Biomedical subjects

C S Tung

Publications and source records attributed to C S Tung.

At least 37 records · Page 2Linked to original sources

PubMed1998

Actinomycin D binds to metastable hairpins in single-stranded DNA.

We have examined the role of DNA composition in the binding of actinomycin D to single-stranded DNA. By using the fluorescent analogue 7-aminoactinomycin D, we were able to monitor binding of the drug to ssDNA with single base changes distant from the 5'-TAGT-3' site previously determined to be a high-affinity site for actinomycin D binding (Wadkins et al. (1996) J. Mol. Biol. 262, 53-68). Our binding studies indicated that secondary structures in the ssDNA were likely to be responsible for binding the drug. A series of six low-melting DNA hairpins containing all or part of the 5'-TAGT-3' binding site were synthesized. The highest Tm observed for the melting of these hairpins was 34.2 +/- 0.3 degrees C, and it depended on the length of the stem region. These metastable hairpins were stabilized by 7-aminoactinomycin D, with the drug shifting the Tm for the drug-hairpin complex to approximately 45 degrees C. The hairpins showed very high affinity (Kd approximately 0.1 microM) for 7-aminoactinomycin D, with some dependence on stem length. Digestion of the hairpins in the presence and absence of drug using mung bean nuclease, which specifically interacts with the loop region of hairpin DNA, revealed that the stable hairpins (i) contain a number of non-Watson-Crick base pairs, and (ii) undergo a conformational change in the loop region upon binding 7-aminoactinomycin D. Our results suggest that stabilization of unusual hairpins by actinomycin D may be an important aspect of the potent transcription inhibition activity of this drug.

Base Composition↗

PubMed1998

Modulation of high affinity hormone binding. Human choriogonadotropin binding to the exodomain of the receptor is influenced by exoloop 2 of the receptor.

The lutropin/choriogonadotropin receptor is a seven-transmembrane receptor and consists of two major domains of similar size, an extracellular exodomain and a membrane-associated endodomain which includes 3 exoloops. The uniquely large exodomain is responsible for high affinity hormone binding whereas receptor activation occurs at the endodomain. However, little is known about the relationship between the exodomain and endodomain. It was reported that hormone binding to the exodomain was improved when the endodomain was truncated. This result suggests that hormone binding to the exodomain was influenced by the endodomain. To test this hypothesis, amino acids of exoloop 2 were examined by Ala substitutions. The binding affinity was enhanced by some Ala substitutions but attenuated by others. These results indicate that exoloop 2 influences the hormone binding to the exodomain. Particularly, the high affinity hormone binding at the exodomain is constrained by a group of amino acids, Ser484, Asn485, Lys488, Ser490, and Ser499. Computer modeling suggests these residues may be positioned on one side of exoloop 2. It also influences the affinity for cAMP induction and the maximal cAMP production in distinct ways, in addition to its influence on the hormone binding affinity. The distinct ways of influencing these functions are sometimes in conflict and compromised to attain the maximal affinity for cAMP induction. As a result, the exodomain attains the maximal affinity for hormone binding when the endodomain is truncated and cAMP induction is disengaged.

Cell Compartmentation↗

PubMed1998

Metabotropic glutamate receptors are involved in calcium-induced LTP of AMPA and NMDA receptor-mediated responses in the rat hippocampus.

Effects of metabotropic glutamate (mGlu) receptors on calcium-induced long-term potentiation (LTP) of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components were investigated in rat hippocampal slices using whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs). Calcium-induced LTP comprises a parallel, long-lasting increase of AMPA and NMDA receptor-mediated components. The calcium-induced LTP of the AMPA receptor-mediated component can be significantly attenuated by the use of a selective NMDA antagonist. (R.S)-alpha-methyl-4-carboxyphenylglycine (MCPG), a selective antagonist of mGlu receptors, abolished the long-lasting increase of both AMPA and NMDA receptor-mediated components observed in calcium-induced LTP. In current clamp mode, the application of a high calcium alone or Schaffer fiber stimulation alone (20 Hz) only generated a short-term increase in the firing rate of evoked action potentials. Conversely, a long-term increase in the firing rate was observed if Schaffer fiber stimulation (20 Hz) accompanied the perfusion of high calcium. These results suggest that calcium-induced LTP involves a parallel, long-lasting enhancement in ionotropic AMPA and NMDA receptor-mediated components. More importantly, the mGlu receptor plays a critical role in the establishment of both AMPA and NMDA receptor-mediated components underlying calcium-induced LTP. In addition, the present study also described an experimental condition in which the coapplication of the high calcium pulse and Schaffer fiber stimulation (20 Hz) can synergistically elicit a long-term increase of neuronal excitability.

