PubMed Health⌕ Search

Biomedical subjects

M Comb

Publications and source records attributed to M Comb.

At least 19 recordsLinked to original sources
PubMed1999

Survival- and death-promoting events after transient cerebral ischemia: phosphorylation of Akt, release of cytochrome C and Activation of caspase-like proteases.

Release of cytochrome c (cyt c) into cytoplasm initiates caspase-mediated apoptosis, whereas activation of Akt kinase by phosphorylation at serine-473 prevents apoptosis in several cell systems. To investigate cell death and cell survival pathways, the authors studied release of cyt c, activation of caspase, and changes in Akt phosphorylation in rat brains subjected to 15 minutes of ischemia followed by varying periods of reperfusion. The authors found by electron microscopic study that a portion of mitochondria was swollen and structurally altered, whereas the cell membrane and nuclei were intact in hippocampal CA1 neurons after 36 hours of reperfusion. In some neurons, the pattern of immunostaining for cyt c changed from a punctuate pattern, likely representing mitochondria, to a more diffuse cytoplasmic localization at 36 and 48 hours of reperfusion as examined by laser-scanning confocal microscopic study. Western blot analysis showed that cyt c was increased in the cytosolic fraction in the hippocampus after 36 and 48 hours of reperfusion. Consistently, caspase-3-like activity was increased in these hippocampal samples. As demonstrated by Western blot using phosphospecific Akt antibody, phosphorylation of Akt at serine-473 in the hippocampal region was highly increased during the first 24 hours but not at 48 hours of reperfusion. The authors conclude that transient cerebral ischemia activates both cell death and cell survival pathways after ischemia. The activation of Akt during the first 24 hours conceivably may be one of the factors responsible for the delay in neuronal death after global ischemia.

Animals↗
PubMed1994

Stress-induced regulation of a human proenkephalin-beta-galactosidase fusion gene in the hypothalamus of transgenic mice.

Transgenic mice expressing an Escherichia coli beta-galactosidase reporter gene under the control of 3 kilobases of human proenkephalin gene 5'-flanking sequence and 1.2 kilobases of 3'-flanking sequence exhibited an anatomically correct pattern of basal and stress-regulated transgene expression within the hypothalamus. Acute osmotic stress and hypovolemia induced transgene expression in neurons within both the paraventricular and supraoptic nuclei. Chronic osmotic stress resulted in dramatic induction of transgene expression in both nuclei. These results demonstrate that the information required for correct hypothalamic expression and stress regulation of the proenkephalin gene is contained within our fusion construct.

Animals↗
PubMed1994

Molecular mechanisms of stress-induced proenkephalin gene regulation: CREB interacts with the proenkephalin gene in the mouse hypothalamus and is phosphorylated in response to hyperosmolar stress.

We have established a transgenic model to facilitate the study of stress-induced gene regulation in the hypothalamus. This model, which uses a human proenkephalin-beta-galactosidase fusion gene, readily permits anatomic and cellular colocalization of stress-regulated immediate early gene products (e.g. Fos) and other transcription factors [e.g. cAMP response element-binding protein (CREB)] with the product of a potential target gene. Moreover, Fos provides a marker of cellular activation that is independent of the transgene. Hypertonic saline stress induced Fos in almost all cells in the PVN that exhibited basal expression of the proenkephalin transgene; however, all cells in which the transgene was activated by stress also expressed Fos. CREB was found in essentially all neurons. Gel shift analysis with and without antisera to Fos and CREB showed that AP-1 binding activity, containing Fos protein, was induced by hyperosmotic stress. However, Fos was not detected binding to the proenkephalin second messenger-inducible enhancer even in hypothalamic cell extracts from stressed animals. In contrast, CREB formed specific complexes with both the proenkephalin enhancer and a cAMP- and calcium-regulated element (CaRE) within the c-fos gene. Moreover, we found that hypertonic saline induced CREB phosphorylation in cells that express the transgene within the paraventricular nucleus and supraoptic nucleus. These results suggest a model in which proenkephalin gene expression in the paraventricular nucleus is regulated by CREB in response to hypertonic stress.

Animals↗
PubMed1994

Opioids modulate stress-induced proenkephalin gene expression in the hypothalamus of transgenic mice: a model of endogenous opioid gene regulation by exogenous opioids.

Stressful stimuli strongly induce proenkephalin gene expression within the paraventricular nucleus (PVN) of the hypothalamus. A human proenkephalin-beta-galactosidase fusion gene has previously been shown to give correct phenotypic expression and appropriate stress regulation within the hypothalamus of transgenic mice; this model provides high sensitivity, cellular resolution, and ready quantification of levels of proenkephalin gene expression. Here we describe use of this transgenic model to study modulation of stress-regulated gene expression in the PVN by opiates. Acute or subacute morphine administration prior to a hypertonic saline stress produced marked superinduction of transgene expression compared with hypertonic saline stress alone. In contrast, chronic morphine administration decreased basal expression of the transgene, and inhibited stress-induced expression of the transgene. The endogenous proenkephalin mRNA was induced in parallel with the transgene as demonstrated by in situ hybridization; the immediate-early gene c-fos was also regulated in parallel with the transgene. These data suggest that acute or subacute morphine administration sensitizes proenkephalin neurons within the PVN and other regions of the hypothalamus to stress and that chronic morphine administration desensitizes this response. Because the molecular mechanisms regulating the expression of the transgene are well understood, this model provides a useful tool for investigating cellular and molecular effects of opioids on the hypothalamus.

