ABSTRACT
BACKGROUND: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial. METHODS: The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. RESULTS: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3ß (p-GSK3ß). CONCLUSION: The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3ß.
Subject(s)
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer/pharmacology , Cell Proliferation/drug effects , Receptors, Opioid, kappa/agonists , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Cell Survival , Drug Resistance, Neoplasm , ErbB Receptors/genetics , Gefitinib , Gene Expression , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Mutation, Missense , Naltrexone/analogs & derivatives , Naltrexone/pharmacology , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Quinazolines/pharmacology , Receptors, Opioid, kappa/antagonists & inhibitors , Receptors, Opioid, kappa/genetics , Receptors, Opioid, kappa/metabolism , STAT3 Transcription Factor/metabolism , Signal TransductionABSTRACT
Advanced magnetic resonance (MR) neuroimaging analysis techniques based on voxel-wise statistics, such as voxel-based morphometry (VBM) and functional MRI, are widely applied to cognitive brain research in both human subjects and in non-human primates. Recent developments in imaging have enabled the evaluation of smaller animal models with sufficient spatial resolution. The common marmoset (Callithrix jacchus), a small New World primate species, has been widely used in neuroscience research, to which voxel-wise statistics could be extended with a species-specific brain template. Here, we report, for the first time, a tissue-segmented, population-averaged standard template of the common marmoset brain. This template was created by using anatomical T(1)-weighted images from 22 adult marmosets with a high-resolution isotropic voxel size of (0.2 mm)(3) at 7-Tesla and DARTEL algorithm in SPM8. Whole brain templates are available at International Neuroinformatics Japan Node website, http://brainatlas.brain.riken.jp/marmoset/.
Subject(s)
Anatomy, Artistic , Atlases as Topic , Brain/anatomy & histology , Callithrix/anatomy & histology , Animals , Female , Image Processing, Computer-Assisted , Magnetic Resonance Imaging , MaleABSTRACT
BACKGROUND: Although matrix metalloproteinases (MMPs) are implicated in tumourigenesis and cancer progression, the role of MMP-13 in melanoma cell metastases is poorly understood. METHODS: Lung metastases of mouse melanoma B16BL6 cells were analysed in MMP-13 knockout (KO) and wild-type (WT) mice after intravenous injection. The mRNA and protein expression of MMP-13 in lung tissues was analysed by RT-PCR, real-time PCR, immunoblotting and immunohistochemistry. The expression of SDF-1α, CXCR4 and endostatin, and effects of endostatin to cultured melanoma cells and lung metastases were also studied. RESULTS: Lung metastases of B16BL6 cells were significantly higher by 2.5-5.7-fold in MMP-13 KO mice than in WT mice. The expression of MMP-13 in WT mouse lung tissue was stimulated on day 1 after intravenous injection of the melanoma cells and MMP-13 was immunolocalised to vascular endothelial cells in the lungs. Endostatin formation, but not degradation of SDF-1α, in the lung tissue was associated with reduced lung metastasis in WT mice. Endostatin significantly inhibited migration of B16BL6 cells in monolayer wounding assay and remarkably suppressed Matrigel invasion and transendothelial invasion of the cells. In addition, lung metastases of melanoma cells in MMP-13 KO mice were reduced by intraperitoneal administration of endostatin. CONCLUSION: Our results suggest that MMP-13 is overproduced by endothelial cells in the lungs with melanoma cells and has a protective role in lung metastasis by local generation of endostatin.
