[Androgen-like and anabolic action of Antheraea pernyi Guerin-Meneville Pas].

It has been found that the ethyl acetate extract isolated from Antheraea pernyi Pas is able to increase the weight of prostate-semina and levator ani muscle-bulbocavarnosus muscle of castrated mice. In addition, the extract also accelerates the growth of younger male mice and enhances the contents of RNA, DNA and protein in the liver tissue of mice. It has been determined that beta-ecdysone is one of the effective constituents for the androgen-like and anabolic action.

Identification and purification of a Bombyx mori homologue of FTZ-F1.

Extracts from embryos and from posterior and middle silk glands of the silkworm, Bombyx mori contain a sequence specific DNA binding factor termed BmFTZ-F1. The factor binds to the recognition site of FTZ-F1, a positive regulator of the fushi tarazu gene in Drosophila melanogaster. BmFTZ-F1 and FTZ-F1 share the same methylation interference patterns, the same chromatographic behaviors and similar protease digestion profiles. Anti-FTZ-F1 cross reacts with BmFTZ-F1. These results indicate that BmFTZ-F1 is a B. mori homologue of FTZ-F1. The mobility of the factor-DNA complex formed in the silk gland extract changes depending on the developmental stages. Purification of BmFTZF1 to an almost homogeneous state reveals that the factor is a 73 kd protein.

The development of identified neurosecretory cells in the tobacco hornworm, Manduca sexta.

The prothoracicotropic hormone (PTTH) is a principal neuropeptide regulator of insect postembryonic molting and metamorphosis. In the tobacco hornworm, Manduca sexta, PTTH is produced by two neurosecretory cells (NSC) located in each protocerebral lobe of the brain. The development of these neurons, the L-NSC III, has been investigated immunocytologically to establish the time course of their morphological differentiation. PTTH may be one of the earliest neuropeptides expressed in insect embryos. PTTH-immunoreactivity was initially detected in the somata at 24 to 30% of embryonic development. Neurites sprouted shortly thereafter and began to grow medially through the brain anlage. By 42% embryonic development, the neurites had decussated to the contralateral brain lobe. As development progressed, the L-NSC III neurites grew along specific tracts through the contralateral brain lobe reaching the ventrolateral regions of the brain by approximately 60% development. The axons exited the brain through a retrocerebral nerve, the nervi corporis cardiaci I + II. At approximately 63% development, the axons innervated the corpus allatum and began branching to form neurohemal terminals for PTTH release. At 60% development, short collaterals began extending in the protocerebral neuropil. During the remainder of embryogenesis, both the dendritic collaterals and the terminal neurohemal varicosities continued to elongate and arborize. By 85% embryonic development, the basic architecture of the L-NSC III was established.

Isolation and structure of cecropins, inducible antibacterial peptides, from the silkworm, Bombyx mori.

1. Cecropins, inducible antibacterial peptides, were purified by simple two step chromatography from immunized larval hemolymph of the silkworm, Bombyx mori. 2. The cecropins were further separated into four major and two minor forms by reverse-phase HPLC. 3. The four major cecropins were sequenced and divided into two types, A and B, whose structures were quite similar to cecropins A and B of Hyalophora cecropia. 4. Three of them contained an unusual amino acid, delta-hydroxylysine. 5. No remarkable difference in antibacterial activity against E. coli was detected among these cecropins.

Amino acid sequence of pheromone-biosynthesis-activating neuropeptide (PBAN) of the silkworm, Bombyx mori.

We have isolated two distinct pheromone-biosynthesis-activating neuropeptides (PBAN), named PBAN-I and -II, as fully oxidized forms of Met residues from adult heads of the silkworm, Bombyx mori. PBAN-I was identical with the PBAN which we had isolated before. The complete amino acid sequence of PBAN-I, a total of 33 amino acid residues, was determined as H-Leu-Ser-Glu-Asp-Met-Pro-Ala-Thr-Pro-Ala-Asp-Gln-Glu-Met-Tyr-Gln-Pro-As p-Pro- Glu-Glu-Met-Glu-Ser-Arg-Thr-Arg-Tyr-Phe-Ser-Pro-Arg-Leu-NH2. Synthetic PBAN-I after oxidation with H2O2 was chromatographically identical with the isolated PBAN-I. Examination of PBAN activity of synthetic analogues indicated that the carboxyl-terminal portion of PBAN-I was important for biological activity.

