Effect of larval brain extracts derived from three swallowtail butterflies,Papilio helenus L., P. machaon L. and P. memnon L., on seasonal morph development of P. xuthus L. (Lepidoptera: Papilionidae)

Adults of the three papilionid butterflies, Papilio helenus L., Papilio machaon L. and Papilio memnon L., exhibit seasonal diphenism comprising spring and summer morphs. To elucidate the physiological mechanism underlying seasonal morph development in papilionid butterflies, we investigated whether a cerebral factor showing summer‐morph‐producing hormone (SMPH) activity is present in the brain of three Papilio species using an assay system with chilled male short‐day pupae of P. xuthus L. When 2% NaCl extracts derived from 20 larval brains of the three species were injected into abdomens of chilled male short‐day pupae of P. xuthus, all recipients destined to develop into spring‐morph adults developed into summer‐ and intermediate‐morph adults. On the other hand, all recipients injected with distilled water as a control developed into spring‐morph adults. These results indicate that a cerebral factor showing SMPH activity is present in the larval brain of the three Papilio species. Additionally, all recipients injected with 2% NaCl extracts derived from 20 adult brains of Bombyx mori L. also developed into summer‐ and intermediate‐morph adults. The results revealed that SMPH or a cerebral factor showing SMPH activity is widely distributed among lepidopteran insects.     

Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions.     

Sorting specificity of spermatogenic cell specific region of mouse hexokinase-s (mHk1-s)

Hexokinase is the first enzyme involved in the glycolysis process that produces glucose phosphorylate. Our previous study reported on our cloning of mouse Hk1-s (mHk1-s) cDNA, which were expressed only in testis cells, and noted that this cDNA has a spermatogenic cell-specific region (SSR) that replaces the porin binding domain (PBD) in the Hk1of somatic cells. Although we know that PBD binds to the outer membrane of a mitochondrion, the role of the SSR is not yet understood. To investigate the intracellular localization of SSR, we constructed expression vectors with the epitope tag (GFP-, HA-), subcloned SSR, or PBD cDNA. We transfected these vectors in mouse fibroblast, NIH3T3 cells, after which we observed the localization of the SSR and PBD in the NIH3T3 cells. Our current study using the immunocytochemical method revealed that PBD is concentrated around the mitochondrion. However, the SSR could not be ascribed to the mitochondrion, ER, or nuclear colocalization. Moreover, subcellular fractionation analysis showed that PBD was detected in the mitochondrial fraction, and that SSR was detected in the cytosolic fraction. Our findings suggest that PBD of Hk1 targets mitochondrion, but the SSR of mHk1-s targets some specific organellae.     

Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3',4,4',5-pentachlorobiphenyl

Coplanar polychlorinated biphenyls included in dioxin-like compounds are bio-accumulated and adversely affect wildlife and human health. Although many researchers have studied the metabolism of PCBs, there have been few reports of the in vitro metabolism of 3,3',4,4',5-pentachlorobiphenyl (PCB126), despite the fact that it has the highest toxicity among PCB congeners. Cytochrome P450 (CYP) 1A1 proteins can metabolize some dioxins and PCBs by hydroxylation, but the activities of human and rat CYP1A1 proteins are very different. The mechanism remains unclear. From our results, rat CYP1A1 metabolized PCB126 into 4-OH-3,3',4',5-tetrachlorobiphenyl and 4-OH-3,3',4',5,5'-pentachlorobiphenyl, but human CYP1A1 did not metabolize. Homology models of the two CYP proteins, and docking studies, showed that differences in the amino acid residues forming their substrate-binding cavities led to differences in the size and shape of the cavities; only the cavity of rat CYP1A1 allowed PCB126 close enough to the haem to be metabolized. Comparison of the amino acid residues of other mammalian CYP1A1 proteins suggested that rats have a unique metabolism of xenobiotics. Our results suggest that it is necessary to be careful in human extrapolation of toxicity data estimated by using the rat as an experimental animal, especially in the case of compounds metabolized by CYP1A1.     

KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.     

Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.     

