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症例は66歳の男性で，心窩部痛，嚥下時つかえ感を主訴に受診した．上部消化管内視鏡検査および造影検査で腹部食道から噴門部にかけて潰瘍を伴う全周性の隆起性病変を認め，造影CTで噴門部および左胃動脈周囲リンパ節腫大を認めたが生検では悪性の診断は得られなかった．経過中に腫瘍による狭窄症状が悪化したため噴門部悪性腫瘍を疑い左開胸開腹下胃全摘，下部食道切除術を行った．摘出標本では食道胃接合部に凹凸の著しい白色上皮に覆われた隆起性病変を認めた．病理組織学的検査ではほとんど異型のない扁平上皮が食道筋層外まで達する病変を形成し，Ki-67免疫染色にて基底細胞層が陽性であることなどから食道に発生したVerrucous carcinoma（以下，VCと略記）と診断した．VCは主に口腔領域に発生する高度に分化した扁平上皮癌の一種で，食道に発生するものはまれとされている．食道に発生したVCの1例を経験したので報告する．

症例は70歳の男性で，主訴は心窩部から右側腹部の痛みであった．腹部USにて胆嚢腫大，胆泥を認め，ERCP，drip infusion cholangiography CT（以下，DIC-CT）で胆嚢は描出されなかった．胃内視鏡検査にて胃角部小彎後壁側に1型胃癌を認めた．さらに，胆泥などによる胆嚢管の閉塞が疑われたため，幽門側胃切除術，胆嚢摘出術を施行した．切除標本にて胆嚢管の硬化を認めた．病理組織学的には胆嚢管に漿膜下まで達する腺癌を認め，切離断端にも腫瘍組織を認めた．後日，肝外胆管切除術，肝十二指腸靭帯周囲リンパ節郭清術，胆管空腸吻合術を施行した．最終的な病理組織学的検査で，切除組織に腫瘍の遺残を認めず，Farrarの診断基準を満たす胆嚢管癌と診断した．術後2年経過したが，再発を認めていない．Farrarの診断基準を満たす症例はまれであり，自験例を含む本邦報告37例の検討を行った．

空腸憩室の頻度は極めて低く，空腸憩室炎から膿瘍を形成した症例の報告は，さらに稀である．症例は52歳の男性で，ネフローゼ症候群のためプレドニンを4mg/2日で内服していた．2006年1月に腹痛，発熱で他院を受診した．腹部CT検査で腹腔内に膿瘍形成を認めたため，当院に紹介となった．心窩部に圧痛，反跳痛，筋性防御と38.5度の発熱を認めた．経皮的膿瘍ドレナージを施行し，造影したところ小腸の一部が造影された．小腸造影を施行したところ，膿瘍ドレナージ部の近傍に空腸憩室を認めた．ドレナージにて膿瘍の縮小を認めたが，食事を開始すると発熱，腹痛を繰り返すため，手術を施行した．開腹するとTreitz靱帯から約10cmのところで小腸が一塊となっていたため，一塊となった小腸を切除した．切除標本では，空腸憩室と考えられる空腸と連続した腔を認め，空腸憩室炎による腹腔内膿瘍と診断した．

診断が困難であった肝膿瘍の1例を経験したので報告する．症例は，42歳の男性で2006年12月，発熱，心窩部痛にて近医より急性胆嚢炎の疑いにて紹介となった．入院時，発熱，右季肋部に圧痛，筋性防御を認めた．腹部超音波検査にて，胆嚢の壁肥厚と軽度腫大を認め，肝S5に低エコー域を認めた．腹部CTでも胆嚢壁の肥厚，肝S5に低吸収域を認め，内部に一部隔壁を伴っていた．肝膿瘍ドレナージを施行したところ内容物は悪臭を伴う粘性の白色膿汁であった．膿培養は陰性で，アメーバ，エキノコッカスなども認めなかった．膿瘍腔造影では肝内胆管との交通を認めた．以上から，胆嚢炎からの逆行性感染による肝膿瘍と診断し，入院から3日後，腹腔鏡下胆嚢摘出術を施行した．切除した胆嚢の粘膜面には炎症所見を認めず，肝膿瘍からの炎症の波及と考えられた．後日，血清アメーバ抗体陽性であったため，アメーバ性肝膿瘍と診断することができた．

