Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A fusion gene containing 3 kilobases of human proenkephalin 5'-flanking sequences and 1 kilobase of human proenkephalin 3'-flanking sequence and the easily visualized histochemical marker, Escherichia coli beta-galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct.
...
PMID:Expression and regulation of a proenkephalin beta-galactosidase fusion gene in the reproductive system of transgenic mice. 143 91

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.
...
PMID:Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture. 230 57

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage lambda. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.
...
PMID:Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms. 246 43

Optimal conditions for formation of calcium phosphate-DNA precipitates and for chromaffin cell transfection by the calcium phosphate method were examined. A relationship was observed between turbidity of calcium phosphate solutions and the ability of calcium phosphate-DNA mixtures to give efficient transfection of bovine chromaffin cells. Under optimal conditions up to 35% of chromaffin cells in cultures transfected with plasmid DNA encoding human proenkephalin or Escherichia coli beta-galactosidase expressed the respective proteins. Important factors for transfection were the pH (6.95) and buffer employed for calcium phosphate-DNA precipitate formation, the amount and type of DNA, and the absence of serum in the cultures. Additionally, phosphate and calcium concentrations in the culture medium during incubation of cells with DNA are critical. Optimal conditions for transfection of chromaffin cells were also useful for transfection of clonal BSC-40 cells, an African green monkey kidney cell line. These results suggest that the optimal conditions described here for chromaffin cells may have broad applicability to other cell types. In addition, the results suggest that it is possible to optimize the solutions used for transfection conditions by monitoring calcium phosphate formation.
...
PMID:Optimization of calcium phosphate transfection for bovine chromaffin cells: relationship to calcium phosphate precipitate formation. 779 20

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.
...
PMID:Opioids modulate stress-induced proenkephalin gene expression in the hypothalamus of transgenic mice: a model of endogenous opioid gene regulation by exogenous opioids. 799 74

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.
...
PMID:Stress-induced regulation of a human proenkephalin-beta-galactosidase fusion gene in the hypothalamus of transgenic mice. 815 26

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.
...
PMID: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. 817 Apr 80

Gonadal steroids and physiological stressors affect the regulation of proenkephalin (PPE) gene expression in the paraventricular (PVN) and ventromedial (VMH) hypothalamic nuclei. To examine the effects of these modulators at the cellular level, the current study utilized a transgenic mouse line that expresses a human proenkephalin promoter/bacterial beta-galactosidase fusion gene (ENK-1). Previous studies have demonstrated that the regulatory sequences included in this transgene are sufficient to support appropriate transcriptional regulation of the reported gene in the PVN of male ENK-1 mice in response to stress. The present experimental paradigm was designed to examine possible interactions of sex and circulating estrogen levels with the opioid responses to acute systemic stressors, an intraperitoneal injection of hypertonic (1.5 M) or isotonic (0.15 M) saline. Adult ENK-1 mice were gonadally intact, gonadectomized, or 21 days postpartum. Forty-eight hours before perfusion, castrated males and ovariectomized females received either 10 micrograms estradiol benzoate or oil vehicle and 4 animals per group received no further treatment. Six h before perfusion, remaining animals received a single intraperitoneal injection of either hypertonic or isotonic saline. Tissues were sectioned through the hypothalamus and processed for X-gal histochemistry. In the VMH of ovariectomized females that received isotonic saline, estrogen significantly elevated transgene expression. This effect was not seen in females that only received estrogen or in those that received the severe systemic stressor of a injection of hypertonic saline. Estrogen and stress did not interact to elevate transgene expression in the VMH of males. A different pattern of expression was observed in the PVN; injection of hypertonic saline induced transgene expression only in gonadally intact males and in castrated males given estrogen. These findings demonstrate that stress and estrogen have sex-specific and site-specific regulatory effects on the expression of a PPE promoter transgene in hypothalamic neurons.
...
PMID:Estrogen and stress interact to regulate the hypothalamic expression of a human proenkephalin promoter-beta-galactosidase fusion gene in a site-specific and sex-specific manner. 914 95

GABA, which is present at high levels within the paraventricular nucleus (PVN) of the hypothalamus, has been implicated in neuroendocrine regulation. Here we use a transgenic mouse model expressing a human proenkephalin-beta-galactosidase fusion gene, in which both up-regulation and down-regulation of gene expression can be measured easily, to study the effects of GABA on basal and stress-induced gene expression within the PVN. This model has been shown previously to be appropriately physiologically regulated by stress within the PVN. Acute systemic administration of GABA, of aminooxyacetic acid, and of the selective GABA(B) receptor agonist, baclofen, induces transgene expression in the PVN. Chronic administration of aminooxyacetic acid inhibits both basal and stress-induced transgene expression. Acute or chronic administration of the selective GABA(A) agonist, muscimol, inhibits both basal and stress-induced expression of the transgene. Muscimol also inhibits transgene expression in hypothalamic slice cultures in which extrahypothalamic afferents are removed. It is surprising that, following chronic aminooxyacetic acid administration, the opiate antagonist naltrexone induces transgene expression in a manner similar to that observed with naloxone-precipitated opioid withdrawal and that expression is accompanied by a behavioral syndrome that partially mimicks opioid withdrawal. Taken together with our previous work, and using gene expression as our read-out, we conclude that transgene-expressing PVN neurons receive tonic inhibitory inputs mediated via GABA(A) receptors. Moreover, these inhibitory inputs can themselves be inhibited via GABA(B) and mu-opioid receptors.
...
PMID:Proenkephalin gene regulation in the paraventricular nucleus by GABA: interactions with opioid systems in a transgenic model. 945 54

Immunologic challenge with lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) produces a functional response within the paraventricular nucleus of the hypothalamus (PVN) and leads to changes in gene expression within PVN neurons. Regulated expression of neuropeptide genes within neurons of the PVN is a potential mechanism by which an organism can adapt to stressful challenges. Here, the authors used a transgenic mouse model in which expression of a readily measurable beta-galactosidase reporter was driven in PVN neurons by human proenkephalin regulatory sequences. This proenkephalin-beta-galactosidase transgene has been demonstrated previously to respond appropriately to a variety of stressors. It is demonstrated that expression of the proenkephalin transgene product was up-regulated significantly in a subset of PVN neurons 6 hours following intraperitoneal LPS (16-400 microg/kg) administration, remained elevated at 12 hours, and fell below basal levels by 24 hours. A more rapid and transient pattern of transgene up-regulation in the PVN followed administration of intraperitoneal IL-1beta (10 microg/kg) with significant induction by 2 hours, peak levels reached by 4 hours, and a return toward basal levels by 6 hours. IL-1beta (10-50 ng/mouse) administered intracerebroventricularly also led to up-regulation of the transgene 6 hours following infusion. Transgene expression was not up-regulated in hypothalamic slice cultures treated directly with IL-1beta (5-10 ng/ml media). Up-regulation of transgene expression does not appear to result from local action of IL-1beta at the level of the PVN but, rather, through as yet unidentified intermediates. The authors demonstrate phosphorylation of the cyclic amino-3-hydroxy-5-methyl-4-isoxazolepropionate response element binding protein, a transcription factor known to interact with proenkephalin regulatory sequences within the transgene, in the PVN following LPS administration. LPS induced up-regulation of the transgene was blocked by pretreatment with naltrexone, indicating an additional role for endogenous opioid systems in regulation of the PVN response to immune challenge.
...
PMID:Proenkephalin transgene regulation in the paraventricular nucleus of the hypothalamus by lipopolysaccharide and interleukin-1beta. 1002 10


1 2 Next >>

---