Establishment of a novel in vivo sex-specific splicing assay system to identify a trans-acting factor that negatively regulates splicing of Bombyx mori dsx female exons

The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.     

Ecdysone-inducible foreign gene expression in stably-transformed lepidopteran insect cells

Cultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change. i.e., cell aggregation, in response to treatment with 1 microM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20-30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.     

Establishment and characterization of a continuous cell line from pupal ovaries of Japanese oak silkworm Antheraea yamamai Guerin-Meneville

Pupal ovaries of the wild oak silkworm Antheraea yamamai Guerin-Meneville were cultured in MGM-448 (Modified Grace Medium-448) medium containing 10% fetal bovine serum. After the primary culture was set up in 1988, a continuous cell line was obtained in 1991, designated as NISES-Anya-0611 (Anya-0611). The population doubling time was 54 hrs. and 19 min. at 96 passages and 88 hrs. and 29 min. at 387 passages. Spindle-shaped and spherical cells coexisted in the cell group. The cell line karyotype line was typical of lepidopteran cell lines, consisting of numerous small chromosomes. The cell line was distinguished from other lepidopteran cell lines by comparing malic enzyme, phosphoglucose isomerase, phosphoglucose mutase, and isocitric dehydrogenase isozyme patterns. The cell line was highly infected to the Antheraea yamamai nuclear polyhedrosis virus (Anya NPV). The luciferase gene of recombinant Bm NPV (BmNPVP6ETL) was able to express in the cell line, too, so that luciferase recombinant products were able to be detected in the cell body and in supernatant. The Anya NPV clone group was isolated on the cell seat using plaque purification.     

Transient in vivo reporter gene assay for ecdysteroid action in the Bombyx mori silk gland

To analyze the molecular mechanisms underlying hormone-regulated gene expression during molt and metamorphosis, we developed a transient reporter gene assay system using the silkworm anterior silk gland. Reporter plasmids were delivered into dissected anterior silk glands by particle bombardment and bombarded glands transplanted into other larvae, to which hormones were then administered. When the green fluorescent protein gene, coupled with the constitutive cytoplasmic actin gene A3 promoter, was introduced into the anterior silk gland, strong green fluorescence was observed a few days later. Bombarded silk glands transplanted into other larvae showed the same morphological changes as intrinsic glands after 20-hydroxyecdysone (20E) alone or 20E plus juvenile hormone (JH) treatment, indicating that the transplanted gland received hormonal signals properly. When a 20E-responsive reporter construct containing four tandemly repeated pal-1 ecdysone response elements upstream from the luciferase gene was delivered into the gland, an approximately 50-fold increase in luciferase activity was detected 30 h after 20E injection. This induction was comparable to that in an ecdysteroid-responsive Bombyx cell line. This in vivo reporter assay system is thus a rapid, effective tool for analyzing gene expression regulated by 20E and probably by JH.     

Maturation of adult peripheral blood CD38(+)CD4(+) T cells demonstrated by cytokine production in response to a superantigen,TSST-1

This study looks at immunoincompetent CD4(+) T cells in adult peripheral blood (APB) using cytokine production in response to a superantigen as a measure of function. We compared the function of APB CD38(+)CD4(+) and CD38(-/low)CD4(+) T cells to that of cord blood (CB) CD4(+) T cells. APB CD4(+) T cell blasts produce substantial amounts of IL-2 in response to TSST-1 restimulation, while CB CD4(+) T cell blasts produce less. APB CD38(+)CD4(+) T cells produce low levels of IL-4 and IFN-gamma in response to TSST-1, even after activation, while APB CD38(-/low)CD4(+) T cells retain their ability to produce high levels of these cytokines despite high CD38 expression. These results suggest that the developmental stage of APB CD38(+)CD4(+) T cells lies between that of CB CD4(+) T cells and APB CD38(-/low)CD4(+) T cells and that APB CD38(+)CD45RO(-)CD4(+) T cells gradually cease to express CD38 as they acquire full function. We reconsider CD4(+) cell maturation and response to TSST-1 and discuss the implications of T cell maturity on infectious diseases.     

