The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation

A phosphorylation balance governed by Ipl1 Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in S. cerevisiae . Deletion of SET1, a histone K4 methyltransferase, suppresses the temperature sensitive phenotype of ipl1-2, and loss the catalytic activity of Set1 is important for this suppression. SET1 deletion also suppresses chromosome loss in ipl1-2 cells. Deletion of other Set1 complex components suppresses the temperature sensitivity of ipl1-2 as well. In contrast, SET1 deletion is synthetic lethal combined with glc7-127. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. I find that Set1 methylates conserved lysines in a kinetochore protein, Dam1, a key mitotic substrate of Ipl1/Glc7. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. My studies demonstrate that Set1 has important, unexpected functions in mitosis through modulating the phosphorylation balance regulated by Ipl1/Glc7. Moreover, my findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function. ^

CARD9 functions as a key regulator in monocyte homeostasis and pathogen clearance

Mononuclear phagocytes are designed to neutralize systemic bacterial and fungal infections. However, the exact regulation of these functions are largely unknown. CARD9 was first identified as an immune-specific adaptor protein of unclear function. Here, we have found that Card9 is specifically expressed in monocyte-origin cell populations. To better understand the biological function of Card9, we have generated Card9-deficient (Card9-/-) mice. Hematologic profiling and histological analysis of Card9-/- mice revealed a decreased leukocyte/myeloid cell count, delayed monocyte maturation in bone marrow as well as monocyte counts in the peripheral blood. Upon M-CSF stimulation, Card9-/- macrophages further exhibit a partial loss in IKK phosphorylation. As a consequence, in vivo challenge with Listeria monocytogenes in Card9-/- mice results in a higher susceptibility to infection-associated inflammation and fatality. Collectively, these data suggest that CARD9 is required for monocyte development and function. ^ At the cellular level, Card9-/- macrophages are defective in killing Listeria and the production of pro-inflammatory cytokines. Molecular characterizations have further demonstrated that CARD9 inducibly interacts with NOD2, controls p38 MAPK activation, and regulates ROS production during Listeria infections. Cytotrap screening showed that CARD9 could physically associate with various g&barbelow;uanine e&barbelow;xchange f&barbelow;actor (GEF) proteins that are essential for regulating ROS production. In summary, we have first identified and provided genetic evidence that CARD9 functions as a novel regulator during monocyte development and serves as an essential protein adaptor for p38 MAPK activation during bacterial clearance processes in macrophages. ^

Rescue of embryonic lethality in mdmx null mice by loss of p53 suggests a non-overlapping pathway with MDM2 to regulate p53

The tumor suppressor p53 is mutated in over 50% of human sporadic tumors originating from diverse tissues. p53 responds to DNA damage and cell stress by activating the transcription of a variety of target genes, the protein products of which then initiate either growth arrest or apoptosis. ^ A p53 target with a particularly intriguing function is the oncogene MDM2. MDM2 functions, in part, by binding to and inhibiting p53's activity. Overexpression of MDM2, by gene amplification, has been found in 30% of human sarcomas harboring a wild type p53, indicating that an increase in MDM2 levels is sufficient for p53 inactivation. Mice carrying a homozygous null allele for mdm2 exhibit an early embryonic lethality that is completely rescued in a p53-null background. These data indicate that MDM2's only critical function in early mouse embryogenesis is the negative regulation of p53. ^ The mdmx gene is the first additional member of the mdm2 gene family to be isolated. MDMX, like MDM2, contains a RING-finger domain, ATP binding domain and a p53 binding domain, which retains the ability to bind and inhibit p53 transactivation in vitro. However, mdmx does not appear to be transcriptionally regulated by p53. We have cloned and characterized the murine mdmx genomic locus from a mouse 129 genomic library. The mdmx gene contains 11 exons, spans approximately 37 Kb of DNA, and is located on mouse chromosome 1. The genomic organization of the mdmx gene is identical to that of mdm2 except at the 5′ end of the gene near the p53 responsive element. Northern expression analysis of mdmx transcripts during mouse embryogenesis and in adult tissues revealed constitutive and ubiquitous expression throughout adult tissues and embryonic development. To determine the in vivo function of MDMX, mice carrying a null allele of mdmx have been generated. Mdmx homozygous null mice are early embryonic lethal. Mdmx null mice do not develop beyond 9.5 dpc and can be discerned by gross dissection as early as 7.5 dpc. Utilizing TUNEL and BrdU assays on 7.5 dpc histological sections we have determined that the mutant embryos are dying due to increased levels of growth arrest, but not apoptosis. Surprisingly, Mdmx homozygous null mice are viable in a p53 null background, indicating that MDMX is also very important in the negative regulation of p53. ^

