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J Douglass

Publications and source records attributed to J Douglass.

At least 37 records · Page 2Linked to original sources
PubMed1995

Precipitated morphine withdrawal stimulates multiple activator protein-1 signaling pathways in rat brain.

Morphine dependence is a long lasting form of neuronal plasticity. Naloxone-precipitated morphine withdrawal, a model of opioid dependence, induces brain region-specific changes in activator protein-1 (AP-1) transcription factor gene expression. Rapid increases in c-fos, fos-B, jun-B, and c-jun mRNA levels accompany withdrawal, with the relative level of induction correlating with the severity of physical dependence. Altered patterns of c-fos mRNA expression were limited to neuronal circuits mediating stress responses, motivation, and cognition. AP-1 DNA-binding activity and dimer composition also exhibited regulation after withdrawal, presumably as a result of both transcriptional and post-translational events. Thus, morphine dependence results in the alteration of diverse, brain region-specific, signal transcription pathways involving AP-1 transcription factors.

Animals↗
PubMed1995

PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine.

Neuronal plasticity associated with both short- and long-term administration of psychomotor stimulants involves alterations in specific patterns of gene expression. In order to screen for brain region specific mRNAs which are transcriptionally regulated by acute cocaine and amphetamine, PCR differential display was employed. This approach identified a previously uncharacterized mRNA whose relative levels in the striatum are induced four- to fivefold by acute psychomotor stimulant administration. Isolation and characterization of corresponding cDNA clones resulted in complete nucleotide sequence analysis, including prediction of the encoded protein product. Alternate polyA site utilization in the predicted 3' noncoding region results in the appearance of an RNA doublet, approximately 700 and 900 bases in length, following Northern analysis. A presumed alternate splicing event further generates diversity within the transcripts, and results in the presence or absence of an in-frame 39 base insert within the putative protein coding region. As a result, the predicted translation products are either 129 or 116 amino acids in length. A common hydrophobic leader sequence at the amino terminus is present within each predicted polypeptide, suggesting that the protein product is targeted for entry into the secretory pathway. Basal expression of the RNA doublet is limited to neuroendocrine tissues, further implying that the protein product plays a functional role in both neuronal and endocrine tissues.

Amino Acid Sequence↗
PubMed1994

Basal and inducible transcriptional activity of an upstream AP-1/CRE element (DYNCRE3) in the prodynorphin promoter.

During chronic pain and inflammation, prodynorphin gene expression is elevated in the spinal cord. To characterize the molecular regulation of prodynorphin gene expression, we examined an AP-1/CRE-like element, TGCGTCA, located at -1545 in the prodynorphin gene (the DYNCRE3 site). Previous work in our laboratory demonstrated by gel shift analysis that Fos and non-Fos-containing complexes formed with oligonucleotides containing this element. To examine the functional significance of this site, constructs containing variable length regions of the prodynorphin promoter were transiently transfected into PC12 or HeLa cells. Constructs containing the DYNCRE3 site consistently permitted higher levels of transcriptional activity than those lacking this site. Furthermore, placement of upstream regions containing the DYNCRE3 site adjacent to the minimal promoter yielded transcriptional activity much greater than that in the presence of the native constructs. PC12 cells transfected with constructs containing the DYNCRE3 site responded to a far greater degree to forskolin stimulation than those transfected with constructs that did not contain this site. Mutation of the DYNCRE3 site (CTcgtca) markedly reduced forskolin-induced increases in transcriptional activity. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate produced little or no change in transcriptional activity. By examining successively more isolated fragments of prodynorphin promoter and by mutational analysis, we identify and characterize a 7-bp site, DYNCRE3, which, though largely unaffected by stimulations of the PKC pathway, dramatically responds to stimulations via the PKA second messenger pathway.

Animals↗
PubMed1994

Ontogeny of kainate-induced gene expression in rat hippocampus.

The ontogeny of kainate induction of AP-1 mRNAs, proteins, and DNA binding activities was examined in the rat hippocampus. In addition, kainate induction of preproenkephalin and preprodynorphin mRNAs was examined; these genes have been shown to be induced by kainate and have been suggested to be targets of AP-1 regulation in adult rat hippocampus. Despite producing seizures at postnatal day (P) 7, kainate failed to induce AP-1 or opiate gene expression and did not increase AP-1 DNA binding activity at this age. Basal levels of AP-1 and opiate mRNAs were low in P7 hippocampus. Basal levels of c-jun protein and AP-1 DNA binding activity were elevated in the P7 hippocampus, to values greater than induced levels in adult hippocampus. Furthermore, AP-1 DNA binding in P7 hippocampal nuclear extract was unaffected by antibodies against fos-related antigens, in contrast to hippocampal extracts from the older rats examined. At P14, induction of AP-1 and preproenkephalin (but not preprodynorphin) mRNAs was observed with kainate treatment, but the time course for inductions was delayed relative to kainate inductions in the adult hippocampus. At P21, responses to kainate were similar to the adult response. Unlike in adult hippocampus, seizure activity caused by kainate treatment does not increase the transcription factor and opioid peptide gene expression in the hippocampi of P7 rats.

