Evaluating the Influence of ipRGCs on Color Discrimination.

To investigate the influence of intrinsically photosensitive retinal ganglion cells (ipRGCs) on color discrimination, it is necessary to create two metameric light stimuli (metameric ipRGC stimuli) with the same amount of cone and rod stimulation, but different amounts of ipRGC stimulation. However, since the spectral sensitivity functions of cones and rods overlap with those of ipRGCs in a wavelength band, it has been difficult to independently control the amount of stimulation of ipRGCs only. In this study, we first propose a method for calculating metameric ipRGC stimulation based on the orthogonal basis functions of human photoreceptor cells. Then, we clarify the controllable range of metameric ipRGC stimulation within a color gamut. Finally, to investigate the color discrimination by metameric ipRGC stimuli, we conduct subjective evaluation experiments on 24 chromaticity coordinates using a multispectral projector. The results reveal a correlation between differences in the amount of ipRGC stimulation and differences in color appearance, indicating that ipRGCs may influence color discrimination.

Immunization method for multi-pass membrane proteins using highly metastatic cell lines.

A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6-3.0×10(8)cells/spleen [n=6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7-11.4×10(8)cells/spleen (n=10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.

A gain-of-function screen to identify genes that reduce lifespan in the adult of Drosophila melanogaster.

BACKGROUND: Several lines of evidence associate misregulated genetic expression with risk factors for diabetes, Alzheimer's, and other diseases that sporadically develop in healthy adults with no background of hereditary disorders. Thus, we are interested in genes that may be expressed normally through parts of an individual's life, but can cause physiological defects and disease when misexpressed in adulthood. RESULTS: We attempted to identify these genes in a model organism by arbitrarily misexpressing specific genes in adult Drosophila melanogaster, using 14,133 Gene Search lines. We identified 39 "reduced-lifespan genes" that, when misexpressed in adulthood, shortened the flies' lifespan to less than 30% of that of control flies. About half of these genes have human orthologs that are known to be involved in human diseases. For about one-fourth of the reduced-lifespan genes, suppressing apoptosis restored the lifespan shortened by their misexpression. We determined the organs responsible for reduced lifespan when these genes were misexpressed specifically in adulthood, and found that while some genes induced reduced lifespan only when misexpressed in specific adult organs, others could induce reduced lifespan when misexpressed in various organs. This finding suggests that tissue-specific dysfunction may be involved in reduced lifespan related to gene misexpression. Gene ontology analysis showed that reduced-lifespan genes are biased toward genes related to development. CONCLUSIONS: We identified 39 genes that, when misexpressed in adulthood, shortened the lifespan of adult flies. Suppressing apoptosis rescued this shortened lifespan for only a subset of the reduced-lifespan genes. The adult tissues in which gene misexpression caused early death differed among the reduced-lifespan genes. These results suggest that the cause of reduced lifespan upon misexpression differed among the genes.

α-1,6-Fucosyltransferase (FUT8) inhibits hemoglobin production during differentiation of murine and K562 human erythroleukemia cells.

Erythropoiesis results from a complex combination of the expression of several transcription factor genes and cytokine signaling. However, the overall view of erythroid differentiation remains unclear. First, we screened for erythroid differentiation-related genes by comparing the expression profiles of high differentiation-inducible and low differentiation-inducible murine erythroleukemia cells. We identified that overexpression of α-1,6-fucosyltransferase (Fut8) inhibits hemoglobin production. FUT8 catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α-1,6 linkage, leading to core fucosylation in mammals. Expression of Fut8 was down-regulated during chemically induced differentiation of murine erythroleukemia cells. Additionally, expression of Fut8 was positively regulated by c-Myc and c-Myb, which are known as suppressors of erythroid differentiation. Second, we found that FUT8 is the only fucosyltransferase family member that inhibits hemoglobin production. Functional analysis of FUT8 revealed that the donor substrate-binding domain and a flexible loop play essential roles in inhibition of hemoglobin production. This result clearly demonstrates that core fucosylation inhibits hemoglobin production. Third, FUT8 also inhibited hemoglobin production of human erythroleukemia K562 cells. Finally, a short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. Our findings define FUT8 as a novel factor for hemoglobin production and demonstrate that core fucosylation plays an important role in erythroid differentiation.

Identification of preferentially reactivated genes during early G1 phase using nascent mRNA as an index of transcriptional activity.

During mammalian mitosis, transcription is silenced due to dissociation of transcription factors from DNA and chromosome condensation. At the end of mitosis, transcription is reactivated through chromosome relaxation and reloading of these factors to the DNA. Early G1 genes, which are preferentially reactivated during M/G1 transition, are important for maintenance of cellular function and are known to be strictly regulated. As only few early G1 genes have been identified to date, screening for early G1 genes by genome-wide analysis using nascent mRNA could contribute to the elucidation of the regulatory mechanisms during early G1. Here, we performed a detailed expression analysis for the M/G1 transition of mammalian cells by microarray analysis of nascent mRNA, and identified 298 early G1 genes. Analysis of these genes provides two important insights. Firstly, certain motifs are enriched in the upstream sequences of early G1 genes; from this we could predict candidate cognate transcription factors, including Sp1, which is recruited to the DNA in the early G1 phase. Secondly, we discovered that neighboring genes of early G1 genes were also frequently up-regulated in the G1 phase. Information about these numerous newly identified early G1 genes will likely contribute to an understanding of the regulatory mechanisms of the early G1 genes.

Phosphorylation of human INO80 is involved in DNA damage tolerance.

Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

Interaction of Hand2 and E2a is important for transcription of Phox2b in sympathetic nervous system neuron differentiation.

