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Biomedical subjects

R Spangler

Publications and source records attributed to R Spangler.

At least 37 records · Page 2Linked to original sources
PubMed1996

Guinea pig preprodynorphin mRNA: primary structure and regional quantitation in the brain.

We isolated and sequenced genomic and cDNA clones of the guinea pig preprodynorphin (ppDyn) mRNA. The sequence of ppDyn mRNA was deduced from a combination of genomic and cDNA clones: The primary structure of two coding exons was derived from a genomic clone and 5' and 3' untranslated sequences were obtained using rapid amplification of cDNA ends (RACE). The predicted mRNA of 2,350 nucleotides coincides well with the size of transcripts in Northern blot analyses of RNA from different brain regions. The deduced amino acid sequence of guinea pig ppDyn shares 70%, 68%, and 61% identity to porcine, human, and rat ppDyn, respectively. The 5' untranslated sequences of guinea pig hippocampal and adrenal ppDyn mRNA are identical; both contain sequences of exon I and, like porcine mRNA, lack an exon (exon II) present in human and rat mRNA. Quantitative solution hybridization RNase protection analysis of total RNA from selected guinea pig brain regions was performed. The nucleus accumbens was found to have the greatest abundance of ppDyn mRNA, followed by caudate putamen, hippocampus, hypothalamus, amygdala, frontal cortex, olfactory bulb, and pons/medulla.

Adrenal Glands↗
PubMed1996

Corticotropin-releasing factor and type 1 corticotropin-releasing factor receptor messenger RNAs in rat brain and pituitary during "binge"-pattern cocaine administration and chronic withdrawal.

Endogenous central corticotropin-releasing factor (CRF) may be involved in the neuroendocrine and behavioral responses to cocaine. In the present study, levels of CRF mRNA were measured in the hypothalamus and in several extrahypothalamic brain regions after different regimens of "binge"-pattern cocaine administration. Male Fischer rats were injected with saline or cocaine (15 mg/kg, 1 hr x 3/day) at the beginning of the light cycle, to approximate the manner in which cocaine is often abused by humans, both in terms of temporal pattern and in relation to circadian rhythm. Cocaine administered in this binge regimen produced time-dependent alterations of CRF mRNA levels in the hypothalamus. There was a significant increase in CRF mRNA levels on day 1, which returned to base-line levels on day 2, with elevated plasma corticosterone levels on both days. CRF receptor type 1 and prooplomeianocortin mRNA levels in the anterior lobe of the pituitary were not significantly altered after acute cocaine injections on day 1 or day 2. On day 14 of chronic binge-pattern cocaine administration, decreased hypothalamic CRF mRNA levels and an attenuated elevation in plasma corticosterone levels were found. After 10 days of withdrawal from 14-day binge cocaine, CRF mRNA returned to basal levels. CRF mRNA levels in the amygdala were also significantly increased on day 1 and returned to basal values on day 2. Chronic (14-day) binge cocaine administration did not alter CRF mRNA levels in the amygdala. These results suggest that the attenuated response in the hypothalamic-pituitary-adrenal axis to chronic binge cocaine administration is coupled to the cocaine-induced decreases in CRF gene expression in the hypothalamus and that activation of CRF gene expression in extrahypothalamic regions may have implications for a molecular understanding of the behavioral responses to cocaine.

Adrenocorticotropic Hormone↗
PubMed1994

Nabumetone, an effective anti-inflammatory agent, lacks gastrointestinal irritancy in the rat when dosed orally for one month: comparison with tiaprofenic acid and etodolac.

OBJECTIVE: To determine the effects of nabumetone, compared with tiaprofenic acid and etodolac, on anti-inflammatory efficacy and gastrointestinal irritancy in the rat when dosed orally for one month at a high anti-inflammatory dose. METHODS: Carrageenan paw edema was used as a model of inflammation. Gastrointestinal mucosal integrity was assessed by concurrently measuring ulcer formation. mucosal and tissue prostanoid production and plasma haptoglobin. Haemoglobin, present in the cecal contents, was used as a measure of blood loss. RESULTS: Nabumetone, tiaprofenic acid and etodolac inhibited inflammation. Etodolac induced marked gastrointestinal damage and blood loss whereas tiaprofenic acid caused only gastric damage. Nabumetone was found not to alter mucosal integrity. CONCLUSION: Nabumetone proved to be an effective anti-inflammatory agent that was devoid of gastrointestinal irritancy.

