A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.

Chromosome biorientation requires Aurora B's spatial separation from its outer kinetochore substrates, but not its turnover at kinetochores.

For correct chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (biorientation). For this, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Once biorientation is formed, tension is applied on kinetochore-microtubule interaction, stabilizing this interaction. The mechanism for this tension-dependent process has been debated. Here, we study how Aurora B localizations at different kinetochore sites affect the biorientation establishment and maintenance in budding yeast. Without the physiological Aurora B-INCENP recruitment mechanisms, engineered recruitment of Aurora B-INCENP to the inner kinetochore, but not to the outer kinetochore, prior to biorientation supports the subsequent biorientation establishment. Moreover, when the physiological Aurora B-INCENP recruitment mechanisms are present, an engineered Aurora B-INCENP recruitment to the outer kinetochore, but not to the inner kinetochore, during metaphase (after biorientation establishment) disrupts biorientation, which is dependent on the Aurora B kinase activity. These results suggest that the spatial separation of Aurora B from its outer kinetochore substrates is required to stabilize kinetochore-microtubule interaction when biorientation is formed and tension is applied on this interaction. Meanwhile, Aurora B exhibits dynamic turnover on the centromere/kinetochore during early mitosis, a process thought to be crucial for error correction and biorientation. However, using the engineered Aurora B-INCENP recruitment to the inner kinetochore, we demonstrate that, even without such a turnover, Aurora B-INCENP can efficiently support biorientation. Our study provides important insights into how Aurora B promotes error correction for biorientation in a tension-dependent manner.

Puf3p, a Pumilio family RNA binding protein, localizes to mitochondria and regulates mitochondrial biogenesis and motility in budding yeast.

Puf3p binds preferentially to messenger RNAs (mRNAs) for nuclear-encoded mitochondrial proteins. We find that Puf3p localizes to the cytosolic face of the mitochondrial outer membrane. Overexpression of PUF3 results in reduced mitochondrial respiratory activity and reduced levels of Pet123p, a protein encoded by a Puf3p-binding mRNA. Puf3p levels are reduced during the diauxic shift and growth on a nonfermentable carbon source, conditions that stimulate mitochondrial biogenesis. These findings support a role for Puf3p in mitochondrial biogenesis through effects on mRNA interactions. In addition, Puf3p links the mitochore, a complex required for mitochondrial-cytoskeletal interactions, to the Arp2/3 complex, the force generator for actin-dependent, bud-directed mitochondrial movement. Puf3p, the mitochore, and the Arp2/3 complex coimmunoprecipitate and have two-hybrid interactions. Moreover, deletion of PUF3 results in reduced interaction between the mitochore and the Arp2/3 complex and defects in mitochondrial morphology and motility similar to those observed in Arp2/3 complex mutants. Thus, Puf3p is a mitochondrial protein that contributes to the biogenesis and motility of the organelle.

Aurora B-INCENP Localization at Centromeres/Inner Kinetochores Is Required for Chromosome Bi-orientation in Budding Yeast.

For proper chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (chromosome bi-orientation) [1, 2]. To promote bi-orientation, Aurora B kinase disrupts aberrant kinetochore-microtubule interactions [3-6]. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. A popular explanation for it is that, upon bi-orientation, sister kinetochores are pulled in opposite directions, stretching the outer kinetochores [7, 8] and moving Aurora B substrates away from Aurora-B-localizing sites at centromeres (spatial separation model) [3, 5, 9]. This model predicts that Aurora B localization at centromeres is required for bi-orientation. However, this notion was challenged by the observation that Bir1 (yeast survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, can become dispensable for bi-orientation [10]. This raised the possibility that Aurora B localization at centromeres is dispensable for bi-orientation. Alternatively, there might be a Bir1-independent mechanism for recruiting Ipl1-Sli15 to centromeres or inner kinetochores [5, 9]. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore, and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres or inner kinetochores is required for bi-orientation. Our results give important insight into how Aurora B disrupts kinetochore-microtubule interaction in a tension-dependent manner to promote chromosome bi-orientation.

[Neonatal neoplasms: a single-centre experience]. / Neoplasias neonatales: experiencia de un centro.

