Obligatory intracellular parasitism by Ehrlichia chaffeensis and Anaplasma phagocytophilum involves caveolae and glycosylphosphatidylinositol-anchored proteins

Obligatory intracellular, human ehrlichiosis agents Ehrlichia chaffeensis and Anaplasma phagocytophilum create unique replicative compartments devoid of lysosomal markers in monocytes/macrophages and granulocytes respectively. The entry of these bacteria requires host phospholipase C (PLC)-gamma2 and protein tyrosine kinases, but their entry route is still unclear. Here, using specific inhibitors, double immunofluorescence labelling and the fractionation of lipid rafts, we demonstrate that bacterial entry and intracellular infection involve cholesterol-rich lipid rafts or caveolae and glycosylphosphatidylinositol (GPI)-anchored proteins. By fluorescence microscopy, caveolar marker protein caveolin-1 was co-localized with both early and replicative bacterial inclusions. Additionally, tyrosine-phosphorylated proteins and PLC-gamma2 were found in bacterial early inclusions. In contrast, clathrin was not found in any inclusions from either bacterium. An early endosomal marker, transferrin receptor, was not present in the early inclusions of E. chaffeensis, but was found in replicative inclusions of E. chaffeensis. Furthermore, several bacterial proteins from E. chaffeensis and A. phagocytophilum were co-fractionated with Triton X-100-insoluble raft fractions. The formation of bacteria-encapsulating caveolae, which assemble and retain signalling molecules essential for bacterial entry and interact with the recycling endosome pathway, may ensure the survival of these obligatory intracellular bacteria in primary host defensive cells.     

The secretory response through electric stimulation of differentiated PC12 rat pheochromocytoma cells transfected with neuropeptide Y fused with enhanced green fluorescent protein

Exocytosis in pheochromocytoma cells was induced by electric stimulation. To chase the movement of vesicles by electric stimulation, dense-core secretory vesicles were visualized by expression of the fusion protein between neuropeptide Y and enhanced green fluorescent protein (EGFP) in these differentiated PC12 rat pheochromocytoma cells. When the cells were stimulated with constant voltage potential at –300 mV, the movement of dense-core secretory vesicles could be regulated.     

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.     

Androgen-dependent atypical fibromas spontaneously arising in the skin of Djungarian hamsters (Phodopus sungorus)

Spontaneous atypical fibromas that arose in the thoracoabdominal skin of one aged female and 31 aged male Djungarian hamsters (Phodopus sungorus) were examined histologically, immunohistochemically, and ultrastructurally. The normal skin from both sexes obtained at various intervals until the age of 12 months was examined, as were the tumors. These tumors were composed of ganglion cell-like (GL) cells that had one or two ovoid nuclei, basophilic foamy cytoplasm, and various amount of collagen fibers between the cells. The tumor cells had positive reaction to vimentin and androgen receptor (AR); the stromal collagen fibers reacted positively with the antibody against collagen type I or III. Ultrastructurally, the tumor cells had abundant rough endoplasmic reticulum in the cytoplasm. On the other hand, small nests of the cells mimicking tumor GL cells were present in the dermal layer to the panniculus of the normal thoracoabdominal skin of adult males, but were seldom found in adult females. The morphologic and immunohistochemical features of these tumor GL cells were basically similar to those of normal skin GL cells, although the former had a certain degree of atypia. These results suggest that atypical skin fibroma in the Djungarian hamster is an androgen-dependent tumor and originates from skin GL cells.     

NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+ -binding motif

Drosophila GCM (glial cell missing) is a novel DNA-binding protein that determines the fate of glial precursors from the neural default to glia. The GCM protein contains the functional domain that is essential for recognition of the upstream sequence of the repo gene. In the DNA-binding region of this GCM protein, there is a cysteine-rich region with which divalent metal ions such as Zn(2+) must bind and other proteins belonging to the GCM family have a corresponding region. To obtain a more detailed insight into the structural and functional features of this DNA-binding region, we have determined the minimal DNA-binding domain and obtained inductively coupled plasma atomic emission spectra and (1)H-(15)N, (1)H-(15)N-(13)C and (113)Cd(2+) NMR spectra, with or without its specific DNA molecule. Considering the results, it was concluded that the minimal DNA-binding domain includes two Zn(2+)-binding sites, one of which is adjacent to the interface for DNA binding. Systematic mutational analyses of the conserved cysteine residues in the minimal DNA-binding domain revealed that one Zn(2+)-binding site is indispensable for stabilization of the higher order structure of this DNA-binding domain, but that the other is not.     

Dipicolinic acid prevents the copper-dependent oxidation of low density lipoprotein

Effect of dipicolinic acid (pyridine 2,6-dicarboxylic acid) and pyridine compounds on the copper-dependent oxidation of human low density lipoprotein was analyzed in relation to the inhibition of copper reduction. Dipicolinic acid inhibited copper-dependent LDL oxidation completely, but the LDL oxidation was slightly inhibited by pyridine compounds with one carboxyl group at 2 or 6-position. Reduction of copper by LDL itself and ascorbate was inhibited completely by dipicolinic acid, but only partially by picolinic acid, quinolinic acid and isocinchomeronic acid with 2- or 6-carboxylic group. Pyridine compounds without 2- or 6-carboxyl group did not show any inhibitory effect on the LDL oxidation and the copper reduction. Protective effect of dipicolinic acid on the LDL oxidation was closely correlated with the copper-reducing activity. Dipicolinic acid shows an antioxidant action by the formation of a chelation complex with copper. This may have implications in understanding mechanisms of preventing LDL oxidation during the early phase of atherosclerosis.     

