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Bombyxin-IV, a molecular species of bombyxin, which is a member of insulin-like heterodimeric peptides of the silkworm Bombyx mori with prothoracicotropic hormone activity, was synthesized. The A- and B-chains of bombyxin-IV containing four and two Cys residues, respectively, were first synthesized separately by solid phase chemistry using Boc protocol. Then they were coupled by stepwise removal of two different protecting groups at the cysteinyl thiols for semiselective formation of disulfide bridges to give bombyxin-IV in 8% yield. The synthetic bombyxin-IV was shown to have chromatographic and biological properties identical with those of natural bombyxin-IV.

すでに,筆者らは化学物質を経口投与することによって,4眠性の交雑種から3眠蚕を出現させうることを,コウジ酸やその関連物質で見い出していたが,今回,アビエチン酸と37種の関連化合物および16種のヒドロフルオレン系化合物の3眠蚕出現活性を調べたところ,それらのあいだに,コウジ酸より強い活性を示す化合物を見い出すことができた。すなわち,供試化合物を200ppm添加した人工飼料を4齢起蚕より連続投与したところ,アビエチン酸誘導体で3種,ヒドロフルオレン系化合物で11種のものが3眠蚕を出現させた。そして,これらの化合物のあいだで,構造-活性相関について若干の知見が得られた。上記14種の化合物のうち,9種類については,100ppmでも3眠蚕出現活性が認められた。最も強い活性を示した〔A-11〕 (methyl 6, 7-dioxo-5α, 10α-podocarpa-8, 11, 13-trien-15-oate)は,50ppmでも3眠蚕を出現させた。また,この化合物の100ppm添加飼料を3齢起蚕より連続投与したところ,無添加区が5齢になった時点でも,多くの幼虫が3齢のままで生存することがわかった。

Rhodotorucine A is a peptidyl factor which induces mating tube formation in Rhodosporidium toruloides . The amino acid sequence of the factor was determined by Edman degradation and enzymatic hydrolysis. Rhodotorucine A was shown to contain a lipophilic amino acid, S-farnesyl cysteine, at C-terminus by proton magnetic resonance, mass spectrometry and chemical synthesis. We proposed the following structure for rhodotorucine A . H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-farnesyl)-OH

The structures of ezomycins A1. and A2, antifungal antibiotics produced by a strain of Streptomyces, were determined as 1 and 2, respectively, by degradative and spectrometric studies.

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.

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.

117種の植物の地上部より得られたメタノール抽出物を人工飼料に混入し,4令起蚕に連続投与して6日間飼育し,カイコの成育におよぼす影響を調べ,その程度によって供試植物を5群に区分けした。6日間の飼育中にすべての幼虫が死亡したI群の植物は34種,成育の著しく劣ったII群は16種であった。ついで,III群は19種,IV群は22種で,ほとんど影響の見られなかったV群は26種であった。植物の分類学的位置とそれらがカイコの成育におよぼす影響との関係を,同一の科で三種以上を試験した植物について調べたところ,ミカン科,ツツジ科,スギ科,モクレン科,ウコギ科には強い悪影響が認められた。これに対して,ユリ科,セリ科,ショウガ科にはほとんど影響が見られなかった。キク科では悪影響の強いものと弱いものとに分かれた。成育に対してはなんらかの影響を示した植物中より25種を選び,それらのメタノール抽出物を分画して活性区を追求したところ,中性区のみに活性のみられたものは10種,水溶性区5種,塩基性区2種,不溶解性区1種で,残り7種は二つ以上の分画に活性が認められた。

From the phytopathogenic fungus, Colletotrichum nicotianae, three phytotoxic substances have been isolated and named colletotrichin, colletotrichins B and C (1, 2 and 3). The structures of these compounds have been elucidated from the physical and chemical evidence. When applied on the tobacco leaves, colletotrichins induced the symptom similar to that of the tobacco anthracnose caused by C. nicotianae. The compounds were also toxic to the lettuce and rice seedlings.

A new insecticidal cyclodepsipeptide, bassianolide (1), was isolated from the mycelia of two entomophagous fungi and its structure was elucidated. Silkworm larvae exhibited an atonic symptom and were killed by administration of 1.

