A pentanucleotide repetitive sequence, (TTAGG)., has been isolated from a silkworm genomic library, using cross-hybridization with a (TTNGGG)5 sequence, which is conserved among most eukaryotic telomeres. Both fluorescent in situ hybridization and Bal 31 exonuclease experiments revealed major clusters of (TTAGG) A telomere is defined as a region of a chromosomal end that is required for complete replication, meiotic pairing, and stability of the chromosome (5, 36). Most eukaryotic telomeres are composed of simple repeated sequences of a Gand C-rich complementary strand with a general structure of (T or A)m(G)n. The G-rich strand is oriented 5' to 3' toward the chromosomal end and is synthesized by an RNA-dependent telomerase activity (13-15, 22, 35). At present, telomeric DNA has been identified in many widely divergent organisms, including yeasts, protozoa, slime molds, flagellates, nematodes, plants, and vertebrates (6, 23).Telomeres for most invertebrates and insects, however, have yet to be isolated and characterized, although some telomeric DNA fragments have been isolated from species of two dipteran genera, Drosophila and Chironomus (3,29,30,32,34). Such dipteran telomeres seem to have complex and exceptional structures in which components can be distinguished with a simple repeat shown in most other eukaryotic organisms. However, the question still remains as to whether the dipteran insects carry a tandem array at the extreme ends of chromosomes. To examine evolutionarily conserved features of the telomeric terminal array in insects, we have identified the telomere sequence in the silkworm Bombyx mori, which is a representative of the lepidopteran order of insects.It is noteworthy that some insects, such as hemipteran and lepidopteran orders, including the silkworm, are believed to have a diffuse type of centromere (24,25). In this case, chromosomes with full-length kinetochores are not sensitive to breakage, induced either spontaneously or by X-ray irradiation, so that chromosome fragments are maintained through cell division (10). Therefore, chromosome fragmentation and de novo telomere formation at the breakage site may provide opportunities to make new chromosomes. Furthermore, translocation of the chromosomal fragment to * Corresponding author.other chromosomes can possibly alter chromosome organization. To study the mechanisms of karyotype evolution, it is of interest to determine the structure and distribution of telomeres in this insect.In this study, we have isolated a five-base repetitive sequence from a silkworm genomic library, based on crosshybridization with a telomeric sequence (TTNGGG) which is highly conserved among a wide variety of eukaryotic organisms. The results of fluorescent in situ hybridization and Bal 31 exonuclease digestion experiments suggest that this (TTAGG)n sequence is located at the extreme terminal region of all Bombyx chromosomes. In addition, we have demonstrated that (TTAGG)n, slightly different from (TTAGGG)n which is conserved among vertebrates and some invertebra...

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Mechanisms for the uptake and transport of carotenoids, essential nutrients for humans, are not well understood in any animal system. The Y (Yellow blood) gene, a critical cocoon color determinant in the silkworm Bombyx mori, controls the uptake of carotenoids into the intestinal mucosa and the silk gland. Here we provide evidence that the Y gene corresponds to the intracellular carotenoid-binding protein (CBP) gene. In the Y recessive strain, the absence of an exon, likely due to an incorrect mRNA splicing caused by a transposon-associated genomic deletion, generates a nonfunctional CBP mRNA, resulting in colorless hemolymph and white cocoons. Enhancement of carotenoid uptake and coloration of the white cocoon was achieved by germ-line transformation with the CBP gene. This study demonstrates the existence of a genetically facilitated intracellular process beyond passive diffusion for carotenoid uptake in the animal phyla, and paves the way for modulating silk color and lipid content through genetic engineering.carotenoid transport ͉ cocoon color ͉ transgenic silkworm

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Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor-independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440-induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1-GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273-84. ©2016 AACR.

The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.All organisms exposed to light contain carotenoids, which are yellow to red C 40 hydrophobic isoprenoid pigments. Carotenoids play pivotal roles in living organisms as precursors of vitamin A, antioxidants, and colorants (1). Their potential roles in medicine have recently been investigated. For example, macular accumulation of the carotenoids lutein and zeaxanthin is associated with a decreased risk of age-related macular degeneration (2), the leading cause of blindness in the developed world. Although plants, certain fungi, and bacteria synthesize carotenoids, animals appear to be incapable of synthesizing these molecules de novo. Therefore, animals must acquire carotenoids from dietary sources, and subsequently transport them to cells of target tissues.The delivery of lipids, including carotenoids, to cells can be divided into three categories: 1) enzyme-mediated processes, such as the action of lipoprotein lipase on very low density lipoproteins, which converts a lipoprotein-bound lipid, triacylglycerol, into a water-soluble product, fatty acid, which diffuses into cells and leaves behind in the blood a lipoprotein product depleted in triacylglycerol (3); 2) receptor-mediated endocytosis, such as the uptake of low density lipoproteins by low density lipoprotein receptor, in which the entire lipoprotein particle is taken into the cell and metabolized (4); and 3) the delivery of specific lipids to specific tissues devoid of lipoprotein degradation, called selective lip...

Dietary intake of lutein and zeaxanthin appears to be advantageous for protecting human retinal and macular tissues from degenerative disorders such as age-related macular degeneration. Selective concentration of just two of the many dietary carotenoids suggests that uptake and transport of these xanthophyll carotenoids into the human foveal region are mediated by specific xanthophyll-binding proteins such as GSTP1 which has previously been identified as the zeaxanthin-binding protein of the primate macula. Here, a membrane-associated human retinal lutein-binding protein (HR-LBP) was purified from human peripheral retina using ion-exchange chromatography followed by size-exclusion chromatography. After attaining 83-fold enrichment of HR-LBP, this protein exhibited a significant bathochromic shift of approximately 90 nm in association with lutein, and equilibrium binding studies demonstrated saturable, specific binding toward lutein with a K(D) of 0.45 muM. Examination for cross-reactivity with antibodies raised against known lutein-binding proteins from other organisms revealed consistent labeling of a major protein band of purified HR-LBP at approximately 29 kDa with an antibody raised against silkworm (Bombyx mori) carotenoid-binding protein (CBP), a member of steroidogenic acute regulatory (StAR) protein family with significant homology to many human StAR proteins. Immunolocalization with antibodies directed against either CBP or GSTP1 showed specific labeling of rod and cone inner segments, especially in the mitochondria-rich ellipsoid region. There was also strong labeling of the outer plexiform (Henle fiber) layer with anti-GSTP1. Such localizations compare favorably with the distribution of macular carotenoids as revealed by resonance Raman microscopy. Our results suggest that HR-LBP may facilitate lutein's localization to a region of the cell subject to considerable oxidative stress.

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.