Molecular and genetic analysis of Myxococcus xanthus early *development

To identify more mutations that can affect the early development of Myxococcus xanthus, the synthetic transposon TnT41 was designed and constructed. By virtue of its special features, it can greatly facilitate the processes of mutation screening/selection, mapping, cloning and DNA sequencing. In addition, it allows for the systematic discovery of genes in regulatory hierarchies using their target promoters. In this study, the minimal regulatory region of the early developmentally regulated gene 4521 was used as a reporter in the TnT41 mutagenesis. Both positive (P) mutations and negative (N) mutations were isolated based on their effects on 4521 expression.^ Four of these mutations, i.e. N1, N2, P52 and P54 were analyzed in detail. Mutations N1 and N2 are insertion mutations in a gene designated sasB. The sasB gene is also identified in this study by genetic and molecular analysis of five UV-generated 4521 suppressor mutations. The sasB gene encodes a protein without meaningful homology in the databases. The sasB gene negatively regulates 4521 expression possibly through the SasS-SasR two component system. A wild-type sasB gene is required for normal M. xanthus fruiting body formation and sporulation.^ Cloning and sequencing analysis of the P52 mutation led to the identification of an operon that encodes the M. xanthus high-affinity branched-chain amino acid transporter system. This liv operon consists of five genes designated livK, livH, livM, livC, and livF, respectively. The Liv proteins are highly similar to their counterparts from other bacteria in both amino acid sequences, functional motifs and predicted secondary structures. This system is required for development since liv null mutations cause abnormality in fruiting body formation and a 100-fold decrease in sporulation efficiency.^ Mutation P54 is a TnT41 insertion in the sscM gene of the ssc chemotaxis system, which has been independently identified by Dr. Shi's lab. The sscM gene encodes a MCP (methyl-accepting chemotaxis protein) homologue. The SscM protein is predicted to contain two transmembrane domains, a signaling domain and at least one putative methylation site. Null mutations of this gene abolish the aggregation of starving cells at a very early stage, though the sporulation levels of the mutant can reach 10% that of wild-type cells. ^

Transcriptional regulation of cell lineage-specific expression of the murine type I collagen genes and of osteoblast differentiation

The aim of my project is to examine the mechanisms of cell lineage-specific transcriptional regulation of the two type I collagen genes by characterizing critical cis-acting elements and trans-acting factors. I hypothesize that the transcription factors that are involved in the cell lineage-specific expression of these genes may have a larger essential role in cell lineage commitment and differentiation. I first examined the proximal promoters of the proα1(I) and the proα2(I) collagen genes for cell type-specific DNA-protein interactions, using in vitro DNaseI and in vivo DMS footprinting. These experiments demonstrated that the cis-acting elements in these promoters are accessible to ubiquitous DNA-binding proteins in fibroblasts that express these genes, but not in other cells that do not express these genes. I speculate that in type I collagen-expressing cells, cell type-specific enhancer elements facilitate binding of ubiquitous proteins to the proximal promoters of these genes. Subsequently, examination of the upstream promoter of the proα(I) collagen gene by transgenic mice experiments delineated a 117 bp sequence (-1656 to -1540 bp) as the minimum element required for osteoblast-specific expression. This 117 bp element contained two segments that appeared to have different functions: (1) the A-segment, which was necessary to obtain osteoblast-specific expression and (2) the C-segment, which was dispensable for osteoblast-specific expression, but was necessary to obtain high-level expression. In experiments to identify trans-acting factors that bind to the 117 bp element, I have demonstrated that the cell lineage-restricted homeodomain proteins, Dlx2, Dlx5 and mHOX, bound to the A-segment and that the ubiquitous transcription factor, Sp1, bound to the C-segment of this element. These results suggested a model where the binding of cell lineage-restricted proteins to the A-segment and of ubiquitous proteins to the C-segment of the 117 bp element of the proα1 (I) collagen gene activated this gene in osteoblasts. These results, combined with additional evidence that Dlx2, Dlx5 and mHOX are probably involved in osteoblast differentiation, support my hypothesis that the transcription factors involved in osteoblast-specific expression of type I collagen genes may have essential role in osteoblast lineage commitment and differentiation. ^