Complex formation with the fibroin gene enhancer through a protein-protein interaction analyzed by a modified DNA-binding assay.

The distal upstream region of the fibroin gene, -238 to -73, is known to contain the cis-acting element(s) responsible for tissue-specific transcription enhancement in vitro. To identify factor(s) trans-acting on this region, we have developed a modified nitrocellulose blot protein-DNA binding procedure that detects DNA-protein complexes via protein-protein interaction. A protein that does not bind to DNA as a monomer cannot be detected by standard Southwestern blotting. To detect protein-DNA complexes involving both protein-DNA and protein-protein interactions, the protein blot was reacted with the extract protein to form a protein complex on the filter, in the presence of the DNA probe. This new method revealed that the 75-kDa protein binds to the enhancer region of the fibroin gene in the presence of the second protein(s). The 75-kDa protein and the factor(s) mediating DNA binding activity were recovered in two different column fractions. The level of the 75-kDa protein appeared to be much higher in silk gland extracts than in nonsilk gland extracts, whereas the mediating factor(s) distributed evenly. These observations suggest that the distal upstream region of the fibroin gene can be specifically recognized by a protein complex composed of at least two distinct proteins, through protein-protein interaction.

Purification of a beta-1,3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm, Bombyx mori.

The plasma fraction (referred to as plasma-CPB) of silkworm hemolymph, from which a protein with affinity to beta-1,3-glucan was specifically removed according to Yoshida et al. (Yoshida, H., Ochiai, M., and Ashida, M. (1986), Biochem. Biophys. Res. Commun. 141, 1177-1184), was used to develop a method for quantitating the beta-1,3-glucan recognition protein of the prophenoloxidase activating system. In principle, a sample was judged to contain beta-1,3-glucan recognition protein when that sample could restore the ability of the system in plasma-CPB to be triggered by beta-1,3-glucan. Purification procedures for the recognition protein from silkworm hemolymph consisted of fractionation with ammonium sulfate, chromatography on DEAE-Toyopearl, Affi-Gel-heparin, and Mono Q and Superose 12 on the fast protein liquid chromatography system of Pharmacia LKB Biotechnology Inc. About 2.03 mg of beta-1,3-glucan recognition protein was obtained from 300 ml of hemolymph. The purified beta-1,3-glucan recognition protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. beta-1,3-Glucan recognition protein had a molecular mass of 62 kDa composed of a single polypeptide and an isoelectric point of pH 4.3. It bound to curdlan beads (composed of beta-1,3-glucan with average particle size of 80 micron) in the absence of divalent cation, whereas its binding to glucans with beta(1----4)- or alpha(1----6)-glycosidic linkages was not detected under the experimental conditions. Elution of the beta-1,3-glucan recognition protein bound to curdlan beads could be achieved under strongly denaturing conditions (after incubation of the beads with sodium dodecyl sulfate and beta-mercaptoethanol in boiling water for 5 min), but elution at room temperature was poor. Since beta-1,3-glucan recognition protein is the only protein in silkworm plasma with strong affinity to beta-1,3-glucan and endows the prophenoloxidase activating system in plasma-CPB with the ability to be triggered by beta-1,3-glucan, it was concluded that binding of the purified beta-1,3-glucan recognition protein with beta-1,3-glucan causes the triggering of the prophenol-oxidase activating system in silkworm plasma. However, the nature of the activity that is generated as the result of binding is not yet known. The purified beta-1,3-glucan recognition protein bound to beta-1,3-glucan did not hydrolyze appreciably any of the 26 commercially available peptidyl-7-amino-4-methylcoumarins, substrates for various proteases.

Interaction of composite protein complex with the fibroin enhancer sequence.