Action Potentials↗

PubMed1998

Arecoline desensitizes carbachol-stimulated phosphatidylinositol breakdown in rat brain cortices.

To understand the effects of arecoline administration on the muscarinic cholinergic signaling pathway, rats were injected with arecoline, 10 mg/kg i.p., and the carbachol-stimulated phosphoinositide breakdown in rat brain cortical slices was examined. In vivo administration of arecoline resulted in inhibition of carbachol-stimulated phosphoinositide turnover in rat brain cortical slices. Arecoline was a partial agonist with peak effects of 30% of the maximum as obtained with carbachol. Coaddition of arecoline inhibited the carbachol-stimulated phosphoinositide breakdown. Pretreatment of rat brain cortical slices with arecoline in vitro resulted in a dose-dependent inhibition of carbachol-stimulated [3H]inositol monophosphate accumulation. The inhibition occurred rapidly, with half-maximal inhibition occurring at 15 min and maximal inhibition achieved within 60 min. The inhibition of phosphoinositide breakdown was recovered 1 h after arecoline was removed. When synaptoneurosomes were used for the ligand binding studies, arecoline pretreatment was found to have decreased the maximal ligand binding (Bmax) without inducing any marked change in binding affinity (K(D)). The influence could be recovered by incubating the synaptoneurosomes in the absence of arecoline for 2 h. Taken together, these data suggest that the underlying mechanism by which phosphoinositide turnover is inhibited is arecoline-induced receptor sequestration.

Animals↗

PubMed1998

Probing site-specific interactions in protein-DNA complexes using heteronuclear NMR spectroscopy and molecular modeling: binding of Cro repressor to OR3.

In this paper, a general method is developed to study site-specific interactions in DNA-protein complexes using heteronuclear NMR spectroscopy and molecular modeling. This method involves two steps: (a) homonuclear 1H NMR and molecular modeling are used to develop a low resolution model and (b) 15N7-guanosine containing oligonucleotides are employed to probe the specific intermolecular interactions predicted in (a). This method is applied to Cro-operator complex due to its small size and extensive prior characterization. Non-exchangeable and exchangeable base protons have been assigned by nuclear Overhauser effect spectroscopy (NOESY) and chemical shift correlation spectroscopy. Extensive line-broadening has been observed in the 1H NMR spectra of the operator DNA in the presence of protein. Differential line-broadening observed in the imino proton region and the comparison of NOESY spectra in the presence and absence of Cro protein show that guanosine-12 and guanosine-14 are involved in the Cro-DNA interaction, while the three A.T base-pairs at the 3'- and 5'-termini play only a minor role in the binding. A model of the Cro-operator DNA complex has been constructed by docking helix-3 of the Cro protein in the major groove and it predicted specific hydrogen bonds between N7 of guanosines-12 and -14 and the side-chain of Lys-32 and Ser-28, respectively. The appearance of a new resonance in the temperature dependent proton detected heteronuclear multiple quantum coherence (HMQC) spectra of the Cro-DNA complex also demonstrates a specific interaction of Cro with guanosine-14 of the operator DNA.

DNA↗

PubMed1998

Intrathecal administration of excitatory amino acid receptor antagonists or nitric oxide synthase inhibitor reduced autotomy behavior in rats.