INTRODUCTION: Malignant tumors are uncommon in the neonatal period and benign tumors may have malignant potential. OBJECTIVES: To describe the neoplasms diagnosed and treated in newborns (

[Study of the introduction of the European Credit Transfer System (ECTS) in pediatrics and modification of the teaching methodology]. / Estudio sobre la introducción del Sistema de Transferencia de Créditos Europeo en Pediatría y modificaciones de la metodología docente.

INTRODUCTION: Spanish medical faculties have initiated the new curriculum reform process within the framework of the European Higher Education Area and are required to incorporate the European Credit Transfer System (ECTS) to new syllabi before 2010. OBJECTIVES: To test the introduction of the ECTS in pediatrics and modify the teaching methodology. STUDY DESIGN: The theoretical and practical programs were adapted; academic objectives and a student evaluation system were established. Students were surveyed on starting the second term of the 2004-05 academic year before the theory examination and again on terminating the academic year: a 5-point Likert-type scale was used for responses. Priorities for generic and specific competencies selected by students were compared with those selected by the National Deans Conference (NDC). The results were analyzed using non-parametric tests. RESULTS: Fifteen credits became 11 ECTS, with 297 student working hours. The theory program was reduced from 80 to 52 lessons. The students prepared 14 tutor-supervised case presentations. The teaching staff considered that learning of theory was similar to previous years (66 %) and that practical learning improved (73.3 %). The students thought the program should continue (73.2 %) but 98.8 % considered the workload excessive. The students believed that their practical training and their ability to prepare and make case presentations significantly improved during the semester. Academic performance was significantly higher than that in students of the previous year. Students agreed with NDC priorities for 9/9 general and 4/17 specific competencies. Estimation of workload by students was significantly higher than that by staff, and 73.3 % of the students believed that workload should be reduced and the examination system improved. CONCLUSIONS: Introducing the ECTS improved academic performance, practical training, and self-directed learning. The project was satisfactory for staff and students. Student workload was underestimated.

Spontaneous speech events in two speech databases of human-computer and human-human dialogs in Spanish.

Previous works in English have revealed that disfluencies follow regular patterns and that incorporating them into the language model of a speech recognizer leads to lower perplexities and sometimes to a better performance. Although work on disfluency modeling has been applied outside the English community (e.g., in Japanese), as far as we know there is no specific work dealing with disfluencies in Spanish. In this paper, we follow a data driven approach in exploring the potential benefit of modeling disfluencies in a speech recognizer in Spanish. Two databases of human-computer and human-human dialogs are considered, which allow the absolute and relative frequencies of disfluencies in the two situations to be compared. The rate of disfluencies in human-human dialogs is found to be very close to that found for similar databases in English. Due to setup factors, the rate of disfluencies found in human-computer dialogs was remarkably higher than that reported for similar databases in English. In any case, from the point of view of speech recognition, the high frequencies of disfluencies and the distinct features of the acoustic events related to them support the need for explicit acoustic models. The regularities observed in the distribution of filled pauses and speech repairs reveal that including them in the language model of the speech recognizer may be also helpful. The extent to which the number of events depends on utterance length and on the speaker is also explored. Statistics are shown that follow previous studies for English, and a sizeable space is devoted to comparing our results with them. Finally, various possible cues for the automatic detection of speech repairs--a key issue from the point of view of speech understanding--are explored: silent pauses, filled pauses, lengthenings, cut off words and discourse markers. As previously observed for English, none of them was found to be reliable by itself. More information, especially at the acoustic-prosodic level, is no doubt needed to reliably detect speech repairs.

Cell integrity signaling activation in response to hyperosmotic shock in yeast.

Current progress highlights the role of the yeast cell wall as a highly dynamic structure that responds to many environmental stresses. Here, we show that hyperosmotic shock transiently activates the PKC signaling pathway, a response that requires previous activation of the HOG pathway. Phosphorylation of Slt2p under such conditions is related to changes in the glycerol turnover and is mostly Mid2p dependent, suggesting that changes in cell turgor, mediated by intracellular accumulation of glycerol, are sensed by PKC sensors to promote the cell integrity response. These observations, together with previous results, suggest that yeast cells respond to changes in cellular turgor by remodeling their cell walls.

[The role of molecular genetics in childhood cancer]. / Papel de la genética molecular en el cáncer infantil.