To characterize proteins that bind to the upstream region of the fibroin gene, we used an electrophoretic mobility assay and a DNase I footprinting assay. A crude nuclear extract from the posterior silk glands forms a sequence-specific complex with the upstream region, -234 to -66, which includes a signal for tissue-specific transcriptional enhancement. This specific complex is composed of at least two factors of proteinous nature and the DNA. The composite proteins specifically protect an approximately 13-base pair (-167 to -155) region in a DNase I footprinting assay. This region contains an interesting repeated sequence, AATTTAATTT. Crude nuclear extracts from the middle silk glands and the ovarian tissues, but not a HeLa whole cell extract, also give the band complexes of similar electrophoretic mobility to the one formed in the posterior silk gland extract. However, under a higher EDTA condition, these complexes are more unstable than the one constructed in the posterior silk gland extract. These results suggest a possibility that these proteinous molecules in the complex, although apparently ubiquitous in Bombyx cells, might be specifically modified in the posterior silk gland cells to play an important role in the specific expression of the fibroin gene.

Small RNAs of the silkworm, Bombyx mori as revealed by in vitro capping and in vitro transcription.

1. We characterized several small RNAs in various tissues and strains of the silkworm, Bombyx mori by the in vitro capping and in vitro transcription. 2. Three subspecies of 7SL RNA, which is believed to be involved in protein secretion, were detected in the commercial strains. Structural differences among the three molecules were due to the nucleotide substitution at two positions or more. 3. A very AU-rich RNA, which is about 500 bases long, was always present in any tissue of Bombyx mori while 55 base RNA was only observed in a substantial amount in the fat body and culture cells. 4. These small RNAs were also observed among the in vitro transcripts of total Bombyx DNA in the cell-free extracts from the silkgland and fat body.

Small-angle x-ray scattering study of insect lipophorin.

The structure of lipophorin in insect blood (hemolymph) was investigated by a small-angle x-ray scattering method over the temperature range 0-45 degrees C. The small-angle x-ray scattering profile of lipophorin exhibited a symmetrical sphere with heterogeneous internal electron density. Cockroach and locust lipophorins, which contain hydrocarbons, demonstrated centrosymmetrical distribution of electron density inside the particles. A previous study suggested that the hydrocarbon-rich region is located in the core of lipophorin particle (Katagiri, C., Kimura, J., and Murase, N. (1985) J. Biol. Chem. 260, 13490-13495). Distance distribution functions, P (r), calculated for a simulated three-layer model (electron-rich shell, middle layer, and electron-deficient core) with radial electron density distribution, show good agreement with those observed experimentally for cockroach and locust lipophorins. The dimensions and electron density obtained for the middle layer reveal that this layer is occupied mainly by diacylglycerol and apolipophorin II. Thus, the present study together with previous reports strongly suggest that insect lipophorin is composed of centrosymmetrical three layers; an outer shell with apolipophorin I and phospholipid, a middle layer with diacylglycerol and apolipophorin II, and a core with hydrocarbons.

A rapid and sensitive method for the estimation of individual tRNA pools.

A rapid and sensitive method is described to quantitatively compare tRNA pools for individual aminoacids in a single experiment. The procedure comprises of: charging of total tRNA with a mixture of radiolabeled aminoacids, deacylation of the esterified tRNA with a volatile base and the recovery of the labeled aminoacid, derivatisation of the aminoacid with phenylisothiocyanate after mixing with excess of nonradioactive aminoacids, baseline separation of the phenylthiocarbamyl aminoacids by reverse phase high performance liquid chromatography monitored by A254nm and quantitation of the radioactivity in individual aminoacid peaks. The radioactivity in the aminoacid peak corresponds to the quantity of the aminoacylated tRNA. The method has been successfully applied to quantitate the individual tRNA pools in the developing silk glands of Bombyx mori, a functionally adapted tissue which undergoes considerable variations in tRNA content.

Amino acid periodicities and their structural implications for the evolutionarily conservative central domain of some silkmoth chorion proteins.

