Eiji Kotani

Kyoto Institute of Technology · Department of Applied Biology

Growth factors, including fibroblast growth factor-7 (FGF-7), are a group of proteins that stimulate various cellular processes and are often used with carriers to prevent the rapid loss of their activities. Sericin with great biocompatibility has been investigated as a proteinaceous carrier to enhance the stability of incorporated proteins. The di...

The δ-endotoxin Cry4Aa from Bacillus thuringiensis israelensis (Bti) has insecticidal characteristics specific to insects of the order Diptera. Although Cry4Aa has shown to have potential as an effective proteinaceous pesticide against mosquitoes, it has a UV-intolerant property that limits its outdoor utilization. Our previous research showed that...

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we establi...

Sericin, which is derived from the outer layer of silk thread proteins of the common silkworm (Bombyx mori), functions as a glue to fix fibers of developing cocoons. Although sericin has potential uses in various fields, including tissue engineering, the biomedical applications of intact sericin have yet to be fully identified. We previously develo...

The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and rele...

Sericins are derived from the outer layer of silk thread proteins of the common silkworm (Bombyx mori) and act as a glue to fix fibers of the developing cocoon. Although sericin has potential uses in various fields, including tissue engineering and the development of cosmetics, biological applications of intact sericin have yet to be identified. We...

Ecdysteroids are mainly synthesised in the prothoracic glands, which regulate larval moulting and metamorphosis in insects. After degeneration of the prothoracic gland during pupal–adult metamorphosis, the ovary becomes the source of ecdysteroids in adult females. Here, we demonstrated that the high ecdysteroid level after adult eclosion decreased...

Cypovirus is an insect virus that is encapsulated in stable cubic protein crystals composed of polyhedrin protein produced in virus-infected cells. Molecular technology developed over the last decade is now able to immobilise proteins of interest on polyhedrin crystals. Modified polyhedrin crystals can be used in cell cultures for implantation in a...

Recombinant hemagglutinin-neuraminidase (HN) based subunit vaccine, which is non-infectious and can be produced using insect cell-culture systems, is a potential alternative to conventional live and inactivated Newcastle disease virus (NDV) vaccines. However, process development for manufacture and efficacy assessment of HN subunit vaccines has bee...

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular...

Significance Specific gene functions have been successfully suppressed by gene silencing or editing in many organisms. However, genetic manipulation to suppress the function of a target tissue has not been achieved using cytotoxin genes. We established transgenic silkworms with posterior silk glands (PSGs) that express the enzymatic domain of the c...

C-type lectin-producing tissue, designated as CLPT, constitutes mouthparts of the flesh fly (Sarcophaga peregrina) and produces a C-type lectin, CLEM 36, which is found to be secreted into saliva. In the present study, CLPT was investigated by immunohistochemical method using a transmission electron microscope (TEM) and scanning electron microscope...

Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human ba...

Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid-binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. In th...

Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 × 10(7) BMP-2 polyhedra, and half were treated with sponges containing 3.6 × 10(6) BMP-2 polyhedra...

Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to r...

Encapsulation of cytokines within protein microcrystals (polyhedra) is a promising approach for the stabilization and delivery of therapeutic proteins. Here, we investigate the influence of vascular endothelial growth factor (VEGF) microcrystals and endostatin microcrystals on angiogenesis. VEGF was successfully encapsulated into microcrystals deri...

To date, two novel freeze-responsive proteins, Fr10 and Li16, have been discovered in the wood frog, Rana sylvatica, and likely support freezing survival. Although previous studies have established tissue distribution of each protein, there have been no studies that explore their functional consequences in intolerant cells. To assess the ability of...

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers...

Supplementary Information

Human leukemia inhibitory factor (LIF) was immobilized into insect virus-derived microcrystals (polyhedra) to generate LIF polyhedra (LIF-PH) that can slowly release LIF into embryonic stem (ES) cell culture media and thus maintain ES cells in an undifferentiated state. Assays of the biological activities of LIF-PH indicated that a single addition...

Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in the crystalline lattice of the viral protein polyhedrin. We have previously demonstrated a novel protein engine...

Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 a...

A complementary DNA encoding a TIA-1-type RNA-binding protein (SfTRN-1) was isolated from cultured cells of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), to characterize its function. The deduced 388-amino acid sequence of SfTRN-1, which possessed three RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain, sh...

BmTRN-1, an RNA-binding protein homologous to mammalian TIA-1/TIAR, which possesses three RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain was shown to be involved in several mechanisms of RNA metabolism including the regulation of transcripts in the cells of the silkworm, Bombyx mori. The present gene analysis revealed that,...

