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Bombyxin-II, an insulin superfamily peptide of the silkmoth Bombyx mori, and its disulfide bond isomers have been synthesized by two ways of stepwise, semiregioselective disulfide bond formation. The disulfide bond CysA20-CysB22 or CysA7-CysB10 was formed first, and then the two other disulfide bonds were formed by iodine oxidation. The conditions for the iodine oxidation were improved to suppress oxidative degradation of unprotected Trp residues. With these conditions, bombyxin-II was synthesized in high yields (26% and 32%). Its disulfide bond isomers were also obtained. Specific activity of the products indicates that the disulfide bond CysA20-CysB22 is important to the bombyxin activity.

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm, Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for alpha-amino group. Three disulfide bonds were bridged step by step (A6-A11, A20-B22, and A7-B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.

Publisher Summary The prothoracicotropic hormone (PTTH) stimulates a paired thoracic endocrine organ to synthesize and release ecdysone, a steroid indispensable for insect development. Ecdysone plays a central role in the endocrine network controlling insect development; however, its chemical structure has long been undetermined. This chapter describes the progress made toward the determination of the primary structure, gene structure, and cellular localization of the PTTH of the silkmoth Bombyx mori , and another Bombyx brain peptide functionally related to PTTH, bombyxin. By using bombyxin antibody, the immunoreactive materials have been detected in the central nervous system of the oligochaete Eisenia foetida , Locusta migratoria , the wax moth Galleria mellonella , and Drosophila. However, studies on physiological and developmental regulations, which are under the control of these peptides, and the mechanism of evolution of the insulin superfamily peptides, which are now thought to occur probably throughout the animal kingdom, may open up new avenues for insulin research.

We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) alone and PEG-IFN-alpha2b plus 5-fluorouracil (5-FU) in vitro on the proliferation of renal cell carcinoma (RCC) cell lines. After the transplantation of RCC cells into nude mice, we administered IFN (PEG-IFN-alpha2b or IFN-alpha2b) alone, 5-FU alone, or IFN (PEG-IFN-alpha2b or IFN-alpha2b) plus 5-FU; and investigated tumor volume, tumor weight, the numbers of apoptotic cells and artery-like blood vessels, relative mRNA expression levels of enzymes which relate to 5-FU metabolism, angiogenesis factor, and type I interferon receptor. RCC cells in vitro were generally and relatively resistant to the anti-proliferative effects of PEG-IFN-alpha2b, but the addition of 5-FU augmented IFN-induced anti-proliferative effects with the induction of apoptosis. PEG-IFN-alpha2b in vivo presented stronger anti-tumor effects than IFN-alpha2b, and its combination with 5-FU augmented the effects. The significant anti-tumor effect of the combination treatment was the increase in apoptotic cell number, but there were no significant differences in the suppression of angiogenesis, expression of IFN receptor, and the actions of metabolic enzymes of 5-FU. In conclusion, PEG-IFN-alpha2b presents stronger anti-tumor effects than non-pegylated IFN, and the effects are augmented in the combination with 5-FU. Our findings suggest the clinical usefulness of PEG-IFN-alpha2b in the treatment of RCC.

Adult eclosion of the silkworm, Bombyx mori, occurs during a specific period of the day under the control of eclosion hormone (EH). Bombyx EH was extracted from the pupal and adult brains (Fugo, et al., 1982), and also the embryos of B. mori (Chen, et al., 1986). In 1985, Bombyx EH was first isolated from the pharate adult heads of B. mori (Nagasawa, et al., 1985). Recently the entire amino acid sequence of Bombyx EH was determined as shown in Fig. 1 through the combination of sequencing the isolated Bombyx EH (Kono, et al., 1987) and cloning the Bombyx EH gene. A monoclonal antibody against the synthetic peptide corresponding to the carboxyl-terminal 13 amino acid residues of Bombyx EH was prepared (Kono, et al., in press).

From 1.2 × 106 male adult heads of the silkworm, Bombyx mori, 0.16 mg of bombyxin-IV was isolated, which was a novel molecular species of bombyxin, previously called 4K-prothoracicotropic hormone. Injection of 0.1 ng of bombyxin-IV into a dauer pupa of the Eri-silkworm, Samia cynthia ricini, evoked adult development. Microsequencing, chemical modification, enzymatic digestion, and fast atom bombardment mass spectrometry showed that bombyxin-IV consisted of two heterogeneous peptides linked by disulfide bonds and had a considerable sequence homology (62.5%) with bombyxin-II.

