Screening for imprinted genes by allelic message display: Identification of a paternally expressed gene Impact on mouse chromosome 18

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation

The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism.

A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas

Molting or ecdysis is the most fundamentally important process in arthropod life history, because shedding of the exoskeleton is an absolute prerequisite for growth and metamorphosis. Although the hormonal mechanisms driving ecdysis in insects have been studied extensively, nothing is known about these processes in crustaceans. During late premolt and during ecdysis in the crab Carcinus maenas, we observed a precise and reproducible surge in hemolymph hyperglycemic hormone (CHH) levels, which was over 100-fold greater than levels seen in intermolt animals. The source of this hormone surge was not from the eyestalk neurosecretory tissues but from previously undescribed endocrine cells (paraneurons), in defined areas of the foregut and hindgut. During premolt (the only time when CHH is expressed by these tissues), the gut is the largest endocrine tissue in the crab. The CHH surge, which is a result of an unusual, almost complete discharge of the contents of the gut endocrine cell, regulates water and ion uptake during molting, thus allowing the swelling necessary for successful ecdysis and the subsequent increase in size during postmolt. This study defines an endocrine brain/gut axis in the arthropods. We propose that the ionoregulatory process controlled by CHH may be common to arthropods, in that, for insects, a similar mechanism seems to be involved in antidiuresis. It also seems likely that a cascade of very precisely coordinated release of (neuro) hormones controls ecdysis.

ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.

Establishment and persistence of photoperiodic memory in hamsters

Long summer days unequivocally stimulate, and short winter days inhibit reproduction in Siberian hamsters. By contrast, intermediate-duration day lengths (12.5–14 h long) either accelerate reproductive development or initiate regression of the reproductive apparatus. Which of these outcomes transpires depends on an animal's photoperiodic history, suggesting that hamsters must encode a representation of prior photoperiods. The duration of nocturnal melatonin secretion is the endocrine representation of day length, but nothing is known about how long it takes to establish photoperiodic histories or how long they endure. Hamsters exposed for 2 or more weeks to long summer day lengths acquired a long-day photoperiodic history that determined subsequent reproductive responses to intermediate-duration day lengths and melatonin signals. The memory for long-day lengths persisted in pinealectomized hamsters for 6.5 weeks, faded significantly after 13 weeks, and was functionally absent after 20 weeks. These findings indicate that hamsters are influenced only by relatively recent day lengths and melatonin signals and ignore earlier ones that might cause them to misinterpret the salience of current day lengths.

The mechanical properties of Saccharomyces cerevisiae

Cell-wall mechanical properties play an integral part in the growth and form of Saccharomyces cerevisiae. In contrast to the tremendous knowledge on the genetics of S. cerevisiae, almost nothing is known about its mechanical properties. We have developed a micromanipulation technique to measure the force required to burst single cells and have recently established a mathematical model to extract the mechanical properties of the cell wall from such data. Here we determine the average surface modulus of the S. cerevisiae cell wall to be 11.1 ± 0.6 N/m and 12.9 ± 0.7 N/m in exponential and stationary phases, respectively, giving corresponding Young's moduli of 112 ± 6 MPa and 107 ± 6 MPa. This result demonstrates that yeast cell populations strengthen as they enter stationary phase by increasing wall thickness and hence the surface modulus, without altering the average elastic properties of the cell-wall material. We also determined the average breaking strain of the cell wall to be 82% ± 3% in exponential phase and 80% ± 3% in stationary phase. This finding provides a failure criterion that can be used to predict when applied stresses (e.g., because of fluid flow) will lead to wall rupture. This work analyzes yeast compression experiments in different growth phases by using engineering methodology.

