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Biomedical subjects

C Hough

Publications and source records attributed to C Hough.

29 records · Page 2Linked to original sources
PubMed1995

Carbamazepine induction of apoptosis in cultured cerebellar neurons: effects of N-methyl-D-aspartate, aurintricarboxylic acid and cycloheximide.

We have previously demonstrated that carbamazepine (CBZ) at concentrations above the therapeutic range is toxic to cultured cerebellar granule cells. Here, we ask whether the effect of CBZ involves neuronal apoptosis or necrosis. Treatment of cultured cerebellar granule cells with CBZ for 3 days resulted in a concentration-dependent fragmentation of DNA revealed as a laddered pattern in agarose gel electrophoresis, a phenomenon characteristic of apoptosis. Pretreatment of cells with N-methyl-D-aspartate (NMDA) blocked CBZ-induced DNA fragmentation and neurotoxicity as assayed by loss of mitochondrial activity with MTT or by [3H]ouabain binding to Na+/K(+)-ATPase. Aurintricarboxylic acid (ATA), a polyanionic dye, also markedly suppressed DNA fragmentation and cell death detected by morphological examination. A considerable level of DNA ladder formation was detected in untreated cells and this basal DNA fragmentation was also blocked by NMDA and ATA. Moreover, NMDA and ATA prevented CBZ-induced chromatin condensation as revealed by DNA binding with the fluorescent dye Hoechst 33258. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, prevented CBZ-induced cell death detected morphologically and attenuated CBZ-induced neurotoxicity assessed by mitochondrial activity and [3H]ouabain binding assays. Taken together, our results suggest that CBZ-induces death of cerebellar granule cells by an apoptotic process that is sensitive to NMDA, ATA and cycloheximide.

Animals↗
PubMed1994

Regulation of beta-adrenergic receptor mRNA in rat C6 glioma cells is sensitive to the state of microtubule assembly.

Microtubule disrupter, colchicine, and microtubule stabilizer, taxol, were used to determine whether microtubules play a role in beta-adrenergic receptor mRNA homeostasis and agonist-induced down-regulation in C6 glioma cells. Colchicine treatment had significant, differential, time-dependent effects on constitutive beta 1- and beta 2-adrenergic receptor mRNA levels. These effects stemmed from the action of colchicine on microtubules, because beta-lumicolchicine, an inactive isomer, had no effect, and nocodazole, a structurally unrelated microtubule disrupter, had similar effects. Colchicine treatment had little effect on the total number of beta-adrenergic receptor binding sites as measured by (-)-[125I]iodopindolol binding, but did alter the relative proportion of beta 1- and beta 2-adrenergic receptor subtypes. Colchicine also had no effect on basal cyclic AMP levels. In contrast to colchicine, taxol treatment had little long-term effect on either beta 1- or beta 2-adrenergic receptor mRNA levels. Taxol antagonized the effects of colchicine on total binding and mRNA levels. Taxol treatment increased basal cyclic AMP levels fourfold and potentiated (-)-isoproterenol-induced cyclic AMP production. Colchicine pretreatment completely inhibited (-)-isoproterenol-induced down-regulation of beta 1-adrenergic receptor mRNA, but not that of beta 2-adrenergic receptor mRNA. Taxol pretreatment had little effect on isoproterenol-induced beta-adrenergic receptor mRNA down-regulation. Colchicine pretreatment also attenuated isoproterenol-induced receptor down-regulation and inhibited agonist-stimulated cyclic AMP production. These effects of colchicine were antagonized by taxol. Whereas the effects of taxol and colchicine on isoproterenol-induced down-regulation of beta-adrenergic receptor mRNA are consistent with their effects on cyclic AMP production, those of colchicine in the absence of stimulation must involve other mechanisms. The data demonstrate that the state of microtubule assembly can affect cyclic AMP levels, beta 1- and beta 2-adrenergic receptor mRNA, and binding site levels in C6 glioma cells.

Animals↗
PubMed1993

Agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells.

Cerebellar granule cells express m2- and m3-muscarinic acetylcholine receptors (mAChRs) and their corresponding mRNA with m3-mAChR being the predominant receptor subtype. After stimulation with the mAChR agonist, carbachol, m2- and m3-mAChR mRNA levels were decreased in a time- and concentration-dependent manner with the maximal down-regulation at 2 and 8 hr, respectively. Immunoprecipitation studies revealed that amounts of m2- and m3-mAChR protein also decreased at 8 and 24 hr, respectively. The carbachol-induced down-regulation of m3-mAChR mRNA was associated with a decrease in the transcription rate, but a substantial enhancement of the mRNA stability. Upon removal of carbachol after treatment for 8 hr, the levels of m3-mAChR mRNA and mAChR binding sites returned to their original values with a t1/2 of approximately 80 min and 6 hr, respectively. The carbachol-elicited loss of m2- and m3-mAChR mRNA was blocked by their corresponding receptor subtype-specific antagonists, AF-DX 116 (m2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (m3-selective), and was concurrent with an increase in c-fos mRNA levels. Exposure of granule cells to the nonselective mAChR antagonist, atropine, caused a time- and concentration-dependent increase in the level of both m2- and m3-mAChR mRNA and mAChR binding sites. At 24 hr, immunoprecipitable m3-mAChR protein was predominantly increased. The atropine-induced up-regulation of m3-mAChR mRNA was concurrent with a marked enhancement of the mRNA stability and its transcription rate. The elevated levels of m3-mAChR mRNA and binding sites declined to their untreated values after the removal of atropine. Treatment with AF-DX 116 and 4-DAMP also produced an increase in the level of m2- and m3-mAChR mRNA and their corresponding immunoprecipitable receptor protein. These results demonstrate that the mAChR agonist and antagonist induce a down- and up-regulation of mAChR expression, respectively, through receptor-mediated mechanisms in cerebellar granule cells. Moreover, at least for m3-mAChR mRNA, the agonist- and antagonist-induced effects are reversible and associated with corresponding changes in the transcription rate of this receptor mRNA species.

