Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.     

Mutation of the SNF2 family member Chd2 affects mouse development and survival

The chromodomain helicase DNA-binding domain (Chd) proteins belong to the SNF2-like family of ATPases that function in chromatin remodeling and assembly. These proteins are characterized by the presence of tandem chromodomains and are further subdivided based on the presence or absence of additional structural motifs. The Chd1-Chd2 subfamily is distinguished by the presence of a DNA-binding domain that recognizes AT-rich sequence. Currently, there are no reports addressing the function of the Chd2 family member. Embryonic stem cells containing a retroviral gene-trap inserted at the Chd2 locus were utilized to generate mice expressing a Chd2 protein lacking the DNA-binding domain. This mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis and in perinatal lethality. Animals heterozygous for the mutation showed decreased neonatal viability and increased susceptibility to non-neoplastic lesions affecting most primary organs. In particular, approximately 85% of the heterozygotes showed gross kidney abnormalities. Our results demonstrate that mutation of Chd2 dramatically affects mammalian development and long-term survival.     

A wide-range phylogenetic analysis of Zic proteins: implications for correlations between protein structure conservation and body plan complexity

We compared Zic homologues from a wide range of animals. Striking conservation was found in the zinc finger domains, in which an exon-intron boundary has been kept in all bilateralians but not cnidarians, suggesting that all of the bilateralian Zic genes are derived from a single gene in a bilateralian ancestor. There were additional conserved amino acid sequences, ZOC and ZF-NC. Combined analysis of the zinc finger, ZOC, and ZF-NC revealed the presence of two classes of Zic, based on the degree of protein structure conservation. The "conserved" class includes Zic proteins from the Arthropoda, Mollusca, Annelida, Echinodermata, and Chordata (vertebrates and cephalochordates), whereas the "diverged" class contains those from the Platyhelminthes, Cnidaria, Nematoda, and Chordata (urochordates). The result indicates that the ancestral bilateralian Zic protein had already acquired an entire set of conserved domains, but that this was lost and diverged in the platyhelminthes, nematodes, and urochordates.     

Discovery of selective PDE4B inhibitors

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 1

We investigated proinflammatory cytokine TNFα production inhibitors in order to develop novel anti-inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetrahydropyridine group at the β-position, showed potent inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS) induced TNFα production in human whole blood, IC₅₀ = 1.86 μM) and in vivo (inhibition of LPS induced TNFα production in mice, ID₅₀ = 5.98 mg/kg).     

Two states of active spermatogenesis switch between reproductive and non-reproductive seasons in the testes of the medaka, Oryzias latipes

Seasonal change in spermatogenesis was examined in the restricted spermatogonium‐type testes of a teleost, Oryzias latipes. Histological observation revealed that the number of each stage of germ cells during most of the non‐reproductive season, from October to January (O–J period) was nearly half of that during the reproductive season, from May to July (M–J period), except for type B spermatogonia (B‐gonia), which was actually equal. As a result, the ratio of primary spermatocytes (P‐cytes) to B‐gonia was remarkably small in the O–J period. Despite the differences between both time periods, the proliferative activity of type A spermatogonia (A‐gonia), B‐gonia, or P‐cytes was at a similar level in both periods. Moreover, in cultured testes treated with bromodeoxyuridine as a cell‐lineage tracer, P‐cytes differentiated to spermatids in 11–15 days in both M–J and O–J periods. These indicate that spermatogenesis is active in each period at a different state. In the spermatogenic testis, A‐gonial proliferation was maintained by human follicle stimulating hormone/luteinizing hormone in culture. Whereas cell death of B‐gonia and/or P‐cytes gradually increased in the M–J period in spite of those cells being constant in population sizes. In transition to the O–J period, A‐gonia and P‐cytes first decreased, which was accompanied by a decrease in proliferative activity of A‐gonia and relative increase of dead cells from B‐gonia and/or P‐cytes against live P‐cytes. These suggest that A‐gonial proliferation and cell death of B‐gonia and/or P‐cytes that is induced coordinately with B‐gonial differentiation are critical for the spermatogenic control.     

Baculovirus actin-rearrangement-inducing factor ARIF-1 induces the formation of dynamic invadosome clusters

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a pathogen of lepidopteran insects, has a striking dependence on the host cell actin cytoskeleton. During the delayed-early stage of infection, AcMNPV was shown to induce the accumulation of actin at the cortex of infected cells. However, the dynamics and molecular mechanism of cortical actin assembly remained unknown. Here, we show that AcMNPV induces dynamic cortical clusters of dot-like actin structures that mediate degradation of the underlying extracellular matrix and therefore function similarly to clusters of invadosomes in mammalian cells. Furthermore, we find that the AcMNPV protein actin-rearrangement-inducing factor-1 (ARIF-1), which was previously shown to be necessary and sufficient for cortical actin assembly and efficient viral infection in insect hosts, is both necessary and sufficient for invadosome formation. We mapped the sequences within the C-terminal cytoplasmic region of ARIF-1 that are required for invadosome formation and identified individual tyrosine and proline residues that are required for organizing these structures. Additionally, we found that ARIF-1 and the invadosome-associated proteins cortactin and the Arp2/3 complex localize to invadosomes and Arp2/3 complex is required for their formation. These ARIF-1–induced invadosomes may be important for the function of ARIF-1 in systemic virus spread.     

Natural killer cell is a major producer of interferon γ that is critical for the IL-12-induced anti-tumor effect in mice

Although the anti-tumor effect of IL-12 is mediated mostly by IFNγ, which cell types most efficiently produce IFNγ and therefore initiate or promote the anti-tumor effect of IL-12 has not been clearly determined. In the present study, we demonstrated hydrodynamic injection of the IL-12 gene led to prolonged IFNγ production, NK-cell activation and complete inhibition of liver metastasis of CT-26 colon cancer cells in wild-type mice, but not in IFNγ knockout mice. NK cells expressed higher levels of STAT4 and upon IL-12 administration displayed stronger STAT4 phosphorylation and IFNγ production than non-NK cells. Adoptive transfer of wild-type NK cells into IFNγ knockout mice restored IL-12-induced IFNγ production, NK-cell activation and anti-tumor effect, whereas transfer of the same number of wild-type non-NK cells did not. In conclusion, NK cells are predominant producers of IFNγ that is critical for IL-12 anti-tumor therapy.     

Fatal exacerbation of type B chronic hepatitis triggered by changes in relaxed circular viral DNA synthesis and virion secretion

Virological features of fulminant liver disease-causing hepatitis B virus (HBV) have not been fully elucidated. We studied longitudinally the viruses obtained before and after fulminant liver disease in a patient with chronic HBV infection showing fatal exacerbation. HBV strains were obtained before and after exacerbation (designated as FEP1 and FEP2). Their virological features were investigated by in vitro transfection. FEP1 and FEP2 possessed higher activity of overall HBV DNA synthesis than the wild-type. FEP1 lacked competence for relaxed circular (RC) HBV DNA synthesis and RC HBV DNA-containing virion secretion, but FEP2 maintained it. Chimeric analysis revealed that the preS/S gene, where FEP1 had a considerable number of mutations and deletions but FEP2 did not, was responsible for impaired RC HBV DNA synthesis and virion secretion. Furthermore, incompetence of FEP1 strain was transcomplemented by the preS/S protein of wild-type strain. In conclusion, the viral strain after exacerbation showed resurgent RC HBV DNA synthesis and virion secretion, which was caused by conversion of the preS/S gene from a hypermutated to hypomutated state. This may have been responsible for disease deterioration in the patient. This is a novel type of HBV genomic variation associated with the development of fulminant liver disease.