鼠径ヘルニア嵌頓を契機に発見されたS状結腸癌の1例を経験したので報告する．症例は89歳の男性，2005年7月，右鼠径部腫大で当院受診となった．右鼠径ヘルニアと診断した．その4日後，右鼠径部痛で再受診し，右鼠径部腫大，圧痛を認め，還納を試みたが不可能であり鼠径ヘルニア嵌頓と診断した．同日ヘルニア根治術施行した．ヘルニア嚢を開放すると，内部に大網が嵌頓しており，一部に結節性変化を認めた．嵌頓していた大網を切除して病理組織学的検査を行ったところ，腹膜播種との診断であった．後日，大腸内視鏡検査でS状結腸に全周性の2型腫瘍を認め，病理組織学的検査でS状結腸癌と診断された．そのため，S状結腸部分切除術を施行したところ，腹腔内に腹水，多発性の腹膜播種結節を認めた．鼠径ヘルニアを契機にして結腸癌が発見される症例は稀であり，自験例を含む本邦報告5例の検討を行った．

Fiber-optic carbon dioxide sensors with a dip-coated sol–gel film containing indicator dye of thymol blue are prepared and characterized. The sensitive film has both organic and inorganic parts with good gas permeability. The difference between attenuations in N 2 gas and in CO 2 /N 2 mixture gas increases with the increase in the CO 2 concentration in the 0.55–0.7 μm range. The attenuation difference increases linearly with the increase in the logalism of CO 2 concentration, although there is no difference under 0.1% CO 2 gas concentration. The sensitivity of the sensor increases with the increase in the dye concentration. The relative humidity and the film composition have an important effect on the attenuation difference.

A 52-year-old Japanese man presented for evaluation and treatment of rectal cancer. Screening computed tomography revealed pancreatic arteriovenous malformations (P-AVMs) and abnormally expanded inferior mesenteric vein (IMV) that resulted from P-AVMs. One-stage surgery for rectal cancer was dangerous so we first performed distal pancreatectomy to cure P-AVM and thus normalize the abnormally expanded IMV. After the operation, the IMV was occluded by the thrombi, and then the IMV became normal. We could perform safely radical laparoscopic surgery for rectal cancer. This is the first case report of P-AVMs combined with rectal cancer.

Two isoforms of prophenoloxidase were isolated from pupae of Oregon-R strain of Drosophila melanogaster. The purification procedure included ammonium sulfate fractionation, Sephacryl S-200 gel chromatography, DEAE-cellulose, and hydroxylapatite column chromatography. The two isoforms, A1 and A3, could be separated by ammonium sulfate fractionation. The isoelectric points of A1 and A3 were determined to be pH 5.8 and 6.7, respectively. The molecular weights of the monomers of A1 and A3 were estimated by SDS-PAGE to be 78 and 77 kDa, respectively. The native states of A1 and A3 are considered to be homodimeric, as judged by gel-filtration chromatography.

Electrophoretic variations of prophenoloxidase were obtained from natural populations of Drosophila melanogaster. A1, one of the 2 isofojrms of the prophenoloxidases, is polymorphic: 10 fast-migrating and 2 slow-migrating types were found in polyacrylamide gel electrophoresis among 271 wild strains. The majority were the intermediate-migrating type. By use of these variants, A1 gene was mapped at 79.6 on the right arm of the second chromosome. By deletion mapping, its cytological position was located at 55A-B. In the electropherograms, hybrids between the types differing in mobility exhibited 3 bands, indicating that A1 is a dimeric protein. The gene for A1 is expressed through larval, pupal and adult stages.

The resin bond strength of plasma-arc curing in restorative dentistry was investigated in comparison to halogen-light curing with respect to two kinds of thickness, shade and opacity of porcelain laminate veneers. The bond strength of the light-cured resin was evaluated by shear tests and SEM observations of the fracture surfaces, and the results were interpreted in terms of the degree of resin polymerization. It was found that plasma-arc curing for 6 s was sufficient to obtain bond strengths similar to those of specimens polymerized with halogen light for 40 s, whereas the plasma-arc curing time needed to be doubled to 12 s in order to achieve similar failure patterns for a darker-shade porcelain of 2 mm thickness. The bond strength achieved by plasma-arc curing was found to be relatively unaffected by the shade or opacity of porcelain.

An activating enzyme for prophenoloxidase A1 was isolated from pupae of Drosophila melanogaster, and the activation of purified prophenoloxidase A1 with this enzyme was analyzed. The purification included ammonium sulfate fractionation, DEAE-cellulose, Superdex 75, arginine-Sepharose and hydroxyapatite column chromatography. The prophenoloxidase activating enzyme was determined to be a 28.5-kDa protein consisting of a single polypeptide. The kinetics of the activation reactions was unusual in that the final levels of phenoloxidase activity varied depending on the initial concentrations of the activating enzyme, not those of the prophenoloxidase. The activation was effectively suppressed by the inhibitors of trypsin-type serine protease. The protein has amidolytic activity, and Boc-Val-Pro-Arg-MCA was the best substrate among the synthetic substrates examined. The molecular mass of the activated phenoloxidase was smaller than that of the prophenoloxidase, indicating that a 5-kDa peptide was released from the prophenoloxidase by limited proteolysis with the activating enzyme. The cleavage site of prophenoloxidase A1 was shown to be between Arg and Phe at positions 52 and 53.