Subject(s)
Endostatins/biosynthesis , Lung Neoplasms/prevention & control , Matrix Metalloproteinase 13/metabolism , Melanoma, Experimental/pathology , Animals , Base Sequence , DNA Primers , Enzyme-Linked Immunosorbent Assay , Lung Neoplasms/enzymology , Lung Neoplasms/secondary , Melanoma, Experimental/enzymology , Mice , Mice, Knockout , Microscopy, Fluorescence , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The radiological modalities that are currently utilized as critical components in clinical medicine have also been adapted to small-animal imaging, among which are ultrasound imaging, X-ray computerized tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Optical imaging techniques such as bioluminescence imaging (BLI) and fluorescence imaging (FLI) are approaches that are commonly used in small animals. Longitudinal surveys of living (i.e., nonsacrificed) animal models with these modalities provide some clues for the development of clinical applications. The techniques are absolutely essential for translational research. However, there are currently few tools available with sufficient spatial or temporal resolution ideal for all experimental studies. In this chapter, we provide a rationale and techniques for visualizing target cells in living small animals and an overview of the advantages and limitations of current imaging technology. Finally, we introduce a humanized mouse and a novel in vivo imaging system that we have developed. We also discuss real-time observations of reconstructs and clinical manifestations.
Subject(s)
Diagnostic Imaging/methods , Models, Animal , Animals , Mice , Mice, SCIDABSTRACT
We have combined genetic and biochemical approaches to analyze the function of the RNA-binding protein Nova-1, the paraneoplastic opsoclonus-myoclonus ataxia (POMA) antigen. Nova-1 null mice die postnatally from a motor deficit associated with apoptotic death of spinal and brainstem neurons. Nova-1 null mice show specific splicing defects in two inhibitory receptor pre-mRNAs, glycine alpha2 exon 3A (GlyRalpha2 E3A) and GABA(A) exon gamma2L. Nova protein in brain extracts specifically bound to a previously identified GlyRalpha2 intronic (UCAUY)3 Nova target sequence, and Nova-1 acted directly on this element to increase E3A splicing in cotransfection assays. We conclude that Nova-1 binds RNA in a sequence-specific manner to regulate neuronal pre-mRNA alternative splicing; the defect in splicing in Nova-1 null mice provides a model for understanding the motor dysfunction in POMA.
Subject(s)
Alternative Splicing/physiology , Antigens, Neoplasm , Motor Neurons/cytology , Motor Neurons/physiology , Nerve Tissue Proteins , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Animals , Apoptosis/genetics , Brain Chemistry/genetics , Brain Stem/cytology , Brain Stem/embryology , Cell Survival/genetics , Exons/genetics , Gene Deletion , Gene Expression Regulation, Developmental , Genotype , In Situ Nick-End Labeling , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Neurons/chemistry , Neuro-Oncological Ventral Antigen , Protein Binding/genetics , RNA Precursors/genetics , RNA-Binding Proteins/metabolism , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Receptors, Glycine/genetics , Receptors, Glycine/metabolism , Ribonucleoproteins/metabolism , Spinal Cord/cytology , Spinal Cord/embryologyABSTRACT
Animal fetuses and embryos may have applications in the generation of human organs. Progenitor cells may be an appropriate cell source for regenerative organs because of their safety and availability. However, regenerative organs derived from exogenous lineage progenitors in developing animal fetuses have not yet been obtained. Here, we established a combination system through which donor cells could be precisely injected into the nephrogenic zone and native nephron progenitor cells (NPCs) could be eliminated in a time- and tissue-specific manner. We successfully achieved removal of Six2+ NPCs within the nephrogenic niche and complete replacement of transplanted NPCs with donor cells. These NPCs developed into mature glomeruli and renal tubules, and blood flow was observed following transplantation in vivo. Furthermore, this artificial nephron could be obtained using NPCs from different species. Thus, this technique enables in vivo differentiation from progenitor cells into nephrons, providing insights into nephrogenesis and organ regeneration.
Subject(s)
Nephrons/metabolism , Stem Cell Transplantation/methods , Stem Cells/metabolism , Transplantation Chimera , Animals , Cell Differentiation , Female , Male , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Nephrons/cytology , Nephrons/embryology , Organogenesis , Rats, Sprague-Dawley , Rats, Transgenic , Species Specificity , Stem Cells/cytologyABSTRACT
Movement dysfunction in Parkinson's disease (PD) is caused by the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Here, we established a method for voxel-based morphometry (VBM) and automatic tissue segmentation of the marmoset monkey brain using a 7-T animal scanner and applied the method to assess DA degeneration in a PD model, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animals, with tyrosine-hydroxylase staining. The most significant decreases of local tissue volume were detected in the bilateral SN of MPTP-treated marmoset brains (-53.0% in right and -46.5% in left) and corresponded with the location of DA neurodegeneration found in histology (-65.4% in right). In addition to the SN, the decreases were also confirmed in the locus coeruleus, and lateral hypothalamus. VBM using 7-T MRI was effective in detecting volume loss in the SN of the PD-model marmoset. This study provides a potential basis for the application of VBM with ultra-high field MRI in the clinical diagnosis of PD. The developed method may also offer value in automatic whole-brain evaluation of structural changes for the marmoset monkey.
