Phenolic plant compounds functioning as reproductive inhibitors in Microtus montanus.

Naturally occurring cinnamic acids and their related vinylphenols were found to inhibit reproductive function in Microtus montanus. When fed on these compounds, the rodents exhibited decreased uterine weight, inhibition of follicular development, and a cessation of breeding activity. It is suggested that these animals utilize plant compounds as a cue to terminate their reproductive effort in natural populations.

Chemical triggering of reproduction in Microtus montanus.

In a replicated experiment, nonbreeding winter populations of Microtus montanus were given supplements of rolled oats coated with 6-methoxybenzoxazolinone, a naturally occurring plant derivative. After 3 weeks of this feeding regime, samples from the populations demonstrated a high incidence of pregnancy in females and testicular hypertrophy in males. Control populations receiving rolled oats coated only with the solvent showed no reproductive activity. These results demonstrate that the presence of 6-methoxybenzoxazolinone in the plant food resource acts as the ultimate cue to trigger reproductive effort in Microtus montanus.

ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43-kildalton protein.

Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.

Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells.

We used transient transfection in COS cells to compare the properties of mouse muscle acetylcholine receptors (AChRs) containing alpha, beta, delta, and either gamma or epsilon subunits. gamma- and epsilon-AChRs had identical association rates for binding 125I-alpha-bungarotoxin, and identical curves for inhibition of toxin binding by d-tubocurarine, but epsilon-AChRs had a significantly longer half-time of turnover in the membrane than gamma-AChRs. A myasthenic serum specific for the embryonic form of the AChR reduced toxin binding to gamma-, but not epsilon-AChRs. The gamma-AChRs had channel characteristics of embryonic AChRs, whereas the major class of epsilon-AChR channels had the characteristics of adult AChRs. Two minor channel classes with smaller conductances were also seen with epsilon-AChR. Thus, some, but not all, of the differences between AChRs at adult endplates and those in the extrasynaptic membrane can be explained by the difference in subunit composition of gamma- and epsilon-AChRs.

Cell type-specific activation of neuronal nicotinic acetylcholine receptor subunit genes by Sox10.

The regulatory factor Sox10 is expressed in neural crest derivatives during development as well as in the adult CNS and peripheral nervous system. Mutations of the human Sox10 gene have been identified in patients with Waardenburg-Hirschsprung syndrome that is characterized by defects in neural crest development. Previous studies suggested that Sox10 might function as an important transcriptional regulator of neural crest development. No natural target genes of Sox10 have yet been identified. Although human Sox10 activates a synthetic promoter consisting of a TATA box and multiple Sox consensus sequences, no transcriptional activity of the rat Sox10 homolog has been detected. Here we report that the neuronal nicotinic acetylcholine receptor beta4 and alpha3 subunit gene promoters are transactivated by rat Sox10 in a cell type-specific manner. The alpha3 and beta4 subunits, in combination with the alpha5 subunit, make up the predominant nicotinic receptor subtype expressed in the peripheral nervous system. Transfections using Sox10 mutants indicate that the C-terminal region is dispensable for its ability to activate the beta4 and alpha3 promoters. Rat Sox10 was originally identified as an accessory protein of the POU domain protein Tst-1/Oct6/SCIP in glial cells. Tst-1/Oct6/SCIP was shown previously to activate the alpha3 promoter. We now demonstrate that it can transactivate the beta4 promoter as well. However, we were unable to detect any synergistic effects of Sox10 and Tst-1/Oct6/SCIP on beta4 or alpha3 promoter activity. Finally, we present data suggesting that recombinant Sox10 protein can directly interact with a previously characterized regulatory region of the beta4 gene.

Synergistic transcriptional activation by Sox10 and Sp1 family members.

