A novel method for isolating specific endocytic vesicles using very fine ferrite particles coated with biological ligands and the high-gradient magnetic separation technique

We have developed a novel method for isolating specific endocytic vesicles using magnetic ligands and high-gradient magnetic separation. Ligands were prepared by coating extremely fine ferrite particles (10-20 nm) with bovine serum albumin and then conjugating asialoglycopeptides. These ligands were introduced into rat liver by perfusion at 16 or 37 degrees C, or by injection through the tail vein. The ligand particles were observed as electron-dense small grains in membrane-bound vesicles in Kupffer as well as parenchymal cells by electron microscopy. Livers were taken out, homogenized and lightly centrifuged. The supernatant was pumped into a separator glass tube filled with very fine ferritic stainless steel fibers and placed in a magnetic field of 0.9-2 T. Vesicles containing ferrite particles were collected with a high efficiency (ca. 70% of endocytosed magnetic ligands). About 70% of uptake appeared to be mediated by the asialoglycoprotein receptors. The captured vesicles were practically free from marker enzymes for plasma membranes, endoplasmic reticulum, and Golgi apparatus. Lysosomal enzyme activity of the vesicles increased with the time of perfusion at 37 degrees C but not at 16 degrees C. Protein composition of the captured vesicles was analyzed by one- and two-dimensional gel electrophoresis. The composition changed characteristically with time on perfusion at 16 and 37 degrees C. The present method provides a powerful tool to collect prelysosomal endocytic vesicles containing specific ligands and lysosomes fused with these specific endocytic vesicles.     

Inhibition and resumption of processing of the staphylokinase in some Escherichia coli prlA suppressor mutants

Escherichia coli strains carrying certain prlA mutations (prlA4 and prlA401) could not support the processing and export of staphylokinase, resulting in the accumulation of the precursor form under high-level synthesis conditions. In order to clarify the cause of the defect in the structure of staphylokinase, we constructed signal peptide mutations of sak which suppressed the processing defect in the prlA4 cells by site-directed mutagenesis. The processing defect was suppressed when glycine or asparagine was introduced in place of the serine residue at position 17 from the amino terminus of the signal peptide. Substitutions of glycine for the leucine residue at position 15 and for the serine residue at position 19 were also effective. Other mutations we constructed had no suppression activity. Taking account of the correlation between the suppression activity and the parameter value of each substituted amino acid for the beta-turn probability, we predict that the staphylokinase signal peptide requires a more bending structure at the end of the hydrophobic core to act efficiently in the prlA4 cells than in the prl+ cells and that a function of the PrlA protein necessary to recognize the staphylokinase signal peptide has become deficient through the prlA4 mutation.     

ATP synthesis kinetics and mitochondrial function in the postischemic myocardium as studied by 31P NMR

The effects of ischemia on mitochondrial function and the unidirectional rate of ATP synthesis (Pi----ATP rate) were studied using a Langendorff-perfused heart preparation and 31P NMR spectroscopy. There was significant postischemic depression of mechanical function assessed as the heart rate pressure product, and the myocardial oxygen consumption rate at a given rate pressure product was elevated. Experiments performed on glucose- and pyruvate-perfused hearts demonstrated the presence of a large contribution to the unidirectional Pi----ATP rate catalyzed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. This rate was much greater than the maximal glucose utilization rate in the myocardium, demonstrating that the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reactions are near equilibrium both before and after ischemia. In the pyruvate-perfused postischemic hearts, the glycolytic contribution was eliminated and the net rate of ATP synthesis by oxidative phosphorylation was measurable. Despite the reduced mechanical function and increased myocardial oxygen consumption rate, the ratio of the net rate of ATP synthesis by oxidative phosphorylation to oxygen consumption rate (the P:O ratio) was not altered subsequent to ischemia (2.34 +/- 0.12 and 2.36 +/- 0.09 in normal and postischemic hearts, respectively). Therefore, mitochondrial uncoupling cannot be the cause of postischemic depression in mechanical function; instead, the data suggest the existence of ischemia-induced inefficiency in ATP utilization.     

