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1.
Proc Natl Acad Sci U S A ; 103(13): 5155-60, 2006 Mar 28.
Article in English | MEDLINE | ID: mdl-16549787

ABSTRACT

The nuclear transcription factor E-26-like protein 1 (Elk-1) is thought to impact neuronal differentiation [Sharrocks, A. D. (2001) Nat. Rev. Mol. Cell Biol. 2, 827-837], cell proliferation [Sharrocks, A. D. (2002) Biochem. Soc. Trans. 30, 1-9], tumorigenesis [Chai, Y. L., Chipitsyna, G., Cui, J., Liao, B., Liu, S., Aysola, K., Yezdani, M., Reddy, E. S. P. & Rao, V. N. (2001) Oncogene 20, 1357-1367], and apoptosis [Shao, N., Chai, Y., Cui, J., Wang, N., Aysola, K., Reddy, E. S. P. & Rao, V. N. (1998) Oncogene 17, 527-532]. In addition to its nuclear localization, Elk-1 is found throughout the cytoplasm, including localization in neuronal dendrites [Sgambato, V., Vanhoutte, P., Pages, C., Rogard, M., Hipskind, R., Besson, M. J. & Caboche, J. (1998) J. Neurosci. 18, 214-226], raising the possibility that Elk-1 may have alternative extranuclear functions in neurons. Using coimmunoprecipitation and reciprocal coimmunoprecipitation from adult rat brain, we found an association between Elk-1 protein and the mitochondrial permeability transition pore complex (PTP), a structure involved in both apoptotic and necrotic cell death. Electron microscopy in adult rat brain sections confirmed this association with mitochondria. Elk-1 was also identified from purified mitochondrial fractions by using Western blotting, and Elk-1 increased its association with mitochondria following proapoptotic stimuli. Consistent with a role for Elk-1 in neuron viability, overexpression of Elk-1 in primary neurons decreased cell viability, whereas Elk-1 siRNA-mediated knockdown increased cell viability. This decrease in viability induced by Elk-1 overexpression was blocked with application of a PTP inhibitor. These results show an association of the nuclear transcription factor Elk-1 with the mitochondrial PTP and suggest an additional extranuclear function for Elk-1 in neurons.


Subject(s)
Ion Channels/metabolism , Neurons/metabolism , ets-Domain Protein Elk-1/metabolism , Animals , Brain/cytology , Brain/drug effects , Brain/metabolism , Camptothecin/pharmacology , Cell Membrane Permeability/drug effects , Cell Survival , Cells, Cultured , DNA Damage/drug effects , Etoposide/pharmacology , Ion Channels/genetics , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Mitochondrial Permeability Transition Pore , Neurons/cytology , Neurons/drug effects , Protein Binding , RNA, Small Interfering/genetics , Rats , ets-Domain Protein Elk-1/genetics
2.
Br J Cancer ; 90(6): 1111-4, 2004 Mar 22.
Article in English | MEDLINE | ID: mdl-15026786

ABSTRACT

Expression profiling of tumours from cancer patients has uncovered several genes that are critically important in the progression of a normal cell to an oncogenic phenotype. Leading the way in these discoveries is the use of microarrays, a technology that is currently in transition from basic science applications to use in the clinic. Microarrays can determine the global gene regulation of an individual cancer, which may be useful in formulating an individualised therapy for the patient. Currently, cells used in breast cancer microarray studies often come from either homogenous cultures or heterogeneous biopsy samples. Both cell sources are at a disadvantage in determining the most accurate gene profile of cancer, which often consists of multiple subspecies of cancerous cells within a background of normal cells. Therefore, acquisition of small, but highly specific biopsies for analysis may be required for an accurate expression analysis of the disease. Amplification methods, such as polymerase chain reaction (PCR) and amplified antisense RNA (aRNA) amplification, have been used to amplify the mRNA signal from very small samples, which can then be used for microarray analysis. In this study, we describe the acquisition, amplification, and analysis of very small samples (<10000 cells) for expression analysis and demonstrate that the ultimate resolution of cancer expression analysis, one cell, is both feasible and practical.


