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R T Boyd

Publications and source records attributed to R T Boyd.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of a rat neuronal nicotinic acetylcholine receptor alpha7 promoter.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the alpha7 subunit are expressed in the central nervous system, autonomic nervous system, retina, adrenal medulla, and PC12 cells. alpha7 nAChRs have been implicated in several important biological activities apart from synaptic transmission such as mediating neurite growth and presynaptic control of neurotransmitter release. A 178-base pair promoter was sufficient to drive high level expression of the alpha7 gene in PC12 cells. The alpha7 promoter was also cell-specific, expressing in PC12 cells but not in L6 rat muscle cells. Within our minimal rat alpha7 nAChR promoter we identified two sequences important for basal level expression. Mutation of a GC-rich sequence at -172 relative to the translational start site led to an increase in activity of the promoter, indicating the presence of a negative regulatory element. Upstream stimulatory factor-1 acted to regulate alpha7 expression positively by binding to an E-box at -116. A site directly adjacent to the upstream stimulatory factor-1 binding site was shown to bind Egr-1. Sp1 and Sp3 binding also occurred downstream from or overlapping the Egr-1 binding site in the rat alpha7 promoter. Several transcription factors interact in close proximity to control expression of the rat alpha7 nicotinic receptor gene.

Adrenal Gland Neoplasms↗
PubMed1999

A novel cytochrome P450 3A isoenzyme in rat intestinal microsomes.

PCR with several pairs of primers facilitates screening for new isoenzymes among highly homologous cytochrome P450s (CYPs). Combinations of two pairs of primers, which amplify N- and C-terminal coding sequences of either CYP3A1/CYP3A23 or CYP3A2 detected the presence of a previously unrecognized CYP3A in enterocyte microsomes isolated from rats. PCR, Northern blot, and immunoblotting with specific antibodies indicated that this isoenzyme is clearly distinguishable from CYP3A1, 3A23 or 3A2. Sequencing of a 285 bp coding fragment of this gene revealed 97% similarity with rat olfactory CYP3A9 (P450olf3).

Animals↗
PubMed1997

The molecular biology of neuronal nicotinic acetylcholine receptors.

The molecular cloning of genes encoding neuronal nicotinic acetylcholine receptors (nAChRs) has made possible a better understanding of the pharmacology and toxicology of cholinergic compounds. Neuronal nAChRs are related in structure to the nAChRs present at the neuromuscular junction. They are composed of multiple subunits designated either alpha and beta. Eight alpha and three beta subunit genes have been cloned. The alpha subunits contain the ligand binding sites, whereas beta subunits are structural subunits that contribute to the function of the receptor. A large number of nAChRs can be formed from different combinations of alpha and beta subunits. Different combinations of alpha and beta subunits can produce receptors in vitro with distinct ion conducting properties. Each subunit gene is expressed in a distinct pattern in the nervous system. The expression of at least some of the nAChR subunit genes is regulated during development and by cell-cell interactions. Each neuronal nAChR subtype has a distinct pharmacology. Both alpha and beta subunits contribute to the pharmacological properties of each subtype. The expression of multiple nAChR subtypes may allow for precise control of neurotransmission mediated by acetylcholine in diverse populations of neurons.

Acetylcholine↗
PubMed1997

Evidence for spare nicotinic acetylcholine receptors and a beta 4 subunit in bovine adrenal chromaffin cells: studies using bromoacetylcholine, epibatidine, cytisine and mAb35.

Relatively little is known about the type and number of nicotinic acetylcholine receptors (nAChRs) that mediate secretion from adrenal chromaffin cells. In these studies, we investigated nAChR reserve pools and their modulation using bromoacetylcholine (brACh) and the anti-nAChR antibody mAb35. By using brACh under acetylating conditions, adrenal catecholamine release was reduced (IC50, approximately 0.3 microM). This effect was slowly reversible. Submaximal concentrations of brACh caused shifts to the right in concentration-response curves of approximately 4-fold, as well as decreases in Emax values for the agonists nicotine and epibatidine. Cytisine is a nAChR agonist (EC50, approximately 46 microM) that was somewhat less efficacious than nicotine (Emax, approximately 85% of 10 microM nicotine) in adrenal chromaffin cells. Submaximal concentrations of brACh caused a small shift to the right in the concentration-response curves for the agonist cytisine, as well as a decrease in the Emax value. mAb35, which causes a slowly developing loss of nAChR-mediated secretion, produced a time-dependent shift to the right in agonist concentration-response curves and a reduction in Emax for nicotine and epibatidine. mAb35 treatment produced only a reduction in the Emax value of cytisine. Finally, we cloned and sequenced a reverse transcription-polymerase chain reaction product from bovine adrenal chromaffin RNA that shares a high degree of homology with beta 4 nAChR subunits. Northern analysis provided evidence for the presence of this transcript in chromaffin cell cultures. Together, these studies support the presence of a nAChR reserve in adrenal chromaffin cells that is down-regulated by mAb35. These studies also support the presence of more than one nAChR population mediating secretion and the presence of beta 4 nAChR subunits.