Transcriptional repression of serum amyloid A1 by AP -2 contributes to its liver -specific expression

Studies on the transcriptional regulation of serum amyloid A1 (SAA1) gene, a liver specific acute-phase gene, identified a regulatory element in its promoter that functioned to repress (SAA1) gene transcription in nonliver cells. This silencer element interacts with a nuclear protein that is detectable in HeLa cells, fibroblasts and placental tissues but not in liver or liver-derived cells. As the expression pattern of this repressor is consistent with its potential regulatory role in repressing SAA1 expression, and that many other liver gene promoters also contain this repressor binding site, we sought to investigate whether this repressor may have a broader functional role in repressing liver genes. ^ We have utilized protein purification, cell culture, transient and stable gene transfection, and molecular biology approaches to identify this protein and investigate its possible function in the regulation of (SAA1) and other liver genes. Analyses of amino acid sequence of the purified nuclear protein, and western blot and gel shift studies identified the repressor as transcription factor AP-2 or AP-2-like protein. Using transient transfection of DNA into cultured cells, we demonstrate that AP-2 can indeed function as a repressor to inhibit transcription of SAA1 gene promoter. This conclusion is supported by the following experimental results: (1) overexpression of AP-2 in hepatoma cells inhibits conditioned medium (CM)-induced expression of SAA1 promoter; (2) binding of AP-2 to the SAA1 promoter is required for AP-2 repression function; (3) one mechanism by which AP-2 inhibits SAA1 may be by antagonizing the activation function of the strong transactivator NFκB; (4) mutation of AP-2 binding sites results in derepression of SAM promoter in HeLa cells; and (5) inhibition of endogenous AP-2 activity by a dominant-negative mutant abolishes AP-2's inhibitory effect on SAM promoter in HeLa cells. In addition to the SAM promoter, AP-2 also can bind to the promoter regions of six other liver genes tested, suggesting that it may have a broad functional role in restricting the expression of many liver genes in nonliver cells. Consistent with this notion, ectopic expression of AP-2 also represses CM-mediated activation of human third component of complement 3 promoter. Finally, in AP-2-expressing stable hepatoma cell lines, AP-2 inhibits not only the expression of endogenous SAA, but also the expression of several other endogenous liver genes including albumin, α-fetoprotein. ^ Our findings that AP-2 has the ability to repress the expression of liver genes in nonliver cells opens a new avenue of investigation of negative regulation of gene transcription, and should improve our understanding of tissue-specific expression of liver genes. In summary, our data provide evidence suggesting a novel role of AP-2 as a repressor, inhibiting the expression of liver genes in nonliver cells. Thus, the tissue-specific expression of AP-2 may constitute an important mechanism contributing to the liver-specific expression of liver genes. ^