Subject(s)
Breast Neoplasms/genetics , Cell Transformation, Neoplastic , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Breast Neoplasms/diagnosis , Female , Humans , Phenotype , Polymerase Chain Reaction , RNA, Antisense , Specimen Handling
3.
Physiol Behav ; 73(5): 841-7, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11566217

ABSTRACT

The molecular substrates of behavior have been difficult to assess because of the large number of messenger RNAs (mRNAs) expressed in a given brain region, the heterogeneous composition of the CNS, and the complexity of mammalian behavior. To gain insight into the molecular components of behavior requires an understanding of the anatomy associated with a specific behavior and the ability to examine multiple gene expression in discrete brain regions. Neuroanatomical and behavioral studies have demonstrated that the amygdaloid complex is an essential component of the neural pathways mediating behaviors, such as fear, anxiety, learning, and memory. The amygdala is composed of several interconnected subnuclei and it is the modulation of information, as it flows through these subnuclei, that underlies amygdala function. To examine the molecular components of the amygdala, we have combined the antisense RNA (aRNA) amplification procedure with microarray technology. This experimental approach permits the simultaneous detection and quantification of numerous mRNAs in fixed tissue sections. Our initial experiment examines region-specific gene expression in naïve mice in order to map the molecular relationship between the subregions of the amygdala. This report provides a general overview of the techniques used to examine regional gene expression, suggests future experiments, and describes a theoretical framework for examining the molecular analysis of behavior.


Subject(s)
Amygdala/physiology , Behavior, Animal/physiology , Gene Expression Profiling , Amygdala/anatomy & histology , Animals , Brain Mapping , Genetics, Behavioral , Mice , Neural Pathways/anatomy & histology , Neural Pathways/physiology , RNA, Messenger/genetics
4.
Restor Neurol Neurosci ; 18(2-3): 105-13, 2001.
Article in English | MEDLINE | ID: mdl-11847433

ABSTRACT

Recent advances in DNA microarray technology have enabled the simultaneous evaluation of thousands of genes and the subsequent generation of massive amounts of biological data relevant to injury or diseases of the central nervous system (CNS). This technology has the potential to bridge the gap between molecular and systems neuroscience by efficiently revealing the discrete molecular aspects underlying the perturbations of complex systemic insults such as those resulting from traumatic brain injury (TBI). One of the more intriguing and as of yet not understood aspects of TBI that can be efficiently explored with DNA microarrays, is the sequence of molecular events that results in pronounced cell death in specific areas of the brain. The elucidation of these changes in gene expression underlying the mechanism of cell death following brain injury is of central importance in the design of future therapeutic agents. This review focuses on the technical aspects of microarray manufacture (photolithography, microspotting, and ink jet technology) and their utility in elucidating the molecular sequelae of brain injury.


Subject(s)
Brain Injuries/genetics , Oligonucleotide Array Sequence Analysis , Animals , Apoptosis/genetics , Gene Expression , Humans
5.
Ann N Y Acad Sci ; 919: 261-77, 2000.
Article in English | MEDLINE | ID: mdl-11083116

ABSTRACT

Neural tube defects (NTDs) are among the most common of all human congenital defects, with multifactorial etiologies comprising both environmental and genetic components. Several murine model systems have been developed in an effort to elucidate genetic factors regulating expression of NTDs. Strain-dependent differences in susceptibility to teratogenic insults and altered patterns of gene expression observed within the neuroepithelium of affected embryos support the hypothesis that subtle genetic changes can result in NTDs. Since several affected genes are folate-regulated, transgenic knockout mice lacking a functional folate receptor were developed. Nullizygous embryos died in utero with significant morphological defects, supporting the critical role of folic acid in early embryogenesis. While epidemiological studies have not established an association between polymorphisms in the human folate receptor gene and NTDs, it is known that folate supplementation reduces infant NTD risk. Continued efforts are therefore necessary to reveal the mechanism by which folate works and the nature of the gene(s) responsible for human NTDs.


