Protein hydrogen exchange in denaturant: quantitative analysis by a two-process model.
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Hydrogen↗
Biomedical subjects
H Qian
Publications and source records attributed to H Qian.
A thermodynamic model for the helix-coil transition coupled to dimerization of short coiled-coil peptides.
A simple thermodynamic formalism is presented to model the conformational transition between a random-coil monomeric peptide and a coiled-coil helical dimer. The coiled-coil helical dimer is the structure of a class of proteins also called leucine zipper, which has been studied intensively in recent years. Our model, which is appropriate particularly for short peptides, is an alternative to the theory developed by Skolnick and Holtzer. Using the present formalism, we discuss the multi-equilibriatory nature of this transition and provide an explanation for the apparent two-state behavior of coiled-coil formation when the helix-coil transition is coupled to dimerization. It is found that such coupling between multi-equilibria and a true two-state transition can simplify the data analysis, but care must be taken in using the overall association constant to determine helix propensities (w) of single residues. Successful use of the two-state model does not imply that the helix-coil transition is all-or-none. The all-or-none assumption can provide good numerical estimates when w is around unity (0.35 < or = w < or = 1.35), but when w is small (w < 0.01), similar estimations can lead to large errors. The theory of the helix-coil transition in denaturation experiments is also discussed.
Macromolecular Substances↗
A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina.
The cinchona alkaloids quinine and quinidine have been shown to block a broad range of voltage-gated membrane conductances in a variety of excitable tissues. Using the whole-cell version of the patch clamp technique, we examined the effects of these compounds on voltage-dependent currents from horizontal cells dissociated enzymatically from the all-rod retina of the skate. We report here a novel and unexpected action of quinine and quinidine on isolated horizontal cells. In addition to blocking several of the voltage-activated currents of these cells, the introduction of the alkaloids evoked a large outward current when the cells were held at depolarized potentials. Using tail current analysis, the reversal potential of the outward current was close to O mV, and the current was markedly suppressed by extracellularly applied cobalt, acetate, and halothane. Depolarization in the presence of quinine also permitted entry into the cells of extracellularly applied Lucifer yellow (MW = 443 D), whereas a 3-kD fluorescein-dextran complex was excluded. These findings suggest that the large, apparently nonselective conductance induced by quinine and quinidine results from the opening of hemi-gap junctional channels.
Animals↗
The effects of quinine and quinidine on isolated retinal horizontal cells.
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Animals↗
The GABAA receptors of Müller (glial) cells in skate retina.
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Animals↗
[Preliminary estimation of malaria transmission potential in areas where Anopheles sinensis is the only vector].
The data, vectorial capacity of Anopheles sinensis in the suburb of Jining city, Shandong Province in 1975, in 1980-1984, and the average gland infection rate of Anopheles sinensis infected with Plasmodium vivax artificially, were calculated by using Macdonald formula (1957). The basic reproduction rates were 4.44, 6.47 and 4.25 in the years 1975, 1980 and 1981, respectively. In 1982-1984, the rate was reduced to 0.69, 0.41 and 0.1, respectively. Since 1982, the incidence rate of malaria in the areas was reduced by 80% annually, indicating that the malaria transmission had been decreased obviously.
Animals↗
Plasmid transfer from Escherichia coli to coryneform bacteria by conjugation.
The mobilizable shuttle plasmids pXZ911, pBZ51, pBZ52, carrying the Mob site and replication origin of the Corynebacteria plasmid pXZ10145, were constructed. In E. coli S17-1, they can be transferred to coryneform bacteria C. glutamicum ATCC13032, C. glutamicum ATCC21543, C. pekinense B3, C. pekinense AS1.299, C. herculis B43, B. flavum ATCC14067 by conjugation and the transfer frequencies are 9 x 10(-5), 1 x 10(-4), 8.5 x 10(-5), 2.3 x 10(-4), 6 x 10(-4), 2.9 x 10(-5), respectively. This plasmid transfer system, which is convenient and highly effective, should greatly facilitate the basic research and optimization of amino acid genetic engineering in coryneform bacteria and related species.
Actinomycetales↗
Pharmacology of novel GABA receptors found on rod horizontal cells of the white perch retina.