Two cDNAs designated BmRelish1 and 2, that encode Relish homologs, were cloned from the silkworm, Bombyx mori. BmRelish1 had an IkappaB-like domain with 5 ankyrin repeats in addition to Rel homology domain (RHD), nuclear localization signal (NLS), and acidic and hydrophobic amino acids (AHAA) rich regions. On the other hand, BmRelish2 lacked the AH...

Some insect virus produces proteinous occlusion bodies named polyhedra, on which it is possible to immobilize functional proteins, such as green fluorescent proteins, antibodies, and growth factors, with keeping their biological activity. In this work, several kinds of polyhedra were micro-patterned by applying a mechanical force induced by femtose...

A double-stranded ribonuclease (Bm-dsRNase) was separated from the digestive juice of the silkworm larvae, Bombyx mori. The full-length cDNA was produced and sequenced using a 20 mer primer designed from the N-terminal sequence of the Bm-dsRNase. The cDNA had an ORF encoding 51 kDa precursor protein which can be divided into three domains: a signal...

A nuclease which degrades double-stranded RNA was purified from the digestive juice of fifth-instar larvae of the silkworm, Bombyx mori, using gel filtration and affinity column chromatography. The enzyme was found to have a molecular weight of 41,000 as estimated by a gel filtration method and detected as a single band with the same molecular weig...

We have isolated a novel gene, CLEM 36, of the flesh fly Sarcophaga peregrina, which shows significant homology to the C-type lectin family. CLEM 36 mRNA was transcribed excessively from the second day after eclosion only in the tip of mouthparts. Whole mount in situ hybridization showed that CLEM 36 mRNA was expressed in the C-type lectin-producin...

A cDNA encoding a 388 amino acid TIA-1-type RNA-binding protein (BmTRN-1) was isolated from midgut cDNAs of the silkworm, Bombyx mori, via homologous cloning, in order to characterize its function. The deduced amino acid sequence, most likely encoded by a single copy gene, has significant homology with human TIA-1 and TIAR known as apoptotic regula...

In order to investigate whether eggs of the black-striped strain (P(S)) of the silkworm, Bombyx mori, represent an appropriate model for estimating the biological effect of cosmic radiation, radiosensitivity of the eggs against X-rays and heavy ion particles was examined as ground-based experiments. The exposure of diapause eggs to X-rays or heavy...

To investigate the effects of cosmic radiation and microgravity on embryogenesis and organogenesis in Bombyx eggs, two different stages of eggs, the early stage after oviposition and the diapause-terminated eggs, were loaded on the US Space Shuttle/Atlantis (STS-84) for a 9 day flight. More than 85% of the early stage eggs hatched in the flight sam...

Sulfur substituted 3-vinylpyrrole 10 was prepared from 3-thio-acetylpyrrole 9 by alkylation with alkyl halide in the presence of propylene oxide. Functionalized 4-alkylthioindoles were made by Diels-Alder reaction of the 3-vinylpyrrole 10 with dienophiles. Chuangxinmycin analogues were synthesized by using some of the functionalized 4-alkylthioindo...

Nishiyama Y, Nejima J, Watanabe A, Kotani E, Sakai N, Hatamochi A, Shinkai H, Kiuchi K, Tamura K, Shimada T, Takano T, Katayama Y (Nippon Medical School, Tokyo, and Chiba University School of Medicine, Chiba, Japan). Ehlers–Danlos syndrome type IV with a unique point mutation in COL3 A1 and familial phenotype of myocardial infarction without organi...

The reaction of oleanane triterpenoid 1b with a FeIII(PA; picolinate)3/H2O2/MeCN system (reagent system A), a simple model system for mono-oxygenase, gave the 11 alpha-hydroxyl derivative 3 as major product, along with 11-oxo derivative 4 and 12-oxo derivative 6. The reaction of lupane triterpenoid 2b with reagent system A gave only oxidative rearr...

A right ventricular outflow tract was partially obstructed by a metastatic tumor from a hepatocellular carcinoma. This tumor was treated via chemoembolization of the right coronary artery, which resulted in tumor regression and improvement of the patient's symptoms.

Direct shoot organogenesis induced from mulberry leaf tissue was observed histologically. In the initial stage of culture, small meristematic nodules were newly formed from subepidermal layer in the midrib and its adjacent zone of cultured leaf tissues, resulting in dome-like protrusions initiated on the adaxial leaf surface. These protrusions were...