The temporal organization of secretion of the prothoracicotropic hormone (PTTH) and ecdysone during larval-pupal development of Samia cynthia ricini was studied by ligations, with particular attention to the circadian control of the timing of hormone release. PTTH and ecdysone are required first for the induction of prodromes of pupation and again later for pupal-cuticle formation. PTTH release in the first step occurs during the second or third photophase after the last-larval ecdysis under a photoperiod of 12 hr light and 12 hr darkness and is thought to be under the control of a circadian clock. Ecdysone release follows 1.5 days later, i.e. during the scotophase that precedes the gut purge. In the second secretory step, PTTH is released 2 days after purging the gut, and ecdysone release follows 6 hr later. The PTTH release at this time occurs at a fixed time after the gut purge irrespective of light conditions, accounting for light insensitivity of the timing for pupal ecdysis. Possible mechanisms relating to the inconsistent association of a circadian clock with PTTH release, and those underlying the determination of timing of the gut purge are discussed.

A simple and highly reproducible procedure for partial purification of prothoracicotropic hormone (PTTH) was established starting with 96,000 male adult Bornbyx heads. Approximately 28,500-fold pulification of PTTH was accomplished with a yield of about 50% and 6 ng of the most purified preparation (“highly purified PTTH”) caused adult development in a brainless Samia pupa. The peptidal nature of PTTH was reconfirmed through the effects of various enzymatic and chemical treatments on the biological activity of “highly purified PTTH”. Gel-filtration indicated the molecular weight of PTTH to be 4,400. Several investigators have previously attempted to isolate the prothoracicotropic hormone (PTTH, brain hormone) (2, 3, 6, 8, 9, 11, 13, 14) and the accumulated data implies that PTTH is a protein or peptide. In 1967, ISHIZAKI and ICHIKAWA (6) achieved about 8,000-fold purification of PTTH from 118,000 pupal brains of Bombyx mori and the most purified preparation caused adult development of Sumiu pupa at a dose of 2ng. However, their purified PTTH still seemed to contain impurities, and consisted of heterogeneous molecules with molecular weights of 9,000 to 31,000. Later, YAMAZAKI and KOBAYASHI (13) also purified PTTH from 100,000 brains of Bombyx pupae and 20 ng of their preparation could cause adult development in a brainless Bombyx pupa, but their preparation appeared impure by electrophoresis. These reports indicate that the brain of Bombyx pupa contains only a small amount of PTTH and that. 100,000 brains is insufficient starting material for isolation of pure PTTH. In 1969, ISHIZAKI (5) reported that the head of the Bombyx moth contains more PTTH than that of the pupa, and later NISHIITSUTSUJI-UWO (1 I) obtained partially purified PTTH from the heads of adult Bombyx which caused adult development in a brainless Samia pupa at a dose of 1.1 pg. We have established a simple procedure for partial purification of PTTH from the heads of Bombyx adults. The preparation termed “crude PTTH” showed activity on a Sumiu pupa at a dose of 1-2 pg (12). In the preceding paper, we reported the effects of various enzymes and chemicals on “crude PTTH”, concluding that PTTH is peptidal and probably contains basic and aromatic amino acids (7). Recently, we prepared “highly purified PTTH” from the 96,000 heads of male Bombyx moths, and found that its specific activity was comparable with that reported by ISHIZAKI and ICHIKAWA (6). This paper describes the preparation of “highly purified PTTH” and studies on its properties.

Under a 12-hr light and 12-hr dark photoperiod, haemolymph ecdysteroid titre of the last(5th)-instar larva of Samia cynthia ricini begins to rise in the early scotophase preceding gut purge, which marks the larval-prepupal transition, to reach a peak titre of 7.6 ng/ml ca. 4.5 hr before gut purge. This profile of ecdysteroid increment is phase-shifted in response to phase shifts of the scotophase immediately before gut purge, in parallel with gut-purge phase shifts, suggesting that ecdysone release is under the control of a circadian clock dictating gut-purge timing. Ecdysone and 20-hydroxyecdysone (10–20 μg), injected no later than 18 hr before the normal gate of gut purge induced well-defined peaks of precocious gut purge ca. 8 hr after injection. Earlier injections caused acceleration of gut purge but the degree of acceleration was unpredictable. These results suggest that the timed surge of ecdysteroids is responsible for the gated occurrence of gut purge and that 18 hr before gut purge larvae acquire the competence to undergo gut purge in a gated fashion provided that they are exposed to a sufficient surge of ecdysteroids.

