Circular double-stranded forms of TT virus DNA in the liver

TT virus (TTV) is an unenveloped, circular, and single-stranded DNA virus commonly infecting human beings worldwide. TTV DNAs in paired serum and liver tissues from three viremic individuals were separated by gel electrophoresis and characterized biophysically. TTV DNAs in sera migrated in sizes ranging from 2.0 to 2.5 kb. TTV DNAs in liver tissues, however, migrated at 2.0 to 2.5 kb as well as at 3.5 to 6.1 kb. Both faster- and slower-migrating forms of TTV DNAs in the liver were found to be circular and of the full genomic length of 3.8 kb. TTV DNAs migrating at 2.0 to 2.5 kb, from either serum or liver tissues, were sensitive to S1 nuclease but resistant to restriction endonucleases, and therefore, they were single-stranded. By contrast, TTV DNAs in liver tissues that migrated at 3.5 to 6.1 kb were resistant to S1 nuclease. They migrated at 3.7 to 4.0 kb after digestion with EcoRI, which suggests that they represent circular, double-stranded replicative intermediates of TTV. When TTV DNAs were subjected to strand-specific primer extension and then amplified by PCR with internal primers, those in serum were found to be minus-stranded DNAs while those in liver tissues were found to be a mixture of plus- and minus-stranded DNAs. These results suggest that TTV replicates in the liver via a circular double-stranded DNA.     

Developmental changes in localization of the main ganglioside during sea urchin embryogenesis

Ganglioside M5 (NeuGcalpha2-6Glcbeta1-1'Cer), the main ganglioside in sea urchin and sand dollar eggs, exists mainly in the endoplasmic reticulum and yolk granules in unfertilized eggs. To study the localization of ganglioside M5 after fertilization, early embryos were stained with an anti-ganglioside M5 monoclonal antibody. Using immunofluorescent and immunoelectron microscopy, intense label was observed in the outer surface and cytoplasm of embryos. These results indicate that ganglioside M5 was secreted during embryogenesis and localized in the extracellular matrix (ECM). When living embryos were incubated in sea water containing 7-nitrobenz-2-oxa-1,3-diazole labeled-ganglioside M5 (NBD-M5), the ECM and plasma membrane were strongly stained. Since the localization of NBD-M5 in the ECM was similar to that of extracellular M5, NBD-M5 was likely to be useful to examine the fate of extracellular ganglioside M5. Interestingly, NBD-M5 was incorporated in subcortical vesicles during embryogenesis, suggesting that the extracellular ganglioside M5 is transported into the cytoplasm. When fertilized eggs were incubated with NBD-M5 and tetramethylrhodamine dextran (a marker dye for endocytotic vesicles), colocalization of the dyes was observed in the vesicles. Thus, it was concluded that NBD-M5 in the ECM and/or plasma membrane was internalized in the cells by endocytosis, suggesting that extracellular M5 is transported from the ECM to endocytotic vesicles.     

Sleep,an integrated physiological function of living body

Sleep and Biological Rhythms -     

Cholesterol‐α‐glucosyltransferase gene is present in most Helicobacter species including gastric non‐Helicobacter pylori helicobacters obtained from Japanese patients

Background

Non‐Helicobacter pylori helicobacters (NHPHs) besides H. pylori infect human stomachs and cause chronic gastritis and mucosa‐associated lymphoid tissue lymphoma. Cholesteryl‐α‐glucosides have been identified as unique glycolipids present in H. pylori and some Helicobacter species. Cholesterol‐α‐glucosyltransferase (αCgT), a key enzyme for the biosynthesis of cholesteryl‐α‐glucosides, plays crucial roles in the pathogenicity of H. pylori. Therefore, it is important to examine αCgTs of NHPHs.

Materials and Methods

Six gastric NHPHs were isolated from Japanese patients and maintained in mouse stomachs. The αCgT genes were amplified by PCR and inverse PCR. We retrieved the αCgT genes of other Helicobacter species by BLAST searches in GenBank.