Neuronal nicotinic acetylcholine receptors (nAChR) are expressed at specific times during development and in discrete neuronal populations. Transcriptional regulation of the receptor genes clearly plays a key role in the molecular pathway underlying the expression of these critical synaptic components. In an effort to understand this regulation, we focus upon the genes encoding three receptor subunits: alpha3, alpha5 and beta4. These subunits are genomically clustered and constitute the predominant nAChR subtype expressed in the peripheral nervous system. We and others demonstrated that the general transcription factors, Sp1 and Sp3, can transactivate the promoter of each subunit gene. Further, we showed that the regulatory factor Sox10 transactivates the alpha3 and beta4 promoters and does so in a cell-type-specific manner. Interestingly, the Sp- and Sox10-binding sites on the beta4 promoter are located immediately adjacent to each other, raising the possibility that the two sets of factors functionally interact to regulate receptor gene expression. Consistent with this hypothesis, we demonstrated that the proteins can directly interact. Here, we extend these observations and show that Sox10 and the Sp factors functionally interact, leading to synergistic transcriptional activation in a cholinergic cell line. Finally, evidence for the existence of cell-type-specific co-regulators for Sp1 and Sox10 is presented.

Transcriptional regulation of nicotinic acetylcholine receptor genes: identification of control elements of a gamma-subunit gene.

The muscle nicotinic acetylcholine receptor undergoes profound changes in abundance and distribution in response to innervation and denervation. As a start towards understanding the detailed mechanisms of acetylcholine receptor gene regulation, we have identified transcriptional regulatory regions of a receptor subunit gene. A region of genomic DNA at the 5' end of the mouse acetylcholine receptor gamma-subunit gene has been shown to promote transcription of the bacterial chloramphenicol acetyltransferase gene in a cell-type specific manner. In addition, the transcriptional activity of this fragment is developmentally regulated in mouse muscle C2C12 cells. We propose that this fragment contains transcriptional control elements of the mouse muscle acetylcholine receptor gamma-subunit gene.

Interactions between regulatory proteins that bind to the nicotinic receptor beta4 subunit gene promoter.

The genes encoding the alpha3, alpha5 and beta4 subunits of nicotinic acetylcholine receptors are tightly clustered within the genome. As these three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system, their genomic proximity suggests a regulatory mechanism ensuring their coordinate expression. We previously identified two transcriptional regulatory elements within the beta4 promoter. One of these elements, a CT box, interacts with the regulatory factors heterogeneous nuclear ribonucleoprotein K and Puralpha. Another element, a CA box, interacts with Sp1 and Sp3. The binding site for a fifth factor, Sox10, overlaps the CT and CA boxes. As the CT and CA boxes are adjacent, we postulated that the proteins that bind to the elements interact. Here we report that the CT box-binding factors interact with each other as do the CA box-binding factors. However, there are no direct associations between the two pairs of proteins. Interestingly though, Sox10 directly interacts with all four proteins, suggesting a central role in beta4 gene expression for this member of the Sox family of regulatory factors.

Localization of preproenkephalin mRNA-expressing cells in rat auditory brainstem with in situ hybridization.

Hair cells and auditory nerve dendrites in the inner ear are innervated by pontine neurons that have been demonstrated by immunochemical techniques to contain several neurotransmitters, including acetylcholine and the opioid peptide enkephalins and dynorphins. The functions of these nerve fibers are not known, but may involve modifying auditory sensitivity to low intensity stimuli. In the guinea pig the opioid pathways originate in the lateral superior olivary region. A recent study in the gerbil has reported cells expressing preproenkephalin mRNA present only in the ventral nucleus of the trapezoid body, and not in the superior olivary region. In the present study, a non-radioisotopically labeled in situ hybridization method was used to identify cells expressing mRNA coding for preproenkephalin in rat pontine neurons, specifically in the ventral nucleus of the trapezoid body. These cells may represent an enkephalin-containing medial olivocochlear system in the rat, the origin of the lateral system in the rat that differs markedly from the better-studied guinea pig and cat, or a non-olivocochlear enkephalin-containing system.