Molecular characterization of a novel gene encoding an 8-kDa-subunit of antigen B from Echinococcus granulosus genotypes 1 and 6

Antigen B in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa which are encoded by a multigene family. Four genes encoding 8-kDa-subunit monomers of the antigen B have been identified from E. granulosus. Recently, we have isolated another novel gene from Echinococcus multilocularis encoding a fifth 8-kDa-subunit of AgB (named EmAgB8/5), predominantly transcribed in the adult worm, but not in vesicles of metacestodes. In this study, we cloned and characterized two EmAgB8/5 homologue genes from E. granulosus genotypes 1 and 6 by PCR, and named as EgG1AgB8/5 and EgG6AgB8/5, respectively. The phylogenetic relationship of these genes with other genes encoding the antigen B 8-kDa-subunit monomers was also discussed.     

The first report on cystic echinococcosis in a cat caused by Echinococcus granulosus sensu stricto (G1)

A case of cystic echinococcosis (CE) in a domestic cat is described from Saint Petersburg, Russia. Ultrasonography showed numerous cysts with hyperechoic walls and anechoic contents within the cat's abdominal cavity. Molecular identification based on mitochondrial DNA genes indicated that the causative agent was Echinococcus granulosus sensu stricto (G1 strain). This is the first report of CE in a cat caused by E. granulosus sensu stricto with molecular confirmation.     

Consequences of linker length alteration of the α7 nicotinic acetylcholine receptor (nAChR) agonist, SEN12333

A series of ligands based on SEN12333, containing either contracted or elongated alkyl chains, were synthesized and evaluated in molecular docking studies against a homology model of the α7 nicotinic acetylcholine receptor (nAChR) subtype. The predicted binding of all ligands was highly similar, with the exception of the analog containing a 5 methylene unit spacer. However, in vitro competition binding assays revealed that the ligands possessed dissimilar binding affinities, with a K(i) range of more than an order of magnitude (K(i)=0.50 to >10 μM), and only SEN12333 itself exhibited functional activity at the α7 nAChR.     

Expanded phylogenies of canonical and non-canonical types of methionine adenosyltransferase reveal a complex history of these gene families in eukaryotes

Most eukaryotes possess the highly-conserved enzyme methionine adenosyltransferase (MAT) that produces S-adenosyl-l-methionine, a molecule essential to a variety of cellular processes. However, a recent study revealed that genomes of a very few eukaryote lineages encode a highly divergent type of MAT (called MATX), instead of the canonical MAT enzyme. Since MATX-containing eukaryotes are phylogenetically interspersed with MAT-containing organisms, it is likely that the MATX gene was spread into the MAT-containing groups via multiple eukaryote-to-eukaryote lateral gene transfer events. Here, we further investigate the evolutionary history of these gene families by vastly increasing the sampling of species containing MAT (22 new taxa) and MATX (8 new taxa). Our expanded analyses reveal the first example of lateral transfer of a MAT gene between the pelagophycean alga Aureococcus anophagefferens and a cryptomonad. The increased MATX sampling also provided new insights into the evolution of MATX. Specifically, our MATX phylogeny robustly grouped the haptophyte homologues with the Aureococcus homologue to the exclusion of the diatom homologues, suggesting a transfer of the MATX gene between haptophytes and pelagophytes. Various scenarios of MAT and MATX gene family evolution in diatoms are re-evaluated in light of the new data.     

Geographic pattern of genetic variation in the fox tapeworm Echinococcus multilocularis

Intraspecific genetic variation of Echinococcus multilocularis, the etiologic agent of human alveolar echinococcosis, has been evaluated among 76 geographic isolates from Europe, Asia and North America by using sequence data of mitochondrial and nuclear DNA. Relatively low genetic variation was found only in the mitochondrial DNA sequence consisting of 3 protein-coding genes. Pairwise divergence among the resultant 18 haplotypes ranged from 0.03 to 1.91%. Phylogenetic trees and parsimony network of these haplotypes depicted a geographic division into European, Asian and North American clades, but 1 haplotype from Inner Mongolia was unrelated to other haplotypes. The coexistence of the Asian and North American haplotypes could be seen, particularly on the St. Lawrence Island in the Bering Sea. These data suggest an evolutionary scenario in which distinct parasite populations derived from glacial refugia have been maintained by indigenous host mammals. The nuclear DNA sequence for the immunodominant B cell epitope region of ezrin/radixin/moesin-like protein (elp) was extremely conservative, indicating that the elp antigen is available for immunodiagnosis in any endemic areas.     

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration

CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.