The central domain is an evolutionarily conservative region that is invariant in length in the A and Hc-A families of silkmoth chorion proteins. This domain shows strong sixfold periodicities for various amino acid residues, such as glycine and large non-polar residues. The periodicities and their phase relationships, together with the documented prevalence of beta-sheets and beta-turns in the chorion, strongly support a secondary structure model in which short (4-residue) beta-sheet strands alternate with beta-turns, forming a compact antiparallel, probably twisted beta-sheet. This structure should be important for the establishment of higher order structure in the chorion.

Identification of estradiol in the ovaries of the silkworm, Bombyx mori.

Estradiol was extracted and partially purified from the ovaries of the silkworm, Bombyx mori. Identification of estradiol was done by use of radioimmunoassay (RIA) and by gas chromatography-mass spectrometry (GC-MS) after derivatization into the ethyldimethylsilyl derivative. Concentration of estradiol in the ovaries was estimated to be 176 pg/g (RIA) and 63 pg/g (GC-MS).

Isolation and partial characterization of U1-U6 small RNAs from Bombyx mori.

We have used a variety of techniques to characterize the U-series small nuclear RNAs from the posterior silk gland of Bombyx mori. Six molecular species have been identified which correspond to the vertebrate U1-U6 RNAs by the following criteria: (a) presence of the RNAs in ribonucleoprotein particles which can be immunoprecipitated by lupus Sm antisera; (b) presence of a 2,2,7-trimethylguanosine cap, as assayed by immunoprecipitation with anti-2,2,7-trimethylguanosine IgG; (c) size, as assayed by acrylamide/urea gel electrophoresis using HeLa cell U-RNA markers; and (d) primary nucleotide sequence, as determined by chemical/enzymatic cleavage of end-labeled molecules. The high conservation of primary sequence (66-81% homology based on partial sequences) relative to the corresponding vertebrate U-RNAs has permitted unambiguous identification of each molecule. With the exception of two subspecies of U3 RNA, the U-snRNAs of Bombyx exhibit a striking conservation of secondary structure relative to the proposed structures of the U-RNAs of vertebrates. This conservation is best exemplified by several compensatory base alterations that result in the maintenance of hairpin structures. These are particularly evident in U1 and U5 RNAs. Bombyx U3 is interesting in that two subspecies (of a total of four that were sequenced) diverge considerably in sequence (and presumably in structure) relative to the U3 RNA of vertebrates. The most abundant U-RNAs in the posterior silk gland appear to be U1 and U2, while U3-U6 are present in relatively small amounts.

Genetic variants of protease inhibitors against fungal protease and alpha-chymotrypsin from hemolymph of the silkworm, Bombyx mori.

Many electrophoretic variants of hemolymph inhibitors of proteases from Aspergillus melleus and pancreatic alpha-chymotrypsin were found using 126 silkworm strains. Six inhibitors of the fungal protease were detected and eight of chymotrypsin; the distribution of inhibitors among Japanese, Chinese, and European races was investigated. Comparison of electrophoretic patterns from F1 hybrids and parents showed that the offspring produce inhibitors of both parental types. Segregation in F2 and backcrossing suggest that the expression of each inhibitor is controlled in most cases by a pair of alleles which are responsible for strong and null bands. Two bands of fungal protease inhibitors C and D were controlled by codominant alleles. These results suggest that polymorphism of hemolymph protease inhibitors in the silkworm would be a useful experimental system for the study of the genetic control of protease inhibitors.

Isolation and some characterization of the prothoracicotropic hormone from Bombyx mori.

The prothoracicotropic hormone (PTTH) was isolated from adult heads of Bombyx mori. Fifty micrograms of pure PTTH was obtained from 648,000 heads through a 15-step purification procedure with a 2 X 10(6)-fold purification and an 8% recovery. Chemical analyses of this PTTH have shown that it is a single-chain peptide consisting of 40-43 amino acid residues (MW, 4330-4740), the N-terminus of which is glycine. As little as 0.1 ng of PTTH elicited adult development in a debrained pupa of Samia cynthia ricini. Five picograms of PTTH directly stimulated the prothoracic glands in vitro so as to enhance ecdysone release. The hemolymph ecdysteroids of brainless Samia pupae that were developed by PTTH injection increased with essentially the same pattern as in developing normal pupae.