Animals↗
PubMed1994

Identification of multiple DNA elements regulating basal and protein kinase A-induced transcriptional expression of the rat prodynorphin gene.

The prodynorphin gene encodes the precursor molecule from which the dynorphin family of opioid peptides is generated. The gene is transcriptionally active in a wide variety of brain regions and endocrine tissues. Much is known regarding the physiological and receptor-mediated events that regulate prodynorphin gene expression in vivo. However, the molecular mechanisms by which specific cis- and trans-acting factors control activity of the prodynorphin promoter are not as clearly defined. In the study described here, transient transfection of prodynorphin promoter-chloramphenicol acetyl transferase plasmid constructs into CV1 cells served to identify three nucleotide sequence elements conforming to cAMP regulatory element motifs which regulate both basal and protein kinase A (PKA)-induced transcription. The three elements are clustered at positions -1543, -1627, and -1659 relative to the RNA cap site. Site-specific mutagenesis further reveals that although the sites can act independently to positively regulate transcription from the prodynorphin promoter, they can also act combinatorially to produce maximal transcriptional efficacy. Gel retention analysis employing rat brain protein extracts also describes the ability of these sequence elements to form sequence-specific DNA/protein complexes.

Animals↗
PubMed1994

A STIMULUS PARADIGM FOR ANALYSIS OF NEAR-FIELD HYDRODYNAMIC SENSITIVITY IN CRUSTACEANS

We present several relatively simple procedures for studying the physiology of near-field mechanoreceptors in crustaceans which extend previous measures of sensitivity. The advantages include the quantitative analysis of range fractionation and directionality of receptors and interneurons in the sensory hierarchy of the central nervous system (CNS), based on a stimulus paradigm that is reproducible and easy to use. The technical considerations for quantitative fluid-coupled stimulation addressed by this paper are the complexity of dipole flow fields, reflected interference from traveling waves, and the underlying stimulus wave form. The techniques described here offer corresponding advantages for physiological experiments using other aquatic organisms. In electrophysiological experiments, crustacean preparations are typically placed in an experimental chamber filled with water or saline solution. For studies on near-field sensory receptors, i.e. those responding to flow fields in the aquatic medium, a dipole or vibrating sphere is frequently used to generate stimulus waves (Tautz et al. 1981; Wiese and Wollnik, 1983; Ebina and Wiese, 1984; Hatt, 1986; Heinisch and Wiese, 1987; Tautz, 1987; Wiese and Marschall, 1990; Killian and Page, 1992b). A dipole stimulator is easily constructed by attaching a spherical probe to an electromechanical device such as a loudspeaker, pen motor or piezo crystal. A periodic signal fed to the transducer generates the oscillating dipole movements. With the sphere immersed in the bathing medium, dipole flow fields are generated (see Kalmijn, 1988, for further discussion of dipole sources), whereas dipole oscillations introduced at the air­saline interface generate traveling surface waves. Numerous additional devices and techniques have been used to stimulate crustacean receptors. Several involve wave motion introduced from one end of the chamber by diaphragms or paddles (Laverack, 1962b, 1963; Flood and Wilkens, 1978), by cylindrical dippers (Wilkens and Larimer, 1972; Wiese et al. 1976; Wiese and Schultz, 1982; Reichert et al. 1983) or by water drops (Laverack, 1962b; Strandburg and Krasne, 1985). Another form of fluid-coupled stimulation involves small jets of saline (Laverack, 1963; Tautz, 1990; Schmitz, 1992). In other studies, receptor hairs have been stimulated directly by using a stylus in place of the dipole, e.g. a needle or glass capillary in contact with the hair (Laverack, 1962a; Killian and Page, 1992a; Yen et al. 1992; Nagayama and Sato, 1993) or a miniature wire loop or capillary tube placed over the hair shaft (Mellon, 1963; Wiese, 1976; Tautz et al. 1981; Killian and Page, 1992b).

Journal Article↗
PubMed1994

Cocaine differentially regulates activator protein-1 mRNA levels and DNA-binding complexes in the rat striatum and cerebellum.