Characterization and cell cycle regulation of the related human telomeric proteins Pin2 and TRF1 suggest a role in mitosis

Telomeres are essential for preserving chromosome integrity during the cell cycle and have been specifically implicated in mitotic progression, but little is known about the signaling molecule(s) involved. The human telomeric repeat binding factor protein (TRF1) is shown to be important in regulating telomere length. However, nothing is known about its function and regulation during the cell cycle. The sequence of PIN2, one of three human genes (PIN1-3) we previously cloned whose products interact with the Aspergillus NIMA cell cycle regulatory protein kinase, reveals that it encodes a protein that is identical in sequence to TRF1 apart from an internal deletion of 20 amino acids; Pin2 and TRF1 may be derived from the same gene, PIN2/TRF1. However, in the cell Pin2 was found to be the major expressed product and to form homo- and heterodimers with TRF1; both dimers were localized at telomeres. Pin2 directly bound the human telomeric repeat DNA in vitro, and was localized to all telomeres uniformly in telomerase-positive cells. In contrast, in several cell lines that contain barely detectable telomerase activity, Pin2 was highly concentrated at only a few telomeres. Interestingly, the protein level of Pin2 was highly regulated during the cell cycle, being strikingly increased in G2+M and decreased in G1 cells. Moreover, overexpression of Pin2 resulted in an accumulation of HeLa cells in G2+M. These results indicate that Pin2 is the major human telomeric protein and is highly regulated during the cell cycle, with a possible role in mitosis. The results also suggest that Pin2/TRF1 may connect mitotic control to the telomere regulatory machinery whose deregulation has been implicated in cancer and aging.

Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.

Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei.

Macronuclei of the ciliated protozoan Tetrahymena thermophila possess a histone acetyltransferase activity closely associated with transcription-related histone acetylation. Nothing definitive is known concerning the polypeptide composition of this activity in Tetrahymena or any comparable activity from any cellular source. An acetyltransferase activity gel assay was developed which identifies a catalytically active subunit of this enzyme in Tetrahymena. This activity gel assay detects a single polypeptide of 55 kDa (p55) in crude macronuclear extracts, as well as in column-purified fractions, which incorporates [3H]acetate from [3H]acetyl-CoA into core histone substrates polymerized directly into SDS polyacrylamide gels. p55 copurifies precisely with acetyltransferase activity through all chromatographic steps examined, including reverse-phase HPLC. Gel-filtration chromatography of this activity indicates a molecular mass of 220 kDa, suggesting that the native enzyme may consist of four identical subunits of 55 kDa. Furthermore, p55 is tightly associated with di- and greater polynucleosomes and therefore may be defined as a component of histone acetyltransferase type A--i.e., chromatin associated.

Ensino-aprendizagem de enfermagem em simulação clínica: desenvolvendo competência profissional para prevenção de úlceras por pressão

O desenvolvimento de competências profissionais em processos de ensino-aprendizagem insere a simulação na perspectiva tecnológica realística de situações clínicas para atingir objetivos educacionais, visando segurança e qualidade. A ação competente operacionaliza saberes pela combinação de conhecimentos, habilidades e atitudes. A competência avaliação de risco para úlcera por pressão é privativa do enfermeiro, o agravo é indicador de qualidade e segurança do paciente. O objetivo do estudo foi avaliar o processo de ensino-aprendizagem ante a estratégia de simulação clínica, visando o desenvolvimento da competência profissional avaliação de risco para úlceras por pressão e seus respectivos conhecimentos, habilidades e atitudes. Fundamentou-se, teoricamente, o estudo, nos preceitos da andragogia, no modelo de Simulação da National League for Nursing/Jeffries, nas concepções de competências de Le Boterf e recomendações para prevenção de úlcera por pressão do NPUAP/ EPUAP. Trata-se de estudo descritivo com análise quanti e qualitativa, desenvolvido no Laboratório de Simulação da Universidade Federal do Piauí, em Teresina, com estudantes de enfermagem do último ano do curso. Constou de três etapas: construção e validação de conteúdo do instrumento para conhecimentos, habilidades e atitudes da competência, elaboração do plano de aula e sequência didática do cenário de simulação, aplicação de pré/pós-teste sobre o componente \"conhecimento\", aula expositiva, execução do cenário de simulação pelos estudantes, avaliação dos cenários pelo comitê de juízes e debriefing por grupo focal, após o cenário. Verificou-se confiabilidade do construto e do instrumento de medida da competência com concordância predominando de 80 a 100% de pertinência. O instrumento constou de 32 itens: conhecimento (14), habilidades (8) e atitudes (10), com escala de resposta de cinco pontos, de nada (1) a extremamente (5). Inicialmente o estudo contou com 35 estudantes. Desses, 29 (82,8%) participaram da simulação. Predominou o sexo feminino 22 (76%), faixa etária de 21 a 24 anos, 24 (82,7%) e procedentes de Universidades Públicas do Estado do Piauí (83%). Os resultados mostraram melhores níveis de combinação de saberes para o componente conhecimentos, após a aplicação das estratégias de ensino autorreferidos pelos estudantes. Na avaliação dos conhecimentos, habilidade e atitudes, durante o cenário, houve discordância entre os juízes: para o juiz 1 predominaram parâmetros negativos da escala (nada e muito pouco) e para o juiz 2 e 3 os positivos (bastante e extremamente). As comparações pelo teste de variância ANOVA mostraram diferenças significativas entre juiz 1 e 2 (p-valor = < 0,01) e juiz 1 e 3 (p-valor=<0,01) para todos os componentes. A análise dos dados obtidos, durante o debriefing, originou cinco categorias com suas respectivas unidades de registro temáticas pela análise de conteúdo de Bardin. Os resultados sugerem que a estratégia é capaz de resgatar o raciocínio operativo dos estudantes durante a ação, desenvolver pensamento crítico-reflexivo sobre a competência, identificar lacunas de aprendizagem, promover satisfação aos estudantes e melhorar a autoimagem profissional. Conclui-se que a estratégia possibilita o desenvolvimento da competência avaliação de risco para úlcera por pressão nas dimensões de saberes (conhecimento), fazeres (habilidades) e querer-agir, saber-agir e poder-agir (atitudes), explorados neste estudo.

