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Biomedical subjects

R C Huang

Publications and source records attributed to R C Huang.

At least 37 records · Page 2Linked to original sources
PubMed1995

Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans.

A plant lignan, 3'-O-methyl nordihydroguaiaretic acid (3'-O-methyl NDGA, denoted Malachi 4:5-6 or Mal.4; molecular weigth 316), was isolated from Larrea tridentata and found to be able to inhibit human immunodeficiency virus (HIV) Tat-regulated transactivation in vivo, induce protection of lymphoblastoid CEM-SS cells from HIV (strain IIIB) killing, and suppress the replication of five HIV-1 strains (WM, MN, VS, JR-CSF, and IIIB) in mitogen-stimulated peripheral blood mononuclear cells, all in a dose-dependent manner. Mal.4 inhibits both basal transcription and Tat-regulated transactivation in vitro. The target of Mal.4 has been localized to nucleotides -87 to -40 of the HIV long terminal repeat. Mal.4 directly and specifically interferes with the binding of Sp1 to Sp1 sites in the HIV long terminal repeat. By inhibiting proviral expression, Mal.4 may be able to interrupt the life cycles of both wild-type and reverse transcriptase or protease mutant viruses in HIV-infected patients.

Animals↗
PubMed1995

Mutational analysis of neurokinin receptor function.

The interactions of the NK1 receptor with peptide agonists or nonpeptide antagonists have been investigated by site-directed mutagenesis and computer modeling. At least 10 residues in the extracellular and transmembrane regions of the receptor are required for the binding of many peptide agonists. The C-terminal amide of peptide agonists is likely to be bound near Asn-85. Residues likely to be involved in the subsequent receptor activation include Glu-78 and Tyr-205. The binding site for nonpeptide antagonists can be defined by at least five residues in transmembrane helices 4-7, and primary contacts between key residues and quinuclidine antagonists have been assigned based on CP-96,345 and its analogs. Analyses of the wild-type and mutant NK1 and NK2 receptors, intact and truncated peptides, and various antagonists suggest that the agonist and antagonist binding sites overlap spatially, even though agonists and antagonists do not interact with the same set of residues on the receptor. Mapping the ligand binding site not only allows us to better understand the ligand--receptor interaction and antagonism but also leads to a refined three-dimensional model of the NK1 receptor.

Amino Acid Sequence↗
PubMed1995

Novel pharmacological properties of transient potassium currents in central neurons revealed by N-bromoacetamide and other chemical modifiers.

The chemical modifiers N-bromoacetamide (NBA), N-bromosuccinimide (NBS), and chloramine-T (ChT) are commonly used to remove fast inactivation of sodium currents and transient potassium currents (IA). In the present study, I examined the effects of these chemical modifiers as well as of others on IA in neurons dissociated from several brain regions. External application of NBA irreversibly inhibited IA, with higher NBA concentrations increasing the rate of inhibition. The current kinetics, however, were not altered by external NBA at any concentration. IA was also inhibited by internal NBA but only at high concentrations, and the rate of inhibition was much slower. The current kinetics were not altered by internal NBA at any concentration. NBA is a nonspecific chemical reagent that can modify a protein at several target amino acids. The NBA-induced irreversible inhibition of IA was reproduced by external diethylpyrocarbonate, a reagent that specifically modifies histidine residues, and by ChT and cyanogen bromide, reagents that modify methionine residues through distinct mechanisms. However, NBS, a reagent that cleaves the peptide bond at tryptophan residues, had no effect on IA, nor did chemical modifiers specific for cysteine and tyrosine residues. Taken together, these results suggest that the conserved, functionally important methionine and/or histidine residues are the likely targets for NBA modifications. These novel pharmacological properties are in sharp contrast to those known previously, despite their similarity in both kinetics and 4-AP sensitivities. Therefore, the pharmacological treatments presented in the present study should be useful for characterizing other IA.

Acetamides↗
PubMed1994

Inhibition of replication of human respiratory syncytial virus by 6-diazo-5-oxo-L-norleucine.

