Keiko Numayama-Tsuruta

Mice lacking histidine decarboxylase exhibit abnormal mast cells

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC‐deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine‐synthesizing activity from histidine. These HDC‐deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC‐deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.

Concise Review: Pax6 Transcription Factor Contributes to both Embryonic and Adult Neurogenesis as a Multifunctional Regulator

Pax6 is a highly conserved transcription factor among vertebrates and is important in various developmental processes in the central nervous system (CNS), including patterning of the neural tube, migration of neurons, and formation of neural circuits. In this review, we focus on the role of Pax6 in embryonic and postnatal neurogenesis, namely, production of new neurons from neural stem/progenitor cells, because Pax6 is intensely expressed in these cells from the initial stage of CNS development and in neurogenic niches (the subgranular zone of the hippocampal dentate gyrus and the subventricular zone of the lateral ventricle) throughout life. Pax6 is a multifunctional player regulating proliferation and differentiation through the control of expression of different downstream molecules in a highly context‐dependent manner.

Role of Fabp7, a Downstream Gene of Pax6, in the Maintenance of Neuroepithelial Cells during Early Embryonic Development of the Rat Cortex

Pax6 is a transcription factor with key functional roles in the developing brain. Pax6 promotes neuronal differentiation via transcriptional regulation of the Neurogenin2 (Ngn2) gene, although Pax6 expression appears in proliferating neuroepithelial cells before the onset of neurogenesis. Here, we identified Fabp7 (BLBP/B-FABP), a member of the fatty acid-binding protein (FABP) family, as a downregulated gene in the embryonic brain of Pax6 mutant rat (rSey2/rSey2) by microarray analysis. Marked reduction of Fabp7 expression was confirmed by quantitative PCR. Spatiotemporal expression patterns of Fabp7 in the wild-type rat embryos from embryonic day 10.5 (E10.5) to E14.5 were similar to those of Pax6, and expression of Fabp7 was undetectable in the rSey2/rSey2 cortex. The expression pattern of Fabp7 in the wild-type mouse embryo at E10.5 (corresponding to E12.5 rat) was different from that in the rat embryo, and no change of expression was observed in the Sey/Sey mouse embryo. Overexpression of exogenous Pax6 mainly induced ectopic expression of Fabp7, rather than of Ngn2, in the early cortical primordium. Interestingly, knocking-down FABP7 function by electroporation of Fabp7 small interfering RNA severely curtailed cell proliferation but promoted neuronal differentiation. We conclude that Fabp7 is a downstream gene of Pax6 transcription factor in the developing rat cortex and essential for maintenance of neuroepithelial cells during early cortical development.

Heterodimerization with LBP-1b is necessary for nuclear localization of LBP-1a and LBP-1c

The LBP‐1 family consists of four proteins, which act as transcription factors in the formation of dimers with a member of this family. LBP‐1a and LBP‐1b are splicing variants from one gene, and LBP‐1c and LBP‐1d also arise from the alternative splicing of another gene. Investigation of subcellular localization of LBP‐1 proteins fused to YFP revealed that the LBP‐1b was localized in the nucleus, whereas LBP‐1a and LBP‐1c were exclusively localized in the cytosol. The peptide of 36 amino acids encoded by exon 6, a specific exon used only for LBP‐1b, possessed the function of a nuclear localization signal (NLS). Nuclear localization of LBP‐1a and LBP‐1c occurred when LBP‐1b was co‐expressed, suggesting that heterodimerization of LBP‐1a and LBP‐1c with LBP‐1b is important for their nuclear transport. Transiently expressed LBP‐1 proteins in COS‐7 cells formed speckles in the nucleus. Most speckles overlapped with the PML body. The activity of LBP‐1a for accumulation in the PML body was mapped in the N‐terminal region.

Collective spreading of red blood cells flowing in a microchannel

Due to recent advances in micro total analysis system technologies, microfluidics provides increased opportunities to manipulate, stimulate, and diagnose blood cells. Controlling the concentration of cells at a given position across the width of a channel is an important aspect in the design of microfluidic devices. Despite its biomedical importance, the collective spreading of red blood cells (RBCs) in a microchannel has not yet been fully clarified. In this study, we experimentally investigated the collective spreading of RBCs in a straight microchannel, and found that RBCs initially distributed in one side of the microchannel spread to the spanwise direction during downstream flow. Spreading increased considerably as the hematocrit increased, though the flow rate had a small effect. We proposed a scaling argument to show that this spreading phenomenon was diffusive and mainly induced by cell-cell interactions. The dispersion coefficient was approximately proportional to the flow rate and the hematocrit. These results are useful in understanding collective behaviors of RBCs in a microchannel and in microcirculation.

Pax6-dependent regulation of the rat Fabp7 promoter activity

Fabp7 gene encodes a brain‐specific fatty acid‐binding protein that is widely used as a marker for neural stem cells. Here, we report that the activity of rat Fabp7 promoter was regulated directly by a transcription factor, Pax6. Deletion analyses identified an essential region (−837 to −64 from transcription start site) in the rat Fabp7 promoter. This region controls promoter activity in rat embryos and in the mouse cultured cell line MEB5. Over‐expressing wild‐type Pax6 or a dominant‐negative Pax6 mutant enhanced and suppressed, respectively, the promoter activity. Pax6 can bind the region directly, although the region contains no clear binding motif for Pax6. The rat Fabp7 promoter also contains conserved binding sites for Pbx/POU (−384 to −377) and CBF1 (−270 to −262). However, specific deletion of the sites showed no significant reduction in the promoter activity, although a gel mobility shift assay confirmed that CBF1 binds the conserved sequence. Taken together, these results suggest that the rat Fabp7 promoter is mainly regulated by Pax6. The Pax6‐dependent regulation of the rat Fabp7 expression might have an evolutionary aspect between rat and mouse; the former may need to efficiently use fatty acids to make the brain bigger than the latter.

Inertial Migration of Cancer Cells in a Microfluidic Device

The circulating tumor cell (CTC) test is used to evaluate the condition of breast cancer patients by counting the number of cancer cells in peripheral blood samples. Although microfluidic systems to detect or separate cells using the inertial migration effect may be applied to this test, the hydrodynamic forces acting on cancer cells are incompletely understood. In this chapter, we explain the inertial migration of cancer cells in microchannels. We also explain fabrication techniques of microchannels used in the experiments. By measuring the cell migration probability, we examined the effects of cell deformability and variations in cell size on the inertial migration of cancer cells. The results clearly illustrate that cancer cells can migrate towards equilibrium positions in the similar manner with rigid spheres. These results will be important for the design of microfluidic devices for the CTC test.