In the last few years molecular genetic studies of childhood cancer have acquired great importance. Advances in these techniques have increased knowledge of the various genes involved in tumoral development. Genetic alterations can occur in three large groups of genes: oncogenes, tumor suppressor genes, and DNA repair genes. Cytogenetic analyses (karyotyping) are complemented by various molecular techniques, such as fluorescence in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and spectral karyotyping (SKY). These are the most reliable techniques and improve the sensitivity of karyotyping. The present article reviews the most representative and best characterized genes involved in the molecular etiology of childhood cancer, both hematologic malignancies (leukemia and lymphoma) and solid tumors (brain tumors, neuroblastoma, Wilms' tumor, hepatoblastoma, rhabdomyosarcoma, Ewing's sarcoma and retinoblastoma). Molecular techniques have enabled more precise diagnosis as well as identification of new prognostic factors and the development of more effective treatments. These techniques can also be useful in identifying minimal residual disease during and after treatment for leukemias, neuroblastomas and sarcomas, with the aim of predicting recurrence.

[Acute perforated sigmoid diverticulitis in childhood]. / Diverticulitis aguda de sigma perforada en la infancia.

Colonic diverticulosis is a pathology of high incidence in normal population during the last decades of life. About 2-5 per 100 of patients admitted because of this disease are below 40 years old, and in this group of patients diverticulitis tends to be very aggressive with a high number of complications. We report a clinical case of acute perforation of sigmoid colon due to diverticulitis in a ten year old child who was treated surgically by means of resection of the sigma and primary end-to-end anastomosis. We did not find in the literature consulted any cases of this illness under twenty years of age.

Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression.

The Chk2-mediated deoxyribonucleic acid (DNA) damage checkpoint pathway is important for mitochondrial DNA (mtDNA) maintenance. We show in this paper that mtDNA itself affects cell cycle progression. Saccharomyces cerevisiae rho(0) cells, which lack mtDNA, were defective in G1- to S-phase progression. Deletion of subunit Va of cytochrome c oxidase, inhibition of F(1)F(0) adenosine triphosphatase, or replacement of all mtDNA-encoded genes with noncoding DNA did not affect G1- to S-phase progression. Thus, the cell cycle progression defect in rho(0) cells is caused by loss of DNA within mitochondria and not loss of respiratory activity or mtDNA-encoded genes. Rad53p, the yeast Chk2 homologue, was required for inhibition of G1- to S-phase progression in rho(0) cells. Pif1p, a DNA helicase and Rad53p target, underwent Rad53p-dependent phosphorylation in rho(0) cells. Thus, loss of mtDNA activated an established checkpoint kinase that inhibited G1- to S-phase progression. These findings support the existence of a Rad53p-regulated checkpoint that regulates G1- to S-phase progression in response to loss of mtDNA.

Mitochondrial inheritance is required for MEN-regulated cytokinesis in budding yeast.

Mitochondrial inheritance, the transfer of mitochondria from mother to daughter cell during cell division, is essential for daughter cell viability. The mitochore, a mitochondrial protein complex containing Mdm10p, Mdm12p, and Mmm1p, is required for mitochondrial motility leading to inheritance in budding yeast. We observe a defect in cytokinesis in mitochore mutants and another mutant (mmr1Delta gem1Delta) with impaired mitochondrial inheritance. This defect is not observed in yeast that have no mitochondrial DNA or defects in mitochondrial protein import or assembly of beta-barrel proteins in the mitochondrial outer membrane. Deletion of MDM10 inhibits contractile-ring closure, but does not inhibit contractile-ring assembly, localization of a chromosomal passenger protein to the spindle during early anaphase, spindle alignment, nucleolar segregation, or nuclear migration during anaphase. Release of the mitotic exit network (MEN) component, Cdc14p, from the nucleolus during anaphase is delayed in mdm10Delta cells. Finally, hyperactivation of the MEN by deletion of BUB2 restores defects in cytokinesis in mdm10Delta and mmr1Delta gem1Delta cells and reduces the fidelity of mitochondrial segregation between mother and daughter cells in wild-type and mdm10Delta cells. Our studies identify a novel MEN-linked regulatory system that inhibits cytokinesis in response to defects in mitochondrial inheritance in budding yeast.

[NBT test evaluation during the neonatal period (author's transl)]. / Valoración del "test" de nitroazul de tetrazolio en el período neonatal.