A transcriptional regulatory element critical for CHRNB4 promoter activity in vivo.

Genome-wide association studies have underscored the importance of the clustered neuronal nicotinic acetylcholine receptor (nAChR) subunit genes with respect to nicotine dependence as well as lung cancer susceptibility. CHRNB4, which encodes the nAChR ß4 subunit, plays a major role in the molecular mechanisms that govern nicotine withdrawal. Thus, elucidating how expression of the ß4 gene is regulated is critical for understanding the pathophysiology of nicotine addiction. We previously identified a CA box regulatory element, (5'-CCACCCCT-3') critical for ß4 promoter activity in vitro. We further demonstrated that a 2.3-kb fragment of the ß4 promoter region containing the 5'-CCACCCCT-3' regulatory element in the ß4 gene promoter (CA box) is capable of directing cell-type specific expression of a reporter gene to a myriad of brain regions that endogenously express the ß4 gene. To test the hypothesis that the CA box is critical for ß4 promoter activity in vivo, transgenic animals expressing a mutant form of the ß4 promoter were generated. Reporter gene expression was not detected in any tissue or cell type at embryonic day 18.5 (ED 18.5). Similarly, we observed drastically reduced reporter gene expression at postnatal day 30 (PD30) when compared to wild type (WT) transgenic animals. Finally, we demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the mutant ß4 promoter. Taken together these results demonstrate that the CA box is critical for ß4 promoter activity in vivo.

Temporally- and spatially-regulated transcriptional activity of the nicotinic acetylcholine receptor beta4 subunit gene promoter.

Signaling through nicotinic acetylcholine (nACh) receptors underlies a diverse array of behaviors. In order for appropriate signaling to occur via nACh receptors, it is necessary for the genes encoding the receptor subunits to be expressed in a highly regulated temporal and spatial manner. Here we report a transgenic mouse approach to characterize the transcriptional regulation of the gene encoding the nACh receptor beta4 subunit. nACh receptors containing this subunit play critical roles in both the central and peripheral nervous systems. We demonstrate that a 2.3-kilobase pair fragment of the beta4 5'-flanking region is capable of directing reporter gene expression in transgenic animals. Importantly, the transcriptional activity of the promoter region is cell-type-specific and developmentally regulated and overlaps to a great extent with endogenous beta4 mRNA expression. These data indicate that the 2.3-kilobase pair fragment contains transcriptional regulatory elements critical for appropriate beta4 subunit gene expression.

From smoking to lung cancer: the CHRNA5/A3/B4 connection.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that modulate key physiological processes ranging from neurotransmission to cancer signaling. These receptors are activated by the neurotransmitter, acetylcholine, and the tobacco alkaloid, nicotine. Recently, the gene cluster encoding the alpha3, alpha5 and beta4 nAChR subunits received heightened interest after a succession of linkage analyses and association studies identified multiple single-nucleotide polymorphisms in these genes that are associated with an increased risk for nicotine dependence and lung cancer. It is not clear whether the risk for lung cancer is direct or an effect of nicotine dependence, as evidence for both scenarios exist. In this study, we summarize the body of work implicating nAChRs in the pathogenesis of lung cancer, with special focus on the clustered nAChR subunits and their emerging role in this disease state.

Respirator fit factor performance while sweating.

The extent to which sweat accumulation inside respirators affects respirator fit has not been quantified. This study represents an attempt to measure facial sweating and to quantify its effects on fit factors of negative pressure, full-facepiece respirators. Respirator fit factor (FF) data were obtained while 14 subjects completed 30 minutes of treadmill walking at an intensity of 75% of age-predicted maximal heart rate in an aerosol test chamber under ambient environmental conditions. Subject facial and whole body sweat production were also measured. Statistical analysis of the treadmill FF results showed that respirator fit was significantly (p < 0.05) degraded after 14 minutes of exercise. Sweat accumulation inside the respirator facepiece averaged 30.9 +/- 15.5 g. However, no significant correlation of subject facial sweat production with overall FF values measured during exercise was found. The results of this study indicate that respirator FFs degrade significantly over time under moderate exercise and environmental conditions and suggest that facial sweat accumulation alone does not account for the reduced FF levels.