UNLABELLED: Peripheral nerve injury may produce neuropathic pain. At the spinal cord level, excitatory amino acid receptors play a role in nociceptive signal modulation. N-methyl-D-aspartate (NMDA) receptor activation initiates the NO-cGMP pathway and further modulates nociceptive signal. Using the autotomy model, we examined the effect of an NMDA and a non-NMDA receptor antagonist, and nitric oxide synthase inhibitor in the treatment of autotomy behavior in rats. A right-side brachial plexus (C5-T1) transection was performed in all rats. In the treatment groups, MK-801, 1-(4-chlorobenzoyl)-piperazine-2,3-dicarboxylic acid (a non-NMDA antagonist), and L-NG-nitro arginine methyl ester (L-NAME) were infused intrathecally via an osmotic pump for 7 days at doses of 10, 4, and 400 microg/h, respectively. Saline was infused to control animals. Autotomy behavior was observed daily for 8 wk. The incidence of autotomy was 85% in the control group. MK-801, the non-NMDA antagonist, and L-NAME reduced the autotomy incidence to 10%, 20%, and 10% (P < 0.0001), respectively. All treatments delayed the onset of the autotomy behavior from 15+/-4.5 days in the control group to 52+/-3.8, 43+/-5.6, and 54+/-2.9 (P < 0.001) days in the treatment groups, respectively. The average autotomy scores were also attenuated significantly by these treatments. We conclude that the excitatory amino acid receptors and their intracellular signal messenger NO play a role in deafferentation behavior development. Inhibition of this signaling pathway may be of use for neuropathic pain relief. IMPLICATIONS: The excitatory amino acid receptors and their intracellular signal messenger NO play a role in deafferentation behavior development. Inhibition of this signaling pathway may be of use for neuropathic pain relief.

Animals↗

PubMed1998

A methodological improvement and system validation to obtain precise behavioral parameters for schedule-induced polydipsia.

A Schedule-Induced Polydipsia (SIP) animal behaviors monitor system was devised. The system included a software package to acquire data and to format the data storage of the animal behavior in the experiment was designed. Data analysis software will also extract the necessary information from recorded data. The SIP experimental apparatus consists of a number of subsystems. They are operant chambers, event counters, signal acquisition and storage system. The operant chamber is equipped with hopper, pellet and lick sensor and locomotion detector. The data acquisition and storage subsystem are custom designed that run on IBM-PC. The discrete time markers for pellet drop, bar pressing and licking water were recorded at 200 Hz. The image of rat locomotion was recorded at 10 Hz. A test of four different stages of animal to verify the accuracy of the system was reported in this article. The food-deprived rats that were exposed to the intermittent food schedule (1 per minute) exhibit an excess drinking behavior. The animal behaviors that were monitored during the scheduled events are adjunctive, facultative and terminal behavior. The volume of water intake was also recorded for reference. The real time data is stored chronologically into two types of data file. Therefore, a special data analysis procedure is designed to extract the results for off-line statistic analysis. The extracted parameters for animal behaviors analyzing including drinking efficiency, inter-lick intervals, number of drinking bursts, size of burst, temporal distribution of licking, temporal distribution of bar pressing and locomotion. The system can be employed in many different SIP studies such as investigating the effect of acute and chronic influence of amphetamine.

Animals↗

PubMed1997

Endothelin-3 reduces C-type natriuretic peptide-induced cyclic GMP formation in C6 glioma cells.

The effect of endothelin-3 (ET-3) on C-type natriuretic peptide (CNP)-induced guanosine 3',5'-cyclic monophosphate (cGMP) was examined in C6 glioma cells, CNP-induced cGMP formation was both time- and dose-dependent, with an EC50 value of about 10 nM. While ET-3 and phorbol 12-myristate 13-acetate (PMA) had no effect on basal cGMP production, both compounds were potent inhibitors of CNP-induced cGMP formation, with IC50 values of approximately 10 and 2 nM, respectively. Although protein kinase C (PKC) inhibitors had no effect on basal cGMP formation, Ro 31-8220, a PKC inhibitor, reversed the ET-3 inhibition on CNP-induced cGMP formation by 63% and that of PMA almost completely. Our findings suggest that stimulation of cGMP formation by CNP in C6 glioma cells is negatively modulated by PKC activation, and that the inhibitory action of ET-3 on CNP-stimulated cGMP formation is mediated partly by PKC.