Acetylcholine↗
PubMed1996

ACTH and AII differentially stimulate steroid hormone orphan receptor mRNAs in adrenal cortical cells.

NGFI-B and Ad4BP are steroid hormone receptor-like transcription factor that may control steroidogenesis, growth and differentiation in the adrenal cortex. We have studied the induction of NGFI-B and Ad4BP and mRNAs by the peptide hormones, ACTH, AII, IGF, FGF, and by KCl depolarization in cultured bovine adrenocortical cells. The mRNAs for these two transcription factors were most effectively but differentially induced by ACTH and AII. mRNA for NGFI-B was typically undetectable in unstimulated cells, but rapidly (< 30 min) accumulated in response to ACTH and AII. Peak increases occurred within 2-3 h after which mRNA levels declined. At maximally effective concentrations, AII produced increases in NGFI-B mRNA 2.7-fold larger than those triggered by ACTH (n = 7). In contrast to NGFI-B, Ad4BP mRNA was readily detectable in unstimulated cells. ACTH and AII induced smaller, slower and more sustained increases in Ad4BP mRNA. Peak values were obtained in 6-8 h and Ad4BP mRNA remained elevated for at least 18 h. ACTH produced increases in Ad4BP that were 2.6-fold larger than those stimulated by AII (n = 8). Antagonists of major signaling pathways that couple ACTH and AII receptors to cortisol secretion, including T-type Ca2+ antagonist Ni2+ and penfluridol, the CaM kinase antagonist KN-62, the A-kinase antagonist H-89 and the non-selective kinase antagonist staurosporine, all failed to suppress increases in NGFI-B and Ad4BP mRNAs triggered by these two peptides. Each of these agents effectively inhibited cortisol production stimulated by the peptides. Further, arguing against their proposed role as transcription factors for steroidogenic enzymes, ACTH- and AII-stimulated increases in steroid orphan receptor mRNAs were not correlated with corresponding increases in cortisol production measured over 24 h. The results show that NGFI-B and Ad4BP mRNAs are differentially regulated by ACTH and AII. Only NGFI-B is rapidly and transiently increased with kinetics common to immediate early genes. The lack of correlation between peptide-stimulated increases in orphan receptor mRNAs and cortisol production in combination with the apparent divergence in the associated signaling pathways argue against a primary role for these transcription factors in ACTH- and AII-stimulated steroidogenesis. The dual function of these peptide hormones as mediators of development and corticosteroid synthesis could necessitate the presence of separate, parallel signaling pathways.

Adrenocorticotropic Hormone↗
PubMed1996

Transcriptional regulation and cell specificity determinants of the rat nicotinic acetylcholine receptor alpha 3 gene.

The effects of increased cAMP level and reduced protein kinase C activity on transcription of the alpha 3 neuronal nicotinic acetylcholine receptor (nAChR) gene in PC12 cells were examined. Two nAChR alpha 3 transcripts (3.9 and 2.4 kb) are expressed in PC12 cells. When PC12 cells were grown in 2 mu m phorbol 12-myristate 13-acetate (PMA) for 2 days to lower protein kinase C activity, the levels of both transcripts were increased. When PC12 cells were grown in 5 mu m forskolin, the level of the 3.9 kb transcript was increased. We previously constructed clones containing promoter elements located upstream of the alpha 3 gene which allow reporter gene expression in PC12 cells. These constructs were transfected into PC12 cells grown in PMA or forskolin. The increase in alpha 3 transcripts in response to PMA or forskolin was shown to be mediated at least in part at the transcriptional level by elements located within 600 nucleotides of the transcriptional start sites. The promoter constructs were also used to demonstrate that elements needed to restrict the expression of alpha 3 in non-neuronal cells lie near to the 5' end of the alpha 3 gene.