The signal transduction pathway underlying ion channel gene regulation by SP1-C-Jun interactions.

During neuronal differentiation, an exquisitely controlled program of signal transduction events takes place, leading to the temporally and spatially regulated expression of genes associated with the differentiated phenotype. A critical class of genes involved in this phenomenon is that made up of genes encoding neurotransmitter-gated ion channels that play a central role in signal generation and propagation within the nervous system. We used the well established PC12 cell line to investigate the molecular details underlying the expression of the neuronal nicotinic acetylcholine receptor class of ion channels. Neuronal differentiation of PC12 cells can be induced by nerve growth factor, leading to an increase in neuronal nicotinic acetylcholine receptor gene expression. Nerve growth factor initiates several signal transduction cascades. Here, we show that the Ras-dependent mitogen-activated protein kinase and phosphoinositide 3-kinase pathways are critical for the nerve growth factor-mediated increase in the transcriptional activity of a neuronal nicotinic acetylcholine receptor gene promoter. In addition, we show that a component of the Ras-dependent mitogen-activated protein kinase pathway, nerve growth factor-inducible c-Jun, exerts its effects on receptor gene promoter activity most likely through protein-protein interactions with Sp1. Finally, we demonstrate that the target for nerve growth factor signaling is an Sp1-binding site within the neuronal nicotinic acetylcholine receptor gene promoter.

A novel regulatory element of a nicotinic acetylcholine receptor gene interacts with a DNA binding activity enriched in rat brain.

Nicotinic acetylcholine receptors are ligand-gated ion channels that play a critical role in signal transmission in the nervous system. The genes encoding the various subunits that comprise functional acetylcholine receptors are expressed in distinct temporal and spatial patterns. Studies to understand the molecular mechanisms underlying the differential expression of the receptor subunit genes have led to the identification, in this report, of a 19-base pair cis-acting element that is required for transcriptional activation of the rat beta 4 subunit gene. Screening of computer data bases with the 19-base pair element revealed the sequence to be unique among known transcriptional regulatory elements. Loss of this element resulted in drastically reduced beta 4 promoter activity in transfected cholinergic SN17 cells. Furthermore, this element specifically interacts with nuclear proteins prepared from both SN17 cells and adult rat brain. UV cross-linking experiments indicated the presence, in SN17 nuclear extracts, of a prominent protein species (approximately 50 kDa) that interacts specifically with the 19-base pair element. These results lead us to hypothesize that interactions between the 50-kDa protein and the novel 19-base pair element are necessary for transcriptional activation of the beta 4 subunit gene.

Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes. A possible role for the DNA-binding protein Puralpha.

Nicotinic acetylcholine receptors constitute a multigene family (alpha2-alpha9, beta2-beta4) expressed in discrete temporal and spatial patterns within the nervous system. The receptors are critical for proper signal transmission between neurons and their targets. The molecular mechanisms underlying receptor gene expression have not been completely elucidated but clearly involve regulation at the level of transcription. We previously identified a novel 19-base pair (bp) transcriptional regulatory element in the promoter region of the rat beta4 subunit gene. This 19-bp element interacts specifically with DNA-binding proteins enriched in nuclear extracts prepared from adult rat brain. Using a combination of cellulose-phosphate, DNA-cellulose, and DNA sequence-specific affinity chromatographies, we purified the 19-bp element binding activity approximately 19,000-fold. Analysis by denaturing gel electrophoresis revealed the presence of four polypeptides in the most purified fraction, ranging in molecular masses between 31 and 114 kDa. Peptide sequence analysis revealed that one of the polypeptides is the bovine homologue of the transcriptional regulatory factor, Puralpha. Electrophoretic mobility shift assays indicated that Puralpha interacts directly and specifically with the 19-bp element. In addition, mobility shift assays using an anti-Puralpha monoclonal antibody revealed the presence of Puralpha, or an immunologically related protein, in nuclear extracts prepared from brain tissue. We hypothesize that the interaction between Puralpha and the 19-bp element is critical for proper expression of the beta4 subunit gene.