Factors influencing the efficiency of gene delivery into mulberry leaf cells using a particle inflow gun were studied by employing a chimeric gene encoding for the production of β-glucuronidase. Two days following bombardment with plasmid-coated micropro-jectiles, transient expression of the β-glucuronidase was detected with the synthetic substrate...

Phosphofructokinase (PFK) was purified from non-diapause eggs (incubated at 25°C) of the silkworm, Bombyx mori, by using hydroxylapatite, Sephacryl 5-300, and ATP-agarose column chromatography. PFK activity separated into two distinct peaks on Sephacryl S-300, the molecular masses being 310 and 185kDa; SDS-PAGE showed that both were composed of a s...

A serine protease of the silkworm, Bombyx mori, with an isoelectric point of pH 10-11 and a pH optimum for succinyl-Leu-Leu-Val-Tyr-MCA degrading activity of about 10, was found in a 0.33 M NaCl-eluted fraction obtained from cation-exchange chromatography of digestive juice. The activity of the enzyme was strongly inhibited by chymostatin and PMSF,...

Large, rounded idioblasts were observed in adaxial leaves of mulberry plants; they were clearly distinguishable from epidermal, trichome and parenchyma cells. The size and density of idioblasts varied according to leaf age. Cytological features of idioblasts were as follows: the outermost region (‘cap’) of idioblasts was situated on the adaxial sur...

Shoot organogenesis from mulberry leaves was examined by using a two-step culture system consisting of seedling culture for obtaining leaf explants and leaf culture for inducing shoot organogenesis. Successful induction of shoots was achieved with an almost perfect induction rate, only when both cultures were carried out in the medium supplemented...

Shoot organogenesis from mulberry leaves was examined by using a two-step culture system consisting of seedling culture for obtaining leaf explants and leaf culture for inducing shoot organogenesis. Successful induction of shoots was achieved with an almost perfect induction rate, only when both cultures were carried out in the medium supplemented...

The injection of synthetic diapause hormone (BomDH-1 [19-Trp]; DH) into pupae of non-diapause producer (polyvoltine race) transformed non-diapause eggs into diapause ones (DH-induced diapause eggs). Single injection of the DH (50~200 pmol/pupa) into 3.5-day-old pupae produced diapause eggs of 18-27% per total laid eggs. The DH-induced diapause eggs...

For callus induction, explants were excised from the roots of mulberry seedlings grown in a medium containing 10⁻⁵ M 6-benzylaminopurine and subject to culture in a solid medium supplemented with 1CT⁻⁶ M 2, 4-dichlorophenoxyacetic acid and 10⁻⁶ M thidiazuron. The isolated calli were repeatedly subcultured in the liquid medium, resulting in a faster...

The adaxial surface of mulberry leaves was surveyed to examine patterns of calcium (Ca) deposition in idioblasts. Histochemical observation showed that the degree of Ca deposition was positively correlated with leaf age. Ca deposition started from the “cap” portion situated in the center of the idioblasts, followed to form a mass of Ca crystal whic...

Shoot bud formation was induced in the basal tissues of mulberry leaves using a two-step culture system: For achieving a high frequency of multiple shoot bud formation, it was essential that seedlings were cultured on a medium supplemented with a combination of 6-benzylaminopurine (BA) and 2, 3, 5-triiodobenzoic acid (TIBA), where after the basal l...

Non-enzymic oxygenation reaction of methyl valproate (2) utilizing a simple model system for mono-oxygenases, Fe(MeCN)2(6+)-H2O2-Ac2O in MeCN, was investigated in connection with stereochemical analyses of the mammalian metabolites of 1. This oxygenation reaction of methyl valproate (2) gave a 92:8 mixture of the anti-isomer 4a and the syn-isomer 4...

Two genomic DNAs encoding cecropin B (CecB), an antibacterial protein from Bombyx mori, were cloned and sequenced. The number of CecB genes was estimated to be more than four copies per haploid by genomic Southern blotting. Two genes, CecB1 and CecB2, were located tandemly within 12 kb in the same orientation. These two genes encoded identical amin...

The effects of 4-phenylalkylpyridines (chain length of 0-5, 7, 9 and 11 carbon atoms) on the induction of hepatic microsomal P450 and its multiple forms (1A1/2 (1A1, 1A2), 3A2, 2B1/2, 2C6 and 2E1) in the male and female rat have been investigated. P450 induction gradually declined with increasing chain length of the 4-phenylalkylpyridines. Immunobl...

A Bombyx mori cDNA was cloned that hybridized with Hyalophora cecropia attacin probe and its nucleotide sequence was determined. This cDNA consisted of 846 nucleotides and the deduced amino acid sequence showed that the cDNA encodes an attacin precursor protein. The putative mature protein of B. mori attacin had 70.4, 68.3 and 18.8% identity in ami...

