Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

Cough provocation with capsaicin is an objective way to test sensory hyperreactivity in patients with asthma-like symptoms

BACKGROUND: A group of patients with asthma-like symptoms and sensitivity to chemical irritants, but without bronchial obstruction, has been found among subjects referred for suspected asthma. They have no well-defined diagnosis, and no objective diagnostic method has previously been available. These patients are more sensitive to inhaled capsaicin than are patients with asthma or healthy controls. The aim was to study cough and other capsaicin-induced symptoms and to test the effect of a drug (lidocaine) that inhibits nerve transmission in sensory nerves. METHODS: Twelve patients were provoked with three different concentrations of inhaled capsaicin solutions in a randomized, double-blind order. They all had asthma-like symptoms and were sensitive to chemical irritants, but had no IgE-mediated allergy or demonstrable bronchial obstruction. Before the provocations, the patients inhaled lidocaine or placebo (saline), also in a double-blind, randomized order. The results were expressed as the number of coughs and scores of various symptoms. RESULTS: The patients reacted in a dose-dependent way with cough, airway, and eye symptoms, which were significantly reduced after preinhalation of lidocaine. CONCLUSIONS: A drug that inhibits transmission in sensory nerves successfully blocked the number of coughs and other symptoms provoked by inhalation of capsaicin. This indicates that the mechanisms underlying chemical sensitivity in these patients may originate in the sensory nervous system, and we call this condition "sensory hyperreactivity".     

Factors modulating the sensitivity of the relaxation to the loading conditions in rat cardiac muscle

The load sensitivity of the relaxation phase was studied in rat papillary muscle, with isotonic afterloaded contractions and stretches applied after the peak of isometric twitches.The tension decay occurred earlier in isotonic than in isometric contractions. When a central region of the preparation was marked with small stainless steel pins, a lengthening of this region could be shown during relaxation of isometric (fixed end) contractions. This lengthening was earlier and faster in isotonic afterloaded contractions. Therefore the sensitivity of relaxation to load or length changes could be described in the context of the general mechanism of relaxation which takes into account the non uniform behaviour of the muscle and the internal movement during contractions.Interventions which decelerate the activation decay rate had different effects on the load dependence of relaxation. Caffeine addition and substitution of strontium for calcium abolished the load sensitivity while a temperature reduction had no influence on it.     

Molecular characterization and determination of the coding capacity of the genome of equine herpesvirus type 2 between the genome coordinates 0.235 and 0.258 (theEcoRI DNA fragment N; 4.2 kbp)

The complete DNA nucleotide sequence of theEcoRI DNA fragment N (0.235 to 0.258 viral map units) of equine herpes virus type 2 (EHV-2) strain T400/3 was determined. This DNA fragment comprises 4237 bp with a base composition of 55.23% G+C and 44.77% A+T. Nineteen open reading frames (ORFs) of 50-287 amino acid (aa) residues were detected. ORF number 10 is located between the nucleotide position 2220 and 2756 coding for a protein of 179 amino acid residues. This protein shows significant homology to the cytokine synthesis inhibitory factor (CSIF; interleukin 10) of human (76.4%) and mouse (68.5%), and to the Epstein-Barr virus (EBV) protein BCRF1 (70.6%). The existence of an interleukin 10 (IL-10) analogous gene within the genome of the EHV-2 was confirmed by screening the genome of nine EHV-2 strains using specific oligonucleotide primers corresponding to the 5 and 3 region of this particular gene by polymerase chain reaction. In all experiments an 870 bp DNA product was amplified. The specifity of the amplified DNA fragments obtained from individual EHV-2 strains was confirmed by DNA-DNA hybridization experiments. The DNA sequence analysis of the amplified DNA products of the EHV-2 strain LK was carried out. This analysis revealed the identity of the corresponding IL-10 gene (540 bp) of this strain to the IL-10 gene of EHV-2 strain T400/3. The presented data indicate that the EHV-2 genome harbors a viral interleukin 10-like gene. This is further evidence that the IL-10 gene can be present in the genomes of members of the Herpesviridae family.     