Subject(s)
Environmental Pollutants/toxicity , Genetic Predisposition to Disease/genetics , Neural Tube Defects/chemically induced , Neural Tube Defects/genetics , Receptors, Cell Surface , Animals , Carrier Proteins/genetics , Cell Cycle/genetics , DNA Fingerprinting , Disease Models, Animal , Embryonic and Fetal Development/genetics , Folate Receptors, GPI-Anchored , Folic Acid/metabolism , Folic Acid/pharmacology , Gestational Age , Growth Substances/genetics , Growth Substances/metabolism , Humans , Hyperthermia, Induced/adverse effects , Mice , Mice, Knockout , Neural Tube Defects/epidemiology , Neural Tube Defects/pathology , Polymorphism, Single-Stranded Conformational , Valproic Acid/pharmacology
6.
Ann Neurol ; 48(1): 77-87, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10894219

ABSTRACT

The pathogenesis of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) is poorly understood, but changes in the expression of specific messenger RNAs (mRNAs) may reflect mechanisms underlying the formation of NFTs and their consequences in affected neurons. For these reasons, we compared the relative abundance of multiple mRNAs in tangle-bearing versus normal CA1 neurons aspirated from sections of AD and control brains. Amplified antisense RNA expression profiling was performed on individual isolated neurons for analysis of greater than 18,000 expressed sequence tagged complementary DNAs (cDNAs) with cDNA microarrays, and further quantitative analyses were performed by reverse Northern blot analysis on 120 selected mRNAs on custom cDNA arrays. Relative to normal CA1 neurons, those harboring NFTs in AD brains showed significant reductions in several classes of mRNAs that are known to encode proteins implicated in AD neuropathology, including phosphatases/kinases, cytoskeletal proteins, synaptic proteins, glutamate receptors, and dopamine receptors. Because cathepsin D mRNA was upregulated in NFT-bearing CA1 neurons in AD brains, we performed immunohistochemical studies that demonstrated abundant cathepsin D immunoreactivity in the same population of tangle-bearing CA1 neurons. In addition, levels of mRNAs encoding proteins not previously implicated in AD were reduced in CA1 tangle-bearing neurons, suggesting that these proteins (eg, activity-regulated cytoskeleton-associated protein, focal adhesion kinase, glutaredoxin, utrophin) may be novel mediators of NFT formation or degeneration in affected neurons. Thus, the profile of mRNAs differentially expressed by tangle-bearing CA1 neurons may represent a "molecular fingerprint" of these neurons, and we speculate that mRNA expression profiles of diseased neurons in AD may suggest new directions for AD research or identify novel targets for developing more effective AD therapies.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/pathology , Hippocampus/pathology , Neurofibrillary Tangles/genetics , Aged , DNA, Complementary/analysis , Female , Humans , In Situ Hybridization , Male , Neurofibrillary Tangles/pathology , RNA, Antisense/analysis , RNA, Messenger/analysis , Transcription, Genetic
7.
J Neurosci ; 20(13): 4821-8, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10864939

ABSTRACT

The cerebral cortex is selectively vulnerable to cell death after traumatic brain injury (TBI). We hypothesized that the ratio of mRNAs encoding proteins important for cell survival and/or cell death is altered in individual damaged neurons after injury that may contribute to the cell's fate. To investigate this possibility, we used amplified antisense mRNA (aRNA) amplification to examine the relative abundance of 31 selected candidate mRNAs in individual cortical neurons with fragmented DNA at 12 or 24 hr after lateral fluid percussion brain injury in anesthetized rats. Only pyramidal neurons characterized by nuclear terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) reactivity with little cytoplasmic staining were analyzed. For controls, non-TUNEL-positive neurons from the cortex of sham-injured animals were obtained and subjected to aRNA amplification. At 12 hr after injury, injured neurons exhibited a decrease in the relative abundance of specific mRNAs including those encoding for endogenous neuroprotective proteins. By 24 hr after injury, many of the mRNAs altered at 12 hr after injury had returned to baseline (sham-injured) levels except for increases in caspase-2 and bax mRNAs. These data suggest that TBI induces a temporal and selective alteration in the gene expression profiles or "molecular fingerprints" of TUNEL-positive neurons in the cerebral cortex. These patterns of gene expression may provide information about the molecular basis of cell death in this region after TBI and may suggest multiple avenues for therapeutic intervention.


