Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT1) and CysLT2 receptors, recently have been characterized and cloned. Because the CysLT1 receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT1 receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT1 receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT1 receptor mRNA is expressed in lung and skin; and reverse transcription–PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT1 receptor was mapped to band XD. Leukotriene (LT) D4 induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT1 receptor cDNA. This agonist effect of LTD4 was fully inhibited by the CysLT1 receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [3H]LTD4; and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD4 >> LTE4 = LTC4 >> LTB4. Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice

The cyclooxygenase (COX) product, prostacyclin (PGI2), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI2 biosynthesis substantially in humans. Because deletion of the PGI2 receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF1α by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI2 biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 ± 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

Remodeling of Yeast Genome Expression in Response to Environmental Changes

We used genome-wide expression analysis to explore how gene expression in Saccharomyces cerevisiae is remodeled in response to various changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity. The results demonstrate that more than half of the genome is involved in various responses to environmental change and identify the global set of genes induced and repressed by each condition. These data implicate a substantial number of previously uncharacterized genes in these responses and reveal a signature common to environmental responses that involves ∼10% of yeast genes. The results of expression analysis with MSN2/MSN4 mutants support the model that the Msn2/Msn4 activators induce the common response to environmental change. These results provide a global description of the transcriptional response to environmental change and extend our understanding of the role of activators in effecting this response.

Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene

We have shown that the DNA demethylation complex isolated from chicken embryos has a G⋅T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a β-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor α. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.

Molecular mechanisms of cocaine reward: Combined dopamine and serotonin transporter knockouts eliminate cocaine place preference

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET). Cocaine reward/reinforcement has been linked to actions at DAT or to blockade of SERT. However, knockouts of neither DAT, SERT, or NET reduce cocaine reward/reinforcement, leaving substantial uncertainty about cocaine's molecular mechanisms for reward. Conceivably, the molecular bases of cocaine reward might display sufficient redundancy that either DAT or SERT might be able to mediate cocaine reward in the other's absence. To test this hypothesis, we examined double knockout mice with deletions of one or both copies of both the DAT and SERT genes. These mice display viability, weight gain, histologic features, neurochemical parameters, and baseline behavioral features that allow tests of cocaine influences. Mice with even a single wild-type DAT gene copy and no SERT copies retain cocaine reward/reinforcement, as measured by conditioned place-preference testing. However, mice with no DAT and either no or one SERT gene copy display no preference for places where they have previously received cocaine. The serotonin dependence of cocaine reward in DAT knockout mice is thus confirmed by the elimination of cocaine place preference in DAT/SERT double knockout mice. These results provide insights into the brain molecular targets necessary for cocaine reward in knockout mice that develop in their absence and suggest novel strategies for anticocaine medication development.

The copper transporter CTR1 provides an essential function in mammalian embryonic development

Copper serves as an essential cofactor for a variety of proteins in all living organisms. Previously, we described a human gene (CTR1;SLC31A1) that encodes a high-affinity copper-uptake protein and hypothesized that this protein is required for copper delivery to mammalian cells. Here, we test this hypothesis by inactivating the Ctr1 gene in mice by targeted mutagenesis. We observe early embryonic lethality in homozygous mutant embryos and a deficiency in copper uptake in the brains of heterozygous animals. Ctr1−/− embryos can be recovered at E8.5 but are severely developmentally retarded and morphologically abnormal. Histological analysis reveals discontinuities and variable thickness in the basement membrane of the embryonic region and an imperfect Reichert's membrane, features that are likely due to lack of activity in the collagen cross-linking cupro-enzyme lysyl oxidase. A collapsed embryonic cavity, the absence of an allantois, retarded mesodermal migration, and increased cell death are also apparent. In the brains of heterozygous adult mice, which at 16 months are phenotypically normal, copper is reduced to approximately half compared with control littermates, implicating CTR1 as the required port for copper entry into at least this organ. A study of the spatial and temporal expression pattern of Ctr1 during mouse development and adulthood further shows that CTR1 is ubiquitously transcribed with highest expression observed in the specialized epithelia of the choroid plexus and renal tubules and in connective tissues of the eye, ovary, and testes. We conclude that CTR1 is the primary avenue for copper uptake in mammalian cells.

