Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.

The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.

IgE receptor-positive non-B/non-T cells dominate the production of interleukin 4 and interleukin 6 in immunized mice.

The phenotype and antigenic specificity of cells secreting interleukin (IL) 4, IL-6, and interferon gamma was studied in mice during primary and secondary immune responses. T lymphocytes were the major source of interferon gamma, whereas non-B/non-T cells were the dominant source of IL-4 and IL-6 in the spleens of immunized animals. Cytokine-secreting non-B/non-T cells expressed surface receptors for IgE and/or IgG types II/III. Exposing these cells to antigen-specific IgE or IgG in vivo (or in vitro) "armed" them to release IL-4 and IL-6 upon subsequent antigenic challenge. These findings suggest that non-B/non-T cells may represent the "natural immunity" analogue of CD4+ T helper type 2 cells and participate in a positive feedback loop involved in the perpetuation of T helper type 2 cell responses.

Patched 1 conditional null allele in mice

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function. genesis 36:158-161, 2003. (C) 2003 Wiley-Liss, Inc.

A polymorphism in the growth hormone (GH)-releasing hormone (GHRH) receptor gene is associated with elevated response to GHRH by human pituitary somatotrophinomas in vitro

A previous study has suggested that a G to A base change at position 169 of the GHRH-receptor gene in human somatotrophinomas is a mutation and confers hypersensitivity to GHRH. The alternative base converts codon 57 from GCG to AGC, resulting in replacement of alanine (Ala) with threonine (Thr). In the present study, two of five human GH-secreting somatotrophinomas were found to possess the codon 57 AGC sequence. The GCG allele was also detected, indicating heterozygosity. However, the patients' normal blood-derived DNA also yielded the same sequence pattern, indicating that the Ala=> Thr amino acid change is a normal polymorphism, and not a somatic mutation. Nevertheless, in vitro, the tumors possessing the Ala=> Thr amino acid change responded very strongly to GHRH in terms of cAMP formation, being increased 40- and 200-fold, in comparison to the 2-fold increases by tumors without the alternative GHRH-receptor sequence. Likewise, the in vitro response of GH secretion to GHRH was elevated. One of the two tumors with the alternative Thr residue, and the highest responder to GHRH, possessed a gsp muration, despite the fact that these defects are thought to reduce responsiveness to GHRH. These results fail to confirm that the GCG => AGC at codon 57 of the GHRH-receptor gene is a mutation, but do support the concept that the alternative form with Thr confers increased sensitivity to GHRH. (C) 2000 Academic Press.

Defective peroxisomal proliferators activated receptor gamma activity due to dominant-negative mutation synergizes with hypertension to accelerate cardiac fibrosis in mice

Aims: Humans with inactivating mutations in peroxisomal proliferators activated receptor gamma (PPAR?) typically develop a complex metabolic syndrome characterized by insulin resistance, diabetes, lipodystrophy, hypertension, and dyslipidaemia which is likely to increase their cardiovascular risk. Despite evidence that the activation of PPAR? may prevent cardiac fibrosis and hypertrophy, recent evidence has suggested that pharmacological activation of PPAR? causes increased cardiovascular mortality. In this study, we investigated the effects of defective PPAR? function on the development of cardiac fibrosis and hypertrophy in a murine model carrying a human dominant-negative mutation in PPAR?. Methods and results: Mice with a dominant-negative point mutation in PPAR? (P465L) and their wild-type (WT) littermates were treated with either subcutaneous angiotensin II (AngII) infusion or saline for 2 weeks. Heterozygous P465L and WT mice developed a similar increase in systolic blood pressure, but the mutant mice developed significantly more severe cardiac fibrosis to AngII that correlated with increased expression of profibrotic genes. Both groups similarly increased the heart weight to body weight ratio compared with saline-treated controls. There were no differences in fibrosis between saline-treated WT and P465L mice. Conclusion: These results show synergistic pathogenic effects between the presence of defective PPAR? and AngII-induced hypertension and suggest that patients with PPAR? mutation and hypertension may need more aggressive therapeutic measures to reduce the risk of accelerated cardiac fibrosis. © The Author 2009.

Cholecystokinin (CCK) receptor and CCK gene expression in human pituitary adenomas and in vitro effects of CCK peptides on GH and gonadotrophin secretion

There is growing evidence that cholecystokinin (CCK) affects growth and differentiation of anterior pituitary cells, via the CCK-B receptor. The possibility of an autocrine / paracrine role for CCK to modulate hormone secretion in human pituitary tumour cells is demonstrated here by RT-PCR and direct sequencing. In support of this conclusion, a neutralising antibody against the CCK peptide exhibited a dose dependent inhibition of hormone secretion by functionless pituitary adenomas. Total RNA was extracted from human pituitary adenomas, reverse transcribed into cDNA and subjected to PCR using primers specific for the gene for CCK, CCK-A and CCK-B receptors. PCR bands of the predicted length were observed in all tumours using human CCK gene and CCK-B receptor primers. Restriction digestion and direct sequence analysis provided further evidence that they represented both the human CCK peptide along with the CCK-A and/B receptor mRNA. CCK-33 and CCK octapeptide sulphate (CCK-8s) both powerfully stimulated phosphatidylinositol hydrolysis, providing evidence for functional activity of the CCK-A and/B receptors. A direct stimulatory effect of CCK peptides on both LH and FSH secretion is reported for the first time, whereas stimulatory effects on GH were blocked by antagonists to CCK. These results may indicate an autocrine role for CCK in the functioning and perhaps development of human pituitary tumours. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart.