A novel type of GABA receptor is present on rod-driven (H4) horizontal cells of the white perch retina (Qian and Dowling, 1993a). These receptors have been tentatively termed GA-BAC receptors. In this study, the pharmacological properties of these receptors were further investigated by applying several conformationally restricted GABAA receptor agonists, GABAA antagonists, and a GABAB agonist to the H4 horizontal cells. GABA analogs locked in a partially folded conformation had a variety of effects. Isonipecotic acid had no effect on these receptors, whereas isoguvacine activated them but with low potency (EC50 = 137 microM). THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) acted as a competitive antagonist on these receptors with an inhibition constant of 82.5 microM. P4S (piperidine-4-sulfonic acid) activated the receptors at high concentrations (> 1 mM), but at lower concentrations it was a competitive antagonist with an inhibition constant of 80.9 microM. 14AA (imidazole-4-acetic acid), a GABA analog with an extended conformation, potently inhibited the GABA responses on H4 horizontal cells with an inhibition constant of 1.67 microM. Muscimol, which can assume both partially folded and extended conformations, acted as a mixed agonist-antagonist. The GABA responses on H4 horizontal cells were resistant to several competitive GABAA receptor antagonists including bicuculline, hydrastine, and SR-95531, but they were very sensitive to picrotoxin (IC50 = 237 nM). The inhibition by picrotoxin was both competitive and noncompetitive in nature. On the other hand, TBPS (tert-butyl-bicyclophosphorothionate), another GABAA receptor channel blocker, had minimal effects on these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The energetics of ion-pair and hydrogen-bonding interactions in a helical peptide.
A single pair of Glu and Lys residues has been placed at four different spacings, and in both orientations, in an otherwise neutral alanine-glutamine peptide helix, and the contribution to helix stability of the different Glu-Lys interactions has been measured. The contribution from the interaction of each charged side chain with the helix macrodipole has also been determined. A side-chain interaction between Gln and Glu, when the spacing is (i,i+4), has been detected and quantified. The interactions have been divided into contributions from hydrogen bonds (independent of the concentration of NaCl) and from electrostatic interactions (present in 10 mM NaCl, absent in 2.5 M NaCl). The major results are as follows: (1) The (i,i+3) and (i,i+4) Glu-Lys interactions are helix-stabilizing and are similar in strength to each other, regardless of the orientation of the side chains. (2) Hydrogen bonds provide the major contribution to these side-chain interactions, as shown by the following facts. First, the major part of the interaction observed in 10 mM NaCl, pH 7, is still present in 2.5 M NaCl. Second, the interaction found at pH 2 is equally as strong as that found in 2.5 M NaCl at pH 7. (3) The (i,i+4) Gln-Glu side-chain hydrogen bond is as strong as the hydrogen-bond component of the Glu-Lys interaction at both pH 2 and pH 7. The Gln-Glu interaction differs from the Glu-Lys interaction in being specific both for the orientation and the spacing of the residues. (4) No significant hydrogen-bonding interaction was found for the (i,i+1) or (i, i+2) Glu-Lys spacings, either at pH 2 or at pH 7, in 2.5 M NaCl. At 10 mM NaCl and pH 7, these spacings show a helix-destabilizing electrostatic interaction which probably results from stabilization of the coil conformation.
Amino Acid Sequence↗
Evidence for hemi-gap junctional channels in isolated horizontal cells of the skate retina.
Prolonged depolarization of isolated, voltage-clamped skate retinal horizontal cells produces an outward current that exhibit a late onset and develops slowly with time. This current, which we refer to as the Q-current, is associated with an increase in membrane conductance, and is present when other voltage-gated conductances have been pharmacologically blocked. The reversal potential for the Q-current, obtained using tail current analysis, was close to 0 mV. The magnitude of the current was greatly reduced by superfusion with 25 mM acetate, and by 4 mM cobalt chloride, 2 mM 1-octanol, and a saturated solution of the general anesthetic halothane. In addition, the low-molecular weight fluorescent dye Lucifer yellow, applied extracellularly, entered the cells during activation of the Q-current, whereas a 3 kD dextran-fluorescein complex did not cross the cell membrane. The effects of divalent cations, the non-specific nature of the ionic current suggested by its reversal potential, the entry of Lucifer yellow, and the ability of acetate, halothane, cobalt, and octanol to block the current lead us to hypothesize that the Q-current results from the opening of hemi-gap junctional channels that mediate electrical coupling between skate horizontal cells.