A novel DNA damage inducible inhibitor of p53

p53 is required for the maintenance of the genomic stability of cells. Mutations in the p53 tumor-suppressor gene occur in more than 50% of human cancers of diverse types. In addition, 70% of families with Li-Fraumeni syndrome have a germline mutation in p53, predisposing these individuals to multiple forms of cancer. In response to DNA damage, p53 becomes stabilized and activated. However the exact mechanism by which DNA damage signals the stabilization and activation of p53 still remains elusive. The biochemical activity of p53 that is required for tumor suppression, and presumably the cellular response to DNA damage, involves the ability of the protein to bind to specific DNA sequences and to function as a transcription factor. For the downstream targets, p53 transactivates many genes involved in growth arrest, apoptosis and DNA repair such as p21, Bax and GADD45, respectively. An open question in the field is how cells can determine the downstream effects of p53. ^ We hypothesize that, through its associated proteins, p53 can differentially transactivate its target genes, which determine its downstream effect. Additionally, p53 interacting proteins may be involved in signaling for the stabilization and activation of p53. Therefore, a key aspect to understanding p53 function is the identification and analysis of proteins that interact with it. We have employed the Sos recruitment system (SRS), a cytoplasmic yeast two-hybrid screen to identify p53 interacting proteins. The SRS is based on the ability of Sos to activate Ras when it becomes localized to the plasma membrane. The system takes advantage of an S. cerevisiae strain, cdc25-2 temperature sensitive mutant, harboring a mutation in Sos. In this strain, fusion proteins containing a truncated Sos will only localize to the membrane by protein-protein interaction, which allows growth at non-permissive temperature. This system allows the use of intact transcriptional activators such as p53. ^ To date, using a modified SRS library screen to identify p53 interacting proteins, I have identified p53 (known to interact with itself) and a novel p53-interacting protein (PIP). PIP is a specific p53 interacting protein in the SRS. The interaction of p53 and PIP was further confirmed by performing in vitro and in vivo binding assays. In the in vivo binding study, the interaction can only be detected in the presence of ionizing radiation suggesting that this interaction might be involved in DNA-damage induced p53-signalling pathway. After screening cDNA and genomic libraries, a full-length PIP-cDNA clone ( ∼ 3kb) was obtained which encodes a protein of 429 amino acids with calculated molecular weight of 46 kDa. The results of genebank search indicated that the PIP is an unidentified gene and contains a conserved ring-finger domain, which is present in a diverse family of regulatory proteins involved in different aspects of cellular function. Northern blot analysis revealed that the size of its messenge is approximately 3 kb preferentially expressed in brain, heart, liver and kidney. The PIP protein is mainly located in the cytoplasm as determined by the cellular localization of a green fluorescence fusion protein. Preliminary functional analysis revealed that PIP downregulated the transactivation activity of p53 on both p21 and mdm2 promoters. Thus, PIP may be a novel negative regulator of p53 subsequent to DNA damage. ^

Functional activation of p53 induces apoptosis in the absence of MDM2

The p53 tumor suppressor gene product is negatively regulated by the product of its downstream target, mdm2. The mdm2 oncogene abrogates p53 transactivation function. Amplification of mdm2 occurs in 36% of human sarcomas, which often retain p53 in wild type form, suggesting that overexpression of mdm2 in tumors results in p53 inactivation. Thus, the relationship of p53 to mdm2 is important in tumorigenesis. The deletion of mdm2 in the mouse results in embryonic lethality by 5.5 days post coitum. Embryonic lethality of the mdm2 null embryos was overcome by simultaneous loss of the p53 tumor suppressor, which substantiates the importance of the negative regulatory function of MDM2 on p53 function in vivo. These data suggest that the loss of MDM2 function allowed the constitutively active p53 protein to induce either a complete G1 arrest or the p53-dependent apoptotic pathway, resulting in the death of the mdm2−/− embryos.^ The present study examines the hypothesis that the absence of mdm2 induces apoptosis due to p53 activation. Viability of the p53−/−mdm2−/− mice has allowed establishment of mouse embryo fibroblasts (MEFs) and a detailed examination of the properties of these cells. To introduce p53 into this system, and essentially recreate a mdm2 null cell, a temperature sensitive p53 (tsp53) point mutant (A135V) was used, which exhibits a nonfunctional, mutant conformation at 39°C and wild type, functional conformation at 32°C. Infected pools of p53−/− and p53−/−mdm2−/− MEFs with the tsp53 gene were established and single-cell clonal populations expressing tsp53 were selected. Shifting the cells from 39°C to 32°C caused p53−/−mdm2 −/− lines expressing tsp53 to undergo up to 80% apoptosis, which did not occur in the p53−/− lines expressing tsp53 nor the parental lines lacking p53 expression. Furthermore, the amount of p53 present in the clonal population determined the extent of apoptosis. Tsp53 is transcriptionally active in this system, however, it discriminates among different target promoters and does not induce the apoptosis effector targets bax or Fas/Apo1. ^ In summary, this study indicates that the presence or absence of mdm2 is the determining factor for the ability of p53 to trigger apoptosis in this system. The loss of mdm2 promotes p53-dependent apoptosis in MEFs in a cell cycle and dose-dependent manner. p53 is differentially phosphorylated in the presence and absence of mdm2, but does not induce the apoptosis effectors, bax or Fas/ Apo1. ^