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit1

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

Integrando o conhecimento de usuários: um estudo sobre a capacidade de absorção de conhecimento de lead users

Os custos elevados de aquisição de conhecimento, a intensificação da concorrência e a necessidade de aproximação do consumidor vêm estimulando empresas a buscar formas alternativas de aumentar seu potencial de inovação pela integração de usuários. No entanto, a literatura e o senso comum convergem ao afirmar que nem todo usuário está habilitado a trazer conhecimentos que sustentem a vantagem competitiva das inovações. Nesse contexto, emerge a figura do lead user que, por definição, é capaz de sentir necessidades de produtos e serviços ainda não expressos por usuários regulares. Esses conhecimentos, quando adequadamente absorvidos, trazem benefícios expressivos às empresas que os incorporam ao DNP. Sabendo que as formas de incorporação de usuários apresentam variações, este estudo se destina a entender como empresas de diferentes setores absorvem conhecimentos de lead users por diferentes práticas de integração. Para tanto, foi escolhido o método de estudo de casos múltiplos incorporados, observados em três multinacionais de grande porte: Natura, Whirlpool e Microsoft (Bing). Ao todo foram avalidados cinco modos de integração distintos, escolhidos a partir de duas formações: individual (conhecimentos isolados de usuários distintos) e coletivo (conhecimentos articulados em discussões em grupo), analisados pelos métodos de indução analítica com síntese cruzada de dados. Os resultados mostraram que as categorias teóricas utilizadas para observação inicial do fenômenol: parâmetro de identificação e técnica de seleção (aquisição); mecanismo de interação (assimilação); mecanismos de socialização (transformação) e sistema de formalização (exploração) apoiaram parcialmente o entendimento das atividades do processo e, por esta razão, precisaram se complementadas pelas categorias emergentes: criação de contexto, motivação (aquisição); estímulos, parâmetro de observação, interpretação (assimilação); definição de papéis, coordenação de processos, combinação de conhecimento (transformação) e gestão do conhecimento (exploração), coletadas na fase empírica Essa complementação aumentou a robustez do modelo inicial e mostrou como a absorção de conhecimentos pode ser avaliada pelas dimensões absortivas. No entanto, as análises intra e intercasos que se seguiram, mostraram que esse entendimento era insuficiente para explicar a capacidade de absorção por diferentes práticas, uma vez que o fenômeno é influenciado por fatores contextuais associados tanto à prática de integração quanto ao modo como cada empresa se organiza para inovar (tipo de acesso ao colaborador). As reflexões teóricas realizadas a partir desses resultados permitiram contribuir com a teoria existente de duas formas: I) pelo entendimento estendido das atividades de absorção necessárias para incorporação de conhecimentos de lead users e III) pela proposição de um modelo conceitual amplo que abarcou diferentes práticas de integração considerando também os antecedentes de inovação, as atividades absortivas e os fatores adjuntos, inerentes a cada prática. Esta pesquisa objetiva contribuir para o conhecimento teórico sobre inovação e motivar reflexões que possam ser úteis para gerentes e executivos interessados em aprimorar suas práticas e processos de captação de conhecimentos de lead users.

Transition bei chronischen Erkrankungen der Kindheit und Jugend

Gesundheitspersonen kommt eine wichtige Aufgabe in der Betreuung von Jugendlichen mit einer chronischen Erkrankung zu. Adoleszenz ist per se keine einfache Zeit, so sind Jugendliche mit einer chronischen Erkrankung zusätzlich damit konfrontiert, Verantwortung für sich und ihre Erkrankung zu übernehmen. Gerade der Transfer von der Kinder- in die Erwachsenenmedizin kann dazu führen, dass Jugendliche die Behandlung abbrechen und es vermehrt zu Notfallsituationen kommt. Somit gehört es zur Verantwortung der betreuenden Kindermediziner, frühzeitig sicherzustellen, dass die Selbstmanagementkompetenzen dieser Jugendlichen gefördert werden und eine adäquate Weiterbetreuung organisiert ist. Dem übernehmenden Erwachsenenteam obliegt es, die Familie willkommen zu heißen und jugendspezifische Themen gemeinsam weiter anzugehen. Diese Themen regelmäßig anzusprechen, sich genügend Zeit für den Jugendlichen zu nehmen und Gespräche unter vier Augen anzubieten, hat sich für eine erfolgreiche Transition als hilfreich erwiesen.

Jie qi ting ji /

Library has: v.1-12.

Collision avoidance and accident survivability. Volume 4: Proposed specification. Final report.

Transportation Systems Center, Cambridge, Mass.

Collision avoidance and accident survivability. Volume 1: collision threat. Final report.

Transportation Systems Center, Cambridge, Mass.

Der deutsche nationalitätskampf in seiner beziehung zu anderen nationalitäten,

Mode of access: Internet.

Le développement de l'idée religieuse dans le judaïsme, le christianisme et l'islamisme /

Antep.

Li Wenzhong gong chi du /

Facsimile ed.