Subject(s)
Callithrix/anatomy & histology , MPTP Poisoning/pathology , Magnetic Resonance Imaging/methods , Substantia Nigra/pathology , Animals , Callithrix/metabolism , MPTP Poisoning/metabolism , Magnetic Resonance Imaging/instrumentation , Male , Organ Size , Pattern Recognition, Automated/methods , Substantia Nigra/metabolism , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Estrogen stimulates uterine epithelial cells to divide, but not estrogen-concentrating neurons in the adult brain. This effect correlates with recent evidence that estrogen can induce the expression of certain growth-related genes in uterus which are not directly induced by estrogen in the adult brain. The possibility that local diffusible factors play a major role in determining tissue-specific effects of estrogen was examined by transplanting uterine tissues into the brain, muscle and kidney of adult rats and then comparing the effects of estrogen on the incorporation of [3H]thymidine and the expression of Fos-, cdc2- and Rb-like immunoreactivity (IR) on native and transplanted uterine tissues, as well as in estrogen-concentrating regions of the brain adjacent to the uterine grafts. In native uteri, estrogen treatment stimulated Fos-, cdc2-, and Rb-like IR, as well as [3H]thymidine incorporation, within lumenal and glandular epithelial cells. All of these effects were estrogen responsive--no immunoreactive staining within uterine epithelial cells and no signs of epithelial cell proliferation were observed in the native uteri of non-estrogen-treated animals. When uterine tissues were transplanted to brain, Fos-, cdc2-, and Rb-like IR epithelial cells, as well as many [3H]thymidine-incorporating uterine epithelial cells, were observed in all estrogen-treated animals and in some non-estrogen-treated animals as well. Identical results were obtained when uterine tissues were transplanted to skeletal muscle, but not to kidney (in the kidney, transplanted epithelial cells expressed all four parameters but only in estrogen-treated animals, comparable to the native uterus). In contrast, estrogen did not stimulate cell division and did not induce Fos-, cdc2-, or Rb-like IR within estrogen-concentrating neuronal regions of the ventromedial hypothalamus. In addition, the presence of uterine tissue in the brain did not confer the ability of estrogen to stimulate any of these parameters within nearby, estrogen-concentrating regions. These data suggest that there are factors in brain and muscle which can allow uterine epithelial cells to divide in the absence of estrogen. There was no evidence of a diffusible factor in brain which inhibits uterine epithelial cell division, nor of a diffusible factor in uterus which can confer estrogenic stimulation of growth-related genes and cell division to central nervous system neurons. In addition, the data provide the first evidence for estrogen regulation of cdc2 and Rb expression in normal uterus.
Subject(s)
Cerebral Ventricles , Estradiol/pharmacology , Gene Expression Regulation/drug effects , Transplantation, Heterotopic , Uterus/transplantation , Animals , CDC2 Protein Kinase/biosynthesis , CDC2 Protein Kinase/genetics , DNA Replication/drug effects , Female , Genes, Retinoblastoma/drug effects , Genes, fos/drug effects , Kidney , Muscles , Organ Specificity , Rats , Rats, Sprague-Dawley , Uterus/metabolismABSTRACT
Intracerebral diffusion, cellular uptake and intracellular localization of oligodeoxynucleotides (ODN) after their microinjection in mouse brains were examined. Using either tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC)- or gamma-33P ATP-labeled ODNs, it was found that both phosphodiester ODNs (D-ODN) and phosphorothioate ODNs (S-ODN) quickly diffused (up to about 500 microns) and were taken up by many cells around the injection site as early as 15 min after administration. Fluorescence labeling intensity and silver grain accumulation of D-ODNs were greatly reduced by 4 h after injection, whereas those of S-ODNs were stable beyond at least 8 h after injection. Most of labeled ODNs were found in neuronal cells as identified by immunocytochemistry for neurofilament, NF 200, and to a much lesser extent in astrocytic cells as identified by immunocytochemistry for glial fibrillary acidic protein.
