Variation in mitochondrial DNA in a cline of allele frequencies and shell phenotype in the dog-whelk Nucella lapillus (L.)

Genetic constitution in the intertidal gastropod Nucella lapillus (L.) influences shell shape, growth rate and physiology. Clinal variation in these traits along a 5 km stretch of coastline in south Devon can be related to environmental variation in temperature and desiccation stress. We have examined mtDNA variation along this shore to investigate whether the cline represents primary or secondary contact. Two distinct mtDNA haplotypes were found which exhibit coincident step clines with karyotypic, allozymic and phenotypic variation and covary with the environmental pressures of temperature and desiccation. These results are interpreted in the context of the wider scale distribution of genetic and phenotypic variation in N. lapillus. It is suggested that the shore studied may represent one of a number of regions of secondary contact within a mosaic hybrid zone in N. lapillus , where coadapted phenotypic variation correlates with habitat and the position of the clines represents an environmental transition.     

Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.      

Crystallization and preliminary crystallographic data for bovine antithrombin III

Crystals of bovine antithrombin III were obtained in the presence of metal ions with ammonium sulphate as precipitating agent. Crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 with cell parameters a = b = 91.4 A, c = 383.1 A; there are two molecules per asymmetric unit. Electrophoresis experiments and amino acid sequence analysis of the N-terminal part of redissolved crystals suggest that the protein molecules are cleaved at the active site.     

Echocardiographic assessment and percutaneous closure of multiple atrial septal defects

Percutaneous coronary intervention can be associated with distal embolization of thrombotic material causing myocardial necrosis and infarction. We discuss the role of intravascular imaging to guide the use of a distal protection device by describing the outcome of a young woman presenting with non-ST elevation myocardial infarction. Coronary angiography demonstrated an isolated minor stenosis in the proximal left anterior descending coronary artery with slight haziness beyond the lesion. Intravascular ultrasound confirmed an extensive thrombus overlying a bulky atherosclerotic plaque. A distal filter wire was therefore successfully used to reduce the risk of distal embolization. The use of intravascular ultrasound in patients presenting with acute coronary syndrome may reveal large thrombi that are difficult to image using conventional angiographic techniques. Intravascular ultrasound can therefore be used as a tool to select lesions requiring distal protection.     

One-Step Purification of a Lipolytic Enzyme from Bacillus pumilus by Mild Hydrophobic Interaction Chromatography on Polyoxyalkylene Glycol-Bound Sepharose 6B

An esterase from Bacillus pumilus was obtained in a highly purified form, starting from a crude extract, by a new chromatographic technique requiring no detergent during the elution step. The stationary phase involved was mildly hydrophobic and was prepared by covalent immobilization of a polyoxyalkylene glycol onto Sepharose 6B.     

S-Adenosyl-N-decyl-aminoethyl, a Potent Bisubstrate Inhibitor of Mycobacterium tuberculosis Mycolic Acid Methyltransferases

