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N Davidson

Publications and source records attributed to N Davidson.

At least 55 records · Page 3Linked to original sources
PubMed1998

IL-7 deficiency prevents development of a non-T cell non-B cell-mediated colitis.

IL-7 is a stromal cell-derived cytokine with a well-established physiologic role in lymphocyte biology. This report describes an unexpected role for IL-7 in the development of colitis in a T and B cell-deficient environment. Recombination-activating gene-2 (RAG-2)-deficient mice (RAG-2(-/-)) were exposed to and subsequently maintained a horizontally transmitted microbial flora that included Helicobacter hepaticus. These animals mounted a strong myeloid cell response and developed both systemic and local signs of a severe colitis. A striking infiltration of F4/80 and MHC class II-positive cells was seen in the colon and cecum of animals undergoing the disease. Mice mutant for both IL-7 and RAG-2 (IL-7/RAG-2(-/-)) that were colonized by the same flora showed no signs of myeloid responses or colitis, indicating that IL-7 plays a critical role in exacerbating a non-T cell/non-B cell-mediated chronic inflammatory response. Recombinant IL-10 protein therapy was able to prevent the occurrence of colitis in susceptible mice, suggesting a pivotal role for macrophages. The implications of a role for IL-7 in this disease model with respect to human inflammatory bowel disease are discussed.

Animals↗
PubMed1998

Expression of a dominant negative TrkB receptor, T1, reveals a requirement for presynaptic signaling in BDNF-induced synaptic potentiation in cultured hippocampal neurons.

We have developed a method to analyze the relative contributions of pre- and postsynaptic actions of a particular gene product in neurons in culture and potentially in slices using adenovirus-mediated gene transfer. A recombinant virus directed the expression of both a GFP reporter protein and TrkB.T1, a C-terminal truncated dominant negative TrkB neurotrophin receptor. When expressed in the presynaptic cell at synapses between embryonic hippocampal neurons in culture, the dominant negative TrkB.T1 inhibited two forms of synaptic potentiation induced by the neurotrophin brain-derived neurotrophic factor (BDNF): (i) greater evoked synaptic transmission and (ii) higher frequency of spontaneous miniature synaptic currents. These inhibition effects are not seen if the transgene is expressed only in the postsynaptic cell. We conclude that BDNF-TrkB signal transduction in the presynaptic terminal leads to both types of potentiation and is therefore the primary cause of synaptic enhancement by BDNF in these neurons.

Animals↗
PubMed1998

The transcriptional corepressor NAB2 inhibits NGF-induced differentiation of PC12 cells.

The PC12 pheochromocytoma cell line responds to NGF by undergoing growth arrest and proceeding to differentiate toward a neuronal phenotype. Among the early genetic events triggered by NGF in PC12 cells are the rapid activation of the zinc finger transcription factor Egr1/NGFI-A, and a slightly delayed induction of NAB2, a corepressor that inhibits Egr1 transcriptional activity. We found that stably transfected PC12 cells expressing high levels of NAB2 do not differentiate, but rather continue to proliferate in response to NGF. Inhibition of PC12 differentiation by NAB2 overexpression was confirmed using two additional experimental approaches, transient transfection, and adenoviral infection. Early events in the NGF signaling cascade, such as activation of MAP kinase and induction of immediate-early genes, were unaltered in the NAB2-overexpressing PC12 cell lines. However, induction of delayed NGF response genes such as TGF-beta1 and MMP-3 was inhibited. Furthermore, NAB2 overexpression led to downregulation of p21(WAF1), a molecule previously shown to play a pivotal role in the ability of PC12 cells to undergo growth arrest and commit to differentiation in response to NGF. Cotransfection with p21(WAF1) restored the ability of NAB2-overexpressing PC12 cells to differentiate in response to NGF.

Animals↗
PubMed1998

Muscle contractility decrement and correlated morphology during the pathogenesis of streptozotocin-diabetic mice.