Subject(s)
Brain/metabolism , Oligonucleotides, Antisense/metabolism , Animals , Base Sequence , Brain/cytology , Diffusion , Female , Glial Fibrillary Acidic Protein/metabolism , Immunohistochemistry , Injections, Intraventricular , Mice , Molecular Sequence Data , Oligonucleotides, Antisense/pharmacokineticsABSTRACT
The present study characterized fetal sulcation patterns and gyrification in the cerebrum of the New World monkey group, common marmosets, using a 3D T2-weighted high-resolution anatomical magnetic resonance imaging (MRI) sequence from the fixed brain at 7-tesla ex vivo. Fetal sulcation in the marmoset cerebrum began to indent the lateral fissure and hippocampal sulcus in gestational week (GW) 12, and then the following sulci emerged: the callosal and calcarine sulci on GW 15; the superior temporal sulcus on GW 17; and the circular and occipitotemporal sulci on GW 18. The degree of cortical convolution was evaluated quantitatively based on 2D MRI slices by the gyrification index (GI) and based on 3D MRI data by sulcation index (SI). Both the mean GI and SI increased from GW 16, and were closely correlated with the cortical volume and the cortical surface area during fetal periods (their correlation coefficients marked more than 0.95). After birth, both the mean GI and SI decreased slightly by 2years of age, whereas the cortical volume and surface area continuously increased. Notably, histological analysis showed that the outer subventricular zone (oSVZ) in non-sulcal regions was thicker than that in the presumptive calcarine sulcal region on GW 13, preceding the infolding of the calcarine sulcus. The present results showed definite sulcal infolding on the cerebral cortical surface of the marmosets, with similar pattern and sequence of their emergences to other higher-order primates such as macaques and humans. Differential expansion of the oSVZ may be involved in gyral convolution and sulcal infolding in the developing cerebrum.
Subject(s)
Brain Mapping , Cerebral Cortex , Magnetic Resonance Imaging , Age Factors , Animals , Animals, Newborn , Callithrix , Cerebral Cortex/anatomy & histology , Cerebral Cortex/embryology , Cerebral Cortex/growth & development , Embryo, Mammalian , Eye Proteins/metabolism , Female , Functional Laterality , Gestational Age , Homeodomain Proteins/metabolism , Image Processing, Computer-Assisted , Male , PAX6 Transcription Factor , Paired Box Transcription Factors/metabolism , Repressor Proteins/metabolism , T-Box Domain Proteins/metabolism , Tubulin/metabolismABSTRACT
The developmental anatomy of the brain is largely directed by neural-based cues. Despite this knowledge, the developmental trajectory of the primate brain has not yet been fully characterized. To realize this goal, the advance in noninvasive imaging methods and new brain atlases are essential. The common marmoset (Callithrix jacchus), a small New World primate, is widely used in neuroscience research. The recent introduction of transgenic techniques has enabled the marmoset to be used as a genetically modifiable primate model for brain development. Here, a magnetic resonance histology technique involving the use of ultra-high-resolution ex vivo magnetic resonance imaging (MRI) was performed to identify the developmental anatomy of the marmoset brain at different time points from gestational week 8 through to birth. The data allowed the generation of a multidimensional atlas of brain structures at different developmental stages. Furthermore, in utero MRI techniques were developed to noninvasively monitor brain development during the embryonic and fetal stages. The multidimensional atlas and the MRI tools developed herein are anticipated to further our understanding of the developing primate brain.