S-Adenosylmethionine-dependent methyltransferases (AdoMet-MTs) constitute a large family of enzymes specifically transferring a methyl group to a range of biologically active molecules. Mycobacterium tuberculosis produces a set of paralogous AdoMet-MTs responsible for introducing key chemical modifications at defined positions of mycolic acids, which are essential and specific components of the mycobacterial cell envelope. We investigated the inhibition of these mycolic acid methyltransferases (MA-MTs) by structural analogs of the AdoMet cofactor. We found that S-adenosyl-N-decyl-aminoethyl, a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain, inhibited MA-MTs from Mycobacterium smegmatis and M. tuberculosis strains, both in vitro and in vivo, with IC₅₀ values in the submicromolar range. By contrast, S-adenosylhomocysteine, the demethylated reaction product, and sinefungin, a general AdoMet-MT inhibitor, did not inhibit MA-MTs. The interaction between Hma (MmaA4), which is strictly required for the biosynthesis of oxygenated mycolic acids in M. tuberculosis, and the three cofactor analogs was investigated by x-ray crystallography. The high resolution crystal structures obtained illustrate the bisubstrate nature of S-adenosyl-N-decyl-aminoethyl and provide insight into its mode of action in the inhibition of MA-MTs. This study has potential implications for the design of new drugs effective against multidrug-resistant and persistent tubercle bacilli.One-third of the world population is infected with the tubercle bacillus, Mycobacterium tuberculosis, and tuberculosis kills one person every 20 s. The inhaled pathogenic bacilli are taken up by phagocytosis by pulmonary macrophages, which, together with lymphocytes and dendritic cells, form granulomas. The bacilli persist in the granuloma until their reactivation, dissemination into the lungs, and the triggering of disease. The natural resistance of persistent tubercle bacilli to drugs and the emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains are two main concerns in the treatment of the disease. A survey carried out by the Centers for Disease Control and Prevention and the World Health Organization between 2000 and 2004 reported that 20% of 17,690 M. tuberculosis isolates from 49 countries were multidrug-resistant, and 2% were extensively drug-resistant (1). The development of new drugs effective against persistent and drug-resistant bacilli has therefore become a priority.The thick lipid-rich envelope of the Mycobacterium genus is characterized by the presence of mycolic acids (MAs),⁴ very long chain (C₆₀–C₉₀) α-alkylated β-hydroxylated fatty acids (2). MAs are the major components of the mycomembrane (3, 4) lipid bilayer, which plays a key role in both the architecture and permeability of the mycobacterial envelope. The MA biosynthetic pathway is essential for mycobacterial survival. MAs are generated by Claisen condensation between two fatty acyl chains as follows: the very long meromycoloyl chain (C₄₀–C₆₀) and a shorter saturated chain (C₂₂–C₂₆) (2). The different types of MAs are defined by the presence of decorations introduced at proximal and distal positions of the meromycolic chain (Fig. 1A) by a family of paralogous S-adenosylmethionine-dependent methyltransferases (AdoMet-MTs), the mycolic acid methyltransferases (MA-MTs). These chemical modifications are known to be important for the pathogenicity, virulence, and persistence of M. tuberculosis. For example, the cis-cyclopropane introduced at the proximal position of α-MAs by PcaA has an impact on the persistence of the tubercle bacillus within infected organisms (5). Furthermore, the keto and methoxy groups, with a vicinal methyl ramification at the distal position of oxygenated MAs, play a role in M. tuberculosis virulence in the mouse model of infection (6) and have recently been reported to be involved in host-pathogen interplay. Indeed, oxygenated MAs have been shown to modulate IL-12p40 production by macrophages (7) and to trigger the in vitro differentiation of monocyte-derived macrophages into foamy macrophages, which house the bacillus in a dormant state, within granulomas (8). Oxygenated MA biosynthesis requires the Hma (MmaA4) methyltransferase (Fig. 1B), as demonstrated by the absence of the oxygenated form in an M. tuberculosis hma knock-out mutant (6, 9). These results suggest that the enzymes responsible for adding the decorations to MAs, including oxygenated groups in particular, may be relevant pharmacological targets for the development of new antituberculous drugs (10).Open in a separate windowFIGURE 1.A, structures of MAs from M. tuberculosis and M. smegmatis. D, distal position; P, proximal position. Enzymes involved in the introduction of decorations on the meromycolic chain are indicated. B, proposed reaction scheme for the introduction of oxygenated groups. m = 17, 19; n, unknown; X, unknown carrier.Based on the essential role played by MA-MTs in the physiopathology of tuberculosis, several studies have investigated the possible inhibition of this family of enzymes. A recent study revealed that the antituberculous drug thiacetazone and its chemical analogs inhibited MA cyclopropanation at concentrations in the micromolar range (11). Another study, based on mixtures of crude extracts of heat-inactivated mycobacteria and recombinant Escherichia coli overproducing MA-MTs, suggested that the incorporation of [³H]AdoMet into growing meromycolic chains is inhibited by a high concentration (1 mg/ml, i.e. 2.6 mm) of S-adenosyl-l-homocysteine (AdoHcy) or sinefungin (12), the demethylated reaction product and a natural structural analog of AdoMet, respectively (Fig. 2). By contrast, AdoHcy and sinefungin are strong inhibitors of other AdoMet-MTs in vitro, including the cyclopropane fatty-acid synthase (CFAS) from E. coli (K_i of 30 and 0.22 μm, respectively) (13, 14). However, they are active only against the isolated enzyme, whereas S-adenosyl-N-decyl-aminoethyl (SADAE), a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain (Fig. 2), is active against CFAS both in vitro (K_i,app = 6 μm) and in vivo (complete inhibition at 150 μm) (15). The broad screening of possible inhibitors of MA-MTs with an in vitro mini-assay poses a major challenge, as these enzymes most likely use very long meromycolic chains as substrates. In this context, the similarity between CFAS and Hma in terms of their sequences (31% sequence identity) and substrates may be useful, as it suggests that SADAE may inhibit MA-MTs (15).Open in a separate windowFIGURE 2.Structure of AdoMet and of the AdoHcy, sinefungin, and SADAE analogs.We report here our investigations of the interactions between Hma and SADAE, as compared with those between Hma and AdoHcy or sinefungin, and the potential impact of these interactions on the activities of Hma and other MA-MTs and mycobacterial growth. Our high resolution crystallographic characterization of the Hma-SADAE interaction illustrates the bisubstrate nature of the ligand, which is strongly correlated with its strong inhibitory properties.     

Patterns of inflammation and the use of reversibility testing in smokers with airway complaints

Background

Although both smoking and respiratory complaints are very common, tools to improve diagnostic accuracy are scarce in primary care. This study aimed to reveal what inflammatory patterns prevail in clinically established diagnosis groups, and what factors are associated with eosinophilia.

Method

Induced sputum and blood plasma of 59 primary care patients with COPD (n = 17), asthma (n = 11), chronic bronchitis (CB, n = 14) and smokers with no respiratory complaints ('healthy smokers', n = 17) were collected, as well as lung function, smoking history and clinical work-up. Patterns of inflammatory markers per clinical diagnosis and factors associated with eosinophilia were analyzed by multiple regression analyses, the differences expressed in odds ratios (OR) with 95% confidence intervals.

Results

Multivariately, COPD was significantly associated with raised plasma-LBP (OR 1.2 [1.04–1.37]) and sTNF-R55 in sputum (OR 1.01 [1.001–1.01]), while HS expressed significantly lowered plasma-LBP (OR 0.8 [0.72–0.95]). Asthma was characterized by higher sputum eosinophilic counts (OR 1.3 [1.05–1.54]), while CB showed a significantly higher proportion of sputum lymphocytic counts (OR 1.5 [1.12–1.9]). Sputum eosinophilia was significantly associated with reversibility after adjusting for smoking, lung function, age, gender and allergy.

Conclusion

Patterns of inflammatory markers in a panel of blood plasma and sputum cells and mediators were discernable in clinical diagnosis groups of respiratory disease. COPD and so-called healthy smokers showed consistent opposite associations with plasma LBP, while chronic bronchitics showed relatively predominant lymphocytic inflammation compared to other diagnosis groups. Only sputum eosinophilia remained significantly associated with reversibility across the spectrum of respiratory disease in smokers with airway complaints.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.