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N Davidson

Publications and source records attributed to N Davidson.

At least 163 records · Page 9Linked to original sources
PubMed1988

Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes.

The properties of rat and rabbit brain sodium (Na) channels expressed in Xenopus oocytes following either unfractionated or high-molecular-weight mRNA injections were compared to assess the relative contribution of different size messages to channel function. RNA was size-fractionated on a sucrose gradient and a high-molecular-weight fraction (7-10 kilobase) encoding the alpha-subunit gave rise to functional voltage-dependent Na channels in the oocyte membrane. Single-channel conductance, mean open time, and time to first opening were all similar to the values for channels following injection of unfractionated RNA. In contrast, inactivation properties were markedly different; Na currents from high-molecular-weight RNA inactivated with a several-fold smaller macroscopic inactivation rate and showed a steady-state voltage dependence that was shifted in the depolarizing direction by at least 10 mV relative to that for unfractionated RNA. Single-channel recording revealed that the kinetic difference arose from a greater probability for high-molecular-weight RNA induced channels to reopen during a depolarizing voltage step. Pooling all gradient fractions and injecting this RNA into oocytes led to the appearance of Na channels with inactivation properties indistinguishable from those following injection of unfractionated RNA. These results suggest that mRNA species not present in the high-molecular-weight fraction can influence the inactivation process of rat brain Na channels expressed in Xenopus oocytes. This mRNA may encode beta-subunits or other proteins that are involved in posttranslational processing of voltage-dependent Na channels.

Animals↗
PubMed1987

The inducible lac operator-repressor system is functional in mammalian cells.

We have investigated the use of the Escherichia coli lac operator-repressor system to regulate expression of transfected genes in mammalian cells. We show that lac repressor produced in mouse L cells by transfection of a lacl expression vector blocks transcription of an MSV-CAT fusion gene when the lac operator is inserted at any one of the following sites within the promoter region: between the initiation codon (ATG) and the transcription start site; between the transcription start and TATA box regions; or upstream of the TATA box region. This last result suggests that the repressor may prevent protein-protein interactions involved in transcription activation. The inducer IPTG causes a marked derepression of CAT expression. The lac repressor-operator complex may be useful as an on/off "switch" in the regulation of gene expression for gene transfer experiments.

Animals↗
PubMed1987

Expression of mouse-Torpedo acetylcholine receptor subunit chimeras and hybrids in Xenopus oocytes.

In this study, in vitro synthesized mRNA encoding mouse and Torpedo nicotinic acetylcholine receptor subunits was injected into Xenopus oocytes, followed by assays for assembly onto the oocyte surface (using [125I]alpha-bungarotoxin binding) and for acetylcholine-induced conductances (using voltage clamp). We constructed hybrid acetylcholine receptors in Xenopus oocytes by injecting all 8 possible combinations of 4 subunit-specific mRNAs in which a single subunit is derived from the other species. For each hybrid combination, there is detectable assembly and conductance. We also constructed cDNA clones that encode chimeric acetylcholine receptor subunits in which part of the gamma subunit from Torpedo was replaced by the homologous region of the delta subunit from mouse. None of the chimeric subunits was able to replace the Torpedo gamma, mouse delta, or Torpedo delta subunit with regard to assembly or function. We therefore conclude that widely spaced (and unknown) parts of the protein chain are required for the intersubunit interactions that eventually lead to functional assembly of the receptor.

Animals↗
PubMed1987

cDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.

We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA.

Animals↗
PubMed1987

Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines.

We report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 125I-labeled PDGF for binding to PDGF receptors on normal human fibroblasts. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by 17 beta-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 degrees C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approximately equal to 30 kDa on NaDodSO4/polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO4/polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain of PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth.

Breast Neoplasms↗
PubMed1987

Tissue-specific expression of the RI and RII sodium channel subtypes.

Anti-peptide antibodies that distinguish between the rat brain sodium channel subtypes referred to as RI and RII were prepared and used to determine their relative expression in nerve and muscle tissues. Sodium channels purified from rat brain are approximately 18% RI and 80% RII. In brain, the RII subtype is preferentially expressed with RI/RII ratios ranging from 0.07 in the hippocampus to 0.17 in the cerebral cortex. The RI subtype is preferentially expressed in more caudal areas of the central nervous system with values of RI/RII of 0.98 for medulla oblongata and 2.2 for spinal cord. Expression of additional unidentified sodium channel subtype(s) is detected in midbrain, medulla, and spinal cord, and expression of unidentified sodium channel subtypes predominates over expression of RI and RII in retina and optic nerve. The RI and RII subtypes are primarily expressed in the central nervous system and are not detected in significant numbers in skeletal or cardiac muscle, sympathetic ganglia, adrenal medulla, sciatic nerve, or cauda equina. The RII subtype appears first in development of both brain and spinal cord but declines in adult spinal cord as the RI subtype increases. The strict regional expression of these two sodium channel subtypes suggests that they may have distinct functional properties or physiological roles.