Subject(s)
Brain Mapping , Brain , Callithrix/anatomy & histology , Magnetic Resonance Imaging , Animals , Animals, Newborn , Brain/anatomy & histology , Brain/embryology , Brain/growth & development , Embryo, Mammalian , Imaging, Three-Dimensional , Time FactorsSubject(s)
Antibodies, Monoclonal/isolation & purification , Hybridomas/immunology , Aequorin/immunology , Animals , Antibodies, Monoclonal/immunology , Ascites/immunology , Hybridomas/transplantation , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Male , Mice , Mice, Inbred BALB C/immunology , Neoplasm TransplantationABSTRACT
The Hu proteins are a group of antigens targeted in an immune-mediated neurodegenerative disorder associated with cancer. We have cloned and characterized four members of the Hu gene family from mouse. We find that the Hu genes encode a large number of alternatively spliced transcripts to produce a series of related neuron-specific RNA binding proteins. Despite this complexity, we have discerned several ordered features of Hu expression. In the embryo, specific Hu genes are expressed in a hierarchy during early neurogenesis. In the E16 developing cortex, mHuB is induced in very early postmitotic neurons exiting the ventricular zone, mHuD is expressed in migrating neurons of the intermediate zone, and mHuC is expressed in mature cortical plate neurons. Such a hierarchy suggests distinct functional roles for each gene in developing neurons. In the adult, all neurons express some set of Hu mRNA and protein. However, specific patterns are evident such that individual neuronal types in the hippocampus, cerebellum, olfactory cortex, neocortex, and elsewhere express from one to several Hu genes. The complexity of potential protein variants within a gene family and of different Hu family members within a neuron suggests a diverse array of function. Given the strong homologies among the Hu proteins, the Drosophila neurogenic gene elav, and the Drosophila splicing factor sxl, we predict that different combinations of Hu proteins determine different neuron-specific aspects of post-transcriptional RNA regulation. Our findings of specific developmental patterns of expression and the correlation between immune targeting of the Hu proteins and adult neurodegenerative disease suggest that the Hu proteins are critical in both the proper development and function of mature neurons.
Subject(s)
Bacterial Proteins/genetics , Carrier Proteins/metabolism , DNA-Binding Proteins/genetics , Neurons/metabolism , RNA/metabolism , Animals , Bacterial Proteins/metabolism , Cloning, Molecular , DNA-Binding Proteins/metabolism , Humans , Immunohistochemistry , In Situ Hybridization , MiceABSTRACT
Expression of the cell cycle regulatory proteins RB and p34cdc2 was examined in the adult rat brain, with special emphasis on proliferation and neuronal differentiation in the hippocampal formation and olfactory bulb. RB-like immunoreactivity (RB-IR) was detected throughout the brain, with particularly intense staining observed in hippocampal pyramidal cells, pyriform cortex, and cerebellar Purkinje cells. Intense RB-IR and cdc2-IR were also detected in proliferating neuronal precursor cells in the subgranular region of the dentate gyrus and in the subependymal region extending from the anterior lateral ventricle into the olfactory bulb. Many of these cells developed into neurons as assessed by the expression of neuron-specific enolase (NSE) and, in the hippocampal formation, the expression of Fos-IR following pentylenetetrazol-induced seizure activity. A good correlation was observed between the number of proliferating cells expressing intense nuclear RB-IR staining and the number of thymidine-labeled cells that had differentiated into functional hippocampal neurons. A substantial decrease in RB-IR during differentiation was also observed and occurred prior to the expression of NSE. The possibility that the loss of RB may be necessary for neuronal differentiation to proceed is discussed.