The effect of 6-diazo-5-oxo-L-norleucine (L-DON), a glutamine analog, on RSV replication was studied. At a concentration of 0.01 mM L-DON, 99% of RSV replication in treated CV-1 cells was inhibited. At this concentration of L-DON, the level of cellular protein synthesis was identical to untreated control cells. Trypan blue staining revealed that all the cells remained viable even at concentrations of L-DON as high as 10 mM. In addition, L-DON added as late as 24 h post infection can effectively suppress viral replication. Analysis of viral mRNA levels by Northern blot revealed that secondary transcription and subsequent steps in the virus life cycle were inhibited. Immunoprecipitation of viral proteins from drug treated or untreated cultures showed that synthesis of all viral proteins was drastically reduced by L-DON, with a slightly greater inhibition of viral glycoproteins. Furthermore, immunofluorescent staining showed that drug treated cells expressed both F and N proteins and that F was inserted into the membrane as the native F protein.

Antiviral Agents↗
PubMed1993

Zinc modulation of a transient potassium current and histochemical localization of the metal in neurons of the suprachiasmatic nucleus.

The effect of Zn2+ on a voltage-dependent, transient potassium current (IA) in acutely dissociated neurons from the suprachiasmatic nucleus was studied with the whole-cell patch-clamp technique. At micromolar concentrations, Zn2+ markedly potentiated IA activated from a holding potential of -60 mV, which is the resting potential of these neurons. This potentiation occurred at a Zn2+ concentration as low as 2 microM and increased with higher Zn2+ concentrations. The Zn2+ action appears to arise from a shift in the steady-state inactivation of IA to more positive voltages. At 30 microM, Zn2+ shifted the half-inactivation voltage by +20 mV (from -80 mV to -60 mV), and 200 microM Zn2+ shifted this voltage by +45 mV (from -80 mV to -35 mV). Histochemically, we have also observed Zn2+ staining throughout the suprachiasmatic nucleus; the staining is particularly intense in the ventrolateral region of the nucleus, which receives the major fiber inputs. Our findings suggest that Zn2+, presumably synaptically released, may modulate the electrical activity of suprachiasmatic nucleus neurons through IA. Because vesicular Zn2+ is fairly widespread in the central nervous system, it is conceivable that this kind of Zn2+ modulation on IA, and possibly on other voltage-activated currents, exists elsewhere in the brain.

Animals↗
PubMed1993

Expression and structural analysis of a teleost homolog of a mammalian zona pellucida gene.

Gene expression in oviparous vertebrates during vitellogenesis is hormonally regulated. Our laboratory has characterized a unique gene (wf female), which is seasonally expressed in the liver of the female winter flounder Pseudopleuronectes americanus. The wf female mRNA is coexpressed with vitellogenin mRNA and reaches a high level during vitellogenesis. The wf female gene is 2554 base pairs in length and encodes a putative protein of 509 amino acids. The gene consists of eight exons separated by seven introns of different sizes. Within exon 1, there are six PQQ-rich repeats. Four of them encode a putative (PQQ)1PKY polypeptide, similar to the repeats found in the extracellular domains of other proteins. Exons 2-7 share homology with the zona pellucida protein genes rc55 of rabbit and zp-2 of mouse, and the positions of intron boundaries are conserved in the wf female and mouse zp-2 genes. In addition, the transcriptional regulatory cis elements (estrogen response element, CCAAT and TATAAA boxes), as found in vertebrate genes, are also conserved for the wf female promoter and mapped upstream from the initiation site of the wf female primary transcript.

Amino Acid Sequence↗
PubMed1993

The mouse intracisternal A particle-promoted placental gene retrotransposition is mouse-strain-specific.