Macrophage inflammatory protein-1α (MIP-1α) enhances a receptor activator of nuclear factor κB ligand (RANKL) expression in mouse bone marrow stromal cells and osteoblasts through MAPK and PI3K/Akt pathways

Osteolytic lesions are rapidly progressive during the terminal stages of myeloma, and the bone pain or bone fracture that occurs at these lesions decreases the patients’ quality of life to a notable degree. In relation to the etiology of this bone destruction, it has been reported recently that MIP-1α, produced in large amounts in myeloma patients, acts indirectly on osteoclastic precursor cells, and activates osteoclasts by way of bone-marrow stromal cells or osteoblasts, although the details of this process remain obscure. In the present study, our group investigated the mechanism by which RANKL expression is induced by MIP-1α and the effects of MIP-1α on the activation of osteoclasts. RANKL mRNA and RANKL protein expressions increased in both ST2 cells and MC3T3–E1 cells in a MIP-1α concentration-dependent manner. RANKL mRNA expression began to increase at 1 h after the addition of MIP-1α; the increase became remarkable at 2 h, and continuous expression was observed subsequently. Both ST2 and MC3T3-E1 cells showed similar levels of increased RANKL protein expression at 1, 2, and 3 days after the addition of MIP-1α. After the addition of MIP-1α, the amount of phosphorylated ERK1/2 and Akt protein expressions showed an increase, as compared to the corresponding amount in the control group. On the other hand, the amount of phosphorylated p38MAPK protein expression showed a decrease from the amount in the control group after the addition of MIP-1α. U0126 (a MEK1/2 inhibitor) or LY294002 (a PI3K inhibitor) was added to ST2 and MC3T3-E1 cells, and was found to inhibit RANKL mRNA and RANKL protein expression in these cells. When SB203580, a p38MAPK inhibitor, was added, RANKL mRNA and RANKL protein expression were increased in these cells. MIP-1α was found to promote osteoclastic differentiation of C7 cells, an osteoclastic precursor cell line, in a MIP-1α concentration-dependent manner. MIP-1α promoted differentiation into osteoclasts more extensively in C7 cells incubated together with ST2 and MC3T3-E1 cells than in C7 cells incubated alone. These results suggested that MIP-1α directly acts on the osteoclastic precursor cells and induces osteoclastic differentiation. This substance also indirectly induces osteoclastic differentiation through the promotion of RANKL expression in bone-marrow stromal cells and osteoblasts. The findings of this investigation suggested that activation of the MEK/ERK and the PI3K/Akt pathways and inhibition of p38MAPK pathway were involved in RANKL expression induced by MIP-1α in bone-marrow stromal cells and osteoblasts. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.     

Effects of leaf quality and microhabitat on the survival of a leaf-rolling weevil (Attelabidae)

Female Cycnotrachelus roelofsi (Coleoptera: Attelabidae) construct two types of leaf roll, i.e., cut-off cradles (CCs) and suspended cradles (SCs), in which to lay eggs; these cradles are generally constructed using older and younger leaves, respectively. We conducted two experiments to determine whether the quality of cradle leaves affects egg and larval survival. In the first experiment, we severed all SCs from a tree and placed them on the ground with unmanipulated CCs in the early breeding season. In the second experiment, we resuspended all CCs in a tree with unmanipulated SCs in the late breeding season. We also compared leaf mass per area (LMA), polyphenol content, and nitrogen content between the two cradle types to determine whether there were any differences in leaf quality. Larval mortality, probably caused by cradle herbivory, was significantly greater in severed SCs than in intact CCs in the early season, suggesting that leaf quality had a profound effect on larval mortality in the terrestrial microhabitat. In contrast, larval mortality did not differ between resuspended CCs and intact SCs in the late season, suggesting that leaf quality had little effect on larval mortality in the arboreal microhabitat. LMA was higher in CCs than in SCs, but there were no differences in the nitrogen and polyphenol contents. These results suggest that cradles constructed using mature, tough leaves were more effective against terrestrial cradle herbivores than those constructed using new, soft leaves.