Modifications of retinal neurons in a mouse model of retinitis pigmentosa

Animal models of retinitis pigmentosa include the rd mouse, in which a mutation of a rod-specific phosphodiesterase leads to the rapid loss of photoreceptors during the early postnatal life. Very little is known about changes occurring in inner retinal neurons after photoreceptor loss. These changes are important in view of the possibility of restoring vision in retinas with photoreceptor degeneration by means of cell transplantation or direct stimulation of inner layers. In this paper, we show that bipolar and horizontal cells of the rd mouse retina undergo dramatic morphological modifications accompanying photoreceptor loss, demonstrating a dependence of second order neurons on these cells. While describing modifications of the rd retina, we also provide quantitative information about neurons of the wild-type mouse retina, useful for future studies on genetically altered animals.

The Mouse Gene Expression Database (GXD)

The Gene Expression Database (GXD) is a community resource of gene expression information for the laboratory mouse. By combining the different types of expression data, GXD aims to provide increasingly complete information about the expression profiles of genes in different mouse strains and mutants, thus enabling valuable insights into the molecular networks that underlie normal development and disease. GXD is integrated with the Mouse Genome Database (MGD). Extensive interconnections with sequence databases and with databases from other species, and the development and use of shared controlled vocabularies extend GXD’s utility for the analysis of gene expression information. GXD is accessible through the Mouse Genome Informatics web site at http://www.informatic s.jax.org/ or directly at http://www.informatics.jax.org/me nus/expression_menu.shtml.

Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange– Nielsen Syndrome

KCNQ1 encodes KCNQ1, which belongs to a family of voltage-dependent K+ ion channel proteins. KCNQ1 associates with a regulatory subunit, KCNE1, to produce the cardiac repolarizing current, IKs. Loss-of-function mutations in the human KCNQ1 gene have been linked to Jervell and Lange–Nielsen Syndrome (JLNS), a disorder characterized by profound bilateral deafness and a cardiac phenotype. To generate a mouse model for JLNS, we created a line of transgenic mice that have a targeted disruption in the Kcnq1 gene. Behavioral analysis revealed that the Kcnq1−/− mice are deaf and exhibit a shaker/waltzer phenotype. Histological analysis of the inner ear structures of Kcnq1−/− mice revealed gross morphological anomalies because of the drastic reduction in the volume of endolymph. ECGs recorded from Kcnq1−/− mice demonstrated abnormal T- and P-wave morphologies and prolongation of the QT and JT intervals when measured in vivo, but not in isolated hearts. These changes are indicative of cardiac repolarization defects that appear to be induced by extracardiac signals. Together, these data suggest that Kcnq1−/− mice are a potentially valuable animal model of JLNS.

The role of antimicrobial peptides in animal defenses

It is becoming clear that the cationic antimicrobial peptides are an important component of the innate defenses of all species of life. Such peptides can be constitutively expressed or induced by bacteria or their products. The best peptides have good activities vs. a broad range of bacterial strains, including antibiotic-resistant isolates. They kill very rapidly, do not easily select resistant mutants, are synergistic with conventional antibiotics, other peptides, and lysozyme, and are able to kill bacteria in animal models. It is known that bacterial infections, especially when treated with antibiotics, can lead to the release of bacterial products such as lipopolysaccharide (LPS) and lipoteichoic acid, resulting in potentially lethal sepsis. In contrast to antibiotics, the peptides actually prevent cytokine induction by bacterial products in tissue culture and human blood, and they block the onset of sepsis in mouse models of endotoxemia. Consistent with this, transcriptional gene array experiments using a macrophage cell line demonstrated that a model peptide, CEMA, blocks the expression of many genes whose transcription was induced by LPS. The peptides do this in part by blocking LPS interaction with the serum protein LBP. In addition, CEMA itself has a direct effect on macrophage gene expression. Because cationic antimicrobial peptides are induced by LPS and are able to dampen the septic response of animal cells to LPS, we propose that, in addition to their role in direct and lysozyme-assisted killing of microbes, they have a role in feedback regulation of cytokine responses. We are currently developing variant peptides as therapeutics against antibiotic-resistant infections.

Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15.