Administration, Oral↗
PubMed1994

Comparison of the anti-inflammatory activity and gastrointestinal irritancy of nabumetone, ibuprofen, and diclofenac in rats following chronic administration.

Nabumetone, a novel nonsteroidal anti-inflammatory drug, has aroused considerable interest due to mounting evidence suggesting a diminished potential for causing gastrointestinal mucosal irritancy. In the present study, rats were administered equally effective oral suspensions of either nabumetone (79mg/kg), ibuprofen (88mg/kg), diclofenac (11.5mg/kg), or a control suspension (n = 8 or 9 per group) daily for one month and subsequently assessed for various indices of anti-inflammatory activity, gastrointestinal irritancy, and prostaglandin inhibitory activity. Daily doses were five times the ID25 for carrageenan-induced paw inflammation as obtained in previous studies. At the end of the administration period, nabumetone maintained effective antiinflammatory activity, but was devoid of gastrointestinal irritancy. In contrast, ibuprofen and diclofenac were associated with marked and significant gastrointestinal mucosal damage. The findings of the present study support published preclinical and clinical studies establishing nabumetone as an effective anti-inflammatory agent with a favourable gastrointestinal safety profile.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

'Binge' cocaine administration induces a sustained increase of prodynorphin mRNA in rat caudate-putamen.

Other workers have established that cocaine injections increase the levels of dynorphin peptides in the caudate putamen and substantia nigra of the rat brain. Using a quantitative solution hybridization protection assay for mRNA, we detected a significant increase in the concentration of prodynorphin mRNA in caudate putamen extracts of rats injected with cocaine following a 'binge' administration pattern designed to mimic human cocaine abuse. Increased prodynorphin mRNA was observed at the earliest time-point studied (50 h) and the lowest dose (10 mg/kg/day) of cocaine tested and persisted through the 14 day period studied. No prodynorphin mRNA was detected in the substantia nigra.

Animals↗
PubMed1992

c-myc is an erythropoietin early response gene in normal erythroid cells: evidence for a protein kinase C-mediated signal.

The proto-oncogene c-myc has been identified as an early response gene for erythropoietin (Epo) in transformed murine erythroleukemia cells. Epo activation of c-myc in these cells requires protein kinase C. We now show the fidelity of this signaling pathway in normal erythroid cells isolated from the spleens of phenylhydrazine-treated mice. Mouse spleen cells rich in erythroid progenitors were washed free of endogenous Epo and then incubated in the absence of Epo. Subsequent addition of Epo for 1 hour led to a dramatic elevation of c-myc transcript. Addition of the protein synthesis inhibitor cycloheximide did not prevent the c-myc response, thus identifying c-myc as an Epo early response gene in normal cells. We used this c-myc response as a reporter for signals initiated by the Epo receptor. Using a series of inhibitors with known specificities and established rank-orders of potency for different kinases, we determined that the c-myc response to Epo was blocked with the following rank order: staurosporine much greater than H7 greater than sangivamycin greater than H8. This sequence is identical to that obtained using transformed cells and is diagnostic of a protein kinase C-dependent signal. Because direct activation of protein kinase by phorbol esters does not induce terminal differentiation of normal cells, the pathway to c-myc established by these studies must represent one part of a signal transduction mechanism.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Erythropoietin induces cytosolic protein phosphorylation and dephosphorylation in erythroid cells.

Erythropoietin, the prime regulator of red blood cell growth and differentiation, causes rapid changes in the phosphorylation of several integral plasma membrane proteins (Choi, H-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936; Choi, H-S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J. (1990) J. Biol. Chem. 265, 4143-4148). In the present study we have demonstrated that erythropoietin's signal is transduced rapidly to the cytosol resulting in specific phosphorylation/dephosphorylation events. Erythropoietin treatment of Rauscher murine erythroleukemia cells previously labeled with [32P]orthophosphate results in a rapid increase in phosphorylation of two cytosolic proteins, designated pp96 and pp80, and a decrease in phosphorylation of another protein, designated pp90. The relative molecular mass and pI of pp80 are virtually identical to those reported for the protein kinase C substrate p80, or "MARCKS protein." Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate also increases pp80 but not pp96 phosphorylation, suggesting that erythropoietin triggers a protein kinase C-dependent pathway to pp80 and a protein kinase C-independent pathway to pp96. The effect of erythropoietin on pp96 phosphorylation was also shown in nontransformed erythroid cells isolated from the spleens of phenylhydrazine-treated mice. In contrast, almost no 32P labeling of pp80 or pp90 was detected, and pp80 and pp90 protein were nearly absent from these normal cells. These differences in expression and phosphorylation of erythropoietin-sensitive phosphoproteins may be related to the growth factor independence or dependence of the erythroid cells.