Crude extracts of Bombyx mori brains can provoke adult development when injected into brain-removed dormant pupae of Bombyx mori and Samia Cynthia ricini. From this fact the prothoracicotropic hormone (PTTH) of Bombyx has long been thought to be species-nonspecifically active on Samia. Chemical fractionation of Bombyx brain or head extracts by fractional precipitation with acetone, Sephadex G-50 gel-filtration, and DEAE-Sepharose CL-6B chromatography, however, separated the fractions which activated Bombyx brainless pupae from those which activated Samia. Those results reveal the existence of two species-specific PTTHs.

Observations on the timing of ecdysis and neck ligation experiments on larvae of Samia cynthia ricini under various light-dark conditions show that an endogenous circadian clock controls the timing of larval ecdysis and prothoracicotropic hormone (PTTH) secretion preceding it. The clock, upon reaching a specific phase point, causes the brain to secrete PTTH provided that the brain has acquired the secretory competence. This time may vary, in relation to a previous ecdysis, according to the light-dark conditions by which the clock phase is specifically determined, but is fixed relative to a subsequent ecdysis. Thus, in the case of the ecdysis to the 5th instar, PTTH is secreted [15+nτ] hr (τ: free-running period, slightly less than 24 hr) after the clock has started when the rhythm is free-running, and in the second and third nights of the 4th instar under a photoperiod of 12 hr light and 12 hr dark. Full secretion of ecdysone occurs 6 hr after PTTH secretion and ecdysis ensues 34 hr thereafter to complete the ultimate sequence of ecdysis.

Vitellogenin was immunohistochemically demonstrated in the ovary and fat body during a reproductive cycle. In terminal oocytes of 4-day adults, vitellogenin began to appear in yolk spherules at the cell periphery. The vitellogenin-containing spherules increased in size and number to occupy the whole cell 2 days later. In the female fat body, vitellogenin began to appear in 3-day adults. It was distributed diffusely in the cytoplasm, at a much lower concentration than in the oocytes, during the vitellogenic period. In 11-day adults whose vitellogenesis had terminated, a higher concentration of vitellogenin was found in the cytoplasmic inclusions of the fat body. Vitellogenin was not detected in the male fat body.

One molecular species of prothoracicotropic hormone with a molecular weight of about 22, 000 (22K-PTTH) of the silkworm, Bombyx mori, was isolated from 5 × 105 adult heads. The purification procedure consisted of 16 steps including defatting, salt-extraction, fractional precipitations, conventional column chromatographies, and high performance liquid chromatographies. An approximately 5 × 106-fold purification was attained to yield 5.4 μg (0.25 nmol) of the pure hormone with a recovery of 3.3%. Injection of 0.11 ng of the purified 22K-PTTH could elicit adult development in a brainless Bombyx pupa. 22K-PTTH is a basic protein (pI 7.7 ~ 8.7) containing disulfide bond(s). The amino-terminal amino acid sequence of 22K-PTTH was determined to be Gly-Asn-Ile-Gln-Val-Glu-Asn-Gln-Ile-Pro-Asp-Pro-.

Bombyxins have been isolated from the heads silkworm, Bombyx a, and stimulate the adult formation of the debrained pupae of Cynthia ricini. bombyxins are t he first insulin-related p eptides isolated from the invertebrate animal. The amino acid sequence of bombyxins has suggested that

Vitellogenin in the eggs of Blattella germanica was solubilized with solutions at high salt concentrations and high pH. This protein was purified by ammonium sulfate precipitation, acetic acid precipitation, DEAE-cellulose chromatography, and hydroxylapatite chromatography, into a chromatographically homogeneous state. By sucrose density gradient centrifugation, the purified vitellogenin was resolved into two components. The relative amounts of the two components varied according to the pH of the solution. An equilibrium seemed to exist in the interconversion between them when the conditions of the solution were fixed. It is suggested that aggregation and disaggregation of the vitellogenin molecules may account for the apparent heterogeneity.

