Homozygosity haplotype allows a genomewide search for the autosomal segments shared among patients

A promising strategy for identifying disease susceptibility genes for both single- and multiple-gene diseases is to search patients' autosomes for shared chromosomal segments derived from a common ancestor. Such segments are characterized by the distinct identity of their haplotype. The methods and algorithms currently available have only a limited capability for determining a high-resolution haplotype genomewide. We herein introduce the homozygosity haplotype (HH), a haplotype described by the homozygous SNPs that are easily obtained from high-density SNP genotyping data. The HH represents haplotypes of both copies of homologous autosomes, allowing for direct comparisons of the autosomes among multiple patients and enabling the identification of the shared segments. The HH successfully detected the shared segments from members of a large family with Marfan syndrome, which is an autosomal dominant, single-gene disease. It also detected the shared segments from patients with model multigene diseases originating with common ancestors who lived 10-25 generations ago. The HH is therefore considered to be useful for the identification of disease susceptibility genes in both single- and multiple-gene diseases.     

Administration of fibroblast growth factor 2 in combination with bone marrow transplantation synergistically improves carbon-tetrachloride-induced liver fibrosis in mice

We previously reported that fibroblast growth factor 2 (FGF2) facilitated the differentiation of transplanted bone marrow cells (BMCs) into hepatocytes. Our earlier study also demonstrated that administration of FGF2 in combination with bone marrow transplantation (BMT) synergistically activated tumor necrosis factor-alpha signaling and significantly improved liver function and prognosis more than BMT alone. However, the way that it affected the extracellular matrix remained unclear. Here, we investigated the effect of FGF2 treatment together with BMT on liver fibrosis in mice treated with carbon tetrachloride (CCl₄). Transplantation of BMCs and concurrent treatment with FGF2 caused a statistically significant reduction in CCl₄-induced liver fibrosis that was accompanied by strong expression of matrix metalloproteinase 9 as compared with FGF2-only treatment or BMT alone. Moreover, in this process, the proliferation of bone-marrow-derived cells was accelerated without causing apoptosis. Thus, the administration of FGF2 in combination with BMT synergistically improves CCl₄-induced liver fibrosis in mice. This treatment has the potential of being an effective therapy for patients with liver cirrhosis. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 16390211 and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5 and H17-Special-015).     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

Unconventional mechanism of mRNA capping by the RNA-dependent RNA polymerase of vesicular stomatitis virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.     

The GID1-mediated gibberellin perception mechanism is conserved in the Lycophyte Selaginella moellendorffii but not in the Bryophyte Physcomitrella patens

In rice (Oryza sativa) and Arabidopsis thaliana, gibberellin (GA) signaling is mediated by GIBBERELLIN-INSENSITIVE DWARF1 (GID1) and DELLA proteins in collaboration with a GA-specific F-box protein. To explore when plants evolved the ability to perceive GA by the GID1/DELLA pathway, we examined these GA signaling components in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. An in silico search identified several homologs of GID1, DELLA, and GID2, a GA-specific F-box protein in rice, in both species. Sm GID1a and Sm GID1b, GID1 proteins from S. moellendorffii, showed GA binding activity in vitro and interacted with DELLA proteins from S. moellendorffii in a GA-dependent manner in yeast. Introduction of constitutively expressed Sm GID1a, Sm G1D1b, and Sm GID2a transgenes rescued the dwarf phenotype of rice gid1 and gid2 mutants. Furthermore, treatment with GA(4), a major GA in S. moellendorffii, caused downregulation of Sm GID1b, Sm GA20 oxidase, and Sm GA3 oxidase and degradation of the Sm DELLA1 protein. These results demonstrate that the homologs of GID1, DELLA, and GID2 work in a similar manner in S. moellendorffii and in flowering plants. Biochemical studies revealed that Sm GID1s have different GA binding properties from GID1s in flowering plants. No evidence was found for the functional conservation of these genes in P. patens, indicating that GID1/DELLA-mediated GA signaling, if present, differs from that in vascular plants. Our results suggest that GID1/DELLA-mediated GA signaling appeared after the divergence of vascular plants from the moss lineage.     

Genetic and epigenetic alteration among three homoeologous genes of a class E MADS box gene in hexaploid wheat

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.     

Nuclear Translocation of Calcium/Calmodulin-dependent Protein Kinase IIδ3 Promoted by Protein Phosphatase-1 Enhances Brain-derived Neurotrophic Factor Expression in Dopaminergic Neurons

We have reported previously that dopamine D₂ receptor stimulation activates calcium/calmodulin-dependent protein kinase II (CaMKII) δ3, a CaMKII nuclear isoform, increasing BDNF gene expression. However, the mechanisms underlying that activity remained unclear. Here we report that CaMKIIδ3 is dephosphorylated at Ser³³² by protein phosphatase 1 (PP1), promoting CaMKIIδ3 nuclear translocation. Neuro-2a cells transfected with CaMKIIδ3 showed cytoplasmic and nuclear staining, but the staining was predominantly nuclear when CaMKIIδ3 was coexpressed with PP1. Indeed, PP1 and CaMKIIδ3 coexpression significantly increased nuclear CaMKII activity and enhanced BDNF expression. In support of this idea, chronic administration of the dopamine D₂ receptor partial agonist aripiprazole increased PP1 activity and promoted nuclear CaMKIIδ3 translocation and BDNF expression in the rat brain substantia nigra. Moreover, aripiprazole treatment enhanced neurite extension and inhibited cell death in cultured dopaminergic neurons, effects blocked by PP1γ knockdown. Taken together, nuclear translocation of CaMKIIδ3 following dephosphorylation at Ser³³² by PP1 likely accounts for BDNF expression and subsequent neurite extension and survival of dopaminergic neurons.     

Prostate Sphere-forming Stem Cells Are Derived from the P63-expressing Basal Compartment

Prostate stem cells (P-SCs) are capable of giving rise to all three lineages of prostate epithelial cells, including basal, luminal, and neuroendocrine cells. Multiple methods have been used to identify P-SCs in adult prostates. These include in vivo renal capsule implantation of a single epithelial cell with urogenital mesenchymal cells, in vitro prostasphere and organoid cultures, and lineage tracing with castration-resistant Nkx3.1 expression (CARN), in conjunction with expression of cell type-specific markers. Both organoid culture and CARN tracing show the existence of P-SCs in the luminal compartment. Although prostasphere cells predominantly express basal cell-specific cytokeratin and P63, the lineage of prostasphere-forming cells in the P-SC hierarchy remains to be determined. Using lineage tracing with P63^CreERT2, we show here that the sphere-forming P-SCs are P63-expressing cells and reside in the basal compartment. Therefore we designate them as basal P-SCs (P-bSCs). P-bSCs are capable of differentiating into AR⁺ and CK18⁺ organoid cells, but organoid cells cannot form spheres. We also report that prostaspheres contain quiescent stem cells. Therefore, the results show that P-bSCs represent stem cells that are early in the hierarchy of overall prostate tissue stem cells. Understanding the contribution of the two types of P-SCs to prostate development and prostate cancer stem cells and how to manipulate them may open new avenues for control of prostate cancer progression and relapse.