Characterization of cellular and molecular functions of the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The p21-activated kinase, Shk1, is an essential serine/threonine kinase required for normal cell polarity, proper mating response, and hyperosmotic stress response, in the fission yeast, Schizosaccharomyces pombe. This study has established a novel role for Shk1 as a microtubule regulator in fission yeast and, in addition, characterized a potential biological substrate of Shk1. Cells defective in Shk1 function were found to exhibit malformed interphase and mitotic microtubules, are hypersensitive to the microtubule disrupting drug thiabendazole (TBZ), and are cold sensitive for growth. Microtubule disruption by TBZ results in a significant reduction of Shk1 kinase activity, which is restored after cells are released from the drug, thus providing a correlation between Shk1 kinase activity and active microtubule polymerization. Consistent with a role for Shk1 as a microtubule regulator, GFP-Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles. Furthermore, loss of Tea1, a presumptive microtubule regulator in fission yeast, exacerbates the growth and microtubule defects of cells deficient in Shk1 function, and results in illicit Shk1 localization. Moreover, loss of the Cdc2 inhibitory kinase Wee1, which has been implicated as a mediator of the Shk1 pathway, leads to significant microtubule defects. Intriguingly, Wee1 protein levels are markedly reduced both by partial loss of Shk1 function and by treatment with TBZ. These results suggest that Shk1 is required for proper regulation of microtubule dynamics in fission yeast and may interact with Tea1 and Wee1 in this regulatory process. ^ To further understand Shk1 function in fission yeast, a yeast two-hybrid screen for proteins that interact with the Shk1 catalytic domain was performed. This screen led to the identification of a novel protein, Skb10 (for S&barbelow;hk1 k&barbelow;inase b&barbelow;inding protein 10). Coprecipitation experiments demonstrated that Skb10 associates with Shk1 in S. pombe cells. (Abstract shortened by UMI.) ^

Estudio de las dinámicas de pérdida de peso corporal en los programas de ejercicio físico y nutrición = Study of the dynamics of body weight loss in exercise and nutritional programs