Cocaine is a psychomotor stimulant that exerts many of its behavioral and physiological effects through alteration of catecholamine reuptake systems. One early cellular response to cocaine administration is a brain region-specific alteration in the transcriptional pattern of immediate early genes belonging to the Fos/Jun family of nucleotide sequence-specific [activator protein-1 (AP-1)] DNA-binding proteins. The work described here compares cocaine-induced transcriptional regulation of immediate early gene mRNA levels, as well as AP-1 DNA-binding activity, within the striatum and cerebellum. In the striatum, acute cocaine administration increases cellular levels of c-fos and jun-B mRNA, whereas transcriptional effects in the cerebellum are limited to c-fos mRNA. After chronic cocaine treatment a desensitization of c-fos mRNA induction is observed in the striatum, with sensitization of the same transcriptional effect occurring in the cerebellum. Pharmacological studies further reveal that the dopamine D1, dopamine D2, gamma-aminobutyric acid type B, and N-methyl-D-aspartate receptor systems mediate the effects of cocaine on cerebellar neurons, whereas striatal effects are modulated through D1 and N-methyl-D-aspartate receptors. Gel retention analysis using antibodies to the various Fos and Jun proteins was used to characterize cocaine-dependent alterations in the composition of striatal and cerebellar AP-1 DNA-binding complexes. In striatum, cocaine increases the relative levels of c-Fos, Fos-B, Jun-B, and Jun-D proteins that bind the AP-1 DNA sequence element, whereas in the cerebellum only c-Fos and Jun-D binding activities are increased. These data suggest two possible neuroanatomical sites where tolerance and sensitization to cocaine can be examined at the genomic level.

Animals↗
PubMed1993

In vivo and in vitro phosphorylation of the T lymphocyte type n (Kv1.3) potassium channel.

The major species of voltage-gated potassium channel found on mammalian T lymphocytes is referred to as the type n channel. This potassium channel exhibits unique functional properties which distinguish it from other species of potassium channels, including a potential role in the onset of cellular events associated with T cell activation. As a first step in characterizing specific biochemical properties of the type n channel, we have generated polyclonal antisera against bacterial fusion proteins containing peptide regions unique to the mouse and human type n channel. From membranes of T cell lines derived from both mouse (SAK 8 cell line) and human (Jurkat cell line), the type n channel can be immunoprecipitated following either surface labeling with 125I or metabolic labeling with 32P. The apparent molecular mass of the immunoprecipitated type n channel is approximately 65 kDa, significantly greater than that of the 58-kDa in vitro translated product, and suggestive of post-translational modification events. Phosphoamino acid analysis of the metabolically labeled Jurkat type n channel reveals phosphorylation of serine residues exclusively. In vitro studies also describe the ability of both protein kinase A and protein kinase C to phosphorylate the Jurkat type n channel. The former kinase also appears to phosphorylate a 40-kDa protein which co-immunoprecipitates with the type n channel. These data suggest that direct phosphorylation of the T lymphocyte type n potassium channel or its associated 40-kDa subunit may serve as a means by which channel activity is regulated.

Amino Acid Sequence↗
PubMed1993

Kainate-induced changes in opioid peptide genes and AP-1 protein expression in the rat hippocampus.

In the rat hippocampus, jun, c-fos, and fos-related antigen immunoreactivity, AP-1 DNA binding, and opioid peptide gene expression were examined after kainate treatment to determine whether the induction and DNA binding of AP-1 transcription factors are correlated with the expression of the opioid peptide genes. One and one-half hours after kainate administration, fos-related antigen and jun immunoreactivity and AP-1 DNA binding were induced; maximal elevation was observed after 4.5 h. Transcription factor expression and DNA binding increased in a dose-dependent manner. Preprodynorphin and preproenkephalin mRNA induction was also dose dependent. The anticonvulsants, pentobarbital and diazepam, effectively blocked electroencephalographic seizure activity caused by kainate treatment, whereas valproic acid was approximately 50% effective. Opioid peptide gene expression, fos-related antigen and jun immunoreactivity, and AP-1 DNA binding all reflected similar reductions after anticonvulsant treatment. Therefore, expression and DNA binding activity of the AP-1 transcription factors are correlated with opioid peptide gene expression in the rat hippocampus.

Animals↗
PubMed1992

Constitutive expression of AP-1 transcription factors in the rat adrenal. Effects of nicotine.

