Fermi surface of CeIn3 above the Néel critical field.

We report measurements of the de Haas-van Alphen effect in CeIn(3) in magnetic fields extending to approximately 90 T, well above the Néel critical field of mu(0)H(c) approximately 61 T. The unreconstructed Fermi surface a sheet is observed in the high magnetic field polarized paramagnetic limit, but with its effective mass and Fermi surface volume strongly reduced in size compared to that observed in the low magnetic field paramagnetic regime under pressure. The spheroidal topology of this sheet provides an ideal realization of the transformation from a "large Fermi surface" accommodating f electrons to a "small Fermi surface" when the f-electron moments become polarized.

The effect of an experimental rhinovirus 16 infection on bronchial lavage neutrophils.

BACKGROUND: Viral respiratory tract infections are the most frequent cause of asthma exacerbations. Of the respiratory viruses associated with these exacerbations, rhinovirus (RV) is the most common. It is proposed that these RV infections may enhance airway inflammation and thus provoke asthma. OBJECTIVE: It is our hypothesis that RV infections generate nasal proinflammatory mediators that are associated with an initial increase in circulating leukocytes and may contribute to later development of neutrophilic airway inflammation. METHODS: To evaluate this hypothesis, subjects with a history of allergic asthma were experimentally inoculated with strain 16 RV (RV16). The effect of this experimental infection was evaluated on circulating leukocytes, nasal-derived mediators, and markers of bronchial inflammation that were obtained by bronchoscopy and lavage. RESULTS: RV16 inoculation was associated with an initial increase in circulating neutrophils. Paralleling these acute changes in circulating neutrophils was an increase in nasal concentrations of IL-8 and granulocyte-colony-stimulating factor (G-CSF). The RV16-associated changes in circulating and nasal G-CSF correlated with increases in peripheral blood neutrophils (r(s) = 0.874, P <. 001 and r(s) = 0.898, P <.001, respectively). Bronchial lavage samples showed no increase in neutrophils 48 hours after RV16 inoculation; however, 96 hours after RV inoculation there was a significant increase in bronchial neutrophils compared with preinoculation values. CONCLUSIONS: These results suggest that the production of nasal mediators associated with the RV infection, particularly G-CSF, may be important to the eventual development of neutrophilic bronchial inflammation and thus contribute to asthma exacerbations.

Relationship between stability and function for isolated domains of troponin C.

Results of spectroscopic thermal and chemical denaturation studies and calcium binding studies are presented for a series of five recombinant chicken troponin C fragments. They were designed to assess the effects of domain isolation, N-helix, and D/E linker helix on stability and calcium affinity. Four of the fragments include the N-terminal regulatory domain and one the C-terminal domain. For the regulatory domain, deletion of the N-helix or the D/E linker decreases the stability of the apo form as measured by delta GN-->U,25. Separation of the domains also decreases the stability. Differences in values of delta GN-->U,25 derived from urea and guanidine hydrochloride studies allowed an estimation of the electrostatic component of the free energy of unfolding. Our measurements provide the first quantitative estimate of the stability for the apo-C-domain (delta GN-->U,25 = -1.8 kcal/mol) which was obtained using the interaction free energy formalism of Schellman. There is an inverse correlation between calcium affinity, binding cooperativity, and stability for all of these homologously structured fragments. The calcium affinity and cooperativity are highest for the unstructured C-domain and lowest for the N-domain which has the highest stability. In view of the direct effects on the folding stability of the apo-N-domain, the N-helix and the bilobed domain organization of TnC are necessarily involved in the fine-tuning of the affinity and cooperativity of calcium binding. Though not directly involved in calcium coordination, these structural features are important for signal transmission by troponin C in the troponin complex.

The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides.

The actin Ser14 hydroxyl is one of a number of ligands that binds to the gamma-phosphate of ATP thereby stabilizing the actin.ATP complex. In yeast actin, conversion of Ser14 to Ala (S14A), causes a temperature-sensitive phenotype in vivo and temperature-sensitive polymerization defects in vitro (Chen, X., and Rubenstein, P. A. (1995) J. Biol. Chem. 270, 11406-11414). Here, using a new luciferase-based procedure, we show that the mutation results in a 40-60-fold decrease in actin's affinity for ATP. The mutation causes a decrease in the intrinsic ATPase activity of both Ca- and Mg-G-actin at 30 degrees C and alters the protease susceptibility of sites on subdomain 2. Ca-S14A-actin but not Mg-S14A-actin binds etheno-ATP at 37 degrees C. Intrinsic tryptophan fluorescence measurements show that at 37 degrees C, Mg-S14A-actin but not the calcium form unfolds. CD measurements show the mutation causes a decrease in the apparent denaturation temperature for Ca-actin from 57 to 45 degrees C and for the magnesium form a decrease from 52 to 40 degrees C. Based on a re-examination of actin's crystal structure coordinates, we propose that the Ser14 hydroxyl forms a polar bridge between the ATP gamma-phosphate and the amide nitrogen of Gly74, thus conferring additional stability on the actin small domain.