Animals↗
PubMed1996

Characterization and localization of adrenal nicotinic acetylcholine receptors: evidence that mAb35-nicotinic receptors are the principal receptors mediating adrenal catecholamine secretion.

Adrenal chromaffin cells contain at least two subtypes of nicotinic acetylcholine receptors (nAChRs). These studies were designed to identify and characterize the subtype of nAChR mediating adrenal catecholamine release using the monoclonal antibody mAb35, which recognizes the alpha-subunit of muscle nAChRs and cross-reacts with some neuronal nAChRs. Immunocytochemical studies demonstrated that mAb35 interacts with specific sites on cultured chromaffin cells. Pretreatment with mAb35 reduced nAChR-stimulated catecholamine release (IC50 of approximately 10nM). mAb35 had no effects on release stimulated through non-nAChR mechanisms. Unlike agonist-induced nAChR desensitization, the mAb35-induced reduction in nAChR-mediated secretion developed slowly. Although not immediately reversible, nAChR-stimulated release recovered after mAB35 removal. However, unlike recovery from agonist pretreatment, recovery from mAb35 pretreatment was relatively slow and was par tially blocked by vinblastine. Hybridization of adrenal chromaffin RNA with a rat alpha3 cDNA revealed two strong bands and two fainter bands: two higher-molecular-weight bands, 6.9 and 8.5 kb; a strong band of 3.2 kb; and a lower amount of 2.3kb RNA. With recovery of nAChR function after agonist or mAb35 treatment, no significant effects on alpha 3 subunit mRNA levels were seen. In summary, these studies demonstrate the presence of mAb35-nAChRs on adrenal chromaffin cells and provide evidence that these receptors represent the major population that regulates secretory events in adrenal chromaffin cells.

Adrenal Glands↗
PubMed1994

Sequencing and promoter analysis of the genomic region between the rat neuronal nicotinic acetylcholine receptor beta 4 and alpha 3 genes.

Nicotinic acetylcholine receptors (nAChRs) found on neurons are composed of ligand binding (alpha) and structural (beta) subunits. Different combinations of alpha and beta subunits produce nAChR subtypes with different pharmacological and ion-conducting properties. Transcriptional regulation may be an important determinant of receptor subtype in a neuronal population and thus influence transmission through a ganglion or group of neurons in the CNS by controlling the nAChR subtype(s) present. In order to understand the transcriptional regulation of neuronal nAChRs by cell contact and electrical activity, it will be first necessary to identify DNA elements that control the expression of members of this family and to identify factors required for the expression of these genes. In this report we have begun to examine the 5'-flanking region of one member of the nAChR family of genes, alpha 3. We have sequenced the region between the beta 4 and alpha 3 genes and have identified two promoter regions in the beta 4-alpha 3 intergenic region. One region is close to the beta 4 gene downstream of exon 6 and has strong promoter activity in both orientations; the other is close to the start of the alpha 3 gene coding region. A region with putative silencer activity is also found near the upstream promoter. This bidirectional promoter region could be involved in the control of alpha 3 and beta 4 gene expression.

Animals↗
PubMed1991

Nicotinic acetylcholine receptor mRNA in dorsal root ganglion neurons.

Nicotinic acetylcholine receptor (AChR) genes are expressed in subpopulations of chick dorsal root ganglion (DRG) neurons. In 18-day embryonic ganglia, 19% of the neurons have material homologous to neuronal AChR alpha 3 gene mRNA, and 8% have material homologous to alpha 4 mRNA as seen with in situ hybridization. RNAase protection experiments confirm that DRG RNA contains alpha 3 and alpha 4 transcripts, and Northern blot analysis establishes the size of the transcripts as being 3.5 and 3.3 kb, respectively. The proportion of DRG neurons containing alpha 3 mRNA does not decline up through 1 year post-hatch, indicating that alpha 3 gene expression is not a developmentally transient event in the ganglion. An antiAChR monoclonal antibody detects cross-reacting material in 16% of the DRG neurons from 18-day embryos, indicating that AChR mRNA is translated into protein. Electrophysiological measurements confirm the presence of functional AChRs on DRG neurons freshly isolated from 18-day embryos: 24% of the neurons have substantial ACh sensitivities, whereas another 23% have small but detectable responses. Staining dorsal root ganglion sections with an anticholine acetyltransferase antiserum reveals cross-reactive material localized in axons in the ganglion; no evidence suggests the presence of cholinergic synaptic structures or AChR clusters on neuronal somata in the ganglion. It is possible that AChRs on DRG neurons participate in a diffuse form of transmission between the cholinergic fibers and a subpopulation of neuronal somata in the ganglion. Alternatively, AChRs on the somata may represent an ectopic distribution of receptors whose primary function is at the terminals of central or peripheral DRG processes.