Animals↗

PubMed1997

Nicotinamide attenuates methamphetamine-induced striatal dopamine depletion in rats.

The aim of the present study was to examine the effects of nicotinamide, a co-factor in the electron transport chain, on the relationship between methamphetamine (MA)-induced striatal dopamine (DA) depletion and energy metabolism change. Four injections of MA (10 mg/kg, i.p.) at 2 h intervals resulted in decreases of 51% and 23%, respectively, in striatal DA and adenosine 5'-triphosphate (ATP) levels 5 days later. Nicotinamide (500 mg/kg, i.p.) treatment prior to each MA injection attenuated the reductions of striatal DA and ATP contents. Nicotinamide had no long-term effects on striatal DA and ATP levels. These findings suggest that energy impairment might play a role in MA-induced DAergic neurotoxicity in the striatum.

Adenosine Triphosphate↗

PubMed1997

A computational approach to modeling nucleic acid hairpin structures.

Hairpin is a structural motif frequently observed in both RNA and DNA molecules. This motif is involved specifically in various biological functions (e.g., gene expression and regulation). To understand how these hairpin motifs perform their functions, it is important to study their structures. Compared to protein structural motifs, structures of nucleic acid hairpins are less known. Based on a set of reduced coordinates for describing nucleic acid structures and a sampling algorithm that equilibrates structures using Metropolis Monte Carlo simulation, we developed a method to model nucleic acid hairpin structures. This method was used to predict the structure of a DNA hairpin with a single-guanosine loop. The lowest energy structure from the ensemble of 200 sampled structures has a RMSD of < 1.5 A, from the structure determined using NMR. Additional constraints for the loop bases were introduced for modeling an RNA hairpin with two nucleotides in the loop. The modeled structure of this RNA hairpin has extensive base stacking and an extra hydrogen bond (between the CYT in the loop and a phosphate oxygen), as observed in the NMR structure.

Algorithms↗

PubMed1997

Amphetamine induces hydroxyl radical formation in the striatum of rats.

Amphetamine-induced hydroxyl radical formation in the striatum of rats was investigated in this study. With the utilization of the microdialysis and HPLC-ECD, the striatal dopamine (DA) release and the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA), derived from the reaction of hydroxyl radicals (.OH) and salicylate in perfusion, were monitored and detected during desipramine and/or amphetamine (AMPH) administration. Our data revealed that after desipramine treatment AMPH injections not only amplified striatal DA release and 2,3-DHBA formation, but also intensified the stereotyped behaviors induced by AMPH. Furthermore, we discovered that alpha-methyl-para-tyrosine (alpha-MT) pretreatment prevented the onset of the above responses. In desipramine-treated rats, the tissue homogenization study demonstrated that a single dose of AMPH produced long-term depletion of striatal DA; this was not seen in saline-treated rats. Moreover, striatal DA depletion could be lessened by pretreatment with mannitol, a .OH scavenger. These results indicate that AMPH-induced striatal .OH formation might be DA-related in desipramine-treated rats, and suggest that .OH formation might be correlated with AMPH-induced neurodegeneration.

Amphetamine↗

PubMed1997

Effects of acute and chronic morphine on DOPAC and glutamate at subcortical DA terminals in awake rats.

Effects of morphine and naloxone on the levels of 3,4-dihydroxy-phenylacetic acid (DOPAC) and glutamate in the striatum and nucleus accumbens of awake rats were studied with in vivo microdialysis. Acute morphine (50 mg/kg, IP) treatment increased the levels of DOPAC and glutamate in the striatum and nucleus accumbens, but both decreased from the elevated levels when naloxone (10 mg/kg, IP) was given 2 h later. Chronic morphine treatment, twice daily for 5 days in incremental doses (5, 10, 20, 40 and 50 mg/kg, IP), increased the level of DOPAC but decreased that of glutamate in the striatum and nucleus accumbens. When naloxone was given 2 h later, the reverse of the above phenomena are found. After given repeated morphine treatment and experiencing naloxone-precipitated withdrawal, the rats with an intact cortex and the rats with ibotenic acid (5 microg/0.5 microl/2.5 min) lesions on the medial prefrontal cortex and sulcal cortex have similar alternations in the levels of DOPAC and glutamate in the striatum. However, in the nucleus accumbens, the level of DOPAC dropped more and the level of glutamate increased more in the intact rats than the lesioned rats during the withdrawal stage. These data suggested that the intact cortex ordinarily exerted an inhibitory role to influence the level of DOPAC in the striatum and nucleus accumbens during chronic morphine treatment. In conclusion, morphine seems to activate different pathways in dependent and non-dependent rats.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1997