Subject(s)
Apoptosis , Brain Injuries/physiopathology , Cerebral Cortex/physiopathology , Nerve Tissue Proteins/genetics , Neurons/pathology , Neurons/physiology , Animals , Brain Injuries/genetics , Brain Injuries/pathology , Caspase 2 , Caspases/genetics , Cell Death , Cerebral Cortex/pathology , Cyclic AMP Response Element-Binding Protein/genetics , Gene Expression Regulation , Genes, fos , In Situ Nick-End Labeling , Inflammation , Male , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Antisense , Rats , Rats, Sprague-Dawley , Receptors, AMPA/genetics , Receptors, N-Methyl-D-Aspartate/genetics , gamma-Aminobutyric Acid/metabolism
8.
Neuroscience ; 96(1): 131-9, 2000.
Article in English | MEDLINE | ID: mdl-10683418

ABSTRACT

The expression of a large panel of selected genes hypothesized to play a central role in post-traumatic cell death was shown to be differentially altered in response to a precisely controlled, mechanical injury applied to an organotypic slice culture of the rat brain. Within 48 h of injury, the expression of nerve growth factor messenger RNA was significantly increased whereas the levels of bcl-2, alpha-subunit of calcium/calmodulin-dependent protein kinase II, cAMP response element binding protein, 65,000 mol. wt isoform of glutamate decarboxylase, 1beta isoform of protein kinase C, and ubiquitin messenger RNA were significantly decreased. Because the expression levels of a number of other messenger RNAs such as the neuron-specific amyloid precursor protein, beta(2) microglobulin, bax, bcl(xl), brain-derived neurotrophic factor, cyclooxygenase-2, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, receptor tyrosine kinase A, and receptor tyrosine kinase B were unaffected, these selective changes may represent components of an active and directed response of the brain initiated by mechanical trauma. Interpretation of these co-ordinated alterations suggests that mechanical injury to the central nervous system may lead to disruption of calcium homeostasis resulting in altered gene expression, an impairment of intracellular cascades responsible for trophic factor signaling, and initiation of apoptosis via multiple pathways. An understanding of these transcriptional changes may contribute to the development of novel therapeutic strategies to enhance beneficial and blunt detrimental, endogenous, post-injury response mechanisms.


Subject(s)
Brain Injuries/genetics , Cell Death/genetics , Gene Expression , Animals , Brain Injuries/metabolism , DNA, Complementary/genetics , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Organ Culture Techniques , RNA, Messenger/metabolism , Rats , Stress, Mechanical
9.
Arch Neurol ; 56(12): 1453-6, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10593299

ABSTRACT

The normal functioning of the central nervous system (CNS) requires complex interactions among numerous biological components. The pathophysiology of perturbations in this system is as complex as that of neurological disease. Many methods exist to examine the biological output of dysfunctional cells from a diseased system (e.g., immunohistochemical analysis, electrophysiology, and microdialysis), with one goal being to understand the mechanisms of cell death. This understanding may allow the design of therapeutic strategies to prevent cell death and ensuing behavioral abnormalities. Analysis of messenger RNA (mRNA) levels for various genes in CNS tissue may enhance understanding of neurological disease, since cells differ in the complement and abundance of genes they express. One popular method for detecting changes in gene expression is the Northern blot technique, in which total RNA from a sample is extracted and the RNA molecules are separated by size on a denaturing gel and transferred or "blotted" onto nylon membranes that are then probed with radiolabeled DNA for subsequent autoradiograpic detection of gene expression.


Subject(s)
Central Nervous System Diseases , Molecular Biology/trends , Neurosciences/trends , Central Nervous System Diseases/genetics , Central Nervous System Diseases/pathology , Central Nervous System Diseases/therapy , Humans , Neurons/pathology
10.
Ann Neurol ; 45(2): 174-81, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9989619

ABSTRACT

The sequestration of RNA in Alzheimer's disease (AD) senile plaques (SPs) and the production of intraneuronal amyloid-beta peptides (Abeta) prompted analysis of the mRNA profile in single immunocytochemically identified SPs in sections of AD hippocampus. By using amplified RNA expression profiling, polymerase chain reaction, and in situ hybridization, we assessed the presence and abundance of 51 mRNAs that encode proteins implicated in the pathogenesis of AD. The mRNAs in SPs were compared with those in individual CA1 neurons and the surrounding neuropil of control subjects. The remarkable demonstration here, that neuronal mRNAs predominate in SPs, implies that these mRNAs are nonproteinaceous components of SPs, and, moreover, that mRNAs may interact with Abeta protein and that SPs form at sites where neurons degenerate in the AD brain.


Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Plaque, Amyloid/metabolism , RNA, Messenger/analysis , Aged , Blotting, Northern , Female , Humans , Immunohistochemistry , In Situ Hybridization , Male , Polymerase Chain Reaction
12.
J Neurosci ; 18(7): 2685-96, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9502826

ABSTRACT

Overexposure to corticosteroid hormones is harmful to hippocampal neuronal integrity, likely by perturbation of calcium homeostasis. To identify molecular mechanisms at the single-cell level, we characterized mRNA expression corresponding to voltage- and ligand-gated Ca channels in individual dissociated CA1 neurons in response to long-term corticosterone (CORT) exposure. Predominant mineralocorticoid receptor occupation (ADC-LO group) resulted in low levels of P/Q- and L-type Ca channel mRNAs, high levels of GluR-2 versus GluR-1, and a high ratio of NMDAR-2A to NMDAR-2B mRNA. Corresponding alterations in protein expression were consistent with the restriction of Ca influx. In contrast, additional glucocorticoid receptor occupation (ADC-HI group) altered the expression of these mRNAs in a manner consistent with enhanced Ca influx; interestingly, qualitatively similar alterations were seen in control ADX neurons. Electrophysiological data from the same neurons indicate that Ca current amplitudes also are modulated by CORT, although on a shorter time scale. Finally, principal components analysis (PCA) suggests that neuronal AMPA and NMDA receptor composition may be regulated by MR and GR activation in a complex manner. Therefore, our data implicate molecular events by which CORT may regulate Ca influx into CA1 hippocampal neurons.


Subject(s)
Adrenal Cortex Hormones/pharmacology , Calcium Channels/genetics , Hippocampus/cytology , Neurons/drug effects , Adrenalectomy , Animals , Calcium/metabolism , Calcium Channels/metabolism , Gene Expression/drug effects , Hippocampus/chemistry , Ion Channel Gating/physiology , Male , Neurons/physiology , Patch-Clamp Techniques , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Receptors, Glucocorticoid/physiology , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism
13.
Biochem Mol Med ; 60(2): 81-91, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9169087

ABSTRACT

The molecular techniques of in situ transcription and antisense RNA amplification (IST/aRNA) have allowed for the monitoring of coordinate changes in the expression of multiple genes simultaneously. However, the analysis of their concurrent behavior during murine embryogenesis has been problematic. Studies involving the investigation of temporal and spatial gene expression during embryogenesis have focused solely on the analysis of isolated, single gene events. Such an approach has failed to provide an integrative picture of genetic control over the varied and complicated cellular processes governing embryogenesis. In order to interpret the enormous amount of gene expression data generated by these procedures, we have attempted to develop an analytical framework by employing the statistical concepts of principal components analysis (PCA). For the current study, we performed IST/aRNA on neural tubes dissected from the highly inbred LM/Bc murine strain collected during four gestational time periods. A subset of these genes, representing a partial signaling pathway in the developing neuroepithelium, was then subjected to PCA. Here, we report that PCA highlighted the transcriptional interplay among the genes p53, wee-1, Tgf beta-2, and bcl-2 such that the combined reciprocal regulation of their gene products is suggestive of a predominant proliferative state for the developing neuroepithelium. The application of PCA to the gene expression data has elucidated previously unknown interrelationships among cell cycle genes, growth, and transcription factors on a transcriptional level during critical stages of neurulation. The information gleaned from this analysis, while not definitive, suggests distinct hypotheses to guide future research.


Subject(s)
Cell Cycle Proteins , Embryonic and Fetal Development/genetics , Gene Expression Regulation, Developmental , Genetic Techniques , Nuclear Proteins , Analysis of Variance , Animals , Cell Cycle/genetics , DNA, Complementary/genetics , Female , Genes, bcl-2/genetics , Genes, fos , Genes, p53 , Gestational Age , Mice , Mice, Inbred Strains , Nervous System/embryology , Pregnancy , Protein-Tyrosine Kinases/genetics , RNA, Antisense/genetics , Transcription, Genetic , Transforming Growth Factor beta/genetics
14.
Ann Neurol ; 41(2): 200-9, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9029069