Insertion of a single long terminal repeat (LTR) of an intracisternal A particle (IAP) gene into a placenta-expressed cellular gene (mouse IAP-promoted placental gene, MIPP) has recently been found for the mouse strains Swiss and CF-1. To test whether such a retrotransposition event has also taken place in other strains of mouse, genomic DNA and total RNAs were analyzed from the outbred CF-1 strain and five inbred strains of mouse, AKR/J, C57BL/6J, DBA/2J, C3H/HeJ, and BALB/c. Specific DNA fragments flanking the LTR region were isolated from MIPP cDNA and genomic clones and used as hybridization probes. Three transcripts [two minor, 4.4 kilobases (kb) and 2.2 kb; one major, 1.2 kb] were detected. The 4.4-kb and 2.2-kb species were found in all strains of mouse studied. The 1.2-kb transcript (promoted by IAP LTR) is present only in placentas of C3H/HeJ and BALB/c mice. Both of these strains contain an IAP LTR in the MIPP gene. In contrast, there is no IAP LTR in this cellular gene in strains DBA/2J, C57BL/6J, and AKR/J. Thus, the IAP MIPP retrotransposition is strain-specific. The parents of the outbred CF-1 mice used for the present studies were both heterozygous at the MIPP locus. Restriction fragment length polymorphism studies using inbred strains and recombinant inbred mice have further established the linkage between the MIPP gene (D4Jhu8) and several loci on distal mouse chromosome 4. The symbol Ipp is being used for this gene in all major mouse data bases and in the comparative section of the human genome data base.

Alleles↗
PubMed1993

Upper excited state photochemistry of DNA.

The quantum yields for cyclobutylpyrimidine dimers, alkali-labile sites, and frank strand breaks in double-stranded DNA have been measured using low-intensity radiation at 199.8, 217.8, and 239.5 nm from a Raman-shifted frequency quadrupled Nd:YAG laser. The quantum yield for cyclobutylpyrimidine dimers was also measured using 254 nm radiation from a low-pressure mercury lamp. The quantum yield for cyclobutylpyrimidine dimers is constant within a factor of two between 254 and 199.8 nm except for 239.5 nm, indicating that upper excited singlet states of bases convert efficiently to the lowest singlet state. The quantum yields for alkali-labile sites and frank strand breaks both increase as the wavelength decreases but follow different patterns. These results indicate that alkali-labile sites from a higher excited state of the base, whereas frank strand breaks form by excitation of the sugar-phosphate backbone.

DNA↗
PubMed1993

Co-regulation of cAMP-activated Na+ current by Ca2+ in neurones of the mollusc Pleurobranchaea.

1. The cAMP-gated Na+ current (INa, cAMP) was studied in axotomized neurons of the pedal ganglion of the sea slug Pleurobranchaea. INa, cAMP responses were elicited by iontophoretic injection of cAMP and recorded in voltage clamp. 2. The current-voltage relation for INa, cAMP was flat between -90 and -50 mV, but declined steeply with depolarization from -50 to -30 mV. Depolarizing pulses also suppressed the INa, cAMP response, which recovered slowly over tens of seconds. 3. The inactivating effects of depolarization on the current were abolished both by blockade of Ca2+ current and intracellular injection of Ca2+ chelator. Thus, Ca2+ influx through voltage-dependent Ca2+ channels probably mediates inactivation of INa, cAMP within its normal physiological range of action. 4. Increasing intracellular cAMP levels antagonized the effects of Ca2+ influx on INa, cAMP. The mutual antagonism of the ions suggests that cAMP and Ca2+ act competitively in regulation of the INa, cAMP channel. 5. Measures of fractional inactivation of INa, cAMP provided evidence for the existence of an appreciable basal level of current, and hence cAMP, in the unstimulated neuron. Since INa, cAMP is a direct function of cAMP activity, measures of fractional inactivation permit quantification of cAMP levels in the living neuron. 6. Calcium inactivation of INa, cAMP completes a negative feedback loop that can contribute to endogenous burst activity. Over the burst cycle, depolarization and action potential activity driven by INa, cAMP would lead to Ca2+ influx, consequent inactivation of the inward current, and hyperpolarization. This mechanism of endogenous bursting resembles other in which the burst cycle has been found to be regulated by kinetics of Ca2+ influx and removal. However, INa, cAMP may vary in its Ca2+ sensitivity in different neurons and these variations may affect the functional expression of endogenous oscillatory activity.