Results

αCgT genes were present in most Helicobacter species and in all Japanese isolates examined. However, we could find no candidate gene for αCgT in the whole genome of Helicobacter cinaedi and several enterohepatic species. Phylogenic analysis demonstrated that the αCgT genes of all Japanese isolates show high similarities to that of a zoonotic group of gastric NHPHs including Helicobacter suis, Helicobacter heilmannii, and Helicobacter ailurogastricus. Of 6 Japanese isolates, the αCgT genes of 4 isolates were identical to that of H. suis, and that of another 2 isolates were similar to that of H. heilmannii and H. ailurogastricus.

Conclusions

All gastric NHPHs examined showed presence of αCgT genes, indicating that αCgT may be beneficial for these helicobacters to infect human and possibly animal stomachs. Our study indicated that NHPHs could be classified into 2 groups, NHPHs with αCgT genes and NHPHs without αCgT genes.     

Visualization of Endoplasmic Reticulum and Mitochondria in <Emphasis Type="Italic">Aurantiochytrium limacinum</Emphasis> by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals

Thraustochytrids are single cell marine eukaryotes that produce large amounts of polyunsaturated fatty acids such as docosahexaenoic acid. In the present study, we report the visualization of endoplasmic reticulum (ER) and mitochondria in a type strain of the thraustochytrid, Aurantiochytrium limacinum ATCC MYA-1381, using the enhanced green fluorescent protein (EGFP) with specific targeting/retaining signals. We expressed the egfp gene with ER targeting/retaining signals from A. limacinum calreticulin or BiP/GRP78 in the thraustochytrid, resulting in the distribution of EGFP signals at the perinuclear region and near lipid droplets. ER-Tracker? Red, an authentic fluorescent probe for the visualization of ER in mammalian cells, also stained the same region. We observed small lipid droplets generated from the visualized ER in the early growth phase of cell culture. Expression of the egfp gene with the mitochondria targeting signal from A. limacinum cytochrome c oxidase resulted in the localization of EGFP near the plasma membrane. The distribution of EGFP signals coincided with that of MitoTracker® Red CMXRos, which is used to visualize mitochondria in eukaryotes. The ER and mitochondria of A. limacinum were visualized for the first time by EGFP with thraustochytrid cell organelle-specific targeting/retaining signals. These results will contribute to classification of the intracellular localization of proteins expressed in ER and mitochondria as well as analyses of these cell organelles in thraustochytrids.     

Six-transmembrane epithelial antigen of the prostate-1 plays a role for in vivo tumor growth via intercellular communication

Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein overexpressed only in the prostate among normal tissues and various types of cancer including prostate, bladder, lung, and ovarian cancer. Although its function in prostate and tumor cells has been remained unclear, due to its unique and restricted expression, STEAP-1 is expected to be an attractive target for cancer therapy. Here, we show that knockdown of STEAP-1 in human cancer cells caused the retardation of tumor growth compared with wild type in vivo. In contrast, STEAP-1 introduced tumor cells augmented the tumor growth compared with STEAP-1-negative wild type cells. Using dye transfer assay, we demonstrate that the STEAP-1 is involved in intercellular communication between tumor cells and adjacent tumor stromal cells and therefore may play a key role for the tumor growth in vivo. These data indicate the inhibition of the STEAP-1 function or expression can be a new strategy for cancer therapy.     

Mesdc2 plays a key role in cell-surface expression of Lrp4 and postsynaptic specialization in myotubes

Low-density lipoprotein receptor-related protein 4 (Lrp4) is essential for pre- and post-synaptic specialization at the neuromuscular junction (NMJ), an indispensable synapse between a motor nerve and skeletal muscle. Muscle-specific receptor tyrosine kinase MuSK must form a complex with Lrp4 to organize postsynaptic specialization at NMJs. Here, we show that the chaperon Mesdc2 binds to the intracellular form of Lrp4 and promotes its glycosylation and cell-surface expression. Furthermore, knockdown of Mesdc2 suppresses cell-surface expression of Lrp4, activation of MuSK, and postsynaptic specialization in muscle cells. These results suggest that Mesdc2 plays an essential role in NMJ formation by promoting Lrp4 maturation.