Animals↗
PubMed1990

CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2.

DNA methylation at HpaII (CmCGG) sites inhibits expression of a human proenkephalin-CAT fusion gene when it is transiently expressed in CV-1 cells or stably expressed in C6-glioma cells. The inhibitory effects of HpaII methylation have been mapped to a site within the human proenkephalin promoter located at position -72 relative to the start site of transcription. This region spans a cAMP and phorbol ester inducible enhancer and methylation at this position inhibits both basal transcription and transcription induced by either cAMP or TPA. The HpaII site is located within an element which binds the transcription factor AP-2. In vitro methylation at this HpaII site inhibits the binding of AP-2. These results suggest that CpG methylation inhibits proenkephalin gene expression by directly interfering with the binding of a positively acting transcription factor previously shown to be essential for maximal basal, cAMP, and TPA inducible transcription.

5-Methylcytosine↗
PubMed1990

The effect of depolarization on expression of the human proenkephalin gene is synergistic with cAMP and dependent upon a cAMP-inducible enhancer.

Membrane depolarization is a critical component of neural signaling; in recent years there also has been a great deal of evidence that membrane depolarization can regulate neural gene expression. Therefore, excitatory neurotransmission may be an important mechanism of neural plasticity. We have investigated the intracellular pathways and DNA regulatory elements through which membrane depolarization activates expression of the neural gene encoding human proenkephalin. In PC12 and C6-glioma cells, depolarization-induced expression of a transfected proenkephalin fusion gene was proportional to extracellular calcium concentration and was inhibited by verapamil. Activation of the gene by KCl-induced depolarization or the calcium ionophore A23187 was dependent upon and synergistic with cAMP in PC12 and C6-glioma cells, but neither depolarization nor treatment with A23187 affected cAMP levels. Trifluoperazine and W7 inhibited depolarization-induced gene expression but did not affect expression induced by the adenylyl cyclase activator forskolin. At the level of the DNA, depolarization-induced activation is conferred on the proenkephalin gene by a previously characterized cAMP-inducible enhancer. Multiple copies of a single component element of that enhancer, containing the CGTCA sequence motif characteristic of cAMP regulatory elements, can reconstitute the entire repertoire of responses to both cAMP and depolarization. These data suggest a model in which membrane depolarization activates gene expression through a calcium-dependent pathway, potentially involving calmodulin, and in which the transcriptional responses to both cAMP and calcium are transduced by the same DNA element.

Base Sequence↗
PubMed1990

Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture.

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5' to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT. A system for transfecting chromaffin cells in primary culture was therefore devised using constructs containing 200 bp of the proenkephalin (ENK) promoter, whose expression characteristics are well known. pENK beta GAL-1, containing the ENK promoter with a lac Z reporter, was introduced into these cells and beta-galactosidase activity was visualized in situ. Approximately 90% of cells transfected were chromaffin; transfection efficiency was 5%. High levels of CAT activity were measured in chromaffin cells transfected with pENKAT12, possessing a CAT reporter. In contrast to tumor cell lines, pENKAT12 induction in these cells by forskolin and phorbol esters did not require a phosphodiesterase inhibitor. In this chromaffin system, both basal and regulated expression of the PNMT fusion genes were detected. Dexamethasone (dex) induced expression of pPNMT3000 and pPNMT900, containing the putative GRE and 3000 bp or 863 bp of PNMT promoter sequence, 4- to 10-fold. Expression of pPNMT300 and pPNMT100, which lack the GRE and contain 273 bp or 99 bp of PNMT promoter sequence, was unaffected by dex. Addition of the PNMT region spanning -490 to -863 bp conferred full dex responsiveness to a thymidine kinase promoter. Deletion of the putative GRE sequence by site-directed mutagenesis abolished the dex response. These data identify the sequence at -513 bp in the rat PNMT gene as a functional, positively acting GRE. Primary cultures of bovine chromaffin cells provide a biologically relevant expression system for transcriptional studies of catecholamine genes and their related neuropeptides.

Animals↗
PubMed1988

Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription.