Introducción. La obesidad puede definirse como una enfermedad metabólica crónica de origen multifactorial, lo que provoca trastornos o problemas físicos y psicológicos a la persona, con patologías asociadas que limitan la esperanza de vida y deterioran la calidad de la misma, siendo determinante para sus áreas sociales y laborales. Este trastorno metabólico crónico se caracteriza por una acumulación excesiva de energía en el cuerpo en forma de grasa, lo que lleva a un aumento de peso con respecto al valor esperado por sexo, edad y altura. La gestión y el tratamiento de la obesidad tienen objetivos más amplios que la pérdida de peso e incluyen la reducción del riesgo y la mejora de la salud. Estos pueden ser alcanzados por la pérdida modesta de peso (es decir, 10.5% del peso corporal inicial), la mejora del contenido nutricional de la dieta y un modesto incremento en la actividad física y condición física. La dieta es uno de los métodos más populares para perder peso corporal. El ejercicio es otra alternativa para perder peso corporal. El aumento de ejercicio provoca un desequilibrio cuando se mantiene la ingesta calórica. También tiene ventajas, como la mejora del tono muscular, la capacidad cardiovascular, fuerza y flexibilidad, aumenta el metabolismo basal y mejora el sistema inmunológico. Objetivos. El objetivo de esta tesis es contribuir en un estudio de intervención para aclarar la evolución del peso corporal durante una intervención de dieta y ejercicio. Para ello, se evaluaron los efectos de la edad, sexo, índice de masa corporal inicial y el tipo de tratamiento en las tendencias de pérdida de peso. Otro objetivo de la tesis era crear un modelo de regresión lineal múltiple capaz de predecir la pérdida de peso corporal después del periodo de intervención. Y, por último, determinar el efecto sobre la composición corporal (peso corporal, índice de masa corporal, la masa grasa, y la masa libre de grasa) de las diferentes intervenciones basadas en ejercicios (fuerza, resistencia, resistencia combinada con fuerza, y las recomendaciones de actividad física (grupo control)) en combinación con dieta de adultos con sobrepeso y obesidad, después de la intervención, así como los cambios de la composición corporal 3 años más tarde. Diseño de la investigación. Los datos empleados en el análisis de esta tesis son parte del proyecto “Programas de Nutrición y Actividad Física para el tratamiento de la obesidad” (PRONAF). El proyecto PRONAF es un estudio clínico sobre programas de nutrición y actividad física para el sobrepeso y la obesidad, desarrollado en España durante varios años de intervención. Fue diseñado, en parte, para comparar diferentes tipos de intervención, con el objetivo de evaluar su impacto en las dinámicas de pérdida de peso, en personas con sobrepeso y obesidad. Como diseño experimental, el estudio se basó en una restricción calórica, a la que, en algunos casos, se le añadió un protocolo de entrenamiento (fuerza, resistencia, o combinado, en igualdad de volumen e intensidad). Las principales variables para la investigación que comprende esta tesis fueron: el peso corporal y la composición corporal (masa grasa y masa libre de grasa). Conclusiones. En esta tesis, para los programas de pérdida de peso en personas con sobrepeso y obesidad con un 25-30% de la restricción calórica, el peso corporal se redujo significativamente en ambos sexos, sin tener en cuenta la edad y el tipo de tratamiento seguido. Según los resultados del estudio, la pérdida de peso realizada por un individuo (hombre o mujer) durante los seis meses puede ser representada por cualquiera de las cinco funciones (lineal, potencial, exponencial, logarítmica y cuadrática) en ambos sexos, siendo la cuadrática la que tiende a representarlo mejor. Además, se puede concluir que la pérdida de peso corporal se ve afectada por el índice de masa corporal inicial y el sexo, siendo mayor para las personas obesas que para las de sobrepeso, que muestran diferencias entre sexos sólo en la condición de sobrepeso. Además, es posible calcular el peso corporal final de cualquier participante involucrado en una intervención utilizando la metodología del proyecto PRONAF sólo conociendo sus variables iniciales de composición corporal. Además, los cuatro tipos de tratamientos tuvieron resultados similares en cambios en la composición corporal al final del período de intervención, con la única excepción de la masa libre de grasa, siendo