Experiments using various light-dark (LD) conditions demonstrated that an endogenous circadian clock controls gut-purge timing in the saturniid mothSamia cynthia ricini. A phase-response curve (PRC) based on the application of brief (15 min) light pulses is used to characterize the underlying pacemaking oscillation. The entrainment of the pacemaker to various LD cycles is interpreted in terms of this PRC. The effect of light immediately preceding gut purge was analyzed to account for the deviation of the actual gut-purge rhythm from the prediction made by considering only the action of the oscillation. Lack of precision in gut-purge timing in LD cycles with a very short scotophase has been explained by the failure of the oscillation in these conditions to attain the specific phase-point at which the clock information dictating gut-purge timing is released.

A genomic DNA encoding bombyxin, a 5kD brain peptide of the silkmoth Bornbyx mori with prothoracicotropic hormone activity, has been isolated. The nucleotide sequence coding for bombyxin shows high homology with insulin-gene family members and the overall organization of the preprobombyxin gene is the same as in preproinsulin genes, indicating that bombyxin shares a common ancestral molecule with insulin-family peptides. The bombyxin gene has no intron contrasting to other members of insulin-gene family.

We have determined the complete amino acid sequence of 4K-PTTH-II, one of three forms of the M r 4400 prothoracicotropic hormone of the silkworm Bombyx mori , active to brainless pupae of Samia cynthia ricini . Like vertebrate insulin, it consists of two nonidentical peptide chains (A and B chains). The A chain consists of 20 amino acid residues. The B chain is a mixture of four microheterogeneous peptides, two of which consist of 28 residues, and the other two, of 26 residues. 4K-PTTH-II has considerable sequence homology (40%) with human insulin, and it resembles porcine relaxin both in the carboxyl-terminal cysteine residue of the A chain and in the amino-terminal pyroglutamic acid residue of the B chain. The identical distribution of the six cysteine residues also indicates that 4K-PTTH-II belongs to the insulin family.

Summary An assay for the prothoracicotropic hormone (PTTH) has been established using in situ activation of the prothoracic glands (PG) of Bombyx mori in its larva-to-larva development. The timing of PTTH release was estimated by examining developmental response of 4th instar larvae to brain removal and neck ligation, and changes in the haemolymph ecdysteroid titer and ecdysone-releasing activity of PG in vitro during the development. Injection of Bombyx brain extracts into 4th instar larvae neck-ligated shortly before full activation of PG elicited larval moulting rather than precocious pupation in headless larvae. This developmental shift was regarded as due to the action of PTTH, and the PTTH unit has been defined from a linear log dose-response relationship. Materials chromatographically fractionated from Bombyx brain extracts were examined for the presence of stage- and species-specific PTTH molecules by using this Bombyx larva assay and Bombyx and Samia pupa assays previously developed. The same fra...

A simple procedure for partial purification of silkworm brain hormone starting with heads of Bombyx moths is described. The method is unique in that it can supply samples with a definite activity without chromatography and bioassay during the purification process. The titer of brain hormone in every batch is 30~40 Samia units/head (specific activity, 1~2μg/Samia unit). Some chromatographic behaviors of the hormone are also reported.

Blockage of adult development by brain removal and its resumption by application of the prothoracicotropic hormone (PTTH) were studied using pupae of a racial hybrid J-122 × C-115 of Bombyx mori. A log-linear dose-response relationship was obrained after injection of a PTTH solution. The Bombyx-unit of PTTH has been defined from this dose-response curve.

The effects of various enzymatic and chemical treatments on the biological activity of the partially purified PTTH are described. PTTH was inactivated by pepsin, trypsin and α-chymotrypsin. Leucine aminopeptidase and carboxypeptidase A did not affect the biological activity of PTTH, suggesting that N- and C-terminus are blocked. Treatments with chemical reagents suggest that the tryptophan residue and disulfide bond are essential for the activity, whereas the sulfhydryl group is not. Tyrosinase exerted no effects. Glycosidases, neuraminidase, and periodate oxidation did not affect the activity, suggesting that carbohydrates are not essential for the biological activity of PTTH.

The occurrence of developmental arrest after brain removal in pupae of Bombyx mori was examined using a racial hybrid Chinese No. 115 × Japanese No. 122. The results are as follows: (1) Adult development was blocked for a long period in most insects debrained shortly after larval-pupal ecdysis; (2) the earlier the brain removal, the more arrested pupae were obtained; (3) the critical period of brain hormone secretion for adult development was earlier in the female than in the male; (4) the developmental arrest which had been induced in female pupae tended to terminate earlier than that in males; (5) the developmental arrest which was induced by extremely early brain removal terminated earlier than that obtained by later brain removal.