BACKGROUND: Peripheral neuropathy of both motor and sensory nerves has been well documented in diabetes mellitus, but the evidence for physiological and correlated morphological changes during the pathogenesis of myopathy is scarce. In the present report, we have chosen the dorsiflexor muscle of adult male mice as a model for studying in situ muscle contraction and neuromuscular ultrastructure during the pathogenesis of streptozotocin-induced diabetes. METHODS: Thirty mice (30 g bodyweight) were injected once i.p. with streptozotocin solution (200 mg/Kg) to induce experimental diabetes mellitus. Comparative analyses of in situ muscle isometric contractile characteristics were studied (at 1 Hz, 5 Hz and 30 Hz nerve stimulation) in urethane-anesthetized (2 mg/g, i.p.) control and diabetic mice at three time points, 2 weeks, 4 weeks, and 8 weeks postinjection. Synaptic delay was also recorded in diabetic and age-matched control mice. RESULTS: There was a significant increase in synaptic delay in both 4-week and 8-week diabetic mice compared with control mice (8.9 +/- 1.2 msec and 7.6 +/- 0.6 msec, respectively, compared with 6.1 +/- 0.5 msec). At all three stimulation frequencies, diabetes did not affect muscle contractile speed but significantly reduced the twitch tension after 8 weeks, with no changes at 2 weeks or 4 weeks. The recorded single-twitch tension values were 2.6 +/- 0.3 g, 2.1 +/- 0.6 g, 2.2 +/- 0.7 g, and 1.2 +/- 0.1 g for control, 2 weeks, 4 weeks, and 8 weeks, respectively. At 30 Hz, the recorded tension values were 4.6 +/- 1.6 g, 3.1 +/- 1.2 g, 3.1 +/- 1.1 g, and 2.1 +/- 1.0 g for control, 2 weeks, 4 weeks, and 8 weeks, respectively. Ultrastructural changes in neuromuscular junctions were similar to those that have been described in disuse and aging. These changes were observed after 8 weeks and included serve loss of synaptic vesicles, electron-dense bodies, and myelin-like figures as well as degeneration of mitochondria. CONCLUSIONS: The results reveal that streptozotocin-induced diabetes affects presynaptically the neuromuscular junction as well as muscle itself. Actions at both sites may contribute to the functional alterations seen in muscle contractile properties and may play a role in the pathogenesis of diabetic neuromyopathy.

Animals↗
PubMed1998

Antidiuretic hormone infusion reduces taurine and NaCl-induced hypernatremia in the rat.

Rats drinking taurine and hypertonic saline (T + S) develop severe hypernatremia, but rats drinking either T or S alone do not. One hypothesis for this disruption of homeostasis is that the T + S combination interferes with the actions of antidiuretic hormone (ADH). Rats drinking T + S developed severe hypernatremia (170 mmol/L) by day 8 when infused with distilled water by osmotic minipumps, but maintained plasma sodium below 150 mmol/L when infused with ADH. Cumulative water balance in T + S drinkers receiving ADH was consistently higher than in those not receiving ADH. However the ratio of cumulative sodium balance to cumulative water balance suggests little uniform advantage to rats receiving ADH nor does comparison of urine osmolality in the two groups. Precisely how ADH administration reduces hypernatremia in T + S drinking rats remains unclear, but the hypothesis that T + S interferes with the action of ADH in its regulation of extracellular fluid volume and osmolality remains viable.

Animals↗
PubMed1997

A C-terminal peptide of the GIRK1 subunit directly blocks the G protein-activated K+ channel (GIRK) expressed in Xenopus oocytes.

1. In order to find out the functional roles of cytosolic regions of a G protein-activated, inwardly rectifying potassium channel subunit we studied block of GIRK channels, expressed in Xenopus laevis oocytes, by synthetic peptides in isolated inside-out membrane patches. 2. A peptide (DS6) derived from the very end of the C-terminus of GIRK1 reversibly blocked GIRK activity with IC50 values of 7.9 +/- 2.0 or 3.5 +/- 0.5 micrograms ml-1 (corresponding to 3.7 +/- 0.9 or 1.7 +/- 0.2 mumol l-1) for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively. 3. Dose dependency studies of GIRK activation by purified beta gamma subunits of the G protein (G beta gamma) showed that DS6 block of GIRK channels is not the result of competition of the peptide with functional GIRK channels for the available G beta gamma. 4. Burst duration of GIRK channels was reduced, whereas long closed times between bursts were markedly increased, accounting for the channel block observed. 5. Block by the DS6 peptide was slightly voltage dependent, being stronger at more negative potentials. 6. These data support the hypothesis that the distal part of the carboxy-terminus of GIRK1 is a part of the intrinsic gate that keeps GIRK channels closed in the absence of G beta gamma.