Animals↗
PubMed1991

Evidence that a G-protein transduces signals initiated by the protein-tyrosine kinase v-Fps.

The protein-tyrosine kinase (PTK) v-Fps induces protein kinase C (PKC)-dependent expression of the transformation-related 9E3 gene in chicken embryo fibroblasts (Spangler, R., Joseph, C., Qureshi, S.A., Berg, K., and Foster, D.A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7017-7021). We present evidence here that a GTP-binding protein (G-protein) is a component of this PKC-dependent signaling pathway. 1) A GTP analogue that stimulates G-protein-mediated signals induced 9E3 gene expression. 2) A GDP analogue that inhibits signaling through G-proteins inhibited expression of 9E3 and phosphorylation of a 67-kDa PKC substrate induced by v-Fps. The GDP analogue had no effect on phosphorylation of the PKC substrate or the expression of 9E3 induced by direct activation of PKC with phorbol ester. 3) Increased v-Fps PTK activity led to increased GTP binding to a 50-kDa protein. The molecular weight of this GTP-binding protein is consistent with the molecular weight of alpha-subunits of G-proteins of the heterotrimeric class. The data suggest that a G-protein functions upstream from PKC in a signaling pathway that connects v-Fps PTK activity to increased 9E3 gene expression.

Animals↗
PubMed1991

Erythropoietin activates the receptor in both Rauscher and Friend murine erythroleukemia cells.

Alterations in the expression of two proto-oncogenes, c-myb and c-myc, have been implicated in the differentiation of transformed erythroid cells induced by chemical inducers, such as dimethyl sulfoxide (Me2SO). In the present study, we compared the expression of c-myb and c-myc during erythropoietin (Epo) and Me2SO induction of Rauscher erythroleukemia cells, which differentiate in response to both inducers, and Friend erythroleukemia cells, in which Epo-induced differentiation is blocked. Our results demonstrate that Epo induces specific changes in expression of c-myb and c-myc in both Rauscher and Friend cells. Epo increases c-myc transcript, in contrast to a decreased caused Me2SO, indicating that the biphasic mode of c-myc regulation seen with Me2SO is not required for erythropoiesis. The Epo-induced changes in c-myb and c-myc do not require new protein synthesis, thus identifying these proto-oncogenes as early response genes for Epo. Both cell types also exhibit rapid changes in membrane protein phosphorylation in response to Epo. Since the signal pathway from Epo receptor activation to the nucleus appears equally functional in both Rauscher and Friend cells, the data suggest that the inability of Friend cells to differentiate in response to Epo is due to a block at a later step in the induction process.

Animals↗
PubMed1991

Erythropoietin increases c-myc mRNA by a protein kinase C-dependent pathway.

The peptide hormone erythropoietin is a major regulator of red blood cell production. While red blood cell development has been studied intensively, little is known about the intracellular signaling events that follow the binding of erythropoietin to its receptor on the target cell. We report here that erythropoietin-induced activation of the immediate early gene c-myc requires protein kinase C and that the binding of erythropoietin causes rapid phosphorylation of the major protein kinase C substrate, p80. Our results also argue for modulation of activity of a second signal transduction element in addition to protein kinase C.

Autoradiography↗
PubMed1991

Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Evidence that v-src and v-fps gene products use a protein kinase C-mediated pathway to induce expression of a transformation-related gene.

Induction of the transformation-related gene 9E3 by the v-src and v-fps gene products (v-Src and v-Fps) is blocked in chicken embryo fibroblasts depleted of protein kinase C (PKC). PKC agonists induce 9E3 gene expression. Protein kinase inhibitors block v-Src- and v-Fps-induced 9E3 gene expression with the same dose-response curves seen for PKC agonist-induced 9E3 gene expression. These data suggest that v-Src and v-Fps use a PKC-mediated signal-transduction pathway to induce expression of the transformation-related 9E3 gene. Consistent with this hypothesis, we find that both v-Src and v-Fps rapidly induce phosphorylation of a 67-kDa PKC substrate.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Purification and biological characterization of an adenovirus type 2 E1A protein expressed in Escherichia coli.