Animals↗
PubMed1993

Sodium and calcium currents in acutely dissociated neurons from rat suprachiasmatic nucleus.

1. Neurons were acutely dissociated from the suprachiasmatic nucleus (SCN) of adult rats and studied with whole-cell and perforated-patch recordings at room temperature. 2. Acutely dissociated SCN neurons had spherical cell bodies of 12 microns in average diameter. The recorded cells were randomly selected and had either no process (38%), one (41%), two (19%), or three processes (2%). They had a resting potential of about -60 mV, an input resistance of approximately 5 G omega, and a cell capacitance of approximately 7 pF. 3. The dissociated neurons had variable spontaneous firing rates, typically (76%) < 1 Hz. 4. Under current clamp, continuous current injection elicited repetitive action potentials. 1 microM tetrodotoxin (TTX) reduced the amplitudes of the action potentials as well as the firing rate, whereas 200 microM Cd2+ stopped repetitive firing altogether. Action potentials were completely eliminated with Cd2+ and TTX present. These results suggest that both Na+ and Ca2+ contribute to the action potential in these cells. 5. With 200 microM Cd2+ present to block calcium currents, a train of brief depolarizing pulses could still elicit repetitive sodium action potentials, but these became attenuated at stimulating frequencies as low as 1 Hz. 6. Under voltage clamp, the sodium current was activated at about -40 mV and peaked at about -10 mV. It inactivated with a time constant of approximately 0.5 ms at 0 mV, and in steady state the current was half-inactivated at about -60 mV. Recovery of the current from inactivation showed two very different phases with time constants of approximately 30 and 600 ms at -60 mV. The slow phase was probably responsible for the very low firing rate of the sodium action responsible for the very low firing rate of the sodium action potential. 7. In the absence of external sodium, depolarization-activated calcium action potentials were preferentially blocked by 20 microM Cd2+, whereas a posthyperpolarizing depolarizing (or anode break) was preferentially reduced by 100 microM Ni2+. These differential effects hinted at the presence of both low-threshold and high-threshold calcium currents in these cells. 8. Voltage-clamp experiments confirmed the presence of a low-threshold, transient calcium current that was activated by depolarizations above -70 mV. It inactivated with a time constant of approximately 25 ms between -50 and -30 mV. Steady-state inactivation was half-complete at about -90 mV and complete at about -70 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1993

cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch.

The cAMP-dependent Na+ current (INa,cAMP) modulates excitability in many molluscan neurons. Rapid activation of INa,cAMP by cyclic nucleotide, its ion dependence, and its blockade by divalent cations resemble cyclic nucleotide-activated cation currents in vertebrate photoreceptors and olfactory receptors, where activation has been found to be independent of kinase activity. We tested the phosphorylation dependence of INa,cAMP in neurons of the feeding and locomotory networks of the predatory marine snail Pleurobranchaea. Identified neurons of pedal and buccal ganglia were axotomized for recording the INa,cAMP response to iontophoretic injection of cAMP under two-electrode voltage clamp. Intracellular injections of specific peptide inhibitor of protein kinase A had no blocking effects on activation of INa,cAMP by iontophoretic injection of cAMP. Inward single-channel currents were activated in excised inside-out patches during exposure to cAMP in salines without added ATP. Sodium was the major current carrying ion. Two distinct types of INa,cAMP channel activity were observed, where opening probability and open times differed, but conductance was similar, 36.7 pS. These observations suggest that INa,cAMP activation occurs by direct binding of cAMP to a regulatory site at the channel, rather than by phosphorylation.

Animals↗
PubMed1992

Nuclear protein binding to the 5' enhancer region of the intracisternal A particle long terminal repeat.