los grupos de entrenamiento los que la mantuvieron durante la restricción calórica. Por otro lado, sólo el grupo combinado logra mantener la reducción de la masa grasa (%) 3 años después del final de la intervención. ABSTRACT Introduction. Obesity can be defined as a chronic metabolic disease from a multifactorial origin, which leads to physical and psychological impacts to the person, with associated pathologies that limit the life expectancy and deteriorate the quality of it, being determinant for the social and labor areas of the person. This chronic metabolic disorder is characterized by an excessive accumulation of energy in the body as fat, leading to increased weight relative to the value expected by sex, age and height. The management and treatment of obesity have wider objectives than weight loss alone and include risk reduction and health improvement. These may be achieved by modest weight loss (i.e. 5–10% of initial body weight), improved nutritional content of the diet and modest increases in physical activity and fitness. Weight loss through diet is one of the most popular approaches to lose body weight. Exercise is another alternative to lose body weight. The increase of exercise causes an imbalance when the caloric intake is maintained. It also has advantages such as improved muscle tone, cardiovascular fitness, strength and flexibility, increases the basal metabolism and improves immune system. Objectives. The aim of this thesis is to contribute with an interventional study to clarify the evolution of the body weight during a diet and exercise intervention. For this, the effects of age, sex, initial body mass index and type of treatment on weight loss tendencies were evaluated. Another objective of the thesis was to create a multiple linear regression model able to predict the body weight loss after the intervention period. And, finally, to determine the effect upon body composition (body weight, body mass index, fat mass, and fat-free mass of different exercise-based interventions (strength, endurance, combined endurance and strength, and physical activity recommendations group (control group)) combined with diet in overweight and obese adults, after intervention as well as body composition changes 3 years later. Research Design. The data used in the analysis of this thesis are part of the project "Programs of Nutrition and Physical Activity for the treatment of obesity" (PRONAF). The PRONAF project is a clinical trial program about nutrition and physical activity for overweight and obesity, developed in Spain for several years of intervention. It was designed, in part, to compare different types of intervention, in order to assess their impact on the dynamics of weight loss in overweight and obese people. As experimental design, the study was based on caloric restriction, which, in some cases, added a training protocol (strength, endurance, or combined in equal volume and intensity). The main research variables comprising this thesis were: body weight and body composition outcomes (fat mass and fat-free mass). Conclusions. In this thesis, for weight loss programs in overweight and obese people with 25-30% of caloric restriction, the body weight was significantly decreased in both sexes, regardless the age and type of followed treatment. According to the results of the study, the weight loss performed by an individual (male or female) during six months can be represented by any of the five functions (linear, power law, exponential, logarithmic and quadratic) in both sexes, being the quadratic one which tends to represent it better. In addition, it can be concluded that the body weight loss is affected by the initial body mass index and sex condition, being greater for the obese people than for the overweight one, showing differences between sexes only in the overweight condition. Moreover, it is possible to calculate the final body weight of any participant engaged in an intervention using the PRONAF Project methodology only knowing their initial body composition variables. Furthermore, the four types of treatments had similar results on body composition changes at the end of the intervention period, with the only exception of fat-free mass, being the training groups the ones that maintained it during the caloric restriction. On the other hand, only the combined group achieved to maintain the fat mass (%) reduced 3 years after the end of the intervention.