La imagen de Argentina hacia los festejos del primer centenario : la mirada germana en los escritos de Johann, Theodor y Ernesto Alemann

Johann, Theodor y Ernesto Alemann pertenecieron a una familia de ascendencia suiza que tuvo un papel muy influyente en la formación de la imagen de la Argentina proyectada hacia la comunidad germanohablante. Fundadores y redactores del Argentinisches Tageblatt (Diario argentino) y promotores de la inmigración germana al país, a través de sus discursos construyen una imagen de la Argentina, pensada sobre todo para un lector situado fuera del país. Este artículo se propone analizar esa imagen, sobre todo en los textos de los autores publicados próximos a la fecha del Primer Centenario de la Revolución de Mayo.

Ingenieros agrónomos, desarrollo rural y cultura política : reflexiones críticas en torno a la práctica de la (ex)tensión rural

En esta nota breve se analiza, desde una perspectiva crítica, la práctica de la extensión rural realizada por profesionales agrónomos en Mendoza, Argentina. En base a una amplia revisión bibliográfica y análisis documental sobre programas de desarrollo rural, analizamos los condicionamientos socio-culturales de dicha práctica, elementos que inciden sobre la concepción, ejecución y orientación de las estrategias de desarrollo promovidas por estos sujetos y sus instituciones. Así argumentamos que a pesar de los profundos cambios ocurridos en el mundo, particularmente los que afectan al mundo rural, las prácticas de los extensionistas y su concepción, continúan arraigadas a los ya viejos supuestos de la modernización y el progreso indefinido. Asimismo sostenemos que la falta de reflexividad crítica institucional retroalimenta la crisis del desarrollo rural con considerables repercusiones en sus promotores, los extensionistas. Finalmente destacamos que la reflexión sobre la práctica de la extensión rural tiene una relevancia social e institucional central, considerando que un análisis crítico de las limitaciones y posibilidades en este campo puede contribuir a orientar -y superar- los desafíos actuales y futuros.

Las muchachas ahorrativas : El rol de las mujeres peronistas en el Plan Económico de Austeridad y el 2° Plan Quinquenal

En este trabajo propongo analizar el rol asignado a las mujeres peronistas y a las unidades básicas femeninas del Partido Peronista Femenino como promotoras del ahorro y la economía doméstica dentro del Plan Económico de Austeridad y el 2° Plan Quinquenal. Luego de la amplia confirmación de la popularidad del gobierno obtenida en las elecciones de noviembre de 1951, el presidente Perón consideró oportuno producir una rectificación y un ajuste en el rumbo de la política económica. Se trataba de una serie de medidas imprescindibles para superar una coyuntura que se tornaba crítica, y que se resumían en el aumento de la producción y la austeridad en el consumo. Esta última responsabilidad recayó en la mujeres - amas de casa amparadas en la acción de las unidades básicas femeninas que actuaron como consejeras y promotoras de los planes de austeridad implementando una serie de medidas que iban desde la cursos de cocina que enseñaban a cocinar con productos alternativos y más económicos hasta la fiscalización de los comercios que no cumplían con los precios máximos oficiales.