The interactions of nuclear proteins from embryonal carcinoma cells (PCC3) with the long terminal repeats (LTRs) of murine intracisternal A particle (IAP) genes were studied. Two protein-DNA complexes were detected between PCC3 nuclear extract and IAP LTRs in a gel mobility shift assay. An additional complex was observed when enriched fractions from a heparin-agarose column were used as the source of proteins. Two regions within the LTR of IAP 81 were identified as the sites of protein interaction by DNase I protection. One region encompasses 43 nucleotides within the U3 region at the 5' end of LTR 81. The other covers a 78 base pair region lying within 100 nucleotides upstream from the transcription initiation site. Studies using constructs containing intact or deleted versions of the LTR fused to the bacterial chloramphenicol acetyltransferase gene indicated that the absence of the 5' 47 base pairs reduced the level of chloramphenicol acetyltransferase transcription to 20% of that driven by the entire LTR. Southwestern analysis of PCC3 nuclear extracts and column fractions revealed that a 28,000- and a 46,000-dalton protein were the major species that interact with the 5' end of IAP LTR 81.

Animals↗
PubMed1991

Identification of a novel murine IAP-promoted placenta-expressed gene.

We have cloned and characterized a novel cellular gene that is promoted by an intracisternal A-particle (IAP) LTR and expressed in the mouse placenta (mouse IAP promoted placental gene, MIPP). A 1067bp cDNA clone containing an IAP LTR U5 region duplicated in its 5' terminus and an ORF coding for a potential 202 amino acids protein was isolated from an 8.5 day old mouse embryo cDNA library. Sequence analysis of the 5' region of a genomic clone revealed the presence of a solo IAP LTR with the same U5 duplication, and primer extension analysis confirmed that transcription of the MIPP gene is under the control of the IAP LTR. Expression of the MIPP gene parallels that of IAP genes in normal mouse tissues with abundant transcripts present in the placenta and also in the myeloma MOPC-315. The MIPP-encoded protein is composed of four 48-amino acid repeat units and shares homology with a vaccinia virus gene product. MIPP-related sequences were also detected in higher eukaryotic genomes including human.

Amino Acid Sequence↗
PubMed1991

Cell lineage-specific expression of the MIPP gene.

The expression of the mouse IAP promoted placental (MIPP) gene was examined in the tissues of the 13.5 day mouse conceptus by Northern hybridization analysis. MIPP transcripts were found in total cellular RNA isolated from the placenta, parietal yolk sac, and visceral yolk sac, but not the embryo or amnion. Expression in the visceral yolk sac was confined to the visceral endoderm cell layer suggesting that the MIPP gene is specifically expressed in tissues derived from the trophectoderm and primitive endoderm cell lineages. The expression of IAP genes paralleled that of MIPP in all tissues studied except for the presence of IAP transcripts in the amnion.

Amnion↗
PubMed1991

Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model.

cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor-induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents.

1-Methyl-3-isobutylxanthine↗
PubMed1989

A self-inducing runaway-replication plasmid expression system utilizing the Rop protein.

A highly efficient prokaryotic expression system has been developed that produces proteins at levels exceeding 150 micrograms/ml of culture medium. The system consists of a temperature-sensitive-copy-number plasmid that carries the rop gene and promoter downstream from the trp promoter. Any sequence cloned into the PvuII site of the rop gene alters Rop protein activity and causes lethal runaway plasmid DNA replication. This plasmid replication can be suppressed in trans by complementation with a similar wild-type plasmid. Cells harboring both plasmids are quite stable, and induction of plasmid DNA synthesis occurs only after cells are grown for several generations under conditions that lead to the loss of the trans-acting repressor. Large amounts of Rop fusion proteins accumulate in the cell as the trp operon is gradually induced via repressor titration. All chimeric proteins accumulate as insoluble aggregates, and are therefore easily purified. They can be solubilized using relatively mild conditions, and the partially purified proteins are highly amenable to cleavage by chemical methods. Using this system we have made Rop fusions with the HIV Tat protein, the herpes simplex virus type-2 38K protein, and Chinese hamster metallothionin.

Amino Acid Sequence↗