Cardiovascular effects of nitric oxide and N-methyl-D-aspartate receptors in the nucleus tractus solitarii of rats.

Nitric oxide (NO) is an endogenously synthesized effector molecule that acts as a neurotransmitter with novel properties in both the central and peripheral nervous systems. We previously reported that NO was involved in central cardiovascular regulation and modulated the baroreflex in the nucleus tractus solitarii (NTS) of rats. The aim of the present study was to determine whether NO and excitatory amino acids reciprocally release each other in the NTS. In normotensive Sprague-Dawley rats, intra-NTS microinjection of L-arginine (1 to 100 nmol/60 nL) produced a dose-dependent decrease in blood pressure and heart rate. Microinjection of excitatory amino acids L-glutamate and NMDA also produced depressor and bradycardic effects. These effects of L-glutamate or NMDA were blocked by prior administration of NO synthase inhibitor N(G)-methyl-L-arginine or N(G)-nitro-L-arginine methyl ester. Similarly, prior administration of N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 and non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione significantly attenuated the depressor and bradycardic effect of L-arginine. These results demonstrated a reciprocal attenuation of NO synthase inhibitor and NMDA receptor antagonist on NMDA and L-arginine responses, respectively, in the NTS and suggest that NO and NMDA receptors may interact in central cardiovascular regulation.

Animals↗

PubMed1996

Effects of NMDA receptor antagonists on inhibition of morphine tolerance in rats: binding at mu-opioid receptors.

Past studies have shown antagonists of excitatory amino acid receptors, both N-methyl-D-aspartate (NMDA) and non-NMDA, to produce an antinociceptive effect in vitro and in vivo. Additionally, NMDA receptor antagonists have been demonstrated to prevent morphine tolerance. We had found that one NMDA receptor antagonist, ketamine, potentiates morphine's analgesic effect in post-operative patients. Our latest experiment was performed to examine the modulatory effect of competitive and non-competitive NMDA receptor antagonists on morphine antinociception and tolerance. A PE10 catheter was intrathecally (i.t.) implanted in male Sprague-Dawley rats for drug administration. The antinociceptive effect of morphine, D-(-)-2-amino-5-phosphonovaleric acid (D-AP5) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate (MK-801) was measured using the hot-water tail immersion test. Neither competitive nor non-competitive NMDA receptor antagonists had an antinociceptive effect by themselves, but they did potentiate the antinociceptive effect of morphine. Both D-AP5 (AD50 = 0.18 micrograms) and MK-801 (AD50 = 0.57 micrograms) shifted the antinociceptive dose-response curve of morphine (AD50 = 4.2 micrograms) to the left. Both D-AP5 (4 micrograms/h) and MK-801 (10 micrograms/h) when co-administered with i.t. morphine infusions (10 micrograms/h) also inhibited the development of tolerance. In [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin ([3H]DAMGO) binding assays, MK-801 (Bmax = 32.90 +/- 3.33 fmol/mg) treatment prevented the down-regulation of mu-opioid receptor high-affinity sites induced by continuous morphine infusions alone (Bmax = 13.97 +/- 1.47 fmol/mg). D-AP5 (Bmax = 20.78 +/- 3.36 fmol/mg) did not prevent the reduction of mu-opioid receptor high-affinity sites. However, high-affinity sites in rats treated with D-AP5 and morphine displayed a higher affinity (KD = 0.45 +/- 0.09 nM) than those of control animals (KD = 0.95 +/- 0.08 nM). Results of this study indicate that competitive as well as non-competitive NMDA receptor antagonists enhance morphine's antinociceptive effect, and prevent the development of morphine tolerance. Thus, in our opinion, there opens a new frontier in clinical pain management, especially for those patients who require long-term opioid treatment for pain relief.