Usher syndrome is a group of diseases with autosomal recessive inheritance, congenital hearing loss, and the development of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and visual field loss over several decades. The causes of Usher syndrome are unknown and no animal models have been available for study. Four human gene sites have been reported, suggesting at least four separate forms of Usher syndrome. We report a mouse model of type I Usher syndrome, rd5, whose linkage on mouse chromosome 7 to Hbb and tub has homology to human Usher I reported on human chromosome 11p15. The electroretinogram in homozygous rd5/rd5 mouse is never normal with reduced amplitudes that extinguish by 6 months. Auditory-evoked response testing demonstrates increased hearing thresholds more than control at 3 weeks of about 30 decibels (dB) that worsen to about 45 dB by 6 months.

Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids Lys-2-Arg-1 and removal of a Lys156-Arg157 dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

Prostate cancer in a transgenic mouse.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.

Investigação da atividade apoptótica na abertura luminal dos ductos das glândulas salivares: análise comparativa entre modelo animal e humano

As glândulas salivares são estruturas essenciais para a manutenção da homeostase da cavidade oral pela síntese e secreção do fluido salivar. A disfunção ou perda permanente das glândulas salivares causadas por radioterapia, doenças inflamatórias ou desordens congênitas elevam principalmente o risco de infecções da mucosa oral e de estruturas dentárias, além de potencialmente prejudicar funções fisiológicas como fala, mastigação e paladar, diretamente interferindo na qualidade de vida dos indivíduos afetados. Os tratamentos atualmente disponíveis são apenas paliativos, ressaltando a necessidade de se compreender melhor os processos embriogênicos a fim de desenvolver novas estratégias terapêuticas capazes de regenerar as glândulas salivares. O princípio da formação das glândulas salivares baseia-se na coordenação de diversos processos morfogenéticos, e este trabalho foca particularmente em investigar a formação do espaço luminal do sistema de ductos, uma vez que a adequada abertura dos lumens é um processo essencial para a secreção salivar. Relata-se que a remoção das células centrais dos cordões sólidos epiteliais por morte celular apoptótica é o principal mecanismo de abertura do espaço luminal dos futuros ductos glandulares em camundongos. Porém, pouco se sabe sobre o controle temporal da apoptose durante o desenvolvimento glandular e sobre seu comportamento em glândulas salivares humanas. Neste trabalho, o perfil de expressão de diversas proteínas envolvidas na cascata apoptótica em glândulas salivares fetais humanas foi analisado de acordo com cada estágio morfogenético por imunoistoquímica (Bax, Bak, Bad, Bid, Bcl-2, Bcl-x, Bcl-xL, caspase-3 clivada, caspases-6, -7 e -9, apaf-1, survivina e citocromo c). As análises semi-qualitativas resultaram em negatividade apenas para as proteínas Bcl-2, Bad, Bid e caspase-3 clivada em todas as fases de desenvolvimento. A expressão nuclear de Bax e Bak foi identificada em presumidos espaços luminais em estágios precoces, enquanto Bcl-xL foi o fator antiapoptótico da família Bcl-2 que exibiu expressão nuclear mais importante. Caspases-6, -7 e -9 foram positivas em todas as fases, e a ausência de caspase-3 clivada sugere caspase-7 como principal caspase efetora da apoptose em desenvolvimento de glândulas salivares humanas. Ambos os componentes do complexo apoptossomo foram positivos durante o desenvolvimento glandular, e o inibidor survivina demonstrou mais positividade nuclear em estágios mais avançados. Ao observar a expressão de reguladores apoptóticos durante o desenvolvimento glandular humano, foram realizados experimentos funcionais com culturas de tecido glandular de camundongos para avaliar o papel das caspases durante a formação desta estrutura. Inicialmente detectou-se a atividade apoptótica em glândulas salivares de camundongos albinos no centro dos cordões epiteliais primários a partir de estágios precoces de desenvolvimento através de TUNEL e caspase-3 clivada. A partir disso, foi realizada a inibição apoptótica funcional in vitro durante o mesmo período, que resultou em ductos significativamente mais amplos e em defeitos morfológicos importantes nas estruturas luminal e acinar. Este trabalho evidenciou portanto atividade apoptótica durante a formação de glândulas salivares humanas e de camundongo, expressando-se em fases mais precoces do que reportadas anteriormente. Além disso, a ausência de Bad e Bid indica que a via intrínseca está mais ativa que a extrínseca, e distintos perfis de expressão da maioria das moléculas sugere adicionais funções não-apoptóticas durante a morfogênese glandular.

