Airway contractility and smooth muscle Ca(2+) signaling in lung slices from different mouse strains.

To investigate the hypothesis that altered Ca2+ signaling in airway smooth muscle cells (SMCs) is responsible for airway hyperreactivity, we compared, with the use of confocal and phase-contrast microscopy, the airway contractility and Ca2+ changes in SMCs induced by acetylcholine (ACh) in lung slices from different mouse strains (A/J, Balb/C, and C3H/ HeJ). The airways from each mouse strain displayed a concentration-dependent contraction to ACh. The contractile response of the airways of the C3H/HeJ mice was found, in contrast to earlier studies, to be much greater and faster than that of A/J and Balb/C mice. This difference in airway reactivity can be, in part, attributable to halothane, a volatile anesthetic that was previously used during in vivo measurements of airway reactivity but found here to significantly alter the ACh contractile response of airways in lung slices. The ACh-induced Ca2+ response of the airway SMCs in all of the various mouse strains was also concentration dependent. The magnitude of the initial Ca2+ increase and the frequency of the subsequent Ca2+ oscillations induced by ACh increased with ACh concentration. However, no differences in the Ca2+ responses to ACh could be distinguished between the mouse strains. These results suggest that the mechanism responsible for airway hyperreactivity in different mouse strains resides with the Ca2+ sensitivity of the contractile apparatus of the SMCs rather than with the Ca2+ signaling itself.     

Heritability of adult body height: a comparative study of twin cohorts in eight countries.

A major component of variation in body height is due to genetic differences, but environmental factors have a substantial contributory effect. In this study we aimed to analyse whether the genetic architecture of body height varies between affluent western societies. We analysed twin data from eight countries comprising 30,111 complete twin pairs by using the univariate genetic model of the Mx statistical package. Body height and zygosity were self-reported in seven populations and measured directly in one population. We found that there was substantial variation in mean body height between countries; body height was least in Italy (177 cm in men and 163 cm in women) and greatest in the Netherlands (184 cm and 171 cm, respectively). In men there was no corresponding variation in heritability of body height, heritability estimates ranging from 0.87 to 0.93 in populations under an additive genes/unique environment (AE) model. Among women the heritability estimates were generally lower than among men with greater variation between countries, ranging from 0.68 to 0.84 when an additive genes/shared environment/unique environment (ACE) model was used. In four populations where an AE model fit equally well or better, heritability ranged from 0.89 to 0.93. This difference between the sexes was mainly due to the effect of the shared environmental component of variance, which appears to be more important among women than among men in our study populations. Our results indicate that, in general, there are only minor differences in the genetic architecture of height between affluent Caucasian populations, especially among men.     

Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active β-lactamase fusion proteins (BlaM⁺) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line Katoill. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.     

The effect of post-hypoxia on roots inSenecio andMyosotis species related to the glutathione system

This paper shows the effect of re-aeration following hypoxic pretreatment on the glutathione system in plants with different flooding tolerance. Re-aeration of hypoxically pretreated roots led to an increase of TBA-rm content indicating an accelerated lipid peroxidation (post-anoxic injury). Re-admission of oxygen resulted in a clear increase in the content of total glutathione in both flooding-intolerant speciesMyosotis arvensis andSenecio jacobaea. Simultaneously, the high ratio between reduced (GSH) and oxidized (GSSG) glutathione decreased in these species upon the onset of re-aeration, while the tolerantMyosotis palustris andSenecio aquaticus showed only little changes in contents of GSH and GSSG. An imbalance in GSH/GSSG ratio reflects oxidative stress. The glutathione reductase (GR) reacted very differently in the investigated genera. The metabolic response to varying oxygen pressure is much stronger in the flooding-intolerant species compared to species naturally growing in wetlands. The present results suggest that glutathione system is an important component in overcoming oxidative stress.     

Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′i−1,Hi α), J(C′i−1,Ci β) and J(C′i−1,C′i) in proteins

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C_i–1,H_i ), ³J(C_i–1,C_i ) and³J(C_i–1,C_i)coupling constants in the indirectly detected ¹³Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C_i–1,H_i ),³J(C_i–1,C_i ) and³J(C_i–1,C_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.     

S-Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography-Mass Spectrometry

Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S -nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S -nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [³⁵S]cysteine-prelabeled slices suggested that S -nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography-mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using ¹⁵N-labeled GSNO ([¹⁵N]-GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.     

Progress in fold recognition

The prediction experiment reveals that fold recognition has become a powerful tool in structural biology. We applied our fold recognition technique to 13 target sequences. In two cases, replication terminating protein and prosequence of subtilisin, the predicted structures are very similar to the experimentally determined folds. For the first time, in a public blind test, the unknown structures of proteins have been predicted ahead of experiment to an accuracy approaching molecular detail. In two other cases the approximate folds have been predicted correctly. According to the assessors there were 12 recognizable folds among the target proteins. In our postprediction analysis we find that in 7 cases our fold recognition technique is successful. In several of the remaining cases the predicted folds have interesting features in common with the experimental results. We present our procedure, discuss the results, and comment on several fundamental and technical problems encountered in fold recognition. © 1995 Wiley-Liss, Inc.