Chromosomes of the antelope genus Kobus (Artiodactyla, Bovidae): karyotypic divergence by centric fusion rearrangements

G- and C-banded karyotypes of four species of the genus Kobus were compared using the standard karyotype of Bos taurus. Chromosomal complements were 2n = 50-54 in K. ellipsiprymnus, 2n = 50 in K. kob, 2n = 48 in K. leche, and 2n = 52 in K. megaceros. The number of autosomal arms in all karyotypes was 58. Fifteen autosomal pairs were conserved among these four species, including the 1;19 and 2;25 centric fusions, and autosomal differences involved eight centric fusion rearrangements. Five centric fusions were each unique to a particular taxon: 3;10 (K. leche), 3;11 and 6;29 (K. kob), and 5;17 and 7;11 (K. ellipsiprymnus). The 4;7 fusion occurred in K. leche and K. megaceros, whereas the 5;13 fusion occurred in K. kob and K. leche; the 6;18 fusion was found in three species but was absent in K. kob. Differences between the X chromosomes of the four Kobus species were attributed to heterochromatic additions or deletions, and Y-chromosome differences may have been the result of pericentric inversion. G-banded karyotypes of putative K. l. leche and K. l. kafuensis appeared identical, as did C-banded karyotypes of the two subspecies. Karyotypes of K. e. ellipsiprymnus and K. e. defassa differed as a result of the 6;18 centric fusion, which was polymorphic in K. e. defassa, and the 7;11 centric fusion, which was polymorphic in K. e. ellipsiprymnus but absent in K. e. defassa. Several centric fusions were related by monobrachial chain-IV complexes; however, records of hybridization indicate that reproductive isolation between at least certain species of Kobus is incomplete. Karyotypic differences between K. ellipsiprymnus (including K. e. ellipsiprymnus and K. e. defassa), K. kob, K. leche, and K. megaceros support the validity of these taxa, as well as the need to manage them as separate populations.     

Novel cap-independent translation with the 5′-noncoding region of L-A virus mRNA

A novel cap-independent translation has been performed where the ribosome entry is regulated by the 5-noncoding region (NCR) of L-A virus mRNA. Despite L-A virus mRNA containing neither cap structure nor a poly(A) tail, the reconstructed mRNA encoding the 5 NCR of L-A virus mRNA and a reporter gene (luciferase) was translated, in yeast lysate, 60 times more efficiently than control mRNA. The 5 NCR from L-A virus was effective in regulating the recruitment of ribosome in vitro. A possible mechanism in Saccharomyces cerevisiae is also suggested, whereby the ribosome entry is regulated by the 5 NCR of L-A virus mRNA.     

Genetic analysis in fungi using restriction-enzyme-mediated integration

Restriction-enzyme-mediated integration (REMI), a method for generating nonhomologous integration of transforming DNA into the chromosomes of eukaryotic cells, has been used for insertion mutagenesis and other genetic studies in diverse organisms. Insertion mutations generated by REMI have facilitated the genetic dissection of developmental pathways in Dictyostelium discoidium and the isolation of virulence factors in several plant pathogenic fungi. Recent work indicates that REMI occurs by nonhomologous end joining.     

DNA barcoding reveals 24 distinct lineages as cryptic bird species candidates in and around the Japanese Archipelago

DNA barcoding using a partial region (648 bp) of the cytochrome c oxidase I (COI) gene is a powerful tool for species identification and has revealed many cryptic species in various animal taxa. In birds, cryptic species are likely to occur in insular regions like the Japanese Archipelago due to the prevention of gene flow by sea barriers. Using COI sequences of 234 of the 251 Japanese‐breeding bird species, we established a DNA barcoding library for species identification and estimated the number of cryptic species candidates. A total of 226 species (96.6%) had unique COI sequences with large genetic divergence among the closest species based on neighbour‐joining clusters, genetic distance criterion and diagnostic substitutions. Eleven cryptic species candidates were detected, with distinct intraspecific deep genetic divergences, nine lineages of which were geographically separated by islands and straits within the Japanese Archipelago. To identify Japan‐specific cryptic species from trans‐Paleartic birds, we investigated the genetic structure of 142 shared species over an extended region covering Japan and Eurasia; 19 of these species formed two or more clades with high bootstrap values. Excluding six duplicated species from the total of 11 species within the Japanese Archipelago and 19 trans‐Paleartic species, we identified 24 species that were cryptic species candidates within and surrounding the Japanese Archipelago. Repeated sea level changes during the glacial and interglacial periods may be responsible for the deep genetic divergences of Japanese birds in this insular region, which has led to inconsistencies in traditional taxonomies based on morphology.