Acetylcholine↗
PubMed1989

Structural and evolutionary comparisons of four alleles of the mouse Igk-J locus which encodes immunoglobulin kappa light chain joining (J kappa) segments.

The Igk-J locus of the mouse encodes the immunoglobulin kappa light chain joining (J) segments. Four Igk-J alleles have been described on the basis of restriction enzyme length polymorphisms. The nucleotide sequences of the Igk-Ja allele (type strain, C.C58), Igk-Jc allele (type strain, SJL/J), and Igk-Jd allele (type strain, SK/CamRk) have been determined and are compared with the previously reported Igk-Jb allele sequence (type strain, BALB/c). The mouse sequences are also compared with published sequences for rat and human J kappa sequences. Far more differences were found between the Igk-Ja allele and the other mouse alleles than between any two of the latter. These result in two amino acid substitutions which distinguish the J2 and J3' segments of the Igk-Ja allele from the other three alleles. Use of the Phylogenetic Analysis Using Parsimony program to generate a phylogenetic tree strongly indicates that after divergence from the rat ancestor, there appears to have been an early split between the Igk-Ja allele and the evolutionary precursor of the other mouse alleles. There also appears to have been far less divergence from the ancestral condition in the Igk-Ja allele than in the other alleles. Also, the presence of only one convergent mutation among the four mouse alleles provides strong evidence against any crossing over within the Igk-J locus during the history of these alleles. Finally, the differences in rates of evolution of the Igk-J alleles are in marked contrast to the relatively uniform rates of divergence of four alleles of a mouse V kappa gene, Igk-VSer.

Alleles↗
PubMed1989

Regulating the number and function of neuronal acetylcholine receptors.

Nicotinic acetylcholine receptors (AChRs) on vertebrate neurons represent a family of receptors distinct from the well-characterized AChR of skeletal muscle. New probes for neuronal AChRs are now being used to examine the regulation of receptor number and function. The results suggest that neuronal AChRs differ from muscle AChRs in regulation by presynaptic input and by at least one second messenger system, and that neuronal AChRs are additionally regulated by retrograde signals from the synaptic target tissue. The forms of regulation provide potential mechanisms by which cell-cell interactions could stabilize synaptic contacts on neurons and modulate synaptic function.

Animals↗
PubMed1988

Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia.

A chicken genomic clone encoding a portion of the neuronal acetylcholine receptor (AChR) alpha 3 subunit was used to identify homologous mRNA in embryonic chick ciliary ganglia. In situ hybridization indicated that the mRNA was neuronal. Northern blot analysis revealed a major hybridizing species of 3.5 kb. Protection experiments confirmed that ganglionic RNA contained material indistinguishable by RNAase digestion from the 300 nucleotide probe used. No transcripts were detected by in situ hybridization or Northern blot analysis for chick neuronal AChR alpha 2 or alpha 4 genes. alpha 3 transcripts were present at all times examined (E6 to 1 year posthatch). Both postganglionic axotomy and preganglionic denervation of ciliary ganglia in newly hatched chicks produced declines in alpha 3 mRNA levels, implying regulation of neuronal AChR mRNA by cell-cell interactions.

Animals↗
PubMed1987

The endogenous retrovirus Mtv-8 on mouse chromosome 6 maps near several kappa light chain markers.