The AP-1 proteins (c-fos and fos-related antigens and jun proteins) are transcription factors that are induced in most cells by various stimuli, but the expression of these proteins is low or undetectable in the basal state. Our data indicate that the rat adrenal gland contains a basally expressed elevated level of AP-1 proteins and mRNAs (40- and 35-kDa fos-related antigens, a 39-kDa c-jun protein, c-jun, junB, and junD mRNA), as well as high basal AP-1 DNA binding activity. Both the medulla and cortex contained equivalent levels of fos-related antigens and c-jun protein; however, the majority of AP-1 DNA binding was detected in the medulla. Handling of rats for several days prior to sacrifice failed to affect protein expression or binding; on the other hand, repeated injections of nicotine increased AP-1 DNA binding. A single nicotine injection 60 min prior to removing the adrenals caused a significant reduction in AP-1 DNA binding without any detectable changes in AP-1 protein expression. Pretreatment of adrenal nuclear extracts with alkaline phosphatase prior to incubation with the AP-1 oligomer decreased binding activity. Thus, unlike most tissues where AP-1 DNA binding activity is controlled by increased expression of AP-1 proteins, DNA binding in the rat adrenal gland appears to be controlled at the post-translational level, possibly through dephosphorylation.

Adrenal Glands↗
PubMed1992

Characterization and functional expression of genomic DNA encoding the human lymphocyte type n potassium channel.

Voltage-gated potassium channels play important functional roles in the development and maintenance of human lymphocyte functions. One such channel, known as the type n channel, has been well defined in human T cells and exhibits unique functional properties that distinguish it from other species of potassium channels. We report the characterization of a human genomic DNA clone, HGK5, encoding a 523-amino-acid potassium channel protein encoded by an open reading frame on a single exon. RNA transcribed in vitro from HGK5 genomic DNA directs expression of functional voltage-dependent potassium currents in Xenopus oocytes. The functional characteristics of the expressed channels are strikingly similar to those of the type n channel on human T lymphocytes. This, together with the presence of significant levels of HGK5 mRNA in human T lymphocytes, supports the notion that HGK5 encodes the human type n voltage-gated potassium channel. The effects of concanavalin A treatment on HGK5 mRNA levels in cultured human T lymphocytes was also examined. Mitogenic concentrations of concanavalin A induced a time-dependent decrease in HGK5 mRNA levels, suggesting that previously observed increases in potassium current density following concanavalin A treatment of human T lymphocytes are not due to increased transcriptional activity of the type n potassium channel gene.

Amino Acid Sequence↗
PubMed1992

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.

Animals↗
PubMed1991

In situ histochemical analysis of region-specific gene expression in the adult rat epididymis.

The epididymal tubule is a dynamic structure, in which spermatozoa undergo distinct physiological and morphological changes. The epithelial cells lining the ductuli vary dramatically in their histochemical and cytological properties according to the region of the tubule in which they are located. Additionally, regional variation is observed regarding the biosynthetic, secretory, and absorptive properties of the epithelial cells. Using in situ histochemical analysis, we document here the region-specific expression of a variety of genes that are transcriptionally active in the adult rat epididymis. Radiolabeled antisense riboprobes were used to localize, within the efferent duct/caput epididymis, transcripts encoding protein B/C, protein D/E (acidic epididymal glycoprotein), sulphated glycoprotein 1, sulphated glycoprotein 2, cellular retinol-binding protein, and the neuroendocrine peptide precursor proenkephalin. Each species of mRNA exhibits a unique pattern of hybridization, revealing that gene transcription within the efferent duct/caput epididymis is also highly region specific. This observation may partially elucidate the molecular basis underlying the phenomenon of regional alterations in the composition of protein factors within the tubule lumen.

Animals↗
PubMed1991

Systemic administration of kainic acid differentially regulates the levels of prodynorphin and proenkephalin mRNA and peptides in the rat hippocampus.

The effects of systemic kainic acid (KA) administration on hippocampal levels of prodynorphin and proenkephalin mRNA, as well as opioid peptides derived from these precursors, were evaluated. A single subcutaneous injection of KA induced a range of seizure states, from mild wet dog shakes to generalized motor seizures. Northern blot analysis of hippocampal mRNA revealed an increase in both prodynorphin and proenkephalin mRNA levels which corresponded to the intensity of the convulsions. Conversely, hippocampal levels of immunoreactive dynorphin A (1-8) and [Met]5-enkephalin were decreased as a function of seizure frequency and intensity. The time course of KA-induced alterations in prodynorphin and proenkephalin mRNA and peptide levels was also investigated. Hippocampal prodynorphin mRNA levels rose at a dramatic rate. At 3 h following KA administration, mRNA levels were maximally elevated approximately 13-fold. The levels decreased over a 48 h period, eventually reaching control values. In contrast, proenkephalin mRNA levels increased more slowly. At 24 h, a maximal 24-fold increase was observed. At 72 h after injection, proenkephalin mRNA levels were still slightly elevated. In the same experiment, immunoreactive enkephalin peptide levels, although somewhat decreased at 3-12 h, began to increase between 12 and 24 h after injection, and were still rising at 72 h. In marked contrast, immunoreactive dynorphin peptide levels ranged from 40% to 80% of control values at all times tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