% men, with an average age of 57 (95% CI, 54.17-59.79), were included. 52.7% were retired; no particular job predominated; 7.3% were off work; 30% had had no examination of arc of movement; 70% evolved satisfactorily. The most common diagnosis was cuff tendinitis, with initial and final diagnosis coinciding well. 77.4% of patients took NSAIDs; 16% were infiltrated; 32.1% did physiotherapy; and 36.8%, exercises at home. 65.1% had no x-ray and only 6.6% had echographies, all of which found something. There was no statistically significant relationship between medication or infiltration and favourable evolution; and there was, between physiotherapy or exercises and favourable evolution. CONCLUSIONS: Most patients were middle-aged, women, pensioners, and had little time off work. Two-thirds evolved well. Examinations were incomplete and few complementary tests were run.

Evolución y características de los pacientes con hombro doloroso en atención primaria / Evolution and characteristics of primary care patients with painful shoulder

Objetivo. Describir el tipo de población que consulta por hombro doloroso en atención primaria en Asturias. Describir la exploración, el diagnóstico, los tratamientos utilizados y la respuesta clínica. Diseño. Estudio descriptivo, longitudinal. Emplazamiento. Centros de salud docentes del Área V de Asturias. Participantes. Pacientes que consultaron por dolor de hombro en octubre y noviembre de 2002. Mediciones principales. Características de los pacientes: edad, sexo, profesión, baja laboral, arco doloroso, flexión, rotación internaexterna e impresión diagnóstica. A los 5 meses se registraron los datos sobre su evolución, radiografías, ecografías, fármacos, infiltraciones, fisioterapia, ejercicios en el domicilio, derivación especializada y diagnóstico final. Resultados. Se recogieron 110 casos (58,2%, mujeres y 41,8%, varones), con una media de edad de 57 años (intervalo de confianza [IC] del 95%, 54,17-59,79). El 52,7% estaba jubilado. El 7,3% se encontraba en situación de baja laboral. En un 30% no se disponía de la exploración de ningún arco de movimiento. El 70% de los pacientes evolucionó satisfactoriamente. El diagnóstico más frecuente fue la tendinitis de manguito, y la concordancia entre los diagnósticos inicial y final fue buena. Un 77,4% tomó antiinflamatorios no esteroideos (AINE). El 16% recibió infiltraciones.Un 32,1% realizó fisioterapia y un 36,8%, ejercicios en el domicilio. En el 65,1% de los pacientes no se hizo ninguna radiografía y sólo se efectuó una ecografía en el 6,6%. La evolución favorable no se asoció estadísticamente con los fármacos o la infiltración, y sí con la fisioterapia y los ejercicios. Conclusiones. La mayoría de pacientes con hombro doloroso son mujeres, de mediana edad y pensionistas. Más del 60% evoluciona satisfactoriamente. La exploración realizada suele ser incompleta y con pocas pruebas complementarias

Objectives. To describe the population attending teaching health centres in Area V, Asturias, to consult for painful shoulder.To describe the examination, diagnosis, treatment and clinical response. Design. Longitudinal, descriptive study. Setting. Teaching health centres in Area V, Asturias. Participants. Patients consulting because of pain in their shoulders in October and November 2002. Main measurements. Two measurements, the first of which in December 2002 appraised age, sex, job, time off work, arc of pain, flection, internal-external rotation, and diagnostic impression. The second, in May 2003, assessed evolution, x-rays, echography, medication, infiltration, physiotherapy, exercises at home, referral to specialist, and final diagnosis. Bivariant and kappa index descriptive analyses were run. Results. 110 cases, 58.2% women and 41.8% men, with an average age of 57 (95% CI, 54.17-59.79), were included. 52.7% were retired; no particular job predominated; 7.3% were off work; 30% had had no examination of arc of movement; 70% evolved satisfactorily. The most common diagnosis was cuff tendinitis, with initial and final diagnosis coinciding well. 77.4% of patients took NSAIDs; 16% were infiltrated; 32.1% did physiotherapy; and 36.8%, exercises at home. 65.1% had no x-ray and only 6.6% had echographies, all of which found something. There was no statistically significant relationship between medication or infiltration and favourable evolution; and there was, between physiotherapy or exercises and favourable evolution. Conclusions. Most patients were middle-aged, women, pensioners, and had little time off work.Two-thirds evolved well. Examinations were incomplete and few complementary tests were run

Temperature and oxygen regulation of oleate desaturation in developing sunflower (Helianthus annuus) seeds.