Clones encoding pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] A1 were isolated from a lambda gt10 library that originated from Drosophila melanogaster strain Oregon-R male adults. The 2294 bp of the cDNA included a 13-bp 5'-noncoding region, a 2070-bp encoding open reading frame of 690 amino acids, and a 211-bp 3'-noncoding region. A hydrophobic NH2-terminal sequence for a signal peptide is absent in the protein. Furthermore, there are six potential N-glycosylation sites in the sequence, but no amino sugar was detected in the purified protein by amino acid analysis, indicating the lack of an N-linked sugar chain. The potential copper-binding sites, amino acids 200-248 and 359-414, are highly homologous to the corresponding sites of hemocyanin of the tarantula Eurypelma californicum, the horseshoe crab Limulus polyphemus, and the spiny lobster Panulirus interruptus. On the basis of the phylogenetic tree constructed by the neighbor-joining method, vertebrate tyrosinases and molluscan hemocyanins constitute one family, whereas pro-POs and arthropod hemocyanins group with another family. It seems, therefore, likely that pro-PO originates from a common ancestor with arthropod hemocyanins, independently to the vertebrate and microbial tyrosinases.

Since estradiol has been detected in Bombyx ovaries effects of estradiol and other steroids on the growth and maturation of the silkworm ovary, rate of oviposition, and embryonic development were examined as a part of a study aimed at the clarification of physiological significance of estradiol in insects. These steroids were injected at various doses into the whole pupae and into the isolated pupal abdomens. No significant effect by the injections was observed on the ovarian development, as judged by increase in protein content or wet weight of ovaries and pattern of protein constituents including vitellin. However, rate of oviposition was considerably affected by the injection of estradiol at high doses. No clear effect was observed on embryonic development or determination of diapause by the injection of estradiol into the pupae. Effects of injection of anti-estrogen, nafoxidine, into the isolated abdomens and whole pupae were also examined. No effects were observed on ovarian development by injection. The relationship between physiological significance of the vertebrate steroids and metabolic activity of the ovary is discussed.

A high activity of molting hormone (ecdysone) was detected in the extract of unfertilized, diapausing and developing silkworm eggs. The active substance in the diapause eggs behaved as a highly polar molecule at partitioning between n-butanol and water, and also in TLC. The majority of the activity seems to be due to a conjugated ecdysone, since the polar active substance was changed to a less polar substance upon treatment with “snail enzymes”.

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.

During pupal-adult development of the silkworm, Bombyx mori, the ecdysone titer changes, exhibiting two maxima in the females: one on the second day of pupal development and the other just before adult emergence. During the second maximum, ecdysone accumulates in the ovaries. It also accumulates in isolated abdomens, which were prepared just after pupal ecdysis and induced to initiate adult development by injection with β-ecdysone. Several lines of evidence suggest that ecdysone is synthesized in the ovary itself.

Activation reaction of prephenoloxidase (pre-enzyme) was analyzed using a system of homogeneous pre-enzyme and highly purified prephenoloxidase-activating enzyme (PPAE) from the silkworm Bombyx mori . When pre-enzyme was activated by PPAE, release of a peptide was demonstrated. Results of polyacrylamide gel electrophoresis revealed that only a single peptide is liberated from pre-enzyme. Several lines of evidence indicated that the released peptide is inhibitory on PPAE.

Six major ecdysteroid conjugates have been isolated from mature ovaries of Bombyx mori by a procedure involving column chromatography on Sephadex G15, silicic acid and Sephadex LH-20, and high-performance liquid chromatography using a reverse-phase column. By analyses including ultraviolet absorption, enzymatic hydrolysis, negative ion fast-atom bombardment mass spectrometry and proton and 31 P nuclear magnetic resonance spectrometry, these conjugates were identified as the following: ecdysone-22-phosphate, 20-hydroxyecdysone-22-phosphate, 2-deoxyecdysone-22-phosphate, 2-deoxy-20-hydroxyecdysone-22-phosphate, 2,22-dideoxy-20-hydroxyecdysone-3-phosphate and bombycosterol-3-phosphate.

