Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.     

The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice

The quality control of sperm is critical for efficient reproduction. In germ cells, cell death involves different processes to those in somatic cells, and in many cases, the trigger to induce cell death in deficient germ cells is still unclear. It is known that the fatty acid composition of sperm is related to fertility. Composition of the fatty acid of germ cells changes dynamically during spermatogenesis, and fatty acid binding protein (FABP) may be involved in these changes. In this study, we developed transgenic mice with a testicular germ-cell-specific FABP (PERF15) transgene, whose expression was controlled by the Cre-LoxP site-specific recombination system. We also developed transgenic mice with the Cre gene under the control of the spermatocyte specific Pgk2 promoter. In double transgenic mice, following Cre-mediated recombination of the PERF15 containing transgene, PERF15 was strongly overexpressed. Its overexpression induced multinucleate symplasts to form, indicating programmed germ cell death occurred at the elongated spermatid stage. As a result, sperm harboring the transgene were significantly decreased, but the surviving sperm demonstrated higher fertility than natural sperm. Therefore, we conclude that PERF15 associate with the direction of germ cell fates and preserve the quality of sperm.     

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.     

Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth

Steroid hormones mediate a wide variety of developmental and physiological events in multicellular organisms. During larval and pupal stages of insects, the principal steroid hormone is ecdysone, which is synthesized in the prothoracic gland (PG) and plays a central role in the control of development. Although many studies have revealed the biochemical features of ecdysone synthesis in the PG, many aspects of this pathway have remained unclear at the molecular level. We describe the neverland (nvd) gene, which encodes an oxygenase-like protein with a Rieske electron carrier domain, from the silkworm Bombyx mori and the fruitfly Drosophila melanogaster. nvd is expressed specifically in tissues that synthesize ecdysone, such as the PG. We also show that loss of nvd function in the PG causes arrest of both molting and growth during Drosophila development. Furthermore, the phenotype is rescued by application of 20-hydroxyecdysone or the precursor 7-dehydrocholesterol. Given that the nvd family is evolutionally conserved, these results suggest that Nvd is an essential regulator of cholesterol metabolism or trafficking in steroid synthesis across animal phyla.     

Permanence of single-species stage-structured models

In this paper, we consider population survival by using single-species stage-structured models. As a criterion of population survival, we employ the mathematical notation of permanence. Permanence of stage-structured models has already been studied by Cushing (1998). We generalize his result of permanence, and obtain a condition which guarantees that population survives. The condition is applicable to a wide class of stage-structured models. In particular, we apply our results to the Neubert-Caswell model, which is a typical stage-structured model, and obtain a condition for population survival of the model.The research is partially supported by the Ministry of Education, Science and Culture, Japan, under Grant in Aid for Scientific Research (A) 13304006.     

Cellular expression of murine Ym1 and Ym2, chitinase family proteins,as revealed by in situ hybridization and immunohistochemistry

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.     

Formation of (R)-2,3-dihydrogeranylgeranoic acid from geranylgeraniol in rat thymocytes

In a metabolic study of [1-(14)C]geranylgeranial involving rat thymocytes, the radioactivity was mainly incorporated into two metabolites, Z1 and Z2, the latter moving slower than the former on a silica-gel thin-layer plate. The time course of Z1 and Z2 formation superficially suggested a precursor-product relationship between Z1 and Z2. The two metabolites were chemically converted to their methyl esters on treatment with trimethylsilyl diazomethane. Z1 was cochromatographed with E,E,E-geranylgeranoic acid (GGA). Z2 was prepared in a large quantity from geranylgeranial using thymocytes, and purified by TLC followed by ESI (negative ion mode) or EI mass-spectrometry. The observation of a negative ion at m/z 305 on ESI and a molecular ion at m/z 306 (C(20)H(34)O(2)) with fragments similar to GGA on EI implied that Z2 was dihydroGGA, which has been detected in the urine and serum of patients with Refsum disease. The EI mass spectrum of (R)-2,3-dihydroGGA was identical to that of Z2. The diastereomeric amide synthesized from metabolite Z2 with (R)-1-(1-naphtyl)ethylamine was cochromatographed with (R acid, R) amide, not with (S acid, R) amide, which were similarly synthesized from (R)- and (S)-2,3-dihydroGGAs, respectively. In another metabolic study on [1-(14)C]geranylgeraniol (GGOH), the radioactivity was similarly incorporated into a metabolite corresponding to (R)-2,3-dihydroGGA. (R)-2,3-DihydroGGA induced DNA ladder formation with a maximum at 15 mciroM in thymocytes. However, 2,3-dihydrofarnesoic acid did not induce it at all.     

Bilateral congenital cataracts result from a gain-of-function mutation in the gene for aquaporin-0 in mice

Cataract Tohoku (Cat(Tohm)) is a dominant cataract mutation that leads to severe degeneration of lens fiber cells. Linkage analysis showed that the Cat(Tohm) mutation is located on mouse chromosome 10, close to the gene for aquaporin-0 (Aqp0), which encodes a membrane protein that is expressed specifically in lens fiber cells. Sequence analysis of Aqp0 revealed a 12-bp deletion without any change in the reading frame, which resulted in a deletion of four amino acids within the second transmembrane region of the AQP0 protein. Targeted expression of the mutated Aqp0 caused lens opacity in transgenic mice, the pathological severity of which depended on the expression level of the transgene. The mutated AQP0 protein was localized to the intracellular and perinuclear spaces rather than to the plasma membranes of the lens fiber cells. The cataract phenotype of Cat(Tohm) is caused by a gain-of-function mutation in the mutated AQP0 protein and not by a loss-of-function mutation.