Biometric authentication of users through iris by using key binding and similarity preserving hash functions = Autenticación biométrica de usuarios a través del iris mediante la ocultación de claves y funciones resumen que preservan la similitud

El extraordinario auge de las nuevas tecnologías de la información, el desarrollo de la Internet de las Cosas, el comercio electrónico, las redes sociales, la telefonía móvil y la computación y almacenamiento en la nube, han proporcionado grandes beneficios en todos los ámbitos de la sociedad. Junto a éstos, se presentan nuevos retos para la protección y privacidad de la información y su contenido, como la suplantación de personalidad y la pérdida de la confidencialidad e integridad de los documentos o las comunicaciones electrónicas. Este hecho puede verse agravado por la falta de una frontera clara que delimite el mundo personal del mundo laboral en cuanto al acceso de la información. En todos estos campos de la actividad personal y laboral, la Criptografía ha jugado un papel fundamental aportando las herramientas necesarias para garantizar la confidencialidad, integridad y disponibilidad tanto de la privacidad de los datos personales como de la información. Por otro lado, la Biometría ha propuesto y ofrecido diferentes técnicas con el fin de garantizar la autentificación de individuos a través del uso de determinadas características personales como las huellas dáctilares, el iris, la geometría de la mano, la voz, la forma de caminar, etc. Cada una de estas dos ciencias, Criptografía y Biometría, aportan soluciones a campos específicos de la protección de datos y autentificación de usuarios, que se verían enormemente potenciados si determinadas características de ambas ciencias se unieran con vistas a objetivos comunes. Por ello es imperativo intensificar la investigación en estos ámbitos combinando los algoritmos y primitivas matemáticas de la Criptografía con la Biometría para dar respuesta a la demanda creciente de nuevas soluciones más técnicas, seguras y fáciles de usar que potencien de modo simultáneo la protección de datos y la identificacíón de usuarios. En esta combinación el concepto de biometría cancelable ha supuesto una piedra angular en el proceso de autentificación e identificación de usuarios al proporcionar propiedades de revocación y cancelación a los ragos biométricos. La contribución de esta tesis se basa en el principal aspecto de la Biometría, es decir, la autentificación segura y eficiente de usuarios a través de sus rasgos biométricos, utilizando tres aproximaciones distintas: 1. Diseño de un esquema criptobiométrico borroso que implemente los principios de la biometría cancelable para identificar usuarios lidiando con los problemas acaecidos de la variabilidad intra e inter-usuarios. 2. Diseño de una nueva función hash que preserva la similitud (SPHF por sus siglas en inglés). Actualmente estas funciones se usan en el campo del análisis forense digital con el objetivo de buscar similitudes en el contenido de archivos distintos pero similares de modo que se pueda precisar hasta qué punto estos archivos pudieran ser considerados iguales. La función definida en este trabajo de investigación, además de mejorar los resultados de las principales funciones desarrolladas hasta el momento, intenta extender su uso a la comparación entre patrones de iris. 3. Desarrollando un nuevo mecanismo de comparación de patrones de iris que considera tales patrones como si fueran señales para compararlos posteriormente utilizando la transformada de Walsh-Hadarmard. Los resultados obtenidos son excelentes teniendo en cuenta los requerimientos de seguridad y privacidad mencionados anteriormente. Cada uno de los tres esquemas diseñados han sido implementados para poder realizar experimentos y probar su eficacia operativa en escenarios que simulan situaciones reales: El esquema criptobiométrico borroso y la función SPHF han sido implementados en lenguaje Java mientras que el proceso basado en la transformada de Walsh-Hadamard en Matlab. En los experimentos se ha utilizado una base de datos de imágenes de iris (CASIA) para simular una población de usuarios del sistema. En el caso particular de la función de SPHF, además se han realizado experimentos para comprobar su utilidad en el campo de análisis forense comparando archivos e imágenes con contenido similar y distinto. En este sentido, para cada uno de los esquemas se han calculado los ratios de falso negativo y falso positivo. ABSTRACT The extraordinary increase of new information technologies, the development of Internet of Things, the electronic commerce, the social networks, mobile or smart telephony and cloud computing and storage, have provided great benefits in all areas of society. Besides this fact, there are new challenges for the protection and privacy of information and its content, such as the loss of confidentiality and integrity of electronic documents and communications. This is exarcebated by the lack of a clear boundary between the personal world and the business world as their differences are becoming narrower. In both worlds, i.e the personal and the business one, Cryptography has played a key role by providing the necessary tools to ensure the confidentiality, integrity and availability both of the privacy of the personal data and information. On the other hand, Biometrics has offered and proposed different techniques with the aim to assure the authentication of individuals through their biometric traits, such as fingerprints, iris, hand geometry, voice, gait, etc. Each of these sciences, Cryptography and Biometrics, provides tools to specific problems of the data protection and user authentication, which would be widely strengthen if determined characteristics of both sciences would be combined in order to achieve common objectives. Therefore, it is imperative to intensify the research in this area by combining the basics mathematical algorithms and primitives of Cryptography with Biometrics to meet the growing demand for more secure and usability techniques which would improve the data protection and the user authentication. In this combination, the use of cancelable biometrics makes a cornerstone in the user authentication and identification process since it provides revocable or cancelation properties to the biometric traits. The contributions in this thesis involve the main aspect of Biometrics, i.e. the secure and efficient authentication of users through their biometric templates, considered from three different approaches. The first one is designing a fuzzy crypto-biometric scheme using the cancelable biometric principles to take advantage of the fuzziness of the biometric templates at the same time that it deals with the intra- and inter-user variability among users without compromising the biometric templates extracted from the legitimate users. The second one is designing a new Similarity Preserving Hash Function (SPHF), currently widely used in the Digital Forensics field to find similarities among different files to calculate their similarity level. The function designed in this research work, besides the fact of improving the results of the two main functions of this field currently in place, it tries to expand its use to the iris template comparison. Finally, the last approach of this thesis is developing a new mechanism of handling the iris templates, considering them as signals, to use the Walsh-Hadamard transform (complemented with three other algorithms) to compare them. The results obtained are excellent taking into account the security and privacy requirements mentioned previously. Every one of the three schemes designed have been implemented to test their operational efficacy in situations that simulate real scenarios: The fuzzy crypto-biometric scheme and the SPHF have been implemented in Java language, while the process based on the Walsh-Hadamard transform in Matlab. The experiments have been performed using a database of iris templates (CASIA-IrisV2) to simulate a user population. The case of the new SPHF designed is special since previous to be applied i to the Biometrics field, it has been also tested to determine its applicability in the Digital Forensic field comparing similar and dissimilar files and images. The ratios of efficiency and effectiveness regarding user authentication, i.e. False Non Match and False Match Rate, for the schemes designed have been calculated with different parameters and cases to analyse their behaviour.

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2–13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 1½ years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.

Mso1p: A yeast protein that functions in secretion and interacts physically and genetically with Sec1p

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1–1 mutation. We now describe a third suppressor of sec1–1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Gene Knockouts Reveal Separate Functions for Two Cytoplasmic Dyneins in Tetrahymena thermophila

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

The Schizosaccharomyces pombe hst4+ Gene Is a SIR2 Homologue with Silencing and Centromeric Functions