ABSTRACT

The polypeptide composition of neurofibrillary tangles (NFTs) and senile plaques (SPs) has been characterized extensively within the Alzheimer's disease (AD) brain. Because few data exist on the nonproteinaceous components of these lesions, we sought to determine if NFTs, neuropil threads (NTs), and SPs contain RNA species. To accomplish this, acridine orange (AO) histofluorescence was employed, alone or in combination with thioflavine S (TS) staining and immunohistochemistry to identify RNAs in paraffin-embedded tissue sections of hippocampus and entorhinal cortex. Postmortem brain samples came from 32 subjects including AD and elderly Down's syndrome (DS) patients, age-matched normal controls, and non-AD diseased controls. AO stained the cytoplasm of normal hippocampal and entorhinal neurons in all of the cases, while NFTs, NTs, and SPs were AO-positive in the same regions of AD and DS brains. Cytoplasmic AO histofluorescence was abolished with RNase, but not DNase or proteinase K, indicating the relative specificity of AO for RNA species. Quantitative analysis of double-labeled sections demonstrated that approximately 80% of TS-positive NFTs also were AO-positive, whereas approximately 55% of TS-stained SPs contained AO labeling. These novel observations demonstrate the presence of RNAs in NFTs, NTs, and SPs.


Subject(s)
Alzheimer Disease/pathology , Hippocampus/pathology , Neurofibrillary Tangles/pathology , tau Proteins/metabolism , Adult , Aged , Female , Humans , Male , Middle Aged , RNA/metabolism
15.
Mol Pharmacol ; 49(5): 852-9, 1996 May.
Article in English | MEDLINE | ID: mdl-8622635

ABSTRACT

The RNA amplification technique was used to examine the pattern of coexpression of mRNAs encoding 16 subtypes/subunits of the glutamate receptor (GluR) in acutely dissociated neurons from adult rat striata. THe signal intensity for each mRNA varied within single neurons, but the general pattern of low versus high expression signals was similar among neurons, except for the GluR4 subunit of the (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. The mRNAs for GluR1-3 subunits of the AMPA receptor were present in all cells, with the signal intensity of GluR1 mRNAs usually the lowest. The kainate receptor subunit mRNAs (GluR5-7) were present in most neurons, and the signal intensity for GluR6 mRNA was the highest. The signals for N-methyl-D-aspartate (NMDA)R1 and NMDAR2B mRNAs were high in most neurons; however, NMDAR2A and NMDAR2C mRNAs gave low or undetectable signals. For mRNAs encoding metabotropic GluRs (mGluRs), signals for mGluR1, mGluR2, and mGluR3 mRNAs were low or undetectable, whereas mGluR4 and mGluR5 mRNA signals were high in most neurons. In most cases (12 of 16 mRNAs), the results agreed with data from in situ hybridization experiments in which individual mRNAs were examined. All neurons expressed subtypes/subunits mRNAs for all four types of GluRs; however, there were differences in the relative intensity of the mRNA signals detected in individual cells, suggesting that these receptors could exist in various combinations within individual neurons and thus confer synapse-specific function for information processing in the striatum.


Subject(s)
Corpus Striatum/metabolism , Receptors, Glutamate/genetics , Animals , Base Sequence , Corpus Striatum/cytology , DNA Primers/chemistry , Male , Molecular Sequence Data , Neurons/metabolism , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptors, Glutamate/classification
16.
DNA Cell Biol ; 14(10): 833-9, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7546289

ABSTRACT

The vertebrate inner ear sensory epithelia contain different types of hair cells and supporting cells. The teleost saccule is anatomically similar to the mammalian saccule and is primarily involved in the detection of translational acceleration and orientation with respect to gravity. To facilitate molecular studies of the teleost saccule cDNA libraries were constructed from microdissected Lepomis macrochirus (bluegill sunfish) saccular maculae. To our knowledge, this is the first report of cDNA libraries constructed from the saccule. In one instance, a non-polymerase chain reaction-based method of amplifying a mRNA population from limited amounts of starting tissue was employed that allowed construction of cDNA libraries from nanogram amounts of tissue mRNA. Conventional cDNA libraries were constructed from the sunfish saccular maculae as well. These cDNA libraries enriched in hair cell and supporting cell transcripts should facilitate molecular biological studies of inner ear sensory epithelia. As an example of their utility, efforts to identify tyrosine kinases expressed in the saccular endorgan using low-stringency hybridization screening of these cDNA libraries and the partial sequence of a cDNA found to encode an erbB-2-related tyrosine kinase are also reported.