Endogenous retroviruses are known to affect expression of cellular genes in the vicinity of their integration sites. The endogenous mouse mammary tumor provirus (Mtv-8) previously has been reported to reside on mouse chromosome 6 near the immunoglobulin kappa chain locus. Using pairs of mouse strains on the BALB/c (Mtv-8 positive) and C58 (Mtv-8 negative) backgrounds which are congenic for chromosome 6 genetic markers, we have confirmed the chromosome assignment of this provirus. Moreover, we have analyzed the N1 progeny of a (B6 X C58) X C58 backcross to determine the segregation of the Mtv-8 provirus with respect to polymorphisms in the Igk-VSer and Igk-J loci. The results with congenic and backcross mice together with results of others suggest that Mtv-8 is located approximately 0.52 cM from several closely linked kappa markers on chromosome 6.

Animals↗
PubMed1986

Genetic polymorphism at the mouse immunoglobulin J kappa locus (Igk-J) as demonstrated by Southern hybridization and nucleotide sequence analysis.

Comparison of the nucleotide sequences of the C.C58 M75 myeloma kappa chain gene and the BALB/c germ-line J kappa segments suggested that the J kappa regions of C.C58 and BALB/c might be distinguished by restriction enzyme polymorphisms. This was shown to be the case in Southern hybridizations of Hinf I and Acc I digests of liver DNA from these and other strains with a J kappa-specific probe. Tests of a wide variety of inbred, congenic, recombinant, and recombinant-inbred strains provided evidence for three alleles, Igk-Ja, Igk-Jb, and Igk-Jc, the type strains for which are C58/J, BALB/c, and SJL/J, respectively. Analysis of the B6.PL(85NS) congenic strain suggests that the Igk-J locus lies in the neighborhood of the Lyt-2/Lyt-3 loci, approximately 0.30 cM from the V gene segment determining the Igk-VSer and Igk-Efl polymorphisms. Finally, nucleotide substitutions lead to amino acid sequence differences between the C.C58 M 75 kappa gene and the BALB/c germ line in J kappa 2 and J kappa 4. Two of these substitutions reflect true germ-line differences, raising the possibility that idiotype differences observed among strains could reflect J kappa as well as V kappa differences.

Amino Acid Sequence↗
PubMed1986

Structural differences in a single gene encoding the V kappa Ser group of light chains explain the existence of two mouse light-chain genetic markers.

Two phenotypic markers of mouse immunoglobulin kappa light chains, the IB-peptide marker and the Ef1a isoelectric focusing marker, are expressed by the C58/J, AKR/J, RF/J, and PL/J strains (called expressor strains) but not by BALB/c and most inbred strains. Expression is linked to the kappa light-chain locus and the Lyt-2/Lyt-3 genes on chromosome 6. Light chains bearing these markers belong to a group of variable region kappa chain (V kappa) regions called V kappa Ser, which has a serine amino terminus and a framework 1 region not observed to date among BALB/c light chains. Southern hybridization of genomic DNA with a V kappa Ser-specific cDNA probe has demonstrated a single strongly hybridizing DNA fragment in all strains of mice tested. Characteristic restriction enzyme polymorphisms define the V kappa Ser alleles of expressor (Igk-VSera) and nonexpressor (Igk-VSerb) strains. In the present study, the unrearranged V kappa Ser gene and its flanking regions from an expressor (C.C58) and nonexpressor (BALB/c) strain have been cloned and their nucleotide sequences determined. The C.C58 V kappa Ser gene isolated (the Igk-VSera allele) was shown to code for the two phenotypic markers described. While the nucleotide sequence of the BALB/c coding region (the Igk-VSerb allele) shows 97% identity with the C.C58 gene, single nucleotide substitutions lead to structural changes in the encoded protein which render it IB-negative and Ef1a-negative. These differences alone can explain the failure of strains containing the BALB/c allele to express these kappa-chain phenotypic markers. Also, the BALB/c gene contains a single substitution in a conserved octamer sequence approximately equal to 100 nucleotides upstream of the coding region, which could affect its expression. Finally, the C.C58 allele contains a BAM5/R repetitive DNA element approximately equal to 1200 nucleotides upstream of the coding regions that is not present in BALB/c. This element gives rise to the EcoRI and BamHI restriction enzyme polymorphisms, which distinguish the Igk-VSera and Igk-VSerb alleles.

Amino Acid Sequence↗
PubMed1985

Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis.

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full-length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain-specific expression of V kappa Ser-associated phenotypic markers are discussed.

Amino Acid Sequence↗