2-Amino-5-phosphonovalerate↗

PubMed1996

Serotonergic serotonin 2 receptor modulation on DOPAC and 5-HIAA levels in rat striatum and nucleus accumbens: microdialysis studies of freely moving rats.

1. In vivo microdialysis with microbore-HPLC/ECD was employed to examine and compare changes of monoaminergic metabolites, dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) in the nucleus accumbens (NuAc) and the neostriatum (Str) of freely moving rats, following systemic administration of the specific 5-HT(2) analogue. 2. The 5-HT(2) agonist decreased DOPAC and 5-HIAA in both the NuAc and Str. However, the effect produced by apomorphine only decreased the DOPAC level in these two areas. 3. This effect did not occur in the rats that had been pretreated with the serotonin (5-HT) depleting agent. However, the effect was found in the rats that the striatal neurons had been damaged. 4. The 5-HT(2) effect regulated the mesencephalic dopamine (DA) synthesis/turnover only when entirely influenced by 5-HT. The striato-nigral feedback loop was not involved in this effect. Additionally, presynaptic relationship probably occurred between the 5-HT and its innervated DA pathways.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1996

Structural prediction of A- and B-DNA duplexes based on coordinates of the phosphorus atoms.

The sequence-dependent structure of DNA double helices was studied extensively during the past 10 years. How the backbone structure correlates with the base structure in a duplex conformation is still an important yet open question. Using a set of reduced coordinates and a least-squares fitting procedure, we have developed a method to predict structures for B-DNA duplexes based on coordinates of the phosphorus atoms. This method can be used to predict all-atom structures for both bent and straight molecules. We estimated the accuracies of the predictions by studying a set of 10 oligonucleotides with their structures available from the Protein Data Bank. We used this method to construct a modeled structure for the bacteriophage lambda cro operator for which the phosphorus coordinates were known from 3.5-angstrum resolution crystal data (4CRO).

Bacteriophage lambda↗

PubMed1996

A geometrical approach in folding a pseudoknot motif within the E. coli 16S-RNA.

Except for tRNA, the tertiary structure of RNA molecules are very little known. The many possibilities in the arrangement of different helices in space and the flexibility in the single-stranded loops that connect the helical regions make the modeling of the tertiary structure of RNA molecule a very complex task. Here, we introduce an approach to fold RNA tertiary structure based only on the information of the secondary structure and the stereochemistry of the molecule. This approach was used to construct an atomic structure of a pseudoknot (bases 500-545) in the E. coli 16S RNA. The resulting structure is a closely packed molecule that is consistent with the predicted secondary structure and stereochemically feasible. This new approach is very general and easily adaptable. Experimental data (e.g., NMR, fluorescence energy transfer, etc.), as they become available, can be incorporated directly into the approach to improve the accuracy of the modeled structure.

Escherichia coli↗

PubMed1996

NAMOT2--a redesigned nucleic acid modeling tool: construction of non-canonical DNA structures.

Using a new set of reduced coordinates developed for describing regular and unusual nucleic acid structures, we have revised our nucleic acid modeling tool NAMOT2. NAMOT2 is general in terms of modeling different nucleic acid structures. A set of modifiable libraries allows users to customize their modeling environment. With this set of libraries, NAMOT2 can be used to model non-canonical structures such as parallel-stranded, triple-stranded and quadruple-stranded nucleic acid molecules. For modeling irregular structures (junctions, hairpin loops, etc.), we introduce a structural recipe approach. The complete procedure using NAMOT2 to construct the structure of a specific molecule is treated as the recipe for that structural motif. The existing recipes can be modified to generate new recipes for different structural motifs. Several examples of nucleic acids with non-canonical structures were modeled using NAMOT2. These examples include a DNA-drug complex, a DNA cube, a six-arm junction and a curved DNA molecule.

Base Sequence↗