Amino Acid Sequence↗
PubMed1997

Site-specific, photochemical proteolysis applied to ion channels in vivo.

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the "signature" Cys128-Cys142 disulfide loop of the nAChR alpha subunit.

Animals↗
PubMed1997

RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels.

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Acetylcholine↗
PubMed1997

Activation of heteromeric G protein-gated inward rectifier K+ channels overexpressed by adenovirus gene transfer inhibits the excitability of hippocampal neurons.

G protein-gated inward rectifier K+ channel subunits 1-4 (GIRK1-4) have been cloned from neuronal and atrial tissue and function as heterotetramers. To examine the inhibition of neuronal excitation by GIRKs, we overexpressed GIRKs in cultured hippocampal neurons from 18 day rat embryos, which normally lack or show low amounts of GIRK protein and currents. Adenoviral recombinants containing the cDNAs for GIRK1, GIRK2, GIRK4, and the serotonin 1A receptor were constructed. Typical GIRK currents could be activated by endogenous GABAB, serotonin 5-HT1A, and adenosine A1 receptors in neurons coinfected with GIRK1+2 or GIRK1+4. Under current clamp, GIRK activation increased the cell membrane conductance by 1- to 2-fold, hyperpolarized the cell by 11-14 mV, and inhibited action potential firing by increasing the threshold current for firing by 2- to 3-fold. These effects were not found in non- and mock-infected neurons, and were similar to the effects of muscarinic stimulation of native GIRK currents in atrial myocytes. Two inhibitory effects of GIRK activation, hyperpolarization and diminution of depolarizing pulses, were simulated from the experimental data. These inhibitory effects are physiologically important in the voltage range between the resting membrane potential and the potential where voltage-gated Na+ and K+ currents are activated; that is where GIRK currents are outward.

Action Potentials↗
PubMed1997

Second messengers, trafficking-related proteins, and amino acid residues that contribute to the functional regulation of the rat brain GABA transporter GAT1.

Recent evidence indicates that several members of the Na+-coupled transporter family are regulated, and this regulation in part occurs by redistribution of transporters between intracellular locations and the plasma membrane. We elucidate components of this process for both wild-type and mutant GABA transporters (GAT1) expressed in Xenopus oocytes using a combination of uptake assays, immunoblots, and electrophysiological measurements of membrane capacitance, transport-associated currents, and GAT1-specific charge movements. At low GAT1 expression levels, activators of protein kinase C (PKC) induce redistribution of GAT1 from intracellular vesicles to the plasma membrane; at higher GAT1 expression levels, activators of PKC fail to induce this redistribution. However, coinjection of total rat brain mRNA with GAT1 permits PKC-mediated modulation at high transporter expression levels. This effect of brain mRNA on modulation is mimicked by coinjection of syntaxin 1a mRNA and is eliminated by injecting synaptophysin or syntaxin antisense oligonucleotides. Additionally, botulinum toxins, which inactivate proteins involved in vesicle release and recycling, reduce basal GAT1 expression and prevent PKC-induced translocation. Mutant GAT1 proteins, in which most or all of a leucine heptad repeat sequence was removed, display altered basal distribution and lack susceptibility to modulation by PKC, delineating one region of GAT1 necessary for its targeting. Thus, functional regulation of GAT1 in oocytes occurs via components common to transporters and to trafficking in both neural and non-neural cells, and suggests a relationship between factors that control neurotransmitter secretion and the components necessary for neurotransmitter uptake.

Amino Acids↗
PubMed1997

Topotecan versus paclitaxel for the treatment of recurrent epithelial ovarian cancer.