Two Consistent Many-Valued Logics for Paraconsistent Phenomena

In this reviewing paper, we recall the main results of our papers [24, 31] where we introduced two paraconsistent semantics for Pavelka style fuzzy logic. Each logic formula a is associated with a 2 x 2 matrix called evidence matrix. The two semantics are consistent if they are seen from 'outside'; the structure of the set of the evidence matrices M is an MV-algebra and there is nothing paraconsistent there. However, seen from "inside,' that is, in the construction of a single evidence matrix paraconsistency comes in, truth and falsehood are not each others complements and there is also contradiction and lack of information (unknown) involved. Moreover, we discuss the possible applications of the two logics in real-world phenomena.

Towards the Consideration of the U.S. Community College Model to Address the Need for Higher Education Reforms in Ghana

ABSTRACT The higher education systems throughout the continent of Africa are undergoing unprecedented challenges and are considered in crisis. African countries, including Ghana, all have in common ties to their colonial legacy whereby they are confronted with weak policies put in place by their colonizers. Having gained their independence, Africans should now take responsibility for the task of reforming their higher education system. To date, nothing substantial has been accomplished, with serious implications for weakening and damaging the structures of the foundation of their educational systems. This qualitative, single case study utilized a postcolonial theory-critical pedagogy framework, providing guidance for coming to grips with the mindset posed by Ghana's colonial heritage in the postcolonial era, especially in terms of its damaging effects on Ghana's higher education system. The study explores alternative pathways for secondary school students to transition to tertiary education--a problematic transition that currently hinders open access to all and equality in educational opportunity, resulting in a tremendous pool of discontinued students. This transitional problem is directly related to Ghana's crisis in higher education with far reaching consequences. The alternative pathway considered in this study is an adaptation of the U.S. community college model or an integration of its applicable aspects into the current structures of the higher education system already in place. In-depth interviews were conducted with 5 Ghanaian professors teaching at community colleges in the United States, 5 Ghanaian professors teaching at universities in Ghana, and 2 educational consultants from the Ghanaian Ministry of Education. Based on their perspectives of the current state of Ghanaian higher education, analyzed in terms of pedagogy, structure/infrastructure, and curriculum, the participants provided their perceptions of salient aspects of the U.S. community college model that would be applicable to Ghana's situation, along with other recommendations. Access to all, including equality of educational opportunity, was considered essential, followed by adaptability, affordability, practicality, and quality of curriculum content and delivery. Canada's successful adaptation of the U.S. model was also discussed. Findings can help guide consideration of alternative pathways to higher education in Ghana and Africa as a whole.