Développement d’un nouveau modèle murin expérimental de sclérodermie

La sclérodermie (SSc) est une maladie rare affectant les personnes génétiquement prédisposées d’une réponse immunitaire défectueuse. Malgré les derniers avancements et développements dans le domaine, l’étiologie et la pathogénèse de la maladie demeurent peu comprises. Par ailleurs, il y a un ralentissement dans la compréhension de cette maladie à cause du manque de modèle animal représentatif de la SSc humaine. Malgré plusieurs lacunes, les souris traitées avec la bléomycine ou portant des modifications génétiques (TSK-1) sont très utilisées dans les études précliniques de la SSc mais elles ne présentent pas toutes les caractéristiques de cette maladie. Pour contribuer à la recherche sur la SSc, la stagiaire postdoctorale Dre Heena Mehta a développé dans le laboratoire du Dre Sarfati en collaboration avec le Dr Senécal, un modèle de souris expérimental induit par l’immunisation de cellules dendritiques (DCs) chargées de peptides de la protéine topoisomérase I (TOPOIA et TOPOIB). Dans le but de caractériser ce modèle murin et d’établir un profil immunitaire, j’ai concentré mes analyses principalement sur les caractéristiques de la SSc telles que la fibrose, l’inflammation, l’hyper-γ-globulinémie polyclonale, la vasculopathie ainsi que de l’expression de cytokines. Brièvement, l’immunisation de souris avec les DCs chargées avec la topoisomérase I (TOPOI) a induit l’inflammation pulmonaire et cutanée, en plus de la fibrose sous forme diffuse (dcSSc). Les souris présentaient également des symptômes de la vasculopathie ainsi que des taux élevés d’anticorps polyclonaux. Les résultats démontraient que les peptides TOPOIA étaient efficaces dans l’induction de la fibrose et de la réponse inflammatoire alors que les peptides TOPOIB étaient surtout impliqués dans la fibrose cutanée. En plus de nos résultats, les observations préliminaires sur le profil de cytokines tissulaires suggéraient que ce modèle pourrait remplacer ou complémenter les autres modèles animaux de SSc.

Effects of exercise training on intrauterine growth restriction in an animal model

Résumé Selon l'OMS, la retard de croissance intra-utérine (RCIU; 10% en dessous du poids normal pendant la grossesse) affecte 5-10% des grossesses et est une cause principale de la morbidité et de la mortalité périnatales. Dans notre étude précédente sur un modèle de souris transgénique de prééclampsie (R+A+), nous avons constaté que l’entraînement physique (ExT) avant et pendant la grossesse réduisait la pression artérielle maternelle et empêchait la RCIU en améliorant le développement placentaire. Dans le cadre de mon projet, nous avons confirmé les bénifices de l’ExT dans un modèle de RCIU (souris déficiente en p57Kip2 (p57-/+). Ainsi, nous avons observé la présence de RCIU, d’une masse placentaire réduite, d’une augmentation de la pathologie placentaire ainsi qu’une plus petite taille des portées chez les souris p57-/+ sédentaire. L’ExT prévient la RCIU ainsi que tous les paramètres mentionnés ci-haut. Nous avons observé que l'expression du facteur de croissance de l’endothélium vasculaire, un régulateur clé de l'angiogenèse lors de la croissance placentaire, était réduite dans le placenta des souris p57-/+ et normalisée par l’ExT. Nous avons également trouvé que l'expression en ARN dans le placenta de 2 facteurs inflammatoires (interleukine-1β et MCP-1) était augmenté chez les souris sédentaires p57-/+ alors que ceci n’était pas présent chez les souris entraînées, ce qui suggère que l'inflammation placentaire peut contribuer à la pathologie placentaire. Toutefois, contrairement aux souris R+A+, le système rénine-angiotensine placentaire chez les souris p57-/+ était normale et aucun effet de l’ExT a été observé. Ces résultats suggèrent que l’ExT prévient la RCIU en normalisant la pathologie placentaire, l’angiogenèse et l’inflammation placentaire.

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Mode of access: Internet.

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Mode of access: Internet.

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