Animals↗
PubMed1987

Equilibrium properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes.

This study used messenger RNA encoding each subunit (alpha, beta, gamma and delta) of the nicotinic acetylcholine (ACh) receptor from mouse BC3H-1 cells and from Torpedo electric organ. The mRNA was synthesized in vitro by transcription with SP6 polymerase from cDNA clones. All 16 possible combinations that include one mRNA for each of alpha, beta, gamma, and delta were injected into oocytes. After allowing 2-3 d for translation and assembly, we assayed each oocyte for (a) receptor assembly, measured by the binding of [125I]alpha-bungarotoxin to the oocyte surface, and (b) ACh-induced conductance, measured under voltage clamp at various membrane potentials. All combinations yielded detectable assembly (30-fold range among different combinations) and ACh-induced conductances (greater than 1,000-fold range at 1 microM). On double-logarithmic coordinates, the dose-response relations all had a slope near 2 for low concentrations of ACh. Data were corrected for variations in efficiency of translation among identically injected oocytes by expressing ACh-induced conductance per femtomole of alpha-bungarotoxin-binding sites. Five combinations were tested for d-tubocurarine inhibition by the dose-ratio method; the apparent dissociation constant ranged from 0.08 to 0.27 microM. Matched responses and geometric means are used for describing the effects of changing a particular subunit (mouse vs. Torpedo) while maintaining the identity of the other subunits. A dramatic subunit-specific effect is that of the beta subunit on voltage sensitivity of the response: gACh(-90 mV)/gACh(+30 mV) is always at least 1, but this ratio increases by an average of 3.5-fold if beta M replaces beta T. Also, combinations including gamma T or delta M usually produce greater receptor assembly than combinations including the homologous subunit from the other species. Finally, EACh is defined as the concentration of ACh inducing 1 microS/fmol at -60 mV; EACh is consistently lower for alpha M. We conclude that receptor assembly, voltage sensitivity, and EACh are governed by different properties.

Animals↗
PubMed1987

Developmentally regulated expression of a truncated myosin light-chain 1F/3F gene.

Fast skeletal muscle myosin light-chain I (MLC1f) and myosin light-chain 3 (MLC3f) mRNAs are both derived from a single rat MLC1/3f gene. MLC1f mRNA begins at the first exon of the gene, while MLC3f mRNA begins with exon 2, 10 kilobases downstream. Both mRNAs require alternate splicing of internal exons for accurate expression. We showed that a truncated rat MLC1f/3f gene lacking exon 1 and the first 6.3 kilobases of the intron separating exons 1 and 2 produced rat MLC3f mRNA in a developmentally regulated manner after introduction into myogenic mouse cells, thus demonstrating in vivo the presence of a functional promoter associated with exon 2. Correctly spliced mRNA was produced after transfer of this truncated gene into both myogenic and nonmyogenic cells, indicating that the pattern of splicing of this complex transcript was due to a structural features of the RNA and was independent of cell type.

Animals↗
PubMed1987

Ca channels induced in Xenopus oocytes by rat brain mRNA.

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2-5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. IBa in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 +/- 4 nA, n = 21). In contrast, injected oocytes showed a peak IBa of 339 +/- 42 nA (n = 33). The threshold for activation of IBa was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (tau = 650 +/- 50 msec at +20 mV). The maintained current was 67 +/- 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward IBa could be observed in BaCl2 solutions at potentials positive to ECl and also in Na-free salines, indicating that neither Cl- nor Na+ was carrying the inward current. Although IBa displayed voltage-independent blockade by Cd (50% inhibition at 6 microM), the peptide Ca channel antagonist, omega-CgTX (1 microM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 microM, or when cells were held at -40 mV) or agonist Bay K-8644. However, IBa was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT2). In contrast, use of forskolin to activate protein kinase A did not alter IBa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Rat brain 5-HT1C receptors are encoded by a 5-6 kbase mRNA size class and are functionally expressed in injected Xenopus oocytes.

Injection of rat brain RNA into Xenopus laevis oocytes induces synthesis of receptors that show an electrophysiological response to bath application of serotonin. While there are at least 4 pharmacologically distinct subtypes of 5-HT binding sites in the rat brain, we find that the pharmacological characteristics of the predominant electrophysiologically active receptor synthesized in Xenopus oocytes are most consistent with those of the 5-HT1C subtype. Additional electrophysiologically active 5-HT receptor types could not be detected. Injection of mRNA isolated from a number of rat brain regions shows that the choroid plexus is particularly enriched for 5-HT1C mRNA. Oocytes injected with RNA isolated from this region respond 16 or 8 times more strongly to serotonin than do oocytes injected with RNA isolated from cortex or substantia nigra, respectively. In addition, by fractionation of rat brain mRNA through agarose gels, we have identified a single RNA size class of about 5-6 kbase that encodes this serotonin receptor.