Although silencing is a significant form of transcriptional regulation, the functional and mechanistic limits of its conservation have not yet been established. We have identified the Schizosaccharomyces pombe hst4+ gene as a member of the SIR2/HST silencing gene family that is defined in organisms ranging from bacteria to humans. hst4Δ mutants grow more slowly than wild-type cells and have abnormal morphology and fragmented DNA. Mutant strains show decreased silencing of reporter genes at both telomeres and centromeres. hst4+ appears to be important for centromere function as well because mutants have elevated chromosome-loss rates and are sensitive to a microtubule-destabilizing drug. Consistent with a role in chromatin structure, Hst4p localizes to the nucleus and appears concentrated in the nucleolus. hst4Δ mutant phenotypes, including growth and silencing phenotypes, are similar to those of the Saccharomyces cerevisiae HSTs, and at a molecular level, hst4+ is most similar to HST4. Furthermore, hst4+ is a functional homologue of S. cerevisiae HST3 and HST4 in that overexpression of hst4+ rescues the temperature-sensitivity and telomeric silencing defects of an hst3Δ hst4Δ double mutant. These results together demonstrate that a SIR-like silencing mechanism is conserved in the distantly related yeasts and is likely to be found in other organisms from prokaryotes to mammals.

Parkin functions as an E2-dependent ubiquitin– protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1

Parkinson's disease is a common neurodegenerative disorder in which familial-linked genes have provided novel insights into the pathogenesis of this disorder. Mutations in Parkin, a ring-finger-containing protein of unknown function, are implicated in the pathogenesis of autosomal recessive familial Parkinson's disease. Here, we show that Parkin binds to the E2 ubiquitin-conjugating human enzyme 8 (UbcH8) through its C-terminal ring-finger. Parkin has ubiquitin–protein ligase activity in the presence of UbcH8. Parkin also ubiquitinates itself and promotes its own degradation. We also identify and show that the synaptic vesicle-associated protein, CDCrel-1, interacts with Parkin through its ring-finger domains. Furthermore, Parkin ubiquitinates and promotes the degradation of CDCrel-1. Familial-linked mutations disrupt the ubiquitin–protein ligase function of Parkin and impair Parkin and CDCrel-1 degradation. These results suggest that Parkin functions as an E3 ubiquitin–protein ligase through its ring domains and that it may control protein levels via ubiquitination. The loss of Parkin's ubiquitin–protein ligase function in familial-linked mutations suggests that this may be the cause of familial autosomal recessive Parkinson's disease.

The interphotoreceptor retinoid binding protein gene in therian mammals: Implications for higher level relationships and evidence for loss of function in the marsupial mole

The subclass Theria of Mammalia includes marsupials (infraclass Metatheria) and placentals (infraclass Eutheria). Within each group, interordinal relationships remain unclear. One limitation of many studies is incomplete ordinal representation. Here, we analyze DNA sequences for part of exon 1 of the interphotoreceptor retinoid binding protein gene, including 10 that are newly reported, for representatives of all therian orders. Among placentals, the most robust clades are Cetartiodactyla, Paenungulata, and an expanded African clade that includes paenungulates, tubulidentates, and macroscelideans. Anagalida, Archonta, Altungulata, Hyracoidea + Perissodactyla, Ungulata, and the “flying primate” hypothesis are rejected by statistical tests. Among marsupials, the most robust clade includes all orders except Didelphimorphia. The phylogenetic placement of the monito del monte and the marsupial mole remains unclear. However, the marsupial mole sequence contains three frameshift indels and numerous stop codons in all three reading frames. Given that the interphotoreceptor retinoid binding protein gene is a single-copy gene that functions in the visual cycle and that the marsupial mole is blind with degenerate eyes, this finding suggests that phenotypic degeneration of the eyes is accompanied by parallel changes at the molecular level as a result of relaxed selective constraints.

Long-term, progressive hippocampal cell loss and dysfunction induced by early-life administration of corticotropin-releasing hormone reproduce the effects of early-life stress

Stress early in postnatal life may result in long-term memory deficits and selective loss of hippocampal neurons. The mechanisms involved are poorly understood, but they may involve molecules and processes in the immature limbic system that are activated by stressful challenges. We report that administration of corticotropin-releasing hormone (CRH), the key limbic stress modulator, to the brains of immature rats reproduced the consequences of early-life stress, reducing memory functions throughout life. These deficits were associated with progressive loss of hippocampal CA3 neurons and chronic up-regulation of hippocampal CRH expression. Importantly, they did not require the presence of stress levels of glucocorticoids. These findings indicate a critical role for CRH in the mechanisms underlying the long-term effects of early-life stress on hippocampal integrity and function.