Subject(s)
Brain/metabolism , CDC2 Protein Kinase/biosynthesis , DNA/biosynthesis , Hippocampus/metabolism , Neurons/metabolism , Olfactory Bulb/metabolism , Retinoblastoma Protein/biosynthesis , Thymidine/metabolism , Animals , Autoradiography , Brain/cytology , Cell Cycle , Cell Differentiation , Hippocampus/cytology , Kinetics , Male , Neurons/cytology , Olfactory Bulb/cytology , Organ Specificity , Pyramidal Tracts/cytology , Pyramidal Tracts/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , TritiumABSTRACT
Immunocytochemical and autoradiographic techniques were used to examine proliferating cells in the adult rat brain along with the expression of specific growth factor receptors and cell cycle proteins. Two hours following an injection of [3H]thymidine ([3H]Thy), dividing cells were detected in the subgranular region of the dentate gyrus and in the subependymal region (SER) extending into the olfactory bulb. Many cells continued to divide over the next 24 h as demonstrated by the ability for thymidine-labeled cells to incorporate bromodeoxyuridine (BrdU); however, the results of BrdU, PSTAIR, and vimentin staining suggest that the majority of the progeny cells detectable by [3H]Thy autoradiography at 3 days and 1 week after injection are postmitotic and at least partially differentiated. Significant numbers of thymidine-labeled cells detected 2 h following thymidine injection in the subgranular region of the dentate gyrus and in the SER of the lateral ventricle stained positively for epidermal growth factor receptor-, vimentin-, and PSTAIR-like immunoreactivity. Significant numbers of thymidine-labeled cells in the SER also stained positively for the low-affinity neurotrophin receptor p75NGFR. No [3H]Thy/p75NGFR-labeled cells were detected in the dentate gyrus. In addition, very few [3H]Thy/PSTAIR- or [3H]Thy/ vimentin-labeled cells were detected in region CA4. These data suggest that proliferating cells located in different regions of the adult brain may not be homogeneous and may be subject to different growth factor regulation.
Subject(s)
CDC2 Protein Kinase/biosynthesis , Epidermal Growth Factor/biosynthesis , Hippocampus/metabolism , Nerve Tissue Proteins/biosynthesis , Peptide Fragments/biosynthesis , Prosencephalon/metabolism , Receptors, Nerve Growth Factor/biosynthesis , Animals , CDC2 Protein Kinase/genetics , Cell Cycle , Cell Differentiation , Cell Division , Dentate Gyrus/cytology , Dentate Gyrus/metabolism , Epidermal Growth Factor/genetics , Gene Expression Regulation , Hippocampus/cytology , Male , Nerve Tissue Proteins/genetics , Olfactory Bulb/cytology , Olfactory Bulb/metabolism , Peptide Fragments/genetics , Prosencephalon/cytology , Rats , Rats, Sprague-Dawley , Receptor, Nerve Growth Factor , Receptors, Nerve Growth Factor/genetics , Stem Cells/cytology , Stem Cells/metabolism , Vimentin/biosynthesis , Vimentin/geneticsABSTRACT
The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor alpha2 (GlyRalpha2) pre-mRNA, and this binding is implicated in Nova-dependent regulation of splicing. Cotransfection assays with a GlyRalpha2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyRalpha2 E3A. brPTB binds to a 90-nt GlyRalpha2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyRalpha2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissue-restricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.
Subject(s)
Alternative Splicing , Antigens, Neoplasm/physiology , Brain/metabolism , Nerve Tissue Proteins/physiology , RNA-Binding Proteins/physiology , Ribonucleoproteins/physiology , Animals , Base Sequence , Male , Molecular Sequence Data , Neuro-Oncological Ventral Antigen , Polypyrimidine Tract-Binding Protein , Rats , Rats, Sprague-Dawley , Receptors, Glycine/geneticsABSTRACT
The TRAP220 component of the TRAP/SMCC complex, a mammalian homologof the yeast Mediator that shows diverse coactivation functions, interacts directly with nuclear receptors. Ablation of the murine Trap220 gene revealed that null mutants die during an early gestational stage with heart failure and exhibit impaired neuronal development with extensive apoptosis. Primary embryonic fibroblasts derived from null mutants show an impaired cell cycle regulation and a prominent decrease of thyroid hormone receptor function that is restored by ectopic TRAP220 but no defect in activation by Gal4-RARalpha/RXRalpha, p53, or VP16. Moreover, haploinsufficient animals show growth retardation, pituitary hypothyroidism, and widely impaired transcription in certain organs. These results indicate that TRAP220 is essential for a wide range of physiological processes but also that it has gene- and activator-selective functions.