Versatile one-piece total artificial heart for bridge to transplantation or permanent heart replacement.

A versatile, one-piece total artificial heart (TAH) system that can be driven by either an electromechanical acutator (EM-TAH) or a pneumatic source (P-TAH) has been developed. The common units for both TAHs are the conically shaped left and right pusher-plate-type pumps (63 ml SV) that sandwich a thin centerpiece (18 mm) having a respective actuator. The EM actuator, mounted in the middle of the centerpiece, consists of a direct-current brushless motor and a roller screw while the pneumatic actuator consists of a low-pressure air source. The outer diameter of the pumping unit is 97 mm with its central thickness being 82 mm; overall volume is 510 cc. The TAH is operated in the left master alternative ejection mode with the left pump fill signal. High-flex-life Hexsyn rubber is used as the diaphragm, and the blood-contacting surface is coated with dry gelatin. The TAH can provide 3-8 L/min flow with a preload of 1-10 mm Hg against 100 mm Hg afterload. Anatomical fit of the pumping unit has been demonstrated in the pericardial space of 26 heart transplant recipients with average body weight of 78 kg. To date, 2 P-TAH and 4 EM-TAH (1 week) implantations were performed in 80-100 kg calves demonstrating excellent anatomical fit, controllability, and biocompatibility. This versatile TAH is suitable for a bridge to transplantation or permanent heart replacement.

Baylor multipurpose circulatory support system for short- to long-term use.

A multipurpose circulatory support system has been developed as both a temporary and permanent device in total artificial hearts (TAHs) and ventricular assist devices (VADs). The multipurpose concept was derived from the development of a totally implantable electromechanical, one-piece TAH. The blood pump is pneumatically driven in short-term use and is electromechanically driven in long-term or permanent use. Both TAH and VAD versions consist of the same components, except for the actuation mechanism. The common components are a compact pumping chamber with the same configuration, a blood contacting surface biolized with gelatin, a pusher-plate, a Hexsyn rubber diaphragm (University of Akron, Akron, OH) and bovine pericardial valves. Both TAHs and VADs have 63 ml of stroke volume, and the VADs are compact compared with other available investigational device exemption devices. Currently, 1 week survival has been achieved using the electromechanical TAH and 2 week survival using the electromechanimcal VAD without anticoagulation. Results suggest that the currently developed system could be applied in varied patients as a temporary device after cardiotomy, a long-term device for bridge to transplantation, or a permanent device for end-stage heart disease.

Interaction of troponin C and troponin C fragments with troponin I and the troponin I inhibitory peptide.

We have quantitated the interactions of two rabbit skeletal troponin C fragments with troponin I and the troponin I inhibitory peptide. The calcium binding properties of the fragments and the ability of the fragments to exert control in the regulated actomyosin ATPase assay have also been studied. The N- and C-terminal divalent metal binding domains of rabbit skeletal troponin C, residues 1-97 and residues 98-159, respectively, were prepared by specific cleavage at cysteine-98 and separation by gel exclusion chromatography. Both of the troponin C fragments bind calcium. The calcium affinity of the weak sites within the N-terminal fragment is about an order of magnitude greater than is reported for these sites in troponin C, suggesting interaction between the calcium-saturated strong sites and the weak sites. Stoichiometric binding (1:1) of the troponin I inhibitory peptide to each fragment and to troponin C increased the calcium affinities of the fragments and troponin C. Complex formation was detected by fluorescence quenching or enhancement using dansyl-labeled troponin C (and fragments) or tryptophan-labeled troponin I inhibitory peptide. The troponin C fragments bind to troponin I with 1:1 stoichiometry and approximately equal affinities (1.6 x 10(6) M-1) which are decreased 4-fold in the presence of magnesium versus calcium. These calcium effects are much smaller than is observed for troponin C. The summed free energies for the binding of the troponin C fragments to troponin I are much larger than the free energy of binding troponin C. This suggests a large positive interaction free energy for troponin C binding to troponin I relative to the fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental rhinovirus 16 infection potentiates histamine release after antigen bronchoprovocation in allergic subjects.

