Slit3 regulates cell motility through Rac/Cdc42 activation in lipopolysaccharide-stimulated macrophages

Three slit genes, slit1 to slit3, have been cloned to date. Slit1 and slit2 act as chemorepellent factors for axon guidance. Slit3 is involved in the formation of the diaphragm and kidney during embryogenesis. However, its molecular function remains unclear. We found that slit3 expression was induced by lipopolysaccharide (LPS)-stimulation in macrophages and that it was localized in the mitochondria and along the plasma membrane. Silencing of slit3 expression by RNA interference reduced cell motility and Rac/Cdc42 activation. These results suggest that slit3 functions as an intracellular signaling molecule for cell motility as part of the LPS-induced signaling cascade.     

Differential gene expression profiling in blood from patients with digestive system cancers

To develop a non-invasive and sensitive diagnostic test for cancer using peripheral blood, we evaluated gene expression profiling of blood obtained from patients with cancer of the digestive system and normal subjects. The expression profiles of blood-derived total RNA obtained from 39 cancer patients (11 colon cancer, 14 gastric cancer, and 14 pancreatic cancer) was clearly different from those obtained from 15 normal subjects. By comparing the gene expression profiles of cancer patients and normal subjects, 25 cancer-differentiating genes (p < 5.0 × 10⁻⁶ and fold differences >3) were identified and an “expression index” deduced from the expression values of these genes differentiated the validation cohort (11 colon cancer, 8 gastric cancer, 18 pancreatic cancer, and 15 normal subjects) into cancer patients and normal subjects with 100% (37/37) and 87% (13/15) accuracy, respectively. Although, the expression profiles were not clearly different between the cancer patients, some characteristic genes were identified according to the stage and species of the cancer. Interestingly, many immune-related genes such as antigen presenting, cell cycle accelerating, and apoptosis- and stress-inducing genes were up-regulated in cancer patients, reflecting the active turnover of immune regulatory cells in cancer patients. These results showed the potential relevance of peripheral blood gene expression profiling for the development of new diagnostic examination tools for cancer patients.     

Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system

We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages. This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system. Eos protein could interact with itself and Ikaros protein through its C-terminal portion in the yeast two hybrid assay. These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.     

MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components

The genomic stability of all organisms depends on the precise partition of chromosomes to daughter cells. The spindle assembly checkpoint (SAC) senses unattached kinetochores and prevents premature entry to anaphase, thus ensuring that all chromosomes attach to opposite spindle poles (bi-orientation) during mitosis. MPS1 is an evolutionarily conserved protein kinase required for the SAC and chromosome bi-orientation. Yet, its primary cellular substrate has remained elusive. We show that fission yeast Mph1 (MPS1 homologue) phosphorylates the kinetochore protein Spc7 (KNL1/Blinkin homologue) at the MELT repeat sequences. This phosphorylation promotes the in vitro binding to the Bub1-Bub3 complex, which is required for kinetochore-based SAC activation (Mad1-Mad2-Mad3 localization) and chromosome alignment. Accordingly, a non-phosphorylatable spc7-12A mutation abolishes kinetochore targeting of Bub1-Bub3, whereas a phospho-mimetic spc7-12E mutation forces them to localize at kinetochores throughout the entire cell cycle, even in the absence of Mph1. Thus, MPS1/Mph1 kinase locating at the unattached kinetochores initially creates a mark, which is crucial for SAC activation and chromosome bi-orientation. This mechanism seems to be conserved in human cells.     

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid‐rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB‐suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB‐suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.     

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.     

Splenic dendritic cells induced by oral antigen administration are important for the transfer of oral tolerance in an experimental model of asthma

Peripheral tolerance can be induced after the feeding of Ag, which is referred to as oral tolerance. We demonstrated in this study that the oral administration of OVA induced tolerance in an experimental model of asthma in mice, and investigated which cells function as the regulatory cells in the transfer of this oral tolerance. In OVA-fed mice, the percentage of eosinophils in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, and mRNA levels of IL-13 and eotaxin were significantly lower than found in nonfed mice. Histological examination of lung tissue showed a suppression of the accumulation of inflammatory cells in the peribronchial area of OVA-fed mice. Feeding after the first immunization or between the first and the second immunization suppressed these findings, whereas feeding just before the airway Ag challenge did not. The suppression of disease in OVA-fed mice was successfully transferred by injection of whole spleen cells of OVA-fed mice. When CD11c+ dendritic cells (DCs) were removed from splenocytes, this transfer of suppression was completely abolished. The injection of splenic DCs purified from OVA-fed mice alone transferred the suppression, whereas the injection of splenic DCs from naive mice that were cocultured with OVA in vitro did not. These data suggest that not only CD4+ T cells, but also CD11c+ DCs induced by Ag feeding are important for the active transfer of oral tolerance in this murine experimental model of asthma.