Subject(s)
Carrier Proteins/metabolism , Pituitary Gland/embryology , Receptors, Thyroid Hormone/metabolism , Thyroid Gland/embryology , Thyroid Hormones/metabolism , Transcription Factors , Animals , Carrier Proteins/genetics , Embryonic and Fetal Development , Fibroblasts/physiology , Genes, Lethal , Heart Defects, Congenital , Liver/embryology , Lung/embryology , Mediator Complex Subunit 1 , Mice , Mice, Knockout , Nervous System Malformations , Pituitary Gland/metabolism , Placenta/embryology , Thyroid Gland/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Tumor Suppressor Protein p53/metabolismABSTRACT
We have identified a target antigen in autoimmune cerebellar degeneration, beta-NAP, that is closely related to the beta-adaptin and beta-COP coat proteins. Beta-NAP is a nonclathrin-associated phosphoprotein expressed exclusively in neurons, from E12 through adulthood. Beta-NAP is present in the neuronal soma and nerve terminal as soluble and membrane-bound pools and is associated with a discrete set of nerve-terminal vesicles. These results establish beta-NAP as a neuron-specific vesicle coat protein. We propose a model in which beta-NAP mediates vesicle transport between the soma and the axon terminus and suggest that beta-NAP may represent a general class of coat proteins that mediates apical transport in polarized cells.
Subject(s)
Adaptor Protein Complex 3/genetics , Adaptor Protein Complex beta Subunits/genetics , Cerebellar Diseases/immunology , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Neurons/chemistry , Phosphoproteins/genetics , Adaptor Protein Complex 3/analysis , Adaptor Protein Complex beta Subunits/analysis , Animals , Antigens/physiology , Capsid , Cerebellum/chemistry , Cerebellum/embryology , Cerebellum/pathology , Fetus/physiology , Gene Expression/physiology , Immunohistochemistry , Membrane Proteins/analysis , Mice , Microscopy, Electron , Molecular Sequence Data , Nerve Tissue Proteins/analysis , Phosphoproteins/analysis , Presynaptic Terminals/chemistry , RNA, Messenger/analysis , Sequence Homology, Amino Acid , Synaptic Vesicles/chemistryABSTRACT
MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. Biochemical evidence demonstrates that these proteins integrate calcium and PKC-dependent signals to regulate actin structure at the membrane. We report here that deletion of the MacMARCKS gene prevents cranial neural tube closure in the developing brain, resulting in anencephaly. This suggests a central role for MacMARCKS and the PKC signal transduction pathway in the folding of the anterior neural plate during the early phases of brain formation, and supports the hypothesis that actin-based motility directs cranial neural tube closure.
Subject(s)
Anencephaly/genetics , Membrane Proteins , Neural Tube Defects/genetics , Proteins/genetics , Animals , Base Sequence , Brain/abnormalities , Brain/embryology , Calmodulin-Binding Proteins , Cell Line , Chimera , DNA Primers , Embryonic and Fetal Development/genetics , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred C57BL , Microfilament Proteins , Molecular Sequence Data , Protein Kinase C/metabolism , Proteins/metabolism , Signal TransductionABSTRACT
Paraneoplastic cerebellar degeneration (PCD) is a disorder in which breast or ovarian tumors express an onconeural antigen termed cdr2, which normally is expressed in cerebellar Purkinje neurons. This leads to an immune response to cdr2 that is associated with tumor immunity and autoimmune cerebellar degeneration. We have found that cdr2, a cytoplasmic protein harboring a helix-leucine zipper (HLZ) motif, interacts specifically with the HLZ motif of c-Myc. Both proteins colocalize in the cytoplasm of adult cerebellar Purkinje neurons, and coimmunoprecipitate from tumor cell lines and cerebellar extracts. cdr2 down-regulates c-Myc-dependent transcription in cotransfection assays, and redistributes Myc protein in the cytoplasm. Disease antisera from six of six PCD patients specifically blocked the interaction between cdr2 and c-Myc in vitro. These data indicate that cdr2 normally sequesters c-Myc in the neuronal cytoplasm, thereby down-regulating c-Myc activity, and suggest a mechanism whereby inhibition of cdr2 function by autoantibodies in PCD may contribute to Purkinje neuronal death.