Mapping transcription start points with T4 DNA polymerase.
Explore the source record for details and available documents.
Actins↗
Biomedical subjects
N Davidson
Publications and source records attributed to N Davidson.
Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes.
Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular gamma-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steady-state GABA currents were increased at higher extracellular [GABA], [Na+], and [Cl-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of GABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na+] and [Cl-], and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-13/s at maximal [GABA] (-80 mV, 96 mM NaCl, 22 degrees C) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GAT1 in synaptic function.
Animals↗
The real costs of emesis--an economic analysis of ondansetron vs. metoclopramide in controlling emesis in patients receiving chemotherapy for cancer.
The cost effectiveness of ondansetron was compared with that of metoclopramide in the prevention of acute emesis due to highly emetogenic chemotherapy in an open, randomised, parallel group pilot study. Ondansetron was given as three 8 mg intravenous doses (0, 4 and 8 h) and metoclopramide as an intravenous loading dose (3 mg/kg) followed by a maintenance dose of 0.5 mg/kg/h for 8 h. Therapeutic outcomes and full utilisation costs, that is nursing time, material costs, in addition to drug acquisition prices were recorded for each antiemetic for 24 h following chemotherapy. The cost per successfully treated patient (< or = 1 emetic episode and no adverse events) was 95.20 pounds for ondansetron and 92.18 pounds for metoclopramide. The results of the study therefore suggest that for the control of acute emesis due to highly emetogenic chemotherapy ondansetron and metoclopramide are equally cost-effective treatments.
Antineoplastic Agents↗
Slow and incomplete inactivations of voltage-gated channels dominate encoding in synthetic neurons.
Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na+ channels only, brief pulses (< 1 ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3 s) depolarizing plateau; this plateau was caused by slow and incomplete Na+ channel inactivation. In cells expressing both Na+ and Drosophila Shaker H4 transient K+ channels, there were neuron-like action potentials. In cells with appropriate Na+/K+ current ratios, maintaining stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to "lock-up" of the potential at a positive value after several seconds of stimulation. The latter effect was due primarily to slow inactivation of the K+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels.
Action Potentials↗
Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.
The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.
Animals↗
Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in Xenopus oocytes.
The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.
Adenylyl Cyclases↗
The role of conserved aspartate and serine residues in ligand binding and in function of the 5-HT1A receptor: a site-directed mutation study.
Wild-type and mutant serotonin 1A receptors were transiently expressed in COS-7 cells using the infection-transfection variant of the vaccinia virus/T7 polymerase vector system. The amino acid substitutions in the transmembrane regions, Asp82-->Asn82, Asp116-->Asn116, and Ser198-->Ala198 all resulted in a decrease in affinity for 5-HT by 60-100-fold, without affecting the affinity for the antagonist, pindolol. The binding of agonist to the additional mutant, Thr199-->Ala199, was too weak to be measured, 5-HT induced GTPase activities for all receptors studied. These findings indicate that the residues mutated play an important role in the binding of the agonist and less critical roles in the binding of the antagonist pindolol.
Aspartic Acid↗
Regions of beta 4.beta 2 subunit chimeras that contribute to the agonist selectivity of neuronal nicotinic receptors.
Fifteen chimeric nicotinic receptor beta subunits were constructed consisting of N-terminal neuronal beta 4 sequences and C-terminal beta 2 sequences. Responses to cytisine, nicotine, or tetramethylammonium were compared to acetylcholine responses for these subunits expressed in Xenopus oocytes with alpha 3 subunits. The results show that (i) two residues in the extracellular domain of chimeric beta 4.beta 2 subunits (108 beta 2F/beta 4V, 110 beta 2S/beta 4T) account for much of the relative cytisine sensitivity; and (ii) four extracellular residues of chimeric beta 4.beta 2 subunits (112 beta 2A/beta 4V, 113 beta 2V/beta 4I and 115 beta 2S/beta 4R, 116 beta 2Y/beta 4S) account for most of the relative tetramethylammonium sensitivity. The data did not permit localization of nicotine sensitivity to any particular region.
Acetylcholine↗
Cloning, expression, and localization of a rat brain high-affinity glycine transporter.