葉を主体とした地上部のメタノール抽出物が,カイコの生育に悪影響を与えることを確認した4種類の植物に含まれる活性成分を単離し,それらの成分のカイコの生育におよぼす影響を調べた。1. キバナオランダセンニチに含まれる活性成分はspilantholであった。spilantholを人工飼料中に200ppm添加すると,すべての幼虫は6日後までに死亡した。2. ジギタリス中の活性成分はdigitoxinにdigitalinが加わったものであると推定された。digitoxinは25ppmで虫体を軟化させ,100ppmで6日後にすべての幼虫を死亡させた。digitalinでは100ppmで虫体の軟化が見られたが,400ppmでも死亡するものはなかった。3. コブシの活性成分としてsesaminとkobusinが単離された。kobusinは新しく見い出された化合物で,400ppm添加飼料を与えると,幼虫に強い生育阻害が見られ,すべてのものが5日後に死亡した。sesaminはkobusinの約1/2の活性を示した。4. キツネノマゴからは活性成分として,justicidin AとBが単離された。justicidin Aでは20ppm添加飼料を与えると6日後にすべての幼虫が,Bではほとんどの幼虫が死亡するという強い活性が認められた。

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.

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-.

A peptidyl factor named α substance-IA, which induces sexual agglutinability in a type cells of the heterothallic Saccharomyces cerevisiae, was found to be composed of twelve amino acid residues. Further, the amino acid sequence was elucidated by the combined application of the Edman-dansyl procedure and the cleavage reaction with cyanogen bromide to be H- His-Trp-Leu-Gln-Leu-Lys-Pro-GIy-Gln-Pro-Met-Tyr-OH.

Summary Earlier investigations had demonstrated that steryl-glucosides may stimulate growth in the Avena coleoptile bioassay ( Kimura et al., 1975). As it was now possible to obtain synthetic substances in larger amounts, the effects of sitosteryl-, campesteryl-, stigmasteryl- and cholesteryl-glucosides were tested over a wide range of concentrations on the growth of Avena -coleoptiles, on the germination of cress seeds ( Lepidium sativum L.) and on the H + -efflux of protoplast suspensions prepared from leaves of Vicia faba L. Avena -assay: concentrations from 0.4 to 200 ppm of steryl-glucoside were tested, in some cases with the addition of 1 ppm IAA. Fig. 2 shows that the dose response curves were mostly biphasic and that the 4 substances tested yielded similar results. Addition of IAA did not markedly change the shape of the curves in relation to the behavior of the control. To explain such dose response curves it is necessary to postulate a multisite system at the site of action, which might be the outer cell membrane. Germination: the percentage of germination was not markedly affected by steryl-glucosides applied in the same concentrations as in the Avena -assay. However the lengths of shoots and roots of 3 days old cress seedlings revealed dose response curves similar to those obtained in the Avena coleoptile test (Fig. 4). Protoplasts: if the mode of action of steryl-glucosides is similar to that of IAA, it must be possible to demonstrate a H + -efflux at the cell membrane. Therefore steryl-glucosides (40 ppm) were applied to a protoplast suspension containing 0.35 M D-mannitol and pH changes were recorded continuously. The results show that already 1.5 hours after application a marked drop in pH was detectable, whereas the pH of the control increased slightly over a period of approximately 7 hours. As the solutions appear to be slightly buffered, it must be born in mind that H + -efflux begins before the first changes can be measured. Addition of 10 −4 M IAA accelerates the efflux of H + , whereas 10 −4 M 2,4-dinitrophenol applied 1 hour prior to the application of the steryl-glucosides inhibits acidification (Fig. 6). As sterols are probably involved in the structure of biological membranes, it is concluded that steryl-glucosides and IAA may act in a similar manner at the outer cell membrane, inducing changes in permeability and hydrogen-ion secretion. It may be that steryl-glucosides or sterols themselves are a kind of secondary messenger affected by hormones, for example by IAA.

The presence of an auxin synergist was confirmed in the neutral fraction of extracts from peanut plants (Arachis hypogaea L.). The main component resposible for the activity was established as stigmasteryl-β-D-glucoside. It promoted markedly the elongation of Avena coleoptile segments induced by indoleacetic acid (IAA), but was only weakly active in the absence of IAA.

