Vivo Approaches (vivo + approach)

Distribution by Scientific Domains


Selected Abstracts


Neuropeptide Y suppresses absence seizures in a genetic rat model primarily through effects on Y2 receptors

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2007
Margaret J. Morris
Abstract Neuropeptide Y (NPY) potently suppresses absence seizures in a model of genetic generalized epilepsy, genetic absence epilepsy rats of Strasbourg (GAERS). Here we investigated the Y-receptor subtype(s) on which NPY exerts this anti-absence effect. A dual in vivo approach was used: the cumulative duration of seizures was quantified in adult male GAERS in 90-min electroencephalogram recordings following intracerebroventricular (i.c.v.) injection of: (i) subtype-selective agonists of Y1 ([Leu31Pro34]NPY, 2.5 nmol), Y2 (Ac[Leu28,31]NPY24,36, 3 nmol), Y5 receptors [hPP1,17,Ala31,Aib32]NPY, 4 nmol), NPY (3 nmol) or vehicle; and following (ii) i.c.v. injection of antagonists of Y1 (BIBP3226, 20 nmol), Y2 (BIIE0246, 20 nmol) and Y5 (NPY5RA972, 20 nmol) receptors or vehicle, followed by NPY (3 nmol). Injection of the Y1 - and Y5 -selective agonists resulted in significantly less mean seizure suppression (37.4% and 53.9%, respectively) than NPY (83.2%; P < 0.05), while the Y2 agonist had similar effects to NPY (62.3% suppression, P = 0.57). Food intake was not increased following injection of the Y2 agonist, while significant increases in food intake were seen following NPY and the other Y-subtype agonists. Compared with vehicle, NPY injection suppressed seizures following the Y1 and Y5 antagonists (45.3% and 80.1%, respectively, P < 0.05), but not following the Y2 antagonist (5.1% suppression, P = 0.46). We conclude that NPY Y2 receptors are more important than Y1 and Y5 receptors in mediating the effect of NPY to suppress absence seizures in a genetic rat model. Y2 receptor agonists may represent targets for novel drugs against genetic generalized epilepsies without resulting in appetite stimulation. [source]


The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex

THE PLANT JOURNAL, Issue 2 2006
Chris A. Helliwell
Summary The Arabidopsis Flowering Locus C (FLC) protein is a repressor of flowering regulated by genes in the autonomous and vernalization pathways. Previous genetic and transgenic data have suggested that FLC acts by repressing expression of the floral integrator genes SOC1 and FT. We have taken an in vivo approach to determine whether the FLC protein interacts directly with potential DNA targets. Using chromatin immunoprecipitation, we have shown that FLC binds to a region of the first intron of FT that contains a putative CArG box, and have confirmed that FLC binds to a CArG box in the promoter of the SOC1 gene. MADS box proteins are thought to bind their DNA targets as dimers or higher-order multimers. We have shown that FLC is a component of a multimeric protein complex in vivo and that more than one FLC polypeptides can be present in the complex. [source]


Chick limbs with mouse teeth: An effective in vivo culture system for tooth germ development and analysis

DEVELOPMENTAL DYNAMICS, Issue 1 2003
Eiki Koyama
Abstract Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4,5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously. © 2002 Wiley-Liss, Inc. [source]


Host immune responses in ex vivo approaches to cutaneous gene therapy targeted to keratinocytes

EXPERIMENTAL DERMATOLOGY, Issue 10 2005
Z. Lu
Abstract:, Epidermal gene therapy may benefit a variety of inherited skin disorders and certain systemic diseases. Both in vivo and ex vivo approaches of gene transfer have been used to target human epidermal stem cells and achieve long-term transgene expression in immunodeficient mouse/human chimera models. Immunological responses however, especially in situations where a neoantigen is expressed, are likely to curtail expression and thereby limit the therapy. In vivo gene transfer to skin has been shown to induce transgene-specific immune responses. Ex vivo gene transfer approaches, where keratinocytes are transduced in culture and transplanted back to patient, however, may avoid signals provided to the immune system by in vivo administration of vectors. In the current study, we have developed a stable epidermal graft platform in immunocompetent mice to analyze host responses in ex vivo epidermal gene therapy. Using green fluorescent protein (GFP) as a neoantigen and an ex vivo retrovirus-mediated gene transfer to mouse primary epidermal cultures depleted of antigen-presenting cells (APCs), we show induction of GFP-specific immune responses leading to the clearance of transduced cells. Similar approach in immunocompetent mice tolerant to GFP resulted in permanent engraftment of transduced cells and continued GFP expression. Activation of transgene-specific immune responses in ex vivo gene transfer targeted to keratinocytes require cross-presentation of transgene product to APCs, a process that is most amenable to immune modulation. This model may be used to explore strategies to divert transgene-specific immune responses to less destructive or tolerogenic ones. [source]