A cDNA clone encoding a glycine transporter has been isolated from rat brain by a combined PCR and plaque-hybridization strategy. mRNA synthesized from this clone (designated GLYT1) directs the expression of sodium- and chloride-dependent, high-affinity uptake of [3H]glycine by Xenopus oocytes. [3H]Glycine transport mediated by clone GLYT1 is blocked by sarcosine but is not blocked by methyl-aminoisobutyric acid or L-alanine, a substrate specificity similar to that described for a previously identified glycine-uptake system called system Gly. In situ hybridization reveals that GLYT1 is prominently expressed in the cervical spinal cord and brainstem, two regions of the central nervous system where glycine is a putative neurotransmitter. GLYT1 is also strongly expressed in the cerebellum and olfactory bulb and is expressed at lower levels in other brain regions. The open reading frame of the GLYT1 cDNA predicts a protein containing 633 amino acids with a molecular mass of approximately 70 kDA. The primary structure and hydropathicity profile of GLYT1 protein reveal that this protein is a member of the sodium- and chloride-dependent superfamily of transporters that utilize neurotransmitters and related substances as substrates.
Amino Acid Sequence↗
Inherited p53 gene mutations in breast cancer.
Recent evidence has implicated germ-line mutations of the p53 gene as the cause of cancer susceptibility in the Li-Fraumeni syndrome, associated with the development of breast cancer and other neoplasms. Furthermore, somatic mutations of the p53 gene have been detected in a high percentage of non-familial breast cancers. We therefore sought to identify potential carriers of p53 gene mutations in a cohort of patients with early onset breast cancer. We examined 126 consecutive patients who developed breast cancer at or before the age of 40 for mutations of p53 within conserved regions of the gene. One patient with an inherited germ-line mutation of the p53 gene was identified but the functional significance of this mutation was not clear. It thus appears that only a small percentage of patients with breast cancer under the age of 40 carry germ-line mutations of the p53 gene, an observation which has implications for potential screening and risk assessment in such patients.
Adult↗
Expression in animal cells of the 5-HT1A receptor by a vaccinia virus vector system.
The co-infection or infection-transfection variants of the T7 RNA polymerase/vaccinia vector system were used to express 5-HT1ARs in COS-7, BSC-40 and GH3 cells, with co-infection giving ca. 3-fold higher level than infection-transfection. Binding affinities were similar to those of the endogenous 5-HT1AR, with highest affinities for 5-HT and 8-OH-DPAT. Functional properties were demonstrated by assays of agonist-stimulated GTPase activity and its inhibition by pertussin toxin. Immunoblot assays showed expression of the unglycosylated and glycosylated receptor protein in the membrane and, surprisingly, in the cytosolic fractions.
8-Hydroxy-2-(di-n-propylamino)tetralin↗
The physical health of children in kinship care.
OBJECTIVE: The objectives of this study were to assess the current and chronic health problems and the adequacy of primary health care of children placed with a relative (kinship care) by a public agency. RESEARCH DESIGN: Population survey. SETTING: Children in kinship care in Baltimore, Md. PARTICIPANTS: Four hundred seven (78%) of 524 children in kinship care in 1989. INTERVENTIONS: None. MEASUREMENTS/MAIN RESULTS: Information on the child's health status and care was obtained via a review of medical records; questionnaires sent to primary care physicians, parents, care givers, and caseworkers; and clinical assessment of the child by a nurse, pediatrician, and child psychologist. The children were found to have health problems similar to those in foster and poor children, but more problems than American children in general. Frequent diagnoses included impaired visual acuity and hearing, obesity, dental caries, and asthma; often, these problems had not been identified or treated. Gaps in the medical records precluded firm conclusions concerning the children's primary care, but suggest an inadequate system for ensuring their health care. CONCLUSIONS: There is a need for improving the system of health care for children in kinship care. There is also a need for additional research on this high-risk group of children.
Adolescent↗
Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.
We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.
Amino Acid Sequence↗
Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.
Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.
Amino Acid Sequence↗
Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.
1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.
Acetylcholine↗
Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells.
The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.
Animals↗
Incubation with horse serum increases viability and decreases background neurotransmitter uptake in Xenopus oocytes.
Explore the source record for details and available documents.
Animals↗
Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.
In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-[gamma-thio]triphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinia virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels.
Acetylcholine↗