Rhodotorucine A, which induces mating tube formation in Rhodospridium toruloides, was found to be a novel peptide composed of eleven amino acid residues with a lipophilic group at the C-terminus. The amino acid sequence of the peptide was determined by Edman degradation and enzymatic hydrolyses. The C-terminus of rhodotorucine A was revealed to be S-farnesylcysteine by proton magnetic resonance, mass spectrometry and chemical synthesis. We proposed the following structure for rhodotorucine A. H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-farnesyl)-OH.

From the antifungal ezomycin complex produced by a strain of Streptomyces were isolated six new components named ezomycins B1, B2, C1, C2, D1 and D2. They are novel pseudouridine-type C-C nucleoside antibiotics, of which B1 is the most active.

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.

After twelve steps of purification, a peptidyl factor named rhodotorucine A*1 was isolated in pure form from a culture filtrate of A type cells of Rhodosporidium toruloides. Rhodotorucine A induced the mating tube formation in a cells, opposite mating type cells of the yeast, at a concentration of 8 ng/ml. A factor in the previous papers is now renamed rhodotorucine A.

Two lignans were isolated from leaves of Magnolia kobus DC. as growth inhibitors on silkworm larvae and structurally elucidated as sesamin (I) and kobusin (II) which has been hitherto unknown.

A new alkaloid named stemospironine (I) was isolated together with stemofoline (II) as the main insecticidal constituents of leaves and stems of Stemona japonica Miq. The absolute stereostructure of I has been determined by X-ray crystallographic analysis. Stemofoline (II) showed a much stronger activity than I against silkworm larvae (Bombyx mori L.) by oral administration.

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.

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.

Cinnzeylanine (1) and cinnzeylanol (2) were isolated from barks of Cinnamonum zeylanicum Nees (ceylon cinnamon). Chemical properties and NMR spectra of the both compounds are disccused in terms of their structures which were determined by the X-ray analysis and chemical reactions. When administered orally with artificial diet, both 1 and 2 killed larvae of silkworm, Bombyx mori L. at a dose of 16 ppm and inhibited larval ecdysis at 2~ 4 ppm.

A new gibberellin C20H26O6, tentatively named “bamboo gibberellin”, was isolated from water extract of bamboo shoots (Phyllostachys edulis) through successive purification procedures: countercurren...

Some different results from previous reports on acid degradation and catalytic reduction studies of the three components were obtained. From gibberellin Al only gibberellin C and no gibberic acid were obtained. From gibberellic acid (namely gibberellin X and A3) only gibberic acid and no gibberellin C were obtained. In addition to this, some experimental results of gibberellin A on animal tissues were also, presented.

A new crystalline steroid conjugate was obtained by partial acid hydrolysis of asterosaponin A, a steroidal saponin from the starfish, Asterias amurensis . The structure of the conjugate was established as 5α-pregn-9(11)-ene-3β,6α-diol-20-one-3-sulfate ( 2 ) on the basis of elemental analysis, IR and PMR spectra measurement and chemical reaction. Solvolysis of compound 2 yielded 5α-pregn-9(11)-ene-3β,6α-diol-20-one. Oxidation with chromium trioxide-pyridine complex followed by solvolysis afforded a new steroid, 5α-pregn-9(11)-ene-3β-ol-6,20-dione, whose structure was deduced by the measurement of ORD curve and PMR spectra. Thus, the location of carbohydrate moiety in asterosaponin A has been assigned to 6α-hydroxy group of the steroid conjugate.

Helminthosporol, a metabolic product of Helminthosporium sativum, remarkably stimulated elongation of the second leaf sheath of rice seedlings of various varieties. It also stimulated the elongation of hypocotyls of cucumber seedlings. This substance did not have any effect on the growth of peas, tomato, sunflower and the dwarf mutants of maize. This substance did not have auxin-like activities.

Helminthosporol (H-ol) promoted the growth of light grown cucumber hypocotyls. The IAA-oxidase activity per dry weight of the hypocotyl of H-ol treated materials was less than that of controls. There was an inverse relationship between IAA-oxidase activity and H-ol induced elongation of the hypocotyl. However, when IAA-oxidase activity per plant was compared between H-ol treated materials and controls there was no essential difference. It is concluded that the apparent inhibitory effect of H-ol on IAA-oxidase activity is not directly related to the mechanism of H-ol action.