161 determinations of NBT performed according to the technique described by Park et al. on 67 patients of ages ranging from 1 hour born to 66 days, with weights between 1,610 to 4,600 g, and gestational ages from 34 to 41 weeks. During neonatal period, reduction of NBT is increased. Birth weight, fetal distress and neonatal asphixia do not seem to affect the NBT index during the first week of life. There is an inverse correlation (r = -0.557, p less than 0.001) between days of age and NBT test. Comparison of NBT test among infected and no infected newborns of various ages, showed significant differences (p less than 0.001). While NBT test may not be the key to detection of infection in neonatal period, according to results, it seems to be a fast and relatively simple test when seriated controls are performed on newborns believed to be infected.

[Electroencephalographic maturation in preterm newborn infants]. / Maduración electroencefalográfica en el recién nacido pretérmino.

We have studied 51 preterm infants [gestational age (GE) less than 32 weeks] by: a neurologic examination at 40 weeks of postconceptional age, serial cranial ultrasonographies, weekly electroencephalograms (EEG) until 42 weeks of postconceptional age, and Brunet-Lezine test of psychomotor developmental at 1 year old. Our objective is the longitudinal study of EEG maturation in preterm infants with or without neurologic injury. In preterm infants with the less gestational age are present the EEG characteristics reported for the older infants, the typical EEG pattern is the "sawtooth" (27-30 weeks GE). If these infants are "normal", the EEG maturation get adjusted to the previous reported pattern, except for the earlier presentation of transitory acute frontal waves and the disappearance of the preterm's discontinuous trace. The EEG maturation of preterm infants with perinatal neurologic injury are significantly delayed in contrast with "normal" preterm infants until 40 weeks of postconceptional age; this fact have a poor prognosis for these infants.

[Hypothermia, respiratory distress and umbilical catheterization as risk factors in necrotizing enterocolitis (author's transl)]. / Enterocolitis necrotizante neonatal. Valoración de la hipotermia, "distress" y cateterización umbilical como factores de riesgo.

Fourteen patients diagnosed of neonatal necrotizing enterocolitis (NNE) admitted into the neonatal unit since August 1977 are presented. They were matched to a control group of 130 newborn infants born during that same period of time for sex, weight and gestational age. All patients were formula fed except for three who followed mixed feeding. NNE was closely associated to hypothermia (p less than 0.005), respiratory distress (p less than 0.001) and umbilical catheterization (p less than 0.005). Umbilical catheters introduced for reasons other than distress did not increase the incidence of NNE. Low birth weight infants were more frequently affected than those weighing over 2,500 gm (ratio, 27:1). Evolution was lethal in six cases. The remaining eight are presently under follow up with no evidence of disease.

[Iron absorption in low birth weight and anemic infants (author's transl)]. / Absorción del hierro en recién nacidos de bajo peso y en lactantes anémicos.

A total of 35 iron balances were performed using a milk formula containing 6.25 +/- 1.23 mcg./dl. of iron. Twenty balances were done during the first 3 days of life (group 1) in low birth weight infants with 36.2 +/- 2.6 weeks of gestation and with birth weights of 2.13 +/- 0.28 kg. Thirteen of these infants received a calcium lactate supplement of 800 mg./kg./day. Ten balances were carried out at 13.9 +/- 4.12 days of life (group II) in LBWI with 36.0 +/- 2.1 weeks of gestation and with birth weights of 2.08 +/- 0.22 kg. Five balances were performed in a heterogeneous group (group III) of patients with iron-deficiency anemia (transferrin saturation: 6.3 +/- 2.9%). In group 1, the absorption coefficient for iron was 48.5 +/- 13.6% and of the 562. +/- 155.9 mcg./kg./day of iron ingested, 577.7 +/- 277.7 mcg./kg./day were retained. There were no statistically significant differences between groups I and II, but there were significant differences for ingestion, excretion and iron retention (p less than 0.001). In group I, iron absorption was not modified by the addition of calcium lactate. There was a positive significant correlation (p less than 0.001) between ingestion and net iron retention in groups I and II. Balances performed on five anemic children showed absorption coefficients of 10.5, 11.5, 41.6, 60.0 and 74.2% respectively. In view of our data, recommendations as to the optimal iron supplementation of cow's milk and as to the most desirable time to initiate it are analyzed.