1. The brain hormone extracted from brains of Bombyx mori was purified by use of heating, ammonium sulfate precipitation, Sephadex gel-filtration, and DEAE-cellulose chromatography. On the basis of protein measurement, about 8000-fold purification was achieved. The most purified preparation was active by 0.002 µg. protein when injected into brainless pupae of Samia cynthia ricini.2. The brain hormone manifested highly heterogeneous molecular forms which were revealed by Sephadex gel-filtration and by DEAE-cellulose chromatography. The molecular weights of the major components were estimated by Sephadex gelfiltration as ranging from 9000 to 31,000.

Electron microscopic observations were made on the fat-body cells of Philosamia cynthia ricini during the development from the early fifth instar to the early stage of adult development. The cells of the young fifth-instar larva possess numerous mitochondria and ribosomes, an endoplasmic reticulum and a Golgi complex. Fat droplets are the only cytoplasmic inclusion which is structurally recognized. About 1 day before the larva matures, glycogen-rich granules begin to appear. During the spinning stage, a drastic disappearance of the cytoplasmic organelles occurs. When larvae finish spinning, most of the cytoplasm becomes filled with such inclusions as fat droplets, glycogen-rich granules, and “dark bodies”. Mitochondria and ribosomes in much decreased numbers are left as well-identifiable cytoplasmic organelles. This condition of the cells, attained at the spinning stage, remains unchanged until the initiation of adult development, when the mitochondria and ribosomes increase in number and the endoplasmic reticulum again develops. Subsequently, the “intercellular matrix” covering the cell surface loses its compact texture, and finally the cell itself disintegrates to liberate its contents into the blood. Remarkable changes in the nuclear morphology which occur during development are described.

Four genes encoding bombyxin have been located in a 14-kilobase Bombyx genomic DNA segment. All of these genes encode preprobombyxin, the precursor molecule for bombyxin, with the domain organization of signal peptide/B chain/C peptide/A chain. Bombyxins are classified as family A or B according to their sequence homology. Two genes, each belonging to a different family, are closely apposed to form a pair with opposite orientation, presumably forming a regulatory unit for transcription. Genomic Southern blot hybridization suggested that there are many such gene pairs in the Bombyx genome. Differences between bombyxin genes and vertebrate insulin-family genes indicate that different mechanisms operate in the evolution of invertebrate and vertebrate insulin-family genes.

Three molecular forms of prothoracicotropic hormone were isolated from the head of the adult silkworm, Bombyx mori, and the amino acid sequence of 19 amino acid residues in the amino terminus of these prothoracicotropic hormones was determined. These residues exhibit significant homology with insulin and insulin-like growth factors.

Prothoracicotropic (PTTH) and eclosion (EH) hormones were extracted from adult heads of Bombyx mori and PTTH was isolated by extensive chromatographic purification. A hormone (melanization and reddish coloration hormone, MRCH) inducing cuticular melanization and epidermal reddish coloration in the armyworm larvae of Leucania separata was also extracted from Bombyx adult heads. The chemical and biological properties of these hormones were investigated.

In the moth Samia cynthia ricini , timing of the release of prothoracicotropic hormone is controlled by a circadian clock in the cephalic organ (brain); while this hormone release is necessary for gut purge, the final timing of this event is controlled by a circadian clock in the prothoracic glands that gates release of ecdysone. The photoreceptor of the prothoracic gland clock(s) is extraocular and evidently in the glands themselves. Demonstration that the clock and its photoreceptor are in the prothoracic glands is based on a mixture of localized illuminations and the transplantation of glands from one larva to another.

Monoclonal antibodies were produced by immunizing mice with a synthetic decapeptide corresponding to the N-terminal portion of the A-chain of bombyxin, a peptide from Bombyx mori which activates the prothoracic glands of the saturniid moth, Samia cynthia ricini, and was previously called 4K-PTTH. We obtained a hybridoma clone secreting an antibody that recognized specifically bombyxin after treatments for disulfide-bond reduction but did not when untreated. Immunoblotting studies demonstrated the presence of highly heterogeneous immunoreactive components in Bombyx brain homogenates. Immunohistochemistry using this antibody indicated that bombyxin was produced by four pairs of mid-dorsal neurosecretory cells of the brain and transferred to and released from the corpora allata of Bombyx.