Animals↗
PubMed1987

Arterial pulse wave velocity, Fourier pulsatility index, and blood lipid profiles.

Increased arterial pulse wave velocity (PWV) and decreased Doppler-shifted Fourier pulsatility index (PI) have been utilized clinically to diagnose the presence and severity of peripheral vascular disease. We have examined the relationships between these two diagnostic indices and several lipoprotein atherogenic risk factors, e.g., serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipo-protein-cholesterol (LDL-C) among 74 healthy male volunteers, aged 20 to 56 yr. The Doppler signal from the radial artery was digitized and processed with the Hamming window and 512-point fast Fourier transform to obtain frequency power spectra and PI index by a desktop computer. PWV was determined on the computer by dividing the distance between two pressure transducers placed over the radial and carotid arteries by the transit time difference between the maximal first derivatives (dP/dt) of the recorded arterial pulse waves. The results indicated that PI was inversely related to TC, LDL-C, and TG (r = -0.50, -0.41, -0.54, respectively, P less than 0.001), but evidenced a positive relationship with the HDL-C/TC ratio (r = 0.69, P less than 0.001). PWV was also significantly correlated to TC (r = 0.40) and TG (r = 0.42), but was inversely associated with the HDL-C/TC ratio (r = -0.45, P less than 0.001). These findings are consistent with the current theory relating lipids to the pathology of atherosclerosis.

Adult↗
PubMed1986

Campylobacter bacteraemia: a report of 10 cases.

Ten cases of campylobacter bacteraemia, which were diagnosed over a three-year period, are described. Four patients presented with acute gastrointestinal illness, three patients had acute febrile illnesses, two patients had cellulitis and one patient presented with ureteric colic. Two patients had lifelong agammaglobulinaemia, two were undergoing corticosteroid therapy and there was one nosocomial infection. In contrast with previous reports, the most common species that was isolated was Campylobacter jejuni, which was found in eight cases, and this includes the first report of cellulitis that was most probably caused by C. jejuni. All of these isolations of campylobacter have occurred since the introduction of the radiometric technique of processing blood cultures.

Adolescent↗
PubMed1986

Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.

Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences.

Amino Acid Sequence↗
PubMed1986

Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes.

Calcium ions flow into cells through several distinct classes of voltage-dependent calcium-selective channels. Such fluxes play important roles in electrical signaling at the cell membrane and in chemical signaling within cells. Further information about calcium channels was obtained by injecting RNA isolated from rat brain, heart and skeletal muscle into Xenopus oocytes. Macroscopic currents through voltage-operated calcium channels were resolved when the endogenous calcium-dependent chloride current was blocked by replacing external calcium with barium and chloride with methanesulfonate. The resulting barium current was insensitive to tetrodotoxin but was completely blocked by cadmium or cobalt. With both heart and brain RNA at least two distinct types of calcium ion conductance were found, distinguishable by their time course and inactivation properties. In oocytes injected with heart RNA, the slowly inactivating component was selectively blocked by the calcium-channel antagonist nifedipine. Barium ion currents induced by heart RNA were modulated by isoproterenol, cyclic adenosine monophosphate, and acetylcholine.

Animals↗
PubMed1986

Gene mapping on human metaphase chromosomes by in situ hybridization with 3H, 35S, and 32P labeled probes and transmission electron microscopy.

A method based on in situ hybridization, autoradiography and transmission electron microscopy for mapping genes on human metaphase chromosomes is presented. Successful mapping of the tandemly repeated rDNA genes and of two nucleic acid probes, N-myc and probe 3 (Kanda et al. 1983), that are amplified in a homogeneously staining region (HSR) of the neuroblastoma cell line, IMR-32 is described. By using sufficiently thin AgBr emulsions, it is possible to obtain observable grains and good resolution with probes radiolabeled with 3H, 35S, or 32P, but the former gives the best results. We observe that neither of the two probes, N-myc and probe 3, has a uniform spatial distribution along the HSR and that the distributions of the two probes differ from each other. These observations support previous studies which indicated that the formation of an HSR is a more complex process than uniform amplification of a single DNA segment to form an n-fold set of perfect tandem repeats. The present study shows that the electron microscopic method is useful for extending the results of light microscopic studies for problems where higher resolution mapping is needed.

Cell Line↗