Viral respiratory infections exacerbate asthma in many patients. We hypothesized that one mechanism by which this effect occurs may include potentiated or altered mediator release by mast cells and/or basophils to favor the development of late-phase asthmatic reaction (LAR). Therefore, we studied eight subjects with allergic rhinitis before and during an experimentally induced rhinovirus 16 (RV16) infection. We determined levels of plasma histamine and tryptase, and we observed the associated patterns of airway obstruction that developed following inhaled antigen challenge. Bronchial responsiveness to histamine, methacholine, and antigen were all significantly increased during the RV16 illness. Further, the incidence of LAR was significantly higher (five of eight) during the infection than before (one of eight; p = 0.014). In addition, in those patients whose pattern of response following antigen challenge converted from an immediate response only before infection to a dual response (immediate + late phase) during infection, plasma histamine concentrations after challenge were significantly greater than in those whose pattern of response did not change. We conclude that one mechanism by which RV16 infection increases the likelihood of LAR could include enhanced mediator release from pulmonary mast cells or from circulating or recruited basophils.

The relationship between plasma histamine concentrations and bronchial obstruction to antigen challenge in allergic rhinitis.

Antigen activation of pulmonary mast cells causes mediator release and airway obstruction in allergic patients with asthma. Concomitant measurements of airway mediators and pulmonary function are technically difficult, even with bronchoalveolar lavage. Thus, a procedure was developed to evaluate further the relationship between mediator release, as measured by plasma histamine concentrations, and airway obstruction in patients with allergic rhinitis challenged with inhaled ragweed antigen. At an initial challenge, the cumulative antigen dose to decrease the FEV1 by approximately 20% was determined. Approximately 4 weeks later, the entire predetermined cumulative antigen dose to decrease the FEV1 by approximately 20% was administered in five consecutive inhalations with simultaneous monitoring of plasma histamine. We found the percent fall in FEV1 (24.3 +/- 2.3 versus 30.4 +/- 4.0; p greater than 0.05; n = 7) was similar whether antigen was administered by a cumulative or single-dose challenge. With the single-dose antigen challenge and monitoring blood samples frequently, we found plasma histamine (picograms per milliliter) values to increase from 73 +/- 17 to 1071 +/- 377 (p = 0.022) with peak value 5 minutes after challenge. Furthermore, we found that the intensity of airway obstruction to antigen corresponded to both the patient's baseline airway responsiveness to histamine and the absolute changes in plasma histamine after antigen challenge. Thus, the degree of airway obstruction to inhaled antigen is determined by both the intensity of the allergic reaction (as reflected by the plasma histamine value) and bronchial responsiveness.

Flexibility of smooth and skeletal tropomyosins.

The rotational relaxation times of nonpolymerizable skeletal and smooth muscle tropomyosin were measured by analysis of the decay of the zero-field birefringence at different temperatures and salt concentrations. Skeletal tropomyosin in solution is equally well modeled as a rigid rod or as a semiflexible rod with a persistence length of 150 nm. Smooth muscle tropomyosin does not fit the rigid rod model but is well approximated by a semiflexible rod model with a persistence length of 55 nm. The results indicate that smooth muscle tropomyosin is either a more flexible molecule than skeletal muscle tropomyosin or is a curved structure with an end-to-end length shorter than the coiled-coil contour length. Smooth muscle tropomyosin controls the actomyosin ATPase differently from skeletal muscle tropomyosin and it had been suggested that the reason is because it is more rigid; clearly, another explanation must be sought.

Rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions.

Although viral upper respiratory infections (URIs) provoke wheezing in many asthma patients, the effect of these illnesses on the airway response to inhaled antigen is not established. The following study evaluated the effect of an experimental rhinovirus (RV) illness on airway reactivity and response to antigen in 10 adult ragweed allergic rhinitis patients. Preinfection studies included measurements of airway reactivity to histamine and ragweed antigen. Furthermore, the patients were also evaluated for late asthmatic reactions (LARs) to antigen (a 15% decrease in forced expiratory volume of the first second approximately 6 h after antigen challenge). 1 mo after baseline studies, the patients were intranasally inoculated with live RV16. All 10 patients were infected as evidenced by rhinovirus recovery in nasal washings and respiratory symptoms. Baseline FEV1 values were stable throughout the study. During the acute RV illness, there was a significant increase in airway reactivity to both histamine and ragweed antigen (P = 0.019 and 0.014, respectively). Before RV inoculation, only 1 of the 10 subjects had an LAR after antigen challenge. However, during the acute RV illness, 8 of 10 patients had an LAR (P less than 0.0085 compared with baseline); the development of LARs was independent of changes in airway reactivity and the intensity of the immediate response to antigen. Therefore, we found that not only does a RV respiratory tract illness enhance airway reactivity, but it also predisposes the allergic patient to develop LARs, which may be an important factor in virus-induced bronchial hyperresponsiveness.