Ventilatory response of prepubertal boys and adults to carbon dioxide at rest and during exercise

Summary The aim of this study was to determine whether the greater ventilation in children at rest and during exercise is related to a greater CO₂ ventilatory response. The CO₂ ventilatory response was measured in nine prepubertal boys [10.3 years (SD 0.1)] and in 10 adults [24.9 years (SD 0.8)] at rest and during moderate exercise ( CO₂ = 20 ml·kg^–1·min^–1) using the CO₂-rebreathing method. Three criteria were measured in all subjects to assess the ventilatory response to CO₂: the CO₂ sensitivity threshold (Th), which was defined as the value of end titalPCO₂ (P _ETCO₂) where the ventilation increased above its steady-state level; the reactivity slope expressed per unit of body mass (SBM), which was the slope of the linear relation between minute ventilation ( _E) andP _ETCO₂ above Th; and the slope of the relationship between the quotient of tidal volume (V _T) and inspiration time (t _I) andP _ETCO₂ (V _T ·t _I ^–1 ·P _ETCO₂ ^–1) values above Th. The _E,V _T, breathing frequency (f _R), oxygen uptake ( O₂), and CO₂ production ( CO₂) were also measured before the CO₂-rebreathing test. The following results were obtained. First, children had greater ventilation per unit body weight than adults at rest (P<0.001) and during exercise (P<0.01). Second, at rest, onlyV _T ·t _I ^–1 ·P _ETCO₂ ^–1 was greater in children than in adults (P<0.001). Third, during exercise, children had a higher S_BM (P < 0.02) andV _T ·t _I ^–1 ·P _ETCO₂ ^–1 (P<0.001) while Th was lower (P<0.02). Finally, no correlation was found between _E/ CO₂ and Th while a significant correlation existed between _E/ CO₂ and S_BM (adults,r=0.79,P<0.01; children,r=0.73,P<0.05). We conclude that children have, mainly during exercise, a greater sensitivity of the respiratory centres than adult. This greater CO₂ sensitivity could partly explain their higher ventilation during exercise, though greater CO₂ production probably plays a role at rest.     

Aspirin Tolerance Induced in Aspirin-Sensitive Asthmatics

Twenty-nine patients with asthma and aspirin-sensitivity were studied in an attempt to induce tolerance to aspirin (ASA). Starting with the smallest ASA doses eliciting bronchial obstruction (threshold doses) we doubled the doses on subsequent days and finally achieved good tolerance of 600 mg ASA per day in 27 patients. It was more difficult to achieve tolerance in patients with low ASA-thresholds than in patients with high ones. Daily ASA administration led to prolongation of the refractory state but when the intervals between consecutive doses were increased, aspirin hypersensitivity recurred. The pause sufficient for a recurrence of sensitivity to ASA was measured in 16 patients and ranged from 24 h to 9 days. Twelve patients challenged with indomethacin after ASA-desensitisation showed good drug tolerance.     

焦虑敏感问卷中文版的信度和效度

目的:翻译和修订焦虑敏感问卷,测试中文版的信效度。方法:翻译焦虑敏感量表,通过对中学生的三次测试,对量表的因素结构、信度和效度进行了检验。结果:修编的焦虑敏感问卷中文版通过探索性因素分析将原量表由36题缩减为15题,共包括三个因素:对生理唤醒的恐惧;对认知失控的恐惧;对社会评价的恐惧;三个因素的累积贡献率为52．29％,项目的因素负荷率在0．63～0．98之间;因素彼此之间的相关在0.37～0．53之间,因素与问卷总分之问的相关在0．72～0．84之间,因素内部的相关在0．68～0．78之间。问卷在各个因素上的内部一致性系数在0.73～0．78之间．分半信度在0．71～0．76之间,重测信度在0．70～0．78之间。结论:问卷的中文简化版在中学生中具有良好的信度和效度。     

Willignes to drive when drunk and personality: A twin study

In a laboratory study of psychomotor sensitivity to alcohol, twins were asked “Would you drive a car now?” at 1, 2, and 3 h after drinking a standard dose of ethanol (0.75 g/kg). Correlations among these binary items, the Eysenck personality scales, and age were investigated using PRELIS and LISREL. Willingness to drive and Extraversion correlate at all three times in both males and females. In males, willingness to drive also correlates with Psychoticism, and in females it correlates negatively with the Lie (or Social Desirability) scale. Most correlations between cotwins in willingness to drive were significant in both monozygotic (MZ) and dizygotic (DZ) male twins but correlations were lower in female twins. Factor and Markovian models were fitted. In males there seem to be both genetic and cultural influences on willingness to drive when drunk. About half the genetic variance seems to be the pleiotropic effects of genes influencing Extraversion. The correlationswith Psychoticism, on the other hand, seem to be largely environmental in origin. The small sample size and lack of proper significance tests mean that these results must be interpreted with caution.     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.