La Revolución de Mayo de 1810 en la historiografía uruguaya de orientación nacionalista

La historiografía oficial uruguaya, articulada por la escuela tradicional de orientación nacionalista, atribuyó a la Revolución de Mayo de 1810 una dimensión subsidiaria. Su crónica es generalmente breve y está contextualizada en relatos geográficamente circunscriptos a la Banda Oriental. Este artículo pretende analizar la interpretación de los acontecimientos de Mayo de 1810 realizada por los principales articuladores de la tesis independentista clásica -Francisco Bauzá, Pablo Blanco Acevedo y Juan Pivel Devoto- e identificar el rol atribuido a la misma en el marco general del discurso nacionalista.

Las muchachas ahorrativas : El rol de las mujeres peronistas en el Plan Económico de Austeridad y el 2° Plan Quinquenal

En este trabajo propongo analizar el rol asignado a las mujeres peronistas y a las unidades básicas femeninas del Partido Peronista Femenino como promotoras del ahorro y la economía doméstica dentro del Plan Económico de Austeridad y el 2° Plan Quinquenal. Luego de la amplia confirmación de la popularidad del gobierno obtenida en las elecciones de noviembre de 1951, el presidente Perón consideró oportuno producir una rectificación y un ajuste en el rumbo de la política económica. Se trataba de una serie de medidas imprescindibles para superar una coyuntura que se tornaba crítica, y que se resumían en el aumento de la producción y la austeridad en el consumo. Esta última responsabilidad recayó en la mujeres - amas de casa amparadas en la acción de las unidades básicas femeninas que actuaron como consejeras y promotoras de los planes de austeridad implementando una serie de medidas que iban desde la cursos de cocina que enseñaban a cocinar con productos alternativos y más económicos hasta la fiscalización de los comercios que no cumplían con los precios máximos oficiales.

La Revolución de Mayo de 1810 en la historiografía uruguaya de orientación nacionalista

La historiografía oficial uruguaya, articulada por la escuela tradicional de orientación nacionalista, atribuyó a la Revolución de Mayo de 1810 una dimensión subsidiaria. Su crónica es generalmente breve y está contextualizada en relatos geográficamente circunscriptos a la Banda Oriental. Este artículo pretende analizar la interpretación de los acontecimientos de Mayo de 1810 realizada por los principales articuladores de la tesis independentista clásica -Francisco Bauzá, Pablo Blanco Acevedo y Juan Pivel Devoto- e identificar el rol atribuido a la misma en el marco general del discurso nacionalista.

Las muchachas ahorrativas : El rol de las mujeres peronistas en el Plan Económico de Austeridad y el 2° Plan Quinquenal

En este trabajo propongo analizar el rol asignado a las mujeres peronistas y a las unidades básicas femeninas del Partido Peronista Femenino como promotoras del ahorro y la economía doméstica dentro del Plan Económico de Austeridad y el 2° Plan Quinquenal. Luego de la amplia confirmación de la popularidad del gobierno obtenida en las elecciones de noviembre de 1951, el presidente Perón consideró oportuno producir una rectificación y un ajuste en el rumbo de la política económica. Se trataba de una serie de medidas imprescindibles para superar una coyuntura que se tornaba crítica, y que se resumían en el aumento de la producción y la austeridad en el consumo. Esta última responsabilidad recayó en la mujeres - amas de casa amparadas en la acción de las unidades básicas femeninas que actuaron como consejeras y promotoras de los planes de austeridad implementando una serie de medidas que iban desde la cursos de cocina que enseñaban a cocinar con productos alternativos y más económicos hasta la fiscalización de los comercios que no cumplían con los precios máximos oficiales.

La Revolución de Mayo de 1810 en la historiografía uruguaya de orientación nacionalista

La historiografía oficial uruguaya, articulada por la escuela tradicional de orientación nacionalista, atribuyó a la Revolución de Mayo de 1810 una dimensión subsidiaria. Su crónica es generalmente breve y está contextualizada en relatos geográficamente circunscriptos a la Banda Oriental. Este artículo pretende analizar la interpretación de los acontecimientos de Mayo de 1810 realizada por los principales articuladores de la tesis independentista clásica -Francisco Bauzá, Pablo Blanco Acevedo y Juan Pivel Devoto- e identificar el rol atribuido a la misma en el marco general del discurso nacionalista.