The adenovirus 2 E1A gene encodes a multifunctional protein of 289 amino acids that can immortalize primary rodent cells and transcriptionally activate a number of viral and cellular genes. To facilitate an understanding of the molecular basis for the various actions of E1A, we have redesigned our bacterial expression vector (Ko, J.-L., and Harter, M. L. (1984) Mol. Cell. Biol. 4, 1427-1439) containing the cloned E1A gene such that a soluble authentic E1A protein now constitutes approximately 1.5% of the total Escherichia coli cellular protein. Further, we have developed a simple rapid purification scheme without the use of detergents or denaturants and show a purity of greater than 98% with a yield of approximately 53%. The E1A so purified is biologically active, stimulating cellular DNA synthesis following microinjection into quiescent NIH 3T3 and REF52 cells. In another report (Spangler, R., Bruner, M., Dalie, B., and Harter, M. L. (1987) Science 237, 1044-1046) we have also shown that our purified E1A protein activates transcription from appropriate promoters in an in vitro system.

Adenovirus Early Proteins↗
PubMed1987

Activation of adenovirus promoters by the adenovirus E1A protein in cell-free extracts.

The primary product of the adenovirus E1A gene is a protein that is sufficient for controlling host-cell proliferation and immortalizing primary rodent cells. The mechanism by which the protein induces these cellular effects is poorly understood, but might be linked to its ability to regulate RNA transcription from a number of viral and cellular genes. The mechanism of E1A's transcriptional-activation (trans-activation) was studied here by monitoring the protein's effect on specific adenovirus promoters in two types of transcriptional systems in vitro. One of these systems consisted of extracts from transformed cells constitutively expressing E1A, and the other consisted of extracts of HeLa cells supplemented with a plasmid-encoded E1A protein purified from Escherichia coli. The results show that the E1A protein specifically stimulates transcription from adenovirus promoters; thus, the induction of cellular transcription factors is not necessary to explain the stimulation of transcription by E1A.

Adenoviridae↗
PubMed1985

Colicin synthesis and cell death.

Colicin E1 is a small plasmid, containing the cea gene for colicin, the most prominent product of the plasmid. Colicin is a 56-kilodalton bacteriocin which is especially toxic to Escherichia coli cells that do not contain the plasmid. Under normal growth conditions very low levels of the plasmid are produced as a result of cea gene repression by the host LexA protein. Conditions that lower the concentration of LexA protein result in elevated levels of colicin synthesis. The LexA protein concentration can be lowered by exposing the cells to DNA-damaging reagents such as UV light or mitomycin C. This is because DNA damage signals the host SOS response; the response leads to activation of the RecA protease which degrades the LexA protein. DNA-damaging reagents result in very high levels of colicin synthesis and subsequent death of plasmid-bearing cells. Elevated levels of colicin are also produced in mutants of E. coli that are deficient in LexA protein. We found that comparably high levels of colicin can be produced in such mutants in the absence of cell death. In lexA strains carrying a defective LexA repressor, colicin synthesis shows a strong temperature dependence. Ten to twenty times more colicin is synthesized at 42 degrees C. This sharp dependence of synthesis on temperature suggests that there are factors other than the LexA protein which regulate colicin synthesis.

Bacterial Proteins↗
PubMed1982

Yucatan miniature swine as a model for the study of human diabetes mellitus.

Two lines of Yucatan miniature swine have been developed by genetic selection for decreased and increased glucose clearance during an i.v. glucose tolerance test. The decreased glucose clearance is due to low peripheral insulin levels usually from a decrease in insulin secretion. In two pigs the decreased peripheral insulin resulted from increased hepatic uptake of insulin. During gestation and lactation, an increased insulin resistance and a diabetic-like state occurs in some animals. When fed a low fiber diet that contains 40% of calories from saturated fat (a typical American diet), Yucatan swine become more glucose intolerant. Seven to ten animals on the diet from the glucose intolerant line showed a glucose tolerance curve that would have been classified as diabetic if they were humans utilizing both intravenous and oral glucose tolerance tests. Miniature swine from this line are available to interested researchers.

Animals↗