Animals↗
PubMed1993

Paradoxical increase of 5-hydroxytryptamine2 receptors and 5-hydroxytryptamine2 receptor mRNA in cerebellar granule cells after persistent 5-hydroxytryptamine2 receptor stimulation.

Rat cerebellar granule cells express 5-hydroxytryptamine (5-HT)2 receptors that mediate phosphoinositide turnover by a pertussis toxin-sensitive mechanism. Prestimulation of these neurons with 10 microM 5-HT or (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane [(+/-)-DOI], a putative 5-HT2 receptor agonist, resulted in a time-dependent desensitization of the phosphoinositide response to 5-HT. The desensitization was detected within 30 min after prestimulation and reached a maximum (about 80%) decrement at 8 hr. However, [3H]ketanserin binding to 5-HT2 receptors in crude membranes or intact cerebellar granule cells was increased by treatment with 5-HT or DOI, in a time- and concentration-dependent manner. The increase occurred after the onset of desensitization and was fully manifest (about 160-190%) at 4 hr after stimulation. Although the Bmax and Kd were unchanged at 1 hr after 5-HT or DOI treatment, both parameters were significantly increased at 4 and 24 hr. The amount of 5-HT2 receptor mRNA detected by Northern blot hybridization using a 5-HT2 receptor-specific riboprobe was increased in parallel with the up-regulation of 5-HT2 receptor binding sites. Thus, an increase in 5-HT2 receptor mRNA was detected within 2 hr after 5-HT or DOI prestimulation, reached a maximum around 4 hr, and remained at a plateau for at least 24 hr. The levels of total RNA, m3 muscarinic acetylcholine receptor mRNA, and beta-actin mRNA were not significantly affected by these treatments. Our results demonstrated that 5-HT2 receptor binding sites and their mRNA undergo a paradoxical induction during persistent agonist stimulation.

Amphetamines↗
PubMed1991

Expression and agonist-induced down-regulation of mRNAs of m2- and m3-muscarinic acetylcholine receptors in cultured cerebellar granule cells.

The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.

Animals↗
PubMed1990

Differential down-regulation of beta 1- and beta 2-adrenergic receptor mRNA in C6 glioma cells.

C6 glioma cells possess both beta 1- and beta 2-adrenergic receptors. In response to exposure to isoproterenol, these cells down-regulated the mRNA for both beta 1- and beta 2-adrenergic receptors in a manner that indicates an independent regulatory mechanism for each subtype. In particular, the mRNA species for the beta 1-adrenergic receptor initially increased two-fold during the first hour of exposure before decreasing to 40% of initial levels at 4 hours after exposure. In contrast, the beta 2 mRNA species decreased rapidly and monotonically to 20% of initial levels by 2 hours. The unique response to isoproterenol of each subtype was blocked by the appropriate subtype-specific antagonists, betaxolol and ICI 118,551. In addition, beta-adrenergic receptor mRNA down-regulation was observed in association with contact inhibition, suggesting that events other than receptor occupancy can regulate beta-adrenergic receptor mRNA levels.

Adrenergic beta-Antagonists↗
PubMed1985

The association of human c-Ha-ras sequences with chromatin and nuclear proteins.

As a step towards the understanding of possible relationship between chromatin organization and regulation of the oncogene expression, we have investigated the chromatin structure of one of the more frequently activated oncogenes, c-Ha-ras, in HeLa-S3 cells. This was accomplished by isolation of the chromatin fractions (soluble and insoluble) after micrococcal nuclease digestion of purified nuclei and probing for the distribution of ras sequences. The polynucleosomal fraction was further resolved by sucrose gradient sedimentation. Southern-blot hybridization of the DNA isolated from various fractions yielded following results: (1) c-Ha-ras sequences segregated predominantly in the lysate fraction. (2) Unlike the B-globin (transcriptionally inactive) sequences, ras-H associated chromatin lacked typical nucleosomal packaging. Furthermore, since post-translational modifications of nuclear proteins have been suggested to modulate the nucleosome structure during DNA transcription and replication, ras sequences, in polynucleosomes immunofractionated on anti-poly (ADP-Ribose) Sepharose were also examined. The data suggested that the major class of this oncogene sequence exists in chromatin more distal to the sites of this particular chromatin modification.

Base Sequence↗
PubMed1984

Antifreeze protein genes of the winter flounder.

A genomic library of the winter flounder in lambda phage Charon 4 was screened with antifreeze protein cDNA. Nine genomic clones were isolated from three haploid genome equivalents of phage and were grouped into three classes on the basis of restriction mapping and heteroduplex analysis. The three chromosomal regions are non-overlapping and each contain two antifreeze protein genes spaced from 3 to 7 kilobase pairs apart. Genomic Southern blots indicate that these six non-allelic genes represent only 10-20% of the complement of this large multigene family. One gene has been identified by sequencing as a variant of the gene encoding the most abundant antifreeze protein. This gene is 1.0 kilobase pair long and contains an intervening sequence of 0.6 kilobase pairs between the coding region for the bulk of the signal sequence and that coding for the proprotein.

Amino Acid Sequence↗