Animals↗
Novel GABA responses from rod-driven retinal horizontal cells.
gamma-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. Two classes of GABA receptors (GABAA and GABAB) have been identified. GABAA receptors are ligand-gated chloride channels that are competitively antagonized by bicuculline, noncompetitively blocked by picrotoxin, and often allosterically modulated by barbiturates and benzodiazepines. GABAB receptors regulate potassium and calcium channels through G-protein and intracellular second-messenger pathways, are selectively activated by baclofen, and are antagonized by phaclofen and 2-hydroxysaclofen. For some years, evidence has accumulated that there are GABA receptors, especially prominent along visual pathways, which are neither antagonized by bicuculline nor activated by baclofen, but are activated by certain conformationally restricted analogues of GABA, including cis-4-aminocrotonic acid (CACA). These receptors have been designated GABAC receptors. As yet, membrane current responses from isolated neurons that reflect this novel pharmacology have not been reported, although such responses have been recorded from oocytes injected with retinal messenger RNA. Here we describe a chloride-mediated current response from isolated rod-driven horizontal cells (H4) of the white perch retina that has this novel pharmacology.
Animals↗
Single-residue substitution in homopolypeptides: perturbative helix-coil theory at a single site.
Based on Lifson-Roig's helix-coil transition theory, substitution of a single heteroresidue into a homopolymer host is studied. This study models recent experiments that substitute a single amino acid into a small peptide in water [A. Chakrabartty, J. A. Schellman, and R. L. Baldwin (1991), Nature, Vol. 351, pp. 586-688]. Our formalism, which is based on a perturbation method, differs from the existing theory for sequenced polymers and is naturally analogous, hence likely to be useful, to substitution experiments in the laboratory. It is shown that the intrinsic helix propensity w is directly proportional to the equilibrium constant for the helix-coil equilibrium of a single residue in a host peptide. This intuitive new result will simplify experimental data interpretations for measurements of the helical conformation on the single amino acid level. It is also shown that substitution affects the total helicity of the host peptide according to two considerations: the helicity of the replaced residue prior to the substitution, and the sensitivity of the site, a measure of neighboring interactions. The relationship between substitution stability and thermal stability is explored.
Chemical Phenomena↗
The effects of lowered extracellular sodium on gamma-aminobutyric acid (GABA)-induced currents of Muller (glial) cells of the skate retina.
1. The effects of external sodium on GABA-induced chloride currents were examined with whole-cell voltage-clamp recordings obtained from enzymatically dissociated solitary Muller cells in culture. Our goal was to determine whether a sodium-dependent GABA uptake mechanism influences the GABAa-mediated responses of skate Muller cells. 2. At low concentrations of GABA (0.01 to 0.5 microM), removal of sodium from the external solution resulted in a marked increase in the ligand-gated currents mediated by activation of GABAa receptors. The enhancement by lowered sodium was greatest at hyperpolarizing potentials and decreased progressively as the cell was depolarized. 3. The reversal potential for the GABA-induced response was not significantly altered by the removal of sodium, suggesting that sodium ions did not directly contribute to the GABAa-mediated current. 4. Lowering external sodium had no effect on the currents induced by the GABAa-agonist muscimol, consistent with its much lower affinity for the GABA transport carrier. 5. Application of the GABA uptake blocker nipecotic acid also abolished the effects of lowered sodium. 6. These findings suggest that the effects of lowered external sodium resulted from a decrease in the uptake of GABA into the Muller cells, thus raising the effective concentration of GABA acting upon the GABAa receptors.
Animals↗
Inactivation of the Streptococcus mutans wall-associated protein A gene (wapA) results in a decrease in sucrose-dependent adherence and aggregation.
A 0.8-kb HindIII-BamHI internal fragment of the Streptococcus mutans wall-associated protein A gene (wapA) was ligated to the 5.1-kb HindIII-BamHI fragment of the chimeric Streptococcus-Escherichia coli plasmid pVA891 (Emr Cmr). The resulting construct was used to transform S. mutans GS-5, and erythromycin-resistant mutants were isolated and analyzed. Directed mutagenesis of the wapA gene by plasmid insertion through homologous recombination was demonstrated by Southern blot hybridization with the wapA and pVA891 probes. Stable mutants were obtained, and the alteration of the wapA gene by insertional inactivation was associated with a significant decrease in S. mutans sucrose-dependent aggregation and binding to smooth surfaces. Thus, WapA may play an important role in the colonization of the tooth surface by S. mutans and in the buildup of dental plaque. These findings provided an explanation for previous studies which indicated that WapA was effective in the prevention of dental caries in animal models. Thus, the use of recombinant WapA in the preparation of a safe and effective human dental vaccine should be investigated further.
Antigens, Bacterial↗
[Effect of controlling the incidence of decubitus ulcers].