Nerve growth factor increases the transcriptional activity of the rat neuronal nicotinic acetylcholine receptor beta 4 subunit promoter in transfected PC12 cells.

Neuronal nicotine acetylcholine receptors play a key role in synaptic transmission in the nervous system. Although complementary DNA clones encoding a family of acetylcholine receptor subunits have been isolated and subsequent anatomical studies indicate differences in the temporal and spatially restricted patterns of expression of each gene, the cellular and molecular mechanisms controlling the expression of these genes are unknown. As part of a long-term goal to elucidate these mechanisms, we have been identifying and characterizing regions of the receptor subunit genes involved in transcriptional regulation. Here, we report the localization of the transcription initiation site of the rat beta 4 subunit gene, demonstrate using transient transfection analysis of PC12 cells that sequences upstream of this site are capable of activating transcription of a heterologous gene, and show that this transcriptional activity is enhanced in PC12 cells by treatment with nerve growth factor.

Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes. Functional interactions between Sp1 and the rat beta4 subunit gene promoter.

To date, 11 members (alpha2-alpha9 and beta2-beta4) of the neuronal nicotinic acetylcholine receptor gene family have been identified. These genes encode subunits that form distinct receptors with different pharmacological and physiological profiles in temporally and spatially restricted patterns within the nervous system. Distinct molecular mechanisms probably orchestrate the expression of various receptor subtypes, yet little is known of specific transcriptional regulatory elements and their associated factors that are responsible for this segregated pattern of expression. Here we report the identification of an element, in the 5'-flanking region of the rat beta4 subunit gene, containing a CA box that is necessary for beta4 promoter activity in a transiently transfected cholinergic cell line, SN17. This element was shown to interact with a protein(s) in SN17 nuclear extracts that is antigenically related to the transcriptional activator Sp1. Furthermore, co-transfection experiments confirmed that Sp1 can transactivate a beta4 promoter-reporter gene construct, indicating that Sp1 is necessary, at least in part, for transcriptional activation of the beta4 subunit gene.

Sp1 and Sp3 regulate expression of the neuronal nicotinic acetylcholine receptor beta4 subunit gene.

Neuronal nicotinic acetylcholine receptors play important roles in signal transduction within the nervous system. The receptors exist in a variety of functionally distinct subtypes that are determined by their subunit structures. The subunits are encoded by 11 genes, alpha2-alpha9 and beta2-beta4. Three of the genes, alpha3, alpha5, and beta4, are tightly clustered, and their encoded proteins make up the predominant receptor subtype in the peripheral nervous system. The tight linkage of the genes suggests there may be a common regulatory mechanism underlying their expression. However, although their expression patterns significantly overlap, they are not identical, indicating that independent regulatory mechanisms must also exist. Our studies have focused upon the gene encoding the beta4 subunit for which we have identified several transcriptional regulatory elements. One of these elements, E2, specifically interacts with the general transcription factor Sp1. Here we show that another member of the Sp family of factors, Sp3, can specifically interact with E2 whereas two other members, Sp2 and Sp4, cannot. Co-transfection experiments indicate that Sp3 can transactivate a beta4 promoter/reporter gene construct and, furthermore, that Sp1 and Sp3 can transactivate the beta4 reporter construct synergistically. The transactivation is dependent upon an intact E2 and may involve direct interactions between Sp1 and Sp3.