Bombyxin, previously referred to as 4K-prothoracicotropic hormone, is a brain peptide of the silkmoth Bombyx mori, the amino acid sequence of which shows considerable homology with vertebrate insulin family peptides. Two independent clones have been isolated from a Bombyx larval brain cDNA library by using a synthetic oligonucleotide probe, one with the complete coding region for preprobombyxin (lambda Bb360) and the other covering the coding region, possibly for bombyxin, only partially (lambda Bb204). lambda Bb360 encodes preprobombyxin in the order of prepeptide/B-chain/proteolytic cleavage signal/C-peptide/proteolytic cleavage signal/A-chain. This domain organization of preprobombyxin is the same as that of preproinsulins, suggesting that the tertiary structure and posttranslational modification mechanism are conserved through the evolution of bombyxin and insulin. Genomic Southern hybridization analyses using this cDNA as probe suggest that the Bombyx genome contains multiple copies of bombyxin gene. Northern hybridization analyses indicate that the concentration of lambda Bb360-type bombyxin mRNA in the bombyxin-producing cells is remarkably high (2.8 x 10(9) molecules/micrograms of total RNA), without undergoing appreciable change during larval-pupal development.

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 × 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.

Changes in titer of brain hormone (BH) extracted from the brain, head, and thorax and abdomen during the development of Bombyx mori from the 4th larval molting up to 6 days after adult emergence were investigated. The hypothesis is presented that high secretory activity of the brain is not reflected in a corresponding change in its BH titer, while low secretory activity brings about an accumulation of BH in the brain. An interesting finding is that extremely high titers of BH were found in the thorax and abdomen, from 4 days after pupation to adult emergence, of females only. This sexual difference was, however, not found in all of the races examined.

THE insect brain hormone which is known to be secreted from the neurosecretory cells of the pars intercerebralis controls metamorphosis by its prothoracotropic action1. We have recently prepared an extract from brains of Bombyx which can stimulate the non-diapausing pupae of Phylosamia cynthia ricini caused to diapause by prompt removal of their brains just after pupation and beginning adult differentiation2. From the fact that extracts of tissues other than brain such as prothoracic, mesothoracic and metathoracic ganglia and fat body possess no, or far less, activity, and that the extract is ineffective in initiating adult development in the isolated abdomen lacking the prothoracic glands (Table 1), one may reasonably infer that the active principle in the brain extract is brain hormone. Subsequent experiments to purify and identify this hormone have yielded evidence supporting the assumption that brain hormone is a protein.

IT is generally accepted that the neurosecretory cells concerning the metamorphosis of insects contain a small amount of a secretory substance at any time of development1,2, notwithstanding a marked effect which they exert when implanted or transplanted. This means that accumulation of the secretory substance released continuously from the cells in the brain is necessary to become effective. This is the reason why a great many brains were used to extract a sufficient amount of the active principle3. However, we have found that only several devitalized brains of Bombyx mori could cause adult differentiation when inserted into the non-diapausing pupae of Philosamia cynthia ricini caused to diapause by prompt removal of their brains just after pupation. Devitalization was brought about by various ways such as subjecting brains to freezing in a deep-freezer at −18° C. for 21 days, desiccating them over silica gel for one day, or heating them at 90° C. for 90 min. After being treated, the brains were all proved to be as effective as live ones, so far as they were tested by five for each test pupa. Needless to say that in this case the brain hormone, enough to stimulate adult differentiation of the recipient, had to be present in the five inserted brains. The possibility that the adult differentiation was caused by the prothoracic gland hormone which contaminated the inserted brains can be ruled out, since neither the same amount of fatty tissue nor blood similarly treated exerted any perceptible effect on the dormant pupae. It seems, however, worthy of mentioning that after treatment brains of Philosamia were inferior to brains of Bombyx in their effectiveness. This presumably indicates that the situation at a given moment is different between the two species, a larger amount of the hormone being contained in the latter.

The presence of so-called sex chromatin has been demonstrated in the interphase nuclei of the cells of the domestic chicken (Gallus domesticus). Definite sex dimorphism was observed for the incidence of this nuclear component; the frequency of its occurrence in females was at least ten times that of its occurrence in the males, ranging from 22 percent in the duodenal muscle cells to 52 percent in the epidermal cells of a growing feather.