Fumaric, palmitic, oleic and abscisic acids and methyl and ethyl hydrogen succinates were isolated from seedlings of dwarf pea (Pisum sativum L., var. Progress No. 9), grown under red light, as growth inhibitors which interfered with the responses of these plants to GA3. Of the six compounds, fumaric acid did not show any inhibitory effect on stem elongation in the absence of GA3.

The structures of seven new dimeric phenylpropanoids isolated from nutmeg, seed kernels of Myristica fragrans Houtt., were established. Two of them were phenylcoumarane derivatives containing a methylenedioxy group and the others were β-aryl ether-type compounds which had an allyl group attached to an aromatic ring.

Two novel gibberellins, GA21 (I) and GA22 (II), were isolated from immature seeds of sword bean, Canavalia gladiata DC. The isolation procedure of these substances as well as their growth-promoting effects on dwarf maize mutants d1 and d5, rice, cucumber and dwarf peas (Progress No. 9) are described.The structures of two new gibberellins, GA21 and GA22, isolated from immature seeds of sword bean, were determined as 4aα, 7α-dihydroxy-8-methylenegibbane-1, 1, 10β-tricarboxylic acid 1→4a lactone (II) and 4aα, 7α-dihydroxy-l β-hydroxymethyl-8-methylenegibb-2-ene-1α, 10β-dicaboxylic acid 1→4a lactone (IV), respectively, on the basis of chemical and physicochemical studies.

A new biologically active substance named fragin was isolated from cultured broth of a bacterial strain, which was identified as Pseudomonas fragi based on taxonomic study. Isolation, characterization and biological activities are described in detail.

The structures of ezomycins A1. and A2, antifungal antibiotics produced by a strain of Streptomyces, were determined as 1 and 2, respectively, by degradative and spectrometric studies.

It has been confirmed that gibberellin A is a mixture of three components, gibberellin A1, gibberellin A2 and gibberellic acid (namely gibberellin X), by treating their methyl ester through the chromatography on A12O3 column. Attempts to separate them in free acid were made. The physical and chemical properties of each gibberellin as well as its physiological properties are described

Piericidin B was isolated from mycellia of Streptomyces mobaraensis besides piericidin A. On the basis of IR, NMR and mass spectral studies together with chemical evidences, its structure was assigned as Id. Its physiological activities are also deescribd.

The elongation of the second leaf sheaths of rice seedlings was promoted strongly by helminthosporol as well as by gibberellic acid. A greater dosage of helminthosporol was required in order to induce the same amount of elongation. Cells of treated plants were somewhat longer than those of controls. The elongation of the second leaf sheath was caused mainly by an increase in cell number. Both helminthosporol and gibberellic acid promoted the elongation of cells in the basal portion of the second leaf sheath.

To confirm the location of the lactone ring at ring A of gihberellin A3, selenium de-hydrogenation experiments were conducted in various ways. The results thus obtained and NMR spectrum studies rather support structure (III).

カイコを被検昆虫として,経口投与により薬用植物中の昆虫生理活性物質を検索する過程で,次のことが明らかになった. (1) 漢方薬ニクズク末がカイコ幼虫の成育を阻害し,ある場合には三眠蚕を出現させた. (2) このような成育阻害活性をもたらすものとして,ニクズク末より単離されたghenylpropanoidsならびに,それらの関連物質のカイコの成育に対する影響をしらべたところ,メチレンジオキシ基や,多数のメトキシル基を持つ化合物が,顕著な阻害活性を示した. (3) これらのphenylpropanoidsのうち,化合物IIIは50ppmで4令カイコ幼虫に対して摂食阻害的作用を示し,化合物Vは100ppmで4令カイコ幼虫に対して毒物的作用をあらわし, 90%致死させた.

Tenuazonic acid has been isolated as a halo-inducing toxin from the culture filtrate of Alternaria longipes, a fungus causative of the tobacco brown-spot disease. Further, its occurrence in leaves of diseased plants was confirmed by a spectrometric method. The role of the acid in the host-parasite interaction was elucidated from the phytopathological standpoint.