Transcription factors play a crucial role in the development of various tissues. In particular, the transcription factors of the basic helix-loop-helix (bHLH) family are crucial regulators of neurodifferentiation. Previous studies suggested that the bHLH transcription factor Hand2 is essential for sympathetic nervous system neuron differentiation in vivo, but the molecular mechanisms involved have not been well elucidated. It is important for understanding their mode of action in cellular differentiation to clarify how these bHLH factors regulate distinct transcriptional targets in a temporally and spatially controlled manner. Recent reports on ES cell differentiation suggested that its molecular mechanism mimics that of in vivo neurogenesis. However, the diverse nature of ES cell populations has prevented efficient analysis. To address this issue, we previously established a cell line in P19 embryonal carcinoma (EC) cells. Efficient sympathetic nervous system (SNS) neuron differentiation is induced in the cell line. Using this cell line, we succeeded in showing that the interaction of bHLH transcription factor Hand2 with E2a is required for transcription of Phox2b, which is essential for autonomic nervous system neuron development, and this binding activates this expression in SNS differentiation. Moreover, we also demonstrated that Hes5 regulated the transcription of Phox2b as a negative regulator and it inhibited the SNS differentiation. These findings have enabled us to determine the novel regulatory mechanism of Phox2b in SNS differentiation.

The mammalian INO80 complex is recruited to DNA damage sites in an ARP8 dependent manner.

Dynamic changes in chromatin structure are essential for efficient DNA processing such as transcription, replication, and DNA repair. Histone modifications and ATP-dependent chromatin remodeling are important for the alteration of chromatin structure. The INO80 chromatin remodeling complex plays an important role in HR-mediated repair of DNA double-strand breaks (DSBs). In yeast, the INO80 complex is recruited to the sites of DSBs via direct interaction with phosphorylated histone H2A and facilitates the processing of DSB ends. However, the function of the mammalian INO80 complex in DNA repair is mostly unknown. Here, we show that the mammalian INO80 complex is recruited to the laser-induced DNA damage sites in a phosphorylated H2AX (γH2AX)-independent manner. We also found that an actin-related protein, ARP8, is an important subunit that is required for the recruitment of the mammalian INO80 complex to the DNA damage sites, although the recruitment of the yeast INO80 complex requires its Nhp10 or Arp4 subunits. These results suggest that the mammalian INO80 complex is also recruited to DNA damage sites similarly to the yeast INO80 complex, but the mechanism of this recruitment may be different from that of the yeast INO80 complex. These findings provide new insights into the mechanisms of DNA repair in mammalian cells.

The bHLH transcription factor Hand2 regulates the expression of nanog in ANS differentiation.

The basic helix-loop-helix transcription factor Hand2 is induced by bone morphogenetic proteins (BMPs) in neural crest-derived precursor cells during the early stage of development of the autonomic nervous system (ANS). Previous studies showed that Hand2 was essential for the ANS differentiation. However, regulatory mechanism of pluripotent genes has not been elucidated in ANS differentiation. Here, we show that Hand2 regulated nanog expression in ANS differentiation. Our studies demonstrated that the forced expression of Hand2 promoted the ANS differentiation program in P19 embryonal carcinoma (EC) cells without aggregation. Furthermore, our results suggested that Hand2 bound to the promoter of nanog, a gene required for embryonic stem cells self-renewal, and suppressed nanog expression after Hand2 induction. The rapid downregulation of nanog mRNA during ANS differentiation correlated with the Hand2 transcriptional activity and nanog promoter methylation. These findings are evidence for a presence of the novel regulatory mechanism of nanog in ANS differentiation.

Development of effective isolation method of ES cells for analysis of differentiation.

Neuroectoderm development is a milestone of vertebrate neurogenesis. However, the molecular mechanism underlying the differentiation of neuroectoderm is still unclear, especially in mammals. ES cells co-cultured with PA6 cells can differentiate to neuroectoderm by the stromal cell-derived inducing activity method (SDIA method), but contamination of PA6 cells is an obstacle to the analysis of molecular mechanisms of differentiation. Here we describe a novel method by which differentiated ES cells are easily isolated from PA6 cells. We attempted to induce the differentiation of ES cells using paraformaldehyde-fixed PA6 cells. RT-PCR and DNA microarray analysis revealed that the background noise derived from contaminated PA6 cells disappeared when fixed PA6 cells were used. Furthermore, genes up-regulated during the differentiation of ES cells were expressed in a developing mouse embryo. Thus, our newly developed method will be very useful for identifying novel genes associated with mouse neuroectoderm development in vitro and in vivo.

Novel DNA microarray system for analysis of nascent mRNAs.

Transcriptional activation and repression are a key step in the regulation of all cellular activities. The development of comprehensive analysis methods such as DNA microarray has advanced our understanding of the correlation between the regulation of transcription and that of cellular mechanisms. However, DNA microarray analysis based on steady-state mRNA (total mRNA) does not always correspond to transcriptional activation or repression. To comprehend these transcriptional regulations, the detection of nascent RNAs is more informative. Although the nuclear run-on assay can detect nascent RNAs, it has not been fully applied to DNA microarray analysis. In this study, we have developed a highly efficient method for isolating bromouridine-labeled nascent RNAs that can be successfully applied to DNA microarray analysis. This method can linearly amplify small amounts of mRNAs with little bias. Furthermore, we have applied this method to DNA microarray analysis from mouse G2-arrested cells and have identified several genes that exhibit novel expression profiles. This method will provide important information in the field of transcriptome analysis of various cellular processes.

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity.

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.