Thrombomodulin is a clock-controlled gene in vascular endothelial cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events.     

Epitope mapping for the anti-rabbit cholesteryl ester transfer protein monoclonal antibody that selectively inhibits triglyceride transfer.

Among the monoclonal antibodies (Mab) against rabbit plasma cholesteryl ester transfer protein (CETP), Mab 14-8F cross-reacted with human CETP and selectively inhibited triglyceride transfer but not cholesteryl ester transfer (Ko, K. W. S., T. Ohnishi, and S. Yokoyama. 1994. J. Biol. Chem. 269: 28206;-28213). The epitope of this antibody was studied by using synthetic fragment peptides of rabbit and human CETP. Mab 14-8F reacted with the peptide R451-Q473 of human CETP near the carboxyl-terminal and not with the peptides representing any other regions, and inhibited the binding of human CETP to the goat antibody against its carboxyl-terminal peptide R451-S476. The experiments with a series of the fragment peptides in this region revealed that the epitope requires the segment 465-473 (EHLLVDFLQ) of human CETP or 485-493 (KHLLVDFLQ) of rabbit CETP (core epitope) though neither peptide by itself binds to the antibody. Both peptides needed extension at least by one residue beyond either amino- or carboxyl-end in order to show the reactivity to the antibody, but the effect was not highly residue-specific at least at the amino-end. Circular dichroism analysis demonstrated the increase of helical conformation by the extension of the "core epitope" peptides to either direction. Thus, the epitope is dependent on conformation of the core epitope induced by the presence of an additional residue(s) in either end. The core epitope occupies the central 64% of the reported linear epitope of Mab TP2, a widely used anti-human CETP monoclonal antibody that inhibits both cholesteryl ester and triglyceride transfer.Therefore, we conclude that the limited interaction of Mab with a common lipid interaction site causes selective inhibition of the transfer of triglyceride that has presumably lower priority than cholesteryl ester for the CETP reaction.     

GRP94 reduces cell death in SH-SY5Y cells perturbated calcium homeostasis

The endoplasmic reticulum (ER) resident-94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.     

Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.     

Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries

During luteolysis, luteal cell apoptosis is induced by the Fas ligand (FasL)/Fas system. In murine luteal bodies, we demonstrated the expression of mRNA of soluble form of Fas (FasB), which binds to FasL and prevents apoptosis induction. By in situ hybridization, strong expression of FasB mRNA was observed in normal luteal bodies, in which no apoptotic cells were detected, but negative/trace expression in regressing luteal bodies, in which many apoptotic cells were observed. Immunohistochemical staining revealed that Fas and TNF-alpha were localized in both normal and regressing luteal bodies, but IFN-gamma was localized only in regressing luteal bodies. Apoptosis was induced in primary cultured luteal cells, when they were pretreated with TNF-alpha and IFN-gamma and then incubated with TNF-alpha, IFN-gamma, and mouse recombinant FasL (rFasL). However, no apoptosis was detected in the cells, when they were treated with rFasL alone, TNF-alpha alone, IFN-gamma alone, TNF-alpha and rFasL, IFN-gamma and rFasL, or TNF-alpha and IFN-gamma. Fas mRNA expression in cultured luteal cells was up-regulated by the treatment of TNF-alpha, IFN-gamma, or TNF-alpha and IFN-gamma. The expression of FasB mRNA was down-regulated, when the cells were treated with TNF-alpha and IFN-gamma, but its expression was not changed by the treatment of TNF-alpha alone or IFN-gamma alone. We conclude that FasB inhibits the apoptosis induction in luteal cells of normal luteal bodies, and that decreased FasB production induced by TNF-alpha and IFN-gamma made possible the apoptosis induction in the luteal cells of regressing luteal bodies.     

A human case of gnathostomiasis nipponica confirmed indirectly by finding infective larvae in leftover largemouth bass meat

A human case of creeping eruption due to Gnathostoma nipponicum was confirmed indirectly by finding infective advanced third-stage larvae in leftover largemouth bass meat. This is the first report indicating that the largemouth bass (Micropterus salmoides) serves as a source of G. nipponicum infection in humans.     

Distinct roles of Rab3B and Rab13 in the polarized transport of apical,basolateral, and tight junctional membrane proteins to the plasma membrane

Regulated transport of proteins to distinct plasma membrane domains is essential for the establishment and maintenance of cell polarity in all eukaryotic cells. The Rab family small G proteins play a crucial role in determining the specificity of vesicular transport pathways. Rab3B and Rab13 localize to tight junction in polarized epithelial cells and cytoplasmic vesicular structures in non-polarized fibroblasts, but their functions are poorly understood. Here we examined their roles in regulating the cell-surface transport of apical p75 neurotrophin receptor (p75NTR), basolateral low-density lipoprotein receptor (LDLR), and tight junctional Claudin-1 using transport assay in non-polarized fibroblasts. Overexpression of Rab3B mutants inhibited the cell-surface transport of LDLR, but not p75NTR and Claudin-1. In contrast, overexpression of Rab13 mutants impaired the transport of Claudin-1, but not LDLR and p75NTR. These results suggest that Rab3B and Rab13 direct the cell-surface transport of LDLR and Claudin-1, respectively, and may contribute to epithelial polarization.     

A genomic timescale for the origin of eukaryotes

Background  

Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes.     

An in vitro approach to the evaluation of the cross talk between intestinal epithelium and macrophages

The intestinal epithelium acts as a mucosal barrier by varying their signals to immune cells within the intestine. To observe the cross talk between intestinal epithelium and macrophages, we establish a Caco-2-THP-1 co-culture system. Using this co-culture system, we suggested that paracrine factors of intestinal epithelium increased the phagocytic capacity of intestinal monocytes/macrophages to be ready for immune and inflammatory responses.     

Identification of a novel microtubule-associated protein that regulates microtubule organization and cytokinesis by using a GFP-screening strategy

Microtubules play critical roles in a variety of cell processes, including mitosis, organelle transport, adhesion and migration, and the maintenance of cell polarity. Microtubule-associated proteins (MAPs) regulate the dynamic organization and stability of microtubules, often through either cell-specific or cell division stage-specific interactions. To identify novel cytoskeletal-associated proteins and peptides that regulate microtubules and other cytoskeletal and adhesive structures, we have developed a GFP cDNA screening strategy based on identifying gene products that localize to these structures. Using this approach, we have identified a novel MAP, GLFND, that shows homology to the Opitz syndrome gene product [6], localizes to a subpopulation of microtubules that are acetylated, and protects microtubules from depolymerization with nocodazole. Expression of an N-terminal deletion binds microtubules but alters their organization. During the cell cycle, GLFND dissociates from microtubules at the beginning of mitosis and then reassociates at cytokinesis. Furthermore, ectopic expression of GLFND inhibits cell division and cytokinesis in CHO cells. These observations make GLFND unique among MAPs characterized thus far.