The future obligations of "project manager" as construction integral director (DIPE) in the law of buildings construction in spain (LOE)

In the year 1999 approves the Law of Construction Building (LOE, in Spanish) to regulate a sector such as construction, which contained some shortcomings from the legal point of view. Currently, the LOE has been in force 12 years, changing the spanish world of the construction, due to influenced by internationalization. Within the LOE, there regulating the different actors involved in the construction building, as the Projects design, the Director of Construction, the developer, The builder, Director of execution of the construction (actor only in Spain, similar as construcion engineer and abroad in), control entities and the users, but lacks figure Project manager will assume the delegation of the promoter helping and you organize, direct and management the process. This figure assumes that the market and contracts are not legally regulated in Spain, then should define and establish its regulation in the LOE. (Spain Construction Law) The translation in spanish of the words "Project Manager is owed to Professor Rafael de Heredia in his book Integrated Project Management, as agent acting on behalf of the organization and promoter assuming control of the project, ie Integraded Project Management . Already exist in Spain, AEDIP (Spanish Association Integrated of Project Construction management) which comprises the major companies in “Project Management” in Spain, and MeDIP (Master in Integrated Construction Project) the largest and most advanced studies at the Polytechnic University of Madrid, in "Construction Project Management" they teach which is also in Argentina. The Integrated Project ("Project Management") applied to the construction process is a methodological technique that helps to organize, control and manage the resources of the promoters in the building process. When resources are limited (which is usually most situations) to manage them efficiently becomes very important. Well, we find that in this situation, the resources are not only limited, but it is limited, so a comprehensive control and monitoring of them becomes not only important if not crucial. The alternative of starting from scratch with a team that specializes in developing these follow directly intervening to ensure that scarce resources are used in the best possible way requires the use of a specific methodology (Manual DIP, Matrix Foreign EDR breakdown structure EDP Project, Risk Management and Control, Design Management, et ..), that is the methodology used by "Projects managers" to ensure that the initial objectives of the promoters or investors are met and all actors in process, from design to construction company have the mind aim of the project will do, trying to get their interests do not prevail over the interests of the project. Among the agents listed in the building process, "Project Management" or DIPE (Director Comprehensive building process, a proposed name for possible incorporation into the LOE, ) currently not listed as such in the LOE (Act on Construction Planning ), one of the agents that exist within the building process is not regulated from the legal point of view, no obligations, ie, as is required by law to have a project, a builder, a construction management, etc. DIPE only one who wants to hire you as have been advanced knowledge of their services by the clients they have been hiring these agents, there being no legal obligation as mentioned above, then the market is dictating its ruling on this new figure, as if it were necessary, he was not hired and eventually disappeared from the building process. As the aim of this article is regular the process and implement the name of DIPE in the Spanish Law of buildings construction (LOE)

Microarray analysis of autoimmune diseases by machine learning procedures

—Microarray-based global gene expression profiling, with the use of sophisticated statistical algorithms is providing new insights into the pathogenesis of autoimmune diseases. We have applied a novel statistical technique for gene selection based on machine learning approaches to analyze microarray expression data gathered from patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS), two autoimmune diseases of unknown genetic origin that share many common features. The methodology included a combination of three data discretization policies, a consensus gene selection method, and a multivariate correlation measurement. A set of 150 genes was found to discriminate SLE and PAPS patients from healthy individuals. Statistical validations demonstrate the relevance of this gene set from an univariate and multivariate perspective. Moreover, functional characterization of these genes identified an interferon-regulated gene signature, consistent with previous reports. It also revealed the existence of other regulatory pathways, including those regulated by PTEN, TNF, and BCL-2, which are altered in SLE and PAPS. Remarkably, a significant number of these genes carry E2F binding motifs in their promoters, projecting a role for E2F in the regulation of autoimmunity.