Synthesis of the endogenous opioid precursor, proenkephalin, is regulated by neurotransmitters and membrane depolarization. These events act through second messenger dependent signal transduction pathways via a short inducible DNA enhancer to regulate transcription of the proenkephalin gene. Two DNA elements located within this enhancer are essential for the transcriptional response to cAMP and phorbol ester. Inactivation of either element by mutation or by alteration of their stereospecific alignment eliminates inducible enhancer activity. The promoter distal element, ENKCRE-1, in the absence of a functional adjacent ENKCRE-2 element, has no inherent capacity to activate transcription. However, in the presence of a functional ENKCRE-2 element, this element synergistically augments cAMP and phorbol ester inducible transcription. The promoter proximal element, ENKCRE-2, is essential for both basal and regulated enhancer function. Four different protein factors found in HeLa cell nuclear extracts bind in vitro to the enhancer region. ENKTF-1, a novel enhancer binding protein, binds to the DNA region encompassing ENKCRE-1. The transcription factors AP-1 and AP-4 bind to overlapping sites spanning ENKCRE-2, and a fourth transcription factor, AP-2, binds to a site immediately downstream of ENKCRE-2. The binding of ENKTF-1 to mutant ENKCRE-1 sequences in vitro correlates with the in vivo inducibility of the mutant elements suggesting that ENKTF-1 acts in combination with factors that recognize the ENKCRE-2 domain to regulate cAMP inducible transcription. Together, the two DNA elements, ENKCRE-1 and ENKCRE-2 and the protein factors with which they interact, play a critical role in the transduction and reception of signals transmitted from cell surface receptors to the proenkephalin nuclear transcription complex.

Cyclic AMP↗
PubMed1988

A common trans-acting factor is involved in transcriptional regulation of neurotransmitter genes by cyclic AMP.

Activation of neurotransmitter receptors can regulate transcription in postsynaptic cells through the actions of second messengers. Trans-synaptic regulation of transcription appears to be an important mechanism controlling the synthesis of molecules involved in neuronal signaling, especially neuropeptides. Proenkephalin, vasoactive intestinal polypeptide, and somatostatin have been shown to be transcriptionally regulated by the second messenger, cyclic AMP (cAMP), as has the catecholamine synthesizing enzyme tryosine hydroxylase. cAMP-inducible elements have been mapped within these genes, and trans-acting factors which bind to several such elements have been identified. With the discovery that individual neurons generally contain multiple transmitters within their synaptic terminals, it has become important to understand in detail the mechanisms by which the synthesis of transmitters can be coregulated. Here we compare the structure and function of the proenkephalin cAMP-inducible enhancer with the mapped cAMP-inducible elements of the vasoactive intestinal polypeptide, somatostatin, and tyrosine hydroxylase genes and a putative cAMP-inducible element in the proto-oncogene c-fos. We have previously shown that the proenkephalin enhancer is composed of two different elements, ENKCRE-1 and ENKCRE-2. We show here that one of these, ENKCRE-2, is structurally similar to elements found within the vasoactive intestinal polypeptide, somatostatin, and tyrosine hydroxylase genes and binds a trans-acting factor that is competed for both in cotransfection experiments (in vivo) and in DNase I footprint assays (in vitro) by these other elements. The c-fos element has similar structural requirements to confer transcriptional induction by cAMP but competes less strongly. Protein purified by affinity chromatography with the ENKCRE-2 sequence binds to each of these elements. A second element within the proenkephalin cAMP-inducible enhancer, ENKCRE-1, binds a factor that is not competed for by these other genes and is therefore distinct. This analysis suggests a potential mechanism of transcriptional coregulation of the neuronally expressed genes investigated in this study and also demonstrates that multiple factors are involved in transcriptional activation by cAMP.

Binding, Competitive↗
PubMed1985

Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing.

A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.

Adrenocorticotropic Hormone↗
PubMed1984

Regulation of expression of opioid peptide genes.

In the past three years it has been shown by recombinant DNA approaches that there are at least three different genes that code for opioid peptides. The basic structures of the three polyprotein precursor molecules from which bioactive opioid peptides are derived are remarkably similar. There are also similarities in the structure of the genes that code for these precursor molecules. Using immunological techniques, it has been shown that the levels of the opioid peptides can be regulated by altering the rates of protein processing or secretion. Recently, complementary DNA clones of the opioid peptide precursor molecules have been used as hybridization probes to determine that regulation also occurs at the level of gene expression (transcription of the opioid peptide genes).

Adrenalectomy↗
PubMed1983

Primary structure of the human proenkephalin gene.

The nucleotide sequence of a 6.8-kb region of human DNA containing the proenkephalin gene and flanking regions is reported. The gene consists of four exons separated by three introns and spans approximately 5.3 kb of DNA. Location and identification of several repetitive DNA sequences within and flanking the gene are also described. The distribution of CpG dinucleotides as well as the extent of CpG methylation at several restriction sites within and surrounding the gene is also presented. The structural organization of the human proenkephalin gene exhibits striking similarities to the organization of the human pro-opiomelanocortin (POMC) gene. Nucleotide sequence homologies suggest that two opioid peptide precursors, proenkephalin and prodynorphin, may have arisen by duplication from a common ancestral gene.

5-Methylcytosine↗