Effects of a dihydropyridine calcium antagonist and agonist on human basophil histamine release.

Calcium influx is important to basophil histamine release, and even though a cromolyn-binding protein has been proposed to constitute a Ca2+ channel, the pathway of Ca2+ influx or involvement of a Ca2+ channel in this process has yet to be established. We evaluated the effects of a dihydropyridine antagonist, nitrendipine, and agonist, BAY k 8644, on human basophil histamine release. Nitrendipine inhibited ragweed antigen E-dependent basophil histamine release in a dose-dependent fashion with a 50% inhibitory dose of 3.7 (+/- 1.1) X 10(-6) mol/L, and maximal inhibition of histamine release (41.8% +/- 7.1%) was achieved with 1.0 X 10(-5) mol/L nitrendipine. Increased extracellular concentrations of Ca2+ reduced nitrendipine inhibition of histamine release. In contrast, the Ca2+ agonist, BAY k 8644, enhanced antigen E-dependent histamine release with an ED50 value of 5.0 (+/- 1.1) X 10(-6) mol/L. BAY k 8644 by itself, however, did not cause basophil histamine release nor did it enhance histamine release to the calcium ionophore A23187. Further, when the effects of BAY k 8644 on basophil histamine release were evaluated in the presence of nitrendipine, the enhancing action of BAY k 8644 was diminished in a competitive fashion. Therefore, even though these compounds act at specific Ca2+ channels in other tissues, our data do not establish either the presence of such channels in the IgE-dependent histamine release process of basophils or the mechanism of action for dihydropyridines in basophil histamine secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced basophil histamine release to concanavalin A in allergic rhinitis.

It has been suggested that IgE-dependent basophil histamine release (HR) does not necessarily relate to the amount of cell-bound IgE and, therefore, basophil "releasability" must be considered an important factor in this secretory process. To compare an IgE-dependent basophil HR process in nonatopic subjects and patients with allergic rhinitis, concanavalin A (Con A) was used as a secretagogue to stimulate mediator secretion. In 1.0 mmol/L of calcium-containing buffer, basophil HR to Con A (3.0 mcg/ml) was 50.2 +/- 8.6% in patients with allergic rhinitis and only 10.1 +/- 3.9% in nonatopic subjects. To evaluate whether this enhanced HR might be related to increased membrane influx of calcium, the following strategy was followed. Strontium (3.0 and 10.0 mmol/L) enhances immunologic (IgE) release of basophil histamine. Although the mechanism for strontium enhancement is not established, strontium may pass through the membrane channel more easily than calcium to increase secretion. We reasoned that if the enhanced release of histamine to Con A was related to increased membrane permeability to calcium, stimulation of basophil histamine secretion in the presence of strontium would reduce this difference. In both nonatopic subjects and patients with allergic rhinitis, strontium (3.0 and 10.0 mmol/L) enhanced HR. Enhanced HR with strontium was greater with basophils from normal subjects than from subjects with allergic rhinitis. Whether our observations with strontium indicate that the enhanced histamine releasability to Con A in subjects with allergic rhinitis may, in part, be due to a greater influx of calcium after immunologic stimulation must await characterization of the strontium effect or direct measurements of calcium ion disposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of troponin and tropomyosin: spectroscopic and calorimetric studies.

The thermodynamic parameters characterizing the interaction between rabbit fast skeletal muscle troponin and tropomyosin have been determined at 25 degrees C for three solution conditions: buffer containing (A) 1 mM CaCl2, simulating a "turned-on" state, (B) 3 mM MgCl2, simulating a "turned-off" state, and (C) 2 mM ethylenediaminetetraacetic acid, a reference state. The enthalpies were measured in two buffers with different heats of ionization to allow correction for dissociation or uptake of protons. The enthalpies corrected for proton effects are -22.1, -25.4, and -23.5 kcal/mol, respectively, in buffers A, B, and C. The interaction between troponin and tropomyosin in the presence of calcium is accompanied by release of 0.9 mol of proton per mole of complex. Proton effects in the presence of magnesium and in the absence of divalent metal ions were too small to quantitate. The association constants were measured by using tropomyosin labeled with the extrinsic fluorescent probe dansylaziridine, and binding was detected by enhancement of the probe fluorescence. The magnitudes of the association constants for unlabeled troponin are 7.5 X 10(5), 4.2 X 10(5), and 9.5 X 10(5) M-1, respectively, for the three solution conditions corresponding to unitary free energies of -10.4, -10.1, and -10.6 kcal/mol. The unitary entropies for the interaction are -39, -51, and -43 cal/(deg X mol), respectively, for the three solution conditions. Under these conditions, the troponin-tropomyosin interaction is enthalpy driven, and a large unfavorable entropy must be overcome in the formation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Binary interactions of troponin subunits.