Calcium-binding proteins: intracellular sensors from the calmodulin superfamily.

In all eukaryotic cells, and particularly in neurons, Ca(2+) ions are important second messengers in a variety of cellular signaling pathways. In the retina, Ca(2+) modulation plays a crucial function in the development of the visual system's neuronal connectivity and a regulatory role in the conversion of the light signal received by photoreceptors into an electrical signal transmitted to the brain. Therefore, the study of retinal Ca(2+)-binding proteins, which frequently mediate Ca(2+) signaling, has given rise to the important discovery of two subfamilies of these proteins, neuronal Ca(2+)-binding proteins (NCBPs) and calcium-binding proteins (CaBPs), that display similarities to calmodulin (CaM). These and other Ca(2+)-binding proteins are integral components of cellular events controlled by Ca(2+). Some members of these subfamilies also play a vital role in signal transduction outside of the retina. The expansion of the CaM-like protein family reveals diversification among Ca(2+)-binding proteins that evolved on the basis of the classic molecule, CaM. A large number of NCBP and CaBP subfamily members would benefit from their potentially specialized role in Ca(2+)-dependent cellular processes. Pinpointing the role of these proteins will be a challenging task for further research.     

Influence of gravity changes induced by parabolic flight on cytokine production in mouse spleen

Dysregulation of the immune system has been a well-documented effect of human exposure to a microgravity environment during space flight. These effects have included altered cytokine production, reduced proliferative responses, altered signal transduction pathways and altered distribution of peripheral immune cells. Recent reports have documented immunologic studies performed in-flight. When tested during space flight, delayed-type hypersensitivity was reduced, indicating a dysregulation of cell-mediated immune function. However, the mechanisms by which this occurs remain unclear. The production of cytokines plays a critical role in the ability of a host to mount an immune response against an invading microorganism. In this study, the alteration of cytokine responses in mice following parabolic flight (PF) was investigated.     

Triplex formation with 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) having C3'-endo conformation at physiological pH

Antigenes, which are substances that inhibit gene expression by binding to double-stranded DNA (dsDNA) in a sequence-specific manner, are currently sought for the treatment of various gene-related diseases. As such antigenes, we developed new nuclease-resistant oligopyrimidine nucleotides that are partially modified with 2′-O,4′-C-ethylene nucleic acids (ENA), which are constrained in the C3′-endo conformation and can form a triplex with dsDNA at physiological pH. It was found that these oligonucleotides formed triplexes similarly to those partially modified with 2′-O,4′-C-methylene nucleic acids (2′,4′-BNA or LNA), as determined by UV melting analyses, electromobility shift assays, CD spectral analyses and restriction enzyme inhibition assays. In our studies, oligonucleotides fully modified with ENA have δ torsion angle values that are marginally higher than those of 2′,4′-BNA/LNA. ENA oligonucleotides present in 10-fold the amount of dsDNA were found to be favorable in forming triplexes. These results provide useful information for the future design of triplex-forming oligonucleotides fully modified with such nucleic acids constrained in the C3′-endo conformation considering that oligonucleotides fully modified with 2′,4′-BNA/LNA do not form triplexes.     

2'-O,4'-C-ethylene-bridged nucleic acid (ENA) for effective antisense formation

ENA antisense oligonucleotides for vascular endothelial growth factor (VEGF) mRNA were synthesized and evaluated in A549 lung cancer cells. It was found that the VEGF ENA-antisense inhibited not only the expression of VEGF, but also the expression of three genes, which were found in Genbank by BLAST and Clustal W search and considered likely to bind to the VEGF ENA-antisense. These results indicate that ENA-antisense oligonucleotides act in a sequence-specific manner, and could be used as effective antisense drugs.