We purified a 450 kDa protein from a low-salt alkaline extract of chicken gizzard smooth muscle. This high molecular mass protein could be extracted with the low-salt alkaline solution at 37 degrees C but not at 4 degrees C. The 450 kDa protein was isolated from the extract by ammonium sulfate fractionation and following sequential column chromatog...

Invertebrate lectins play an important role in a non-specific self-defense mechanism, as invertebrates do not synthesize specific antibodies. We report the cloning of several overlapping cDNAs encoding the entire silkworm (Bombyx mori) lectin, which we propose to call hemocytin. The sequence (10477 bp) encoded 3133 amino acids. The characteristics...

Invertebrate lectins play an important role in a non-specific self-defense mechanism, as invertebrates do not synthesize specific antibodies. We report the cloning of several overlapping cDNAs encoding the entire silkworm (Bombyx mori) lectin, which we propose to call hemocytin. The sequence (10477 bp) encoded 3133 amino acids. The characteristic f...

We examined the effect of 1-benzylimidazole on the induction of cytochrome P-450 1A1/2 (P-450 1A1/2) and cytochrome P-450 2B1/2 (P-450 2B1/2) in normal, castrated, ovariectomized and hypophysectomized male rats, and in castrated rats treated with testosterone. 1-Benzylimidazole markedly increased P-450 content in male and female rats. Parallel to t...

We investigated the structural requirements for the induction of hepatic microsomal cytochrome P450 2B1/2 (P450 2B1/2) and cytochrome P450 1A1/2 (P450 1A1/2) by imidazole- and pyridine-containing compounds in rats. Clotrimazole, an azole antifungal drug, and 1-diphenylmethylimidazole preferentially induced P450 2B1/2 in a dose-dependent manner, and...

We examined the abilities of 4-, 3- and 2-benzylpyridine and 4-tert-butylpyridine to induce hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in male and female rats in order to define the effects of pyridine-containing compounds on drug metabolism. 4-Benzylpyridine (0.4 mmol/kg, for 2 consecutive days) induced total cytochrome P450...

In the hemolymph of the silkworm, Bombyx mori, lectin with hemagglutinating activity against sheep red blood cells increases at larval-larval ecdysis and at spinning stage (Suzuki and Natori, 1983) and is induced by infection with cytoplasmic polyhedrosis virus. A Bombyx lectin polypeptide with molecular weight approx 280K is responsible for hemagg...

We investigated the structure-activity relationship in the induction of hepatic microsomal cytochrome P450 by clotrimazole and its structurally related compounds. For this purpose, we synthesized various compounds structurally analogous to clotrimazole and injected them into rats at a fixed dose of 0.2 mmol/kg. We found that the chlorine atom in cl...

Cyclohexene (I) is oxidized by the reagent system (Fe(Me-CN)6)2+/H2O2/ Ac2O generated in situ to give the diacetoxycyclohexanes (III) and (IV) as the main products.

Oxygenation of methyl O-acetyl-α-cyclohexyl-α-phenylglycolate (4c) with the reagent system Fe(MeCN)2+6-H2O2-Ac2O, a model enzyme system for mono-oxygenase, was investigated in connection with the metabolism of the drug oxybutynin hydrochloride (1) in humans and dogs. Among the oxygenation products 5d, 7, 8, and 9, 5d, 7, and 8 correspond to the mam...

Treatment of the thioacetals (I), (IV), and (VI) with the iron(III) complex (II) yields the corresponding carbonyl compounds (III), (V), and (VII) respectively.

The diaryl propanes (I) undergo oxidative C-C-coupling in the presence of Fe(III) complexes (II) to yield the spiro cyclohexadienones (III).

Mit dem ausEisenperchlorat, Acetonitril, H2O2 und Acetanhydrid gebildeten Reagens werden aromatische und cycloaliphatische Kohlenwasserstoffe wie (I), (V) und (IXa) zu entsprechenden Acetaten und Diacetaten oxidiert.

Mit dem aus (II) und (III) in Acetonitril und Acetanhydrid gebildeten Reagens werden Kohlenwasserstoffe wie (I), (Va), (VI) oder (VIII) zu den entsprechenden Acetoxy- bzw. Acetamino-Derivaten (IV), (Vb), (VII) bzw. (IX) oxidiert.

Die Oxidation primärer Alkylbenzole (I) mit zwei Moläquivalenten des im Titel genannten Reagens (II) führt zu den entsprechenden Acetamiden (III).