The association constants for the formation of the binary complexes of rabbit fast skeletal muscle troponin subunits have been determined for three solution conditions: (a) 1 mM CaCl2, (b) 3 mM MgCl2 and 1 mM EGTA, and (c) 2 mM EDTA. The subunits were labeled with extrinsic fluorescence probes, either 5-(iodoacetamido)eosin (IAE) or dansylaziridine (DANZ), and the binding was detected by enhancement or quenching of the probe fluorescence. The association constant for the TnI X TnT (where TnI and TnT are the inhibitory subunit and the tropomyosin-binding subunit, respectively, of troponin) complex was measured with two different probes, IAE-TnI and IAE-TnT. The measured values were not affected by the presence of Ca2+ or Mg2+, and the mean values for the three buffer conditions are, respectively, 8.0 X 10(6) and 9.0 X 10(6) M-1 for the two probes. The association constant for TnC-TnI (where TnC is the Ca2+-binding subunit of troponin) interaction was measured with three probes, IAE-TnC, DANZ-TnC, and IAE-TnI. Values of 1.7 X 10(9), 1.2 X 10(8), and 1.0 X 10(6) M-1 were obtained, respectively, in the presence of calcium ion, in the presence of magnesium ion (no calcium), and in the absence of divalent metal ions. A mean value of 4.0 X 10(7) M-1 was obtained for the association constant of TnC X TnT using DANZ-TnC and IAE-TnC as probes in the presence of calcium or magnesium ions. A value of 4.5 X 10(6) M-1 was obtained in the absence of divalent metal ions. The results show that the presence of magnesium ion in the Ca2+-Mg2+ sites strengthens the TnC-TnI and the TnC-TnT interactions and suggest that the troponin structure would be stabilized. This likely results from the effect of magnesium ion on the Ca2+-Mg2+ domains of TnC. The presence of calcium ion in the Ca2+-specific sites provides an additional binding free energy for the TnC-TnI interaction which presumably reflects the changes in the subunit interactions required for the calcium regulatory switch.

Effect of influenza A virus on leukocyte histamine release.

Viral respiratory infections provoke asthma in many patients. In the following study we examined the effect of an in vitro incubation of influenza A on leukocyte histamine release. After incubation with a live influenza A (H3N2) virus, calcium ionophore A23187 (0.5, 1.0, and 1.5 microgram/ml)-induced leukocyte histamine release (HR) was enhanced (p less than 0.05). This effect was also found with heat- or ether-inactivated virus. Similarly, influenza A-exposed leukocytes had augmented leukocyte HR during subsequent incubation with ragweed AgE. Incubation of the leukocyte suspension with interferon (800 IU/ml) for 24 hr was also associated with enhanced HR to ragweed AgE. In contrast, interferon did not alter the calcium ionophore A23187 HR. Therefore, although interferon may mediate the enhanced leukocyte HR when ragweed AgE is the inciting stimulus, it does not change HR to the calcium ionophore.

Stability of the Ca2+-specific and Ca2+-Mg2+ domains of troponin C. Effect of pH.

The unfolding of rabbit fast skeletal muscle troponin C has been examined as a function of pH using differential scanning calorimetry as a probe. Studies were conducted between pH 7.2 and 5.25 in the absence of both Ca2+ and Mg2+. At pH 7.2, a single unfolding transition, with a tm of 58 degrees C, is observed. Decreasing the pH to 5.5 increases the tm for this transition to 67 degrees C. At pH values less than or equal to 5.5, a second unfolding transition is observed, which has a tm of 51 degrees C. These studies suggest that troponin C consists of two types of structural domains: one has a compact structure throughout the pH range examined; the other has a poorly ordered structure at pH 7.2 which becomes more compact as the pH is decreased. We conclude that stabilization of both types of domains occurs through protonation of carboxylate groups clustered in each of the four Ca2+-binding loops. Our data are consistent with assignment of the higher-temperature transition to unfolding of the N-terminal portion of the molecule, which contains the Ca2+-specific domains and of the lower-temperature transition to unfolding of the C-terminal portion, which contains the Ca2+-Mg2+ domains.