The effect of low (10 degrees C) and high (30 degrees C) temperature on in vivo oleate desaturation has been studied in developing sunflower (Helianthus annuus L.) seeds under conditions of different oxygen availability (capitulum, detached achenes or peeled seeds). In seeds remaining in the capitulum, only a part of the oleate newly synthesized at high temperature was desaturated to linoleate, whereas more oleate than that synthesized de novo was desaturated at low temperature. Achenes were only able to significantly desaturate oleate at low temperatures. In contrast, oleate desaturation was detected in peeled seeds incubated at low and high temperatures, showing the highest rate at 20 degrees C. Hull removing dramatically increased the activity of the microsomal oleate desaturase (FAD2, EC 1.3.1.35) at all studied temperatures, although a long-term inactivation of the enzyme was observed at high temperatures. Low oxygen concentration (1-2%) obtained by respiration of peeled seeds incubated in sealed vials, brought about the inactivation of the enzyme. All these data suggest that temperature regulates oleate desaturation controlling the amount of oleate and the FAD2 activity. In addition, this enzyme seems to be also regulated by the availability of oxygen, which is affected inside the achene by its diffusion through the hull, and the competition with respiration, both factors being temperature-dependent.

Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation.

We have identified a novel polymerase beta (Pol beta)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol beta, shows a nuclear localization that contrasts with the mitochondrial localization of Pol beta from Crithidia fasciculata, a closely related parasite, the only polymerase beta described so far in Trypanosomatidae. Li Pol beta, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol beta-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol beta, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol beta mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol beta at the infective phase of the parasite. The data suggest a role of Li Pol beta in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells.

Identification of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in human DNA polymerase lambda: a possible role in base excision repair.

Base excision repair (BER) is a major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic (AP) sites in DNA. Pivotal to this process is the 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity of DNA polymerase beta (Pol beta). DNA polymerase lambda (Pol lambda) is a recently identified eukaryotic DNA polymerase that is homologous to Pol beta. We show here that human Pol lambda exhibits dRP lyase, but not AP lyase, activity in vitro and that this activity is consistent with a beta-elimination mechanism. Accordingly, a single amino acid substitution (K310A) eliminated more than 90% of the wild-type dRP lyase activity, thus suggesting that Lys(310) of Pol lambda is the main nucleophile involved in the reaction. The dRP lyase activity of Pol lambda, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitro reconstituted BER reaction. These results suggest that Pol lambda may participate in "single-nucleotide" base excision repair in mammalian cells.

DNA polymerase mu, a candidate hypermutase?

A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining.

DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis.

A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19. Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells. It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X. Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis. Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity. The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair. Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes. These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis.

DNA polymerase mu (Pol mu), homologous to TdT, could act as a DNA mutator in eukaryotic cells.

A novel DNA polymerase has been identified in human cells. Human DNA polymerase mu (Pol mu), consisting of 494 amino acids, has 41% identity to terminal deoxynucleotidyltransferase (TdT). Human Pol mu, overproduced in Escherichia coli in a soluble form and purified to homogeneity, displays intrinsic terminal deoxynucleotidyltransferase activity and a strong preference for activating Mn(2+) ions. Interestingly, unlike TdT, the catalytic efficiency of polymerization carried out by Pol mu was enhanced by the presence of a template strand. Using activating Mg(2+) ions, template-enhanced polymerization was also template-directed, leading to the preferred insertion of complementary nucleotides, although with low discrimination values. In the presence of Mn(2+) ions, template-enhanced polymerization produced a random insertion of nucleotides. Northern-blotting and in situ analysis showed a preferential expression of Pol mu mRNA in peripheral lymphoid tissues. Moreover, a large proportion of the human expressed sequence tags corresponding to Pol mu, present in the databases, derived from germinal center B cells. Therefore, Pol mu is a good candidate to be the mutator polymerase responsible for somatic hyper- mutation of immunoglobulin genes.

Rat liver nucleotide pyrophosphatase/phosphodiesterase is an efficient adenylyl transferase.

Rat liver nucleotide pyrophosphatase/phosphodiesterase I (NPP/PDE) catalysed efficiently the transfer of adenylate from ATP to alcohols (methanol, ethanol, propanol, ethylene glycol, glycerol, 2, 2-dichloroethanol and glycerol 2-phosphate), which acted as adenylate acceptors competing with water with different efficiencies. NPP/PDE kinetics in alcohol/water mixtures were accounted for by rate equations for competitive substrates, modified to include alcohol negative co-operativity and, depending on the nature of the alcohol, enzyme denaturation by high alcohol concentrations or activation by low alcohol concentrations. The correlation of alcohol efficiencies with alcohol acidities, the comparison of rat liver with snake venom NPP/PDE, and the different effects of ionic additives on the efficiencies of glycerol 2-phosphate and glycerol provided evidence for interaction of the alcohols with a base catalyst, a non-polar and a cationic subsite in the active centre of rat liver NPP/PDE. The enzyme thus appears to be well suited to act as transferase, and we propose that NPP/PDE could be an adenylylating agent in the membrane.

The purified colicin S8 is a multimeric protein.

Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc. Colicins are commonly inducible and extracellular. Their molecular masses range from 30 to 90 kDa. Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure. In our hands, purified colicin S8 was an aggregation of extremely related polypeptides. Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa. Behavior on molecular filtration indicated a molecular weight higher than 200 kDa. Similar results were obtained when purification was carried out through FPLC. Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide. We conclude that more than one form of colicin S8 exists. The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides. Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.