PURPOSE: Topotecan and paclitaxel were evaluated in a randomized, multicenter study of patients with advanced epithelial ovarian carcinoma who had progressed during or after one platinum-based regimen. PATIENTS AND METHODS: Patients received either topotecan (1.5 mg/m2) as a 30-minute infusion daily for 5 days every 21 days (n = 112) or paclitaxel (175 mg/m2) infused over 3 hours every 21 days (n = 114). Patients had bidimensionally measurable disease and were assessed for efficacy and toxicity. RESULTS: Response rate was 23 of 112 (20.5%) in topotecan-treated patients and 15 of 114 (13.2%) in paclitaxel-treated patients (P = .138). Disease stabilization for at least 8 weeks was noted in 30% of patients with topotecan and 33% of patients with paclitaxel. Median durations of response to topotecan and paclitaxel were 32 and 20 weeks, respectively (P = .222) and median times to progression were 23 and 14 weeks, respectively (P = .002). Median survival was 61 weeks for topotecan and 43 weeks for paclitaxel (P = .515). Response rates for topotecan and paclitaxel were 13.3% versus 6.7% (P = .303) in resistant patients (not responded to prior platinum-based therapy or progressed within 6 months of an initial response) and 28.8% versus 20.0% (P = .213) in sensitive patients (progressed > 6 months after response). Neutropenia was significantly more frequent on the topotecan arm 79% versus paclitaxel arm 23% (P < .01). It was short-lasting and noncumulative in both arms. Nonhematologic toxicities were generally mild (grades 1 to 2) for both agents. CONCLUSION: Topotecan has efficacy at least equivalent to paclitaxel manifested by the higher response rate and significantly longer time to progression.

Adult↗
PubMed1996

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation.

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G beta gamma subunits, but instead shows constitutive activation, and (ii) is no longer a K(+)-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na(+)-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.

Acetylcholine↗
PubMed1996

Desensitization of inositol 1,4,5-trisphosphate/Ca2+-induced Cl- currents by prolonged activation of G proteins in Xenopus oocytes.

Expression of G protein alpha subunits of the Gq family with various G protein-coupled receptors induces activation of an inositol 1,4, 5-trisphosphate (IP3)/Ca2+-mediated Cl- conductance in Xenopus oocytes. Our present data show that two members of this family, the human Galpha16 subunit and the murine homologue Galpha15, can induce both activation and inhibition of these agonist-induced currents. Although extremely low amounts (10-50 pg) of injected Galpha16 subunit cRNA cause modest ( approximately 2-fold) enhancement of ligand-induced Cl- currents in oocytes co-injected with thyrotropin-releasing hormone (TRH) receptor cRNA 48 h postinjection, larger Galpha16 and Galpha15 cRNA injections cause >10-fold inhibition of TRH or 5HT2c receptor responses. The inhibition is analyzed in this study. The inhibited currents are recovered if various Gbetagamma subunit combinations are also expressed with the Galpha subunits. The constitutively active mutant, Galpha16Q212L, also causes a strong attenuation of the ligand-induced Cl- currents, but this inhibition is not recovered by co-expression of Gbetagamma subunits. These results indicate that the free Galpha subunit is responsible for the inhibitory signal. Although expression of TRH receptor alone produces maximum responses approximately 48 h after injection, co-expression of TRH receptor with Galpha16 results in enhanced responses 6-12 h postinjection, followed by complete attenuation at 36 h. Furthermore, injection of Galpha16 cRNA alone at comparable levels gives rise to spontaneous Cl- currents within 6-12 h postinjection, suggesting that the early spontaneous activation underlies the later suppression. Expression of other G protein alpha subunits of the Gq family, at cRNA levels considerably higher than effective for Galpha16, produces both analogous spontaneous Cl- currents and, later, inhibition of ligand-induced Cl- currents. Experiments with direct injection of IP3 and of Ca2+ suggest that this inhibition is consistent with the down-regulation of IP3 receptors. These data indicate that both enhancement and inhibition of signaling through G protein-coupled receptors can be mediated by the expression level and/or activity of an individual G protein.

Animals↗
PubMed1996

A role for endothelial NO synthase in LTP revealed by adenovirus-mediated inhibition and rescue.

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction.

Adenoviridae↗
PubMed1996

Ion binding and permeation at the GABA transporter GAT1.

This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [3H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3H]GABA and [3H]tiagabine binding. Voltage jumps, Na+ and Cl- concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na+ ions interact with the transporter even in the absence of GABA, and (2) that Cl- facilitates the binding of Na+. Comparison between the charge movements and the transport-associated current shows that this initial Na(+)-transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation--treatment with high-affinity uptake blockers and the W68L mutation-"lock" Na+ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na+ substitutes Li+ and Cs+ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na+ and Cl-, and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.

Animals↗