As concepções de professores de química sobre a utilização de elementos da história e filosofia da ciência no ensino

Nas últimas décadas, investigações têm destacado o papel da história e filosofia da ciência (HFC) na educação científica. O reconhecimento da pluralidade e complexidade da ciência tem originado estudos, os quais apontam que o ensino sobre ciências deve levar em conta as diferenças entre as disciplinas científicas. Assim, tem-se discutido a importância da consideração de elementos da história e da filosofia da química no aprimoramento do processo de ensino e aprendizagem dessa ciência. Apesar disso, investigações têm constatado que pouco ou nada se discute acerca desses assuntos nas aulas de ciências. Diante disso, esta dissertação tem como intuito investigar a concepção de professores de química sobre a inclusão de elementos da HFC no ensino, com foco nas características inerentes às práticas e aos conhecimentos químicos. Foram investigadas as concepções de cinco professores de química, que atuam em diferentes escolas públicas da região metropolitana de São Paulo, acerca de cinco temas, elaborados com base em estudos da área de ensino de ciências e em diretrizes curriculares oficiais: relações com a sociedade, modelos e modelagem, experimentação, desenvolvimento histórico e reducionismo. Por meio da análise qualitativa dos dados, as concepções docentes, para cada tema, foram classificadas em dois subeixos temáticos: prática docente e relevância. Ademais, foram identificados fatores externos e internos que influenciam as práticas e concepções dos sujeitos concernentes a considerações filosóficas, históricas e sociológicas sobre a química. Os resultados evidenciam que, de modo geral, os sujeitos consideram relevantes determinados assuntos relacionados à natureza da química e, em certa medida, buscam inseri-los em suas práticas cotidianas. Contudo, mostraram também que os motivos pelos quais os docentes conferem importância a algumas discussões, e o modo como as desenvolvem, podem limitar a compreensão dos estudantes sobre a complexidade da construção do conhecimento científico. Além disso, foram raras as referências a aspectos específicos da química, à importância de evidenciar e discutir esses aspectos com os estudantes, assim como à preparação de situações didáticas próximas à prática química autêntica.

The Jewish Law Firm: Past and Present

The rise and growth of large Jewish law firms in New York City during the second half of the twentieth century was nothing short of an astounding success story. As late as 1950, there was not a single large Jewish law firm in town. By the mid-1960s, six of the largest twenty law firms were Jewish, and by 1980, four of the largest ten prestigious law firms were Jewish firms. Moreover, the accomplishment of the Jewish firms is especially striking because, while the traditional large White Anglo-Saxon Protestant law firms grew at a fast rate during this period, the Jewish firms grew twice as fast, and they did so in spite of experiencing explicit discrimination. What happened? This book chapter is a revised, updated study of the rise and growth of large New York City Jewish law firms. It is based on the public record, with respect to both the law firms themselves and trends in the legal profession generally, and on over twenty in-depth interviews with lawyers who either founded and practiced at these successful Jewish firms, attempted and failed to establish such firms, or were in a position to join these firms but decided instead to join WASP firms. According to the informants interviewed in this chapter, while Jewish law firms benefited from general decline in anti-Semitism and increased demand for corporate legal services, a unique combination of factors explains the incredible rise of the Jewish firms. First, white-shoe ethos caused large WASP firms to stay out of undignified practice areas and effectively created pockets of Jewish practice areas, where the Jewish firms encountered little competition for their services. Second, hiring and promotion discriminatory practices by the large WASP firms helped create a large pool of talented Jewish lawyers from which the Jewish firms could easily recruit. Finally, the Jewish firms benefited from a flip side of bias phenomenon, that is, they benefited from the positive consequences of stereotyping. Paradoxically, the very success of the Jewish firms is reflected in their demise by the early twenty-first century: because systematic large law firm ethno-religious discrimination against Jewish lawyers has become a thing of the past, the very reason for the existence of Jewish law firms has been nullified. As other minority groups, however, continue to struggle for equality within the senior ranks of Big Law, can the experience of the Jewish firms serve as a “separate-but-equal” blueprint for overcoming contemporary forms of discrimination for women, racial, and other minority attorneys? Perhaps not. As this chapter establishes, the success of large Jewish law firms was the result of unique conditions and circumstances between 1945 and 1980, which are unlikely to be replicated. For example, large law firms have become hyper-competitive and are not likely to allow any newcomers the benefit of protected pockets of practice. While smaller “separate-but-equal” specialized firms, for instance, ones exclusively hiring lawyer-mothers occasionally appear, the rise of large “separate-but-equal” firms is improbable.