Subject(s)
Gene Library , Perciformes/genetics , Saccule and Utricle/physiology , Amino Acid Sequence , Animals , Consensus Sequence , DNA, Complementary/genetics , Enzyme Induction , Gene Amplification , Hair Cells, Auditory/physiology , Mammals/anatomy & histology , Molecular Sequence Data , Nucleic Acid Hybridization , Perciformes/anatomy & histology , Phosphorylation , Protein Processing, Post-Translational , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor, ErbB-2/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
17.
Reprod Toxicol ; 8(5): 383-95, 1994.
Article in English | MEDLINE | ID: mdl-7841657

ABSTRACT

SWV mouse embryos collected on gestational days (GD) 9:12 and 10:00 following chronic in utero exposure to teratogenic concentrations of phenytoin were utilized for in situ transcription studies of gene expression. The substrate cDNA obtained from the frozen embryo sections was amplified into radiolabelled antisense RNA (RT/aRNA) and used as a probe to screen a panel of 20 cDNA clones representing genes that are important regulators of craniofacial and neural development. The magnitude of alteration in gene expression following phenytoin treatment was determined densitometrically by changes in the hybridization intensity of the aRNA probes to the cDNA clones immobilized to the slot blots. We found that both Wnt-1 and the calcium channel gene were developmentally regulated, as their level of expression decreased significantly between the two collection times. Phenytoin treatment produced a significant downregulation in the level of expression for 25% of the genes examined in the GD 9:12 embryos, including the growth factors TGF-beta and NT3, the proto-oncogene Wnt-1, the nicotinic receptor, and the voltage sensitive calcium channel gene. Additional changes in the coordinate expression of several of the growth and transcription factors were observed at both gestational timepoints. The application of RT/aRNA technology has extended our appreciation of the normal patterns of gene expression during craniofacial and neural development, and provided the first demonstration of multiple coordinate changes in transcription patterns following teratogenic insult.


Subject(s)
Gene Expression Regulation, Developmental/drug effects , Phenytoin/toxicity , Analysis of Variance , Animals , Brain-Derived Neurotrophic Factor , Calcium Channels/drug effects , Ciliary Neurotrophic Factor , Cloning, Molecular , DNA, Complementary/genetics , Disease Models, Animal , Down-Regulation , Embryonic and Fetal Development/drug effects , Embryonic and Fetal Development/genetics , Female , Gene Expression Regulation, Developmental/genetics , Genes , Gestational Age , Mice , Nerve Growth Factors/drug effects , Nerve Growth Factors/genetics , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , Neurotrophin 3 , Nucleic Acid Hybridization , Phenytoin/administration & dosage , Pregnancy , Proto-Oncogene Proteins/drug effects , Proto-Oncogene Proteins/metabolism , RNA, Antisense/genetics , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/genetics , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Transforming Growth Factor beta/drug effects , Transforming Growth Factor beta/genetics
18.
Proc Natl Acad Sci U S A ; 91(1): 385-9, 1994 Jan 04.
Article in English | MEDLINE | ID: mdl-7506420

ABSTRACT

The chronic use of several drugs, including opiates, results in the stereotypical behaviors characteristic of addiction. Alterations in gene expression have been associated with the use of these addictive drugs. Previous studies, however, have been limited to describing changes in amounts of individual mRNAs from single tissue samples. Cellular adaptation to opiates, reflected in the regulation of the expression of many different mRNAs, seems likely to contribute to the complicated behaviors of addiction. The present studies examined coordinate alterations in the amounts of multiple mRNAs in the rat striatum and in NG108-15 cells after opioid stimulation or the precipitated withdrawal of opioid use. The experimental approach combined amplification of the poly(A)+ RNA population with reverse Northern blot analysis to simultaneously characterize the relative changes in several mRNAs. Morphine treatment of rats for 5 days was associated with a reduction in the amount of striatal RNA for the voltage-sensitive K+ channel without significant changes in other ion channels. In NG108-15 cells stimulation with the delta-opiate receptor agonist [D-Ala2,D-Leu5]enkephalin (DADLE) alone and followed by naloxone (precipitated withdrawal) caused relative changes in the abundances of several mRNAs. The composite effects of alterations in the abundance of multiple mRNAs (and the proteins they encode) in response to opioid use likely contribute to the development and maintenance of opiate-mediated behaviors.