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.

Transgenic Expression of Endothelin Receptor-B Rescues the Aganglionosis Phenotype of Piebald Lethal Mice

Neural crest cells (NCC) are a unique population of cells in vertebrates that arise between the presumptive epidermis and the dorsal most region of the neural tube. During neurulation, NCC migrate to many regions of the body to give rise to a wide variety of cell types. NCC that originate from the neural tube at the levels of somite 1-7 colonize the gut and give rise to the enteric ganglia. The endothelin signaling pathway has been shown to be crucial for proper development of some neural crest derivatives. Mice and humans with mutations in the Endothelin receptor b (Ednrb) gene exhibit similar phenotypes characterized by hypopigmentation, hearing loss, and megacolon. Thesephenotypes are due to lack of melanocytes in the skin, inner ear and enteric ganglia in the distal portion of the colon, respectively. It is well established that Ednrb is required early during the embryonic development for normal innervation of the gut. However, it is not clear if Ednrb acts on enteric neuron precursor cells or in pre-committed NC precursors. Additionally, it is controversial whether the action of Ednrb is cell autonomous or non- autonomous. We generated transgenic mice that express Ednrb under the control of the Nestin second intron enhancer (Nes) which drives expression to pre-migrating NCC. These mice were crosses to the spontaneous mouse mutant piebald lethal, which carriers a null mutation in Ednrb and exhibits enteric aganglionosis. The Nes-Ednrb was capable of rescuing the aganglianosis phenotype of piebald lethal mutants demonstrating that expression of Ednrb in pre-committed precursors is sufficient for normal enteric ganglia development. This study provides insight in early embryonic development of NCC and could eventually have potential use in cellular therapies for Hirschsprung's disease.

Mechanical and material properties of cortical and trabecular bone from cannabinoid receptor-1-null (Cnr1-/-) mice

Funding ABK was funded by a studentship from the University of Aberdeen, Institute of Medical Sciences, and the Overseas Research Students Awards Scheme Acknowledgments We are grateful to Dr J.S. Gregory for assistance with Image J and Mr K. Mackenzie for assistance with Micro-CT analysis.

Gene expression profiling in hearts of transgenic mice overexpressing guanylyl cyclase domain of the GC-A receptor

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Gene expression profiling in hearts of transgenic mice overexpressing guanylyl cyclase domain of the GC-A receptor

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Double incretin receptor knock-out (DIRKO) mice present with alterations of trabecular and cortical micromorphology and bone strength.

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Absence of LPA1 receptor results in altered pattern of limbic activation after tail suspension test

Stress serves as an adaptive mechanism and helps organisms to cope with life-threatening situations. However, individual vulnerability to stress and dysregulation of this system may precipitate stress-related disorders such as depression. The neurobiological circuitry in charge of dealing with stressors has been widely studied in animal models. Recently our group has demonstrated a role for lysophosphatidic acid (LPA) through the LPA1 receptor in vulnerability to stress, in particular the lack of this receptor relates to robust decrease of adult hippocampal neurogenesis and induction of anxious and depressive states. Nevertheless, the specific abnormalities in the limbic circuit in reaction to stress remains unclear. The aim of this study is to examine the differences in the brain activation pattern in the presence or absence of LPA1 receptor after acute stress. For this purpose, we have studied the response of maLPA1-null male mice and normal wild type mice to an intense stressor: Tail Suspension Test. Activation induced by behaviour of brain regions involved in mood regulation was analysed by stereological quantification of c-Fos immunoreactive positive cells. We also conducted multidimensional scaling analysis in order to unravel coativation between structures. Our results revealed hyperactivity of stress-related structures such as amygdala and paraventricular nucleus of the hypothalamus in the knockout model and different patterns of coactivation in both genotypes using a multidimensional map. This data provides further evidence to the engagement of the LPA1 receptors in stress regulation and sheds light on different neural pathways under normal and vulnerability conditions that can lead to mood disorders.

Antiangiogenic And Finasteride Therapies: Responses Of The Prostate Microenvironment In Elderly Mice.

The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416+TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-β and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-β reactivity were verified in the finasteride, SU5416 groups and especially in SU5416+TNP-470 group. The TNP-470 group showed reduced AR and ER-β protein levels. The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416+TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-β.