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Aged↗
Clinical evaluation of radioimmunoimaging with 131I-C0C183B2 monoclonal antibody against ovarian carcinoma by intraperitoneal injection.
Forty-eight cases subjected to radioimmunoimaging (RII) by intraperitoneal injection with 131I-C0C183B2 monoclonal antibody (MAb) prepared in our laboratory were studied. Thirteen of 14 cases of proved primary ovarian carcinoma were positive. In 11 follow-up cases of ovarian carcinoma after initial surgery and chemotherapy, 5 recurrences were positive and 6 cases without recurrence were negative; all were confirmed histopathologically after a second operation. One false negative was ovarian mucinous adenocarcinoma, which also negatively stained with C0C183B2 by the peroxidase anti-peroxidase method. Twenty of 23 cases of nonepithelial or metastatic carcinoma of the ovary, benign tumors, and benign diseases were negative. The sensitivity and specificity were 94.7 and 89.7%, respectively. If patients had complications with ascites, the MAb which positively stained with the cancer cells in the ascites was chosen for RII. For follow-up cases PAP staining with the tumor tissue from the initial surgery and the MAb should be done before RII. These are the principal factors that increase the positive rate and accuracy of RII. The intraperitoneal route seems to be a valuable method for clinical staging and tumor localization as well as for follow-up use.
Adult↗
Studies on the structure of actin gels using time correlation spectroscopy of fluorescent beads.
Fluorescence correlation spectroscopy (FCS) has been used to measure the diffusion of fluorescently labeled beads in solutions of polymerized actin or buffer. The results, obtained at actin concentrations of 1 mg/ml, show that small beads (0.09 micron in diameter) diffuse nearly as rapidly in the actin gel as in buffer, whereas the largest beads tested (0.5 micron in diameter) are immobilized. Measured autocorrelation times for motions of beads with intermediate sizes show that the diffusion is retarded (relative to buffer) and that the time behavior cannot be represented as a single diffusive process. In addition to the retarded diffusion observed over distances > 1 micron, 0.23-micron beads also show a faster motion over smaller distances. Based on the measured rate of this faster motion, we estimate that the beads may be constrained within a cage approximately 0.67 micron on a side, equal to a filament length of approximately 250 subunits. Fluorescence correlation spectroscopy measurements made in the same small spot (radius of 1.4 microns) of the gel vary over time. From the variations of both the autocorrelation functions and the mean fluorescence, we conclude that, corresponding to a spatial scale of 1.4 microns, the actin gel is a dynamic structure with slow rearrangement of the gel occurring over periods of 20-50 s at 21-22 degrees C. This rearrangement may result from local reorganization of the actin matrix. Data for the retardation of beads by the actin gel are consistent with a detailed theory of the diffusion of particles through solutions of rigid rods that have longitudinal diffusion coefficients much less than that of the particles (Ogston, A. G., B. N. Preston, and J. D. Wells. 1973. Proc. R. Soc. Lond. A. 333:297-316).
Actins↗
Receptive field properties of rod-driven horizontal cells in the skate retina.
The large receptive fields of retinal horizontal cells result primarily from extensive intercellular coupling via gap (electrical) junctions; thus, the extent of the receptive field provides an index of the degree to which the cells are electrically coupled. For rod-driven horizontal cells in the dark-adapted skate retina, a space constant of 1.18 +/- 0.15 mm (SD) was obtained from measurements with a moving slit stimulus, and a comparable value (1.43 +/- 0.55 mm) was obtained with variation in spot diameter. These values, and the extensive spread of a fluorescent dye (Lucifer Yellow) from the site of injection to neighboring cells, indicate that the horizontal cells of the all-rod retina of skate are well coupled electrically. Neither the receptive field properties nor the gap-junctional features of skate horizontal cells were influenced by the adaptive state of the retina: (a) the receptive field organization was unaffected by light adaptation, (b) similar dye coupling was seen in both dark- and light-adapted retinae, and (c) no significant differences were found in the gap-junctional particle densities measured in dark- and light-adapted retinas, i.e., 3,184 +/- 286/microns 2 (n = 8) and 3,073 +/- 494/microns 2 (n = 11), respectively. Moreover, the receptive fields of skate horizontal cells were not altered by either dopamine, glycine, GABA, or the GABAA receptor antagonists bicuculline and picrotoxin. We conclude that the rod-driven horizontal cells of the skate retina are tightly coupled to one another, and that the coupling is not affected by photic and pharmacological conditions that are known to modulate intercellular coupling between cone-driven horizontal cells in other species.
Animals↗