Subject(s)
Corpus Striatum/metabolism , Ion Channels/genetics , Morphine Dependence/genetics , Animals , Enkephalin, Leucine-2-Alanine/pharmacology , GTP-Binding Proteins/genetics , Gene Expression , Genes, fos , Genes, jun , Male , Morphine/pharmacology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Receptors, Retinoic Acid/genetics
19.
Mol Pharmacol ; 44(2): 308-15, 1993 Aug.
Article in English | MEDLINE | ID: mdl-7689143

ABSTRACT

Glutamate-mediated neurotransmission occurs through the activation of multimeric postsynaptic receptors. One mechanism by which functional diversity of glutamate responsiveness may occur is by a single cell expressing multiple receptors containing different subunits. In a direct test of this hypothesis, we examined the glutamate receptor subunit mRNA composition of several individual CA1 neurons in hippocampal slices. Experiments used amplified antisense RNA coupled with expression profiling and polymerase chain reaction amplification to identify and determine the relative amounts of subunit mRNAs co-localized in single cells. The results demonstrate that each CA1 neuron contains varying amounts of most glutamate receptor mRNAs. In addition to relative mRNA levels, the single-cell approach also highlighted other possible sources of receptor diversity. This included the existence of novel, alternatively spliced forms of the N-methyl-D-aspartate receptor type 1 and glutamate-kainate receptor type 2 subunits. Surprisingly, levels of N-methyl-D-aspartate receptor type 1 mRNA were relatively low, compared with those of other glutamate receptor mRNAs. One postulated source of potential heterogeneity, RNA editing, was not a general cellular mechanism. There was no evidence that glutamate receptor type 5 mRNA was edited in any of the cells that were examined. These data show that individual CA1 neurons, in the intact synaptic network of hippocampal slices, generate glutamate receptor mRNA diversity in several ways, which together contribute to the diversity of functional receptors observed electrophysiologically.


Subject(s)
Hippocampus/metabolism , Neurons/metabolism , Poly A/metabolism , RNA/metabolism , Receptors, Glutamate/genetics , Receptors, N-Methyl-D-Aspartate/genetics , Animals , Blotting, Southern , Electrophysiology , In Vitro Techniques , Oligonucleotides, Antisense , Polymerase Chain Reaction , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Glutamate/chemistry , Receptors, Glutamate/metabolism , Receptors, Kainic Acid , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism
20.
Microsc Res Tech ; 25(1): 19-28, 1993 May 01.
Article in English | MEDLINE | ID: mdl-8353304

ABSTRACT

The secondary and tertiary structure of RNA, in situ, is thought to be involved in distinct functions such as directing association of the RNA with the cytoskeleton, enzymatic activity of some RNAs, and the control of translation. In situ transcription (IST), a procedure by which cDNA is synthesized in situ, has been used to assess mRNA structure in situ using fixed cells or tissues. Distinct banding patterns were noted for mouse and rat POMC. Unique IST banding patterns were observed when an oligonucleotide complementary to a putative POMC stem-loop structure was used to prime IST. Indeed local changes in banding patterns could be elicited by pharmacological agents which modulate POMC translation. Inhibition of POMC synthesis with NaF or dexamethasone decreased the number of POMC mRNAs in the polysome fractions and increased the intensity of high molecular weight IST-derived bands. Forskolin, a stimulator of POMC synthesis, had the opposite effect. One mechanism by which translational control is thought to occur is by regulation of ribosome movement down the mRNA by specific binding of cytosolic proteins to RNA structure. Cytosolic protein fractions from AtT20 pituitary cells have been shown to specifically bind to the IST-predicted RNA structure. These findings suggest that 1) mRNA structure can be assessed in situ, 2) translation may be altered by the secondary and tertiary structure of mRNAs, and 3) a predicted stem-loop structure exists in situ in the 5'-end of POMC mRNA.


Subject(s)
Pro-Opiomelanocortin/chemistry , RNA, Messenger/chemistry , Animals , Base Sequence , Cell Line , Cells, Cultured , Dexamethasone/pharmacology , Electrophoresis, Polyacrylamide Gel , Male , Mice , Microscopy/methods , Molecular Sequence Data , Oligonucleotide Probes , Pituitary Gland/chemistry , Pro-Opiomelanocortin/genetics , Rats , Rats, Sprague-Dawley , Sodium Fluoride/pharmacology , Transcription, Genetic
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