Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an approximately 42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062-8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.     

Phospholipid composition of myocardium in children with normoxemic and hypoxemic congenital heart diseases

Samples of myocardial tissue were obtained during cardiac surgery from children operated for different types of normoxemic and hypoxemic congenital heart diseases. The phospholipid composition was analyzed by thin layer chromatography. The concentration of total phospholipids (PL), phosphatidylcholine and phosphatidylethanolamine (PE) was found lower in atrial tissue of both normoxemic and hypoxemic groups in comparison with the ventricles. When comparing the difference between hypoxemic and normoxemic defects, hypoxemia was found to increase the concentration of total PL, PE and phosphatidylserine in ventricles and total PL and PE in the atria. The increased level of particular phospholipid species may represent adaptive mechanisms to hypoxemia in children with congenital heart diseases.     

McRAPD as a new approach to rapid and accurate identification of pathogenic yeasts

Despite advances in antifungal prophylaxis and therapy, morbidity and mortality incurred by yeasts remain a significant burden. As pathogenic yeast species vary in their susceptibilities to antifungal agents, clinical microbiology laboratories face an important challenge to identify them rapidly and accurately. Although a vast array of phenotyping and genotyping methods has been developed, these are either unable to cover the whole spectrum of potential yeast pathogens or can do this only in a rather costly or laborious way. Random amplified polymorphic DNA (RAPD) fingerprinting was repeatedly demonstrated to be a convenient tool for species identification in pathogenic yeasts. However, its wider acceptance has been limited mainly due to special expertise and software needed for analysis and comparison of the resulting banding patterns. Based on a pilot study, we demonstrate here that a simple and rapid melting curve analysis of RAPD products can provide data for identification of five of the most medically important Candida species. We have termed this new approach melting curve of random amplified polymorphic DNA (McRAPD) to emphasize its rapidity and potential for automation, highly desirable features for a routine laboratory test.     

Biogeographically interesting planktonic Nostocales (Cyanobacteria) in the Czech Republic and their polyphasic evaluation resulting in taxonomic revisions of Anabaena bergii Ostenfeld 1908 (Chrysosporum gen. nov.) and A. tenericaulis Nygaard 1949 (Dolichospermum tenericaule comb. nova)

Questions of biogeography of freshwater cyanobacteria and their ability to colonize new areas have been recently discussed in connection with increasing occurrence of some formerly rare morphospecies in temperate zones. Nevertheless, the general knowledge about the distribution of cyanobacterial species is still fragmentary, and any new findings on cyanobacterial biogeography and spread are valuable. In this study, we provide updated information on the occurrence of Anabaena bergii, Raphidiopsis mediterranea, and Sphaerospermopsis aphanizomenoides in the Czech Republic. In addition, more nostocacean morphospecies are newly reported from the Czech Republic (A. fusca, A. tenericaulis, Dolichospermum curvum, D. mucosum, and S. reniformis). All of these morphospecies were characterized from a morphological point of view, and their phylogenetic affiliations were assessed on the basis of their 16S rRNA gene sequences. Based on these results, Anabaena bergii was reclassified into Chrysosporum gen. nov., and D. tenericaule comb. nova was established.     

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.