FGF and FGFR signaling in chondrodysplasias and craniosynostosis

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2005
P.J. Marie
Abstract The first experimental mouse model for FGF2 in bone dysplasia was made serendipitously by overexpression of FGF from a constitutive promoter. The results were not widely accepted, rightfully drew skepticism, and were difficult to publish; because of over 2,000 studies published on FGF-2 at the time (1993), only a few reported a role of FGF-2 in bone growth and differentiation. However, mapping of human dwarfisms to mutations of the FGFRs shortly, thereafter, made the case that bone growth and remodeling was a major physiological function for FGF. Subsequent production of numerous transgenic and targeted null mice for several genes in the bone growth and remodeling pathways have marvelously elucidated the role of FGFs and their interactions with other genes. Indeed, studies of the FGF pathway present one of the best success stories for use of experimental genetics in functionally parsing morphogenetic regulatory pathways. What remains largely unresolved is the pleiotropic nature of FGF-2. How does it accelerate growth in one cell then stimulate apoptosis or retard growth for another cell in the same type of tissue? Some of the answers may come through distinguishing the FGF-2 protein isoforms, made from alternative translation start sites, these appear to have substantially different functions. Although we have made substantial progress, there is still much to be learned regarding FGF-2 as a most complex, enigmatic protein. Studies of genetic models in mice and human FGFR mutations have provided strong evidence that FGFRs are important modulators of osteoblast function during membranous bone formation. However, there is some controversy regarding the effects of FGFR signaling in human and murine genetic models. Although significant progress has been made in our understanding of FGFR signaling, several questions remain concerning the signaling pathways involved in osteoblast regulation by activated FGFR. Additionally, little is known about the specific role of FGFR target genes involved in cranial bone formation. These issues need to be addressed in future in in vitro and in vivo approaches to better understand the molecular mechanisms of action of FGFR signaling in osteoblasts that result in anabolic effects in bone formation. J. Cell. Biochem. © 2005 Wiley-Liss, Inc. [source]


Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradation

JOURNAL OF NEUROCHEMISTRY, Issue 1 2005
Jeganathan Ramesh Babu
Abstract Inclusions isolated from several neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by ubiquitin-positive proteinaceous aggregates. Employing confocal and immunoelectron microscopy, we find that the ubiquitin-associating protein sequestosome1/p62, co-localizes to aggregates isolated from AD but not control brain, along with the E3 ubiquitin ligase, TRAF6. This interaction could be recapitulated by co-transfection in HEK293 cells. Employing both in vitro and in vivo approaches, tau was found to be a substrate of the TRAF6, possessing lysine 63 polyubiquitin chains. Moreover, tau recovered from brain of TRAF6 knockout mice, compared with wild type, was not ubiquitinated. Tau degradation took place through the ubiquitin,proteasome pathway and was dependent upon either the K63-polyubiquitin chains or upon p62. In brain lysates of p62 knockout mice, tau fails to co-interact with Rpt1, a proteasomal subunit, thereby indicating a requirement for p62 shuttling of tau to the proteasome. Our results demonstrate that p62 interacts with K63-polyubiquitinated tau through its UBA domain and serves a novel role in regulating tau proteasomal degradation. We propose a model whereby either a decline in p62 expression or a decrease in proteasome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau. [source]


Methodology for the study of the hypothalamic-pituitary hormone secretion in cattle

ANIMAL SCIENCE JOURNAL, Issue 1 2009
Tsutomu HASHIZUME
ABSTRACT Studies on the neuroregulatory mechanisms on the secretion of anterior pituitary (AP) hormones in domestic animals are important because nearly all complex physiological and metabolic processes are regulated by the AP hormones. To examine them, this article considers in vivo approaches such as the techniques of intrahypothalamic injection, intracerebroventricular injection, push-pull perfusion, and microdialysis, which have been employed in our own research group for the study in cattle. Also, in vitro approaches such as bovine AP cell culture and the AP explants superfusion system are described. This article clarifies the potential of neuroendocrine study techniques in cattle. [source]


2264: Expression and role of aquaporins in diabetic retinopathy

ACTA OPHTHALMOLOGICA, Issue 2010
E MOTULSKY
Purpose Aquaporins (AQP) are involved in water movements but also in cell proliferation. The aim of our study is to investigate their potential role in diabetic retinopathy Methods In vitro, human and mouse RPE cell line will be grown under different conditions. Expression of AQPs will be investigated by RT-PCR and Western blot. Immunofluorescence will be used to determine AQP expression on retinal section from normal and NOD diabetic mice, and on on vitreoretinal membranes removed from patients with proliferative retinopathy Results Human ARPE-19 cells expressed AQP4, while mouse B6-RPE07 cells expressed AQP1. Hypertonic conditions strongly decrease AQP4 expression in ARPE-19, but not AQP1 expression in B6-RPE07 cells. In B6-RPE07 cells, significantly decrease in AQP1 expression was obtained following TNF, treatment. In normal mice, AQP1 and AQP4 expression were restricted to the photoreceptor layer and to the Müller cells, respectively. Retinal endothelial cells did not express AQP1. No AQP4 expression was detected in RPE cells. AQP1 was strongly expressed by choroidal endothelial cells, rendering difficult the evaluation of AQP1 expression by RPE cells in vivo Conclusion Our preliminary data confirms that blood retinal barrier cells express AQPs in vitro and in vivo. More studies are needed to precise the nature and regulation of this expression in normal and diabetic conditions. We hope that our combined in vitro and in vivo approaches might help to better understand this complex aspect of retinal biology [source]