Ecdysteroids were studied in relation to embryonic development and diapause of the silkworm, Bombyx mori. The majority of the ecdysteroids was found to be in the conjugated form, and minor part, in the free form. In the developing eggs, 2-deoxyecdysone, 2-deoxy-20-hydroxy-ecdysone and 3-epi-ecdysone were found to have the free ecdysteroid form as well as the conjugated ecdysteroid form. Ecdysone and 20-hydroxyecdysone almost exclusively had the conjugated form. The concentration of ecdysteroids in the embryo was higher than that in yolkplasm in the early embryonic stages. During the embryonic diapause, the concentration of free ecdysteroids decreased to a low level while the conjugated form maintained the original level.

Protein kinase has been partially purified from silkworm eggs about 70-fold. The apparent substrate specificity, K m values for ATP and cyclic nucleotides, pH-dependence, metal requirements and other properties of the enzyme have been examined. The molecular weight of the enzyme was 72,000 by gel filtration. The role of the protein kinase in the control mechanisms of carbohydrate metabolism in silkworm eggs is discussed.

The mechanism of polyol accumulation in diapausing Bombyx eggs, conversion of [6-14C] glucose-6-phosphate into polyols and other neutral sugars was investigated in in vitro reaction systems. When a crude homogenate or a press juice of the eggs was incubated with [6-14C]glucose-6-P, the labelled trehalose, sorbitol and glycerol accumulated in the reaction mixture. In the press juice incubation system of developing eggs at day 1, 14C-sorbitol was detected in appreciable amounts, but it decreased rapidly with the development of the embryos. When the press juice was prepared from eggs in diapause, the formation of 14C-sorbitol was 3–5 times greater in eggs at early stages (day 2 to day 4) than in developing eggs.

2-Deoxy-20-hydroxyecdysone was identified in the ovaries of pharate adults of the silkworm, Bombyx mori, by mass spectrometry and high performance liquid chromatography. Together with 2-deoxyecdysone, 2,22-bisdeoxy-20-hydroxy-ecdysone and ecdysone, this ecdysteroid constitutes one of the four major moulting hormones of Bombyx mori ovaries. Accumulation in ovary of these ecdysteroids suggests a forked pathway of biosynthesis, one leading to ecdysone and the other to 20-hydroxyecdysone.

1. 1. Malic enzyme (malate dehydrogenase (decarboxylating) E.C. 1.1.1.40) was purified from cytosol of diapausing silkworm eggs by ammonium sulphate fractionation, ion exchange chromatography on DEAE- and phosphocellulose, and calcium phosphate gel negative absorption. 2. 2. The enzyme has an optimum pH at 8.5 and requires Mn or Mg ions for its maximum activity. 3. 3. Pyruvate is the main end product of the reaction. 4. 4. The Km for l -malate and NADP was determined to be 253 μM and 3.3 μM respectively. 5. 5. The enzyme has a mol. wt of 61,000 as determined by gel filtration.

Two cyclic nucleotide-dependent protein kinases, designated as protein kinase-I and -II, were obtained from the eggs of the silkworm, Bombyx mori. Protein kinase-I is highly dependent on cGMP, whereas protein kinase-II is dependent on cAMP. In developing non-diapause eggs, the level of cyclic nucleotide-dependent protein kinase activity is quite high but that in the diapause eggs is not. The developmental changes in the two protein kinases and the level of cyclic nucleotides were also studied during the development of the eggs.

From the ligation experiments, it is indicated that the secretion from prothoracic glands during early pupal period induces the ovarian development in female pupa of the silkworm, Bombyx mori. The exogenous β-ecdysone injected into isolated pupal abdomen also induces the initiation of the ovarian development.

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).

In the swallowtails of the genus Papilio, there are two pupal types, green and brown. These types are determined just before pupation by environmental stimuli ( Ohnishi and Hidaka , 1955). The pigment composition of the pupal cuticles of these types was analysed. In the green pupae three carotenoids, β-carotene, lutein, and an unknown carotenoid were found. Neither β-carotene nor lutein was detected in the brown pupae, but an astaxanthin-like carotenoid and a group of previously unknown carotenoids, which were called “papilioerythrins”, were found. A blue bile pigment, mesobiliverdin, was extracted from both types of pupae. Its content in the brown pupae was 66 per cent (P. xuthus) or 34 per cent (P. protenor demetrius) of that in the green pupae. Three pterins, xantho-, isoxantho-, and leucopterin, were detected in the cuticles of both types by paper chromatography. These findings are discussed in connexion with a hormone, which is secreted from the prothoracic ganglion and is the immediate factor controlling these pupal types.

It has been reported that tyrosinase of Drosophila or Musca occurs as a proenzyme, which becomes active by the action of a protein activator, and that the level of tyrosinase activity finally attained was considerably lower when the activation was allowed to take place at 25°C, instead of 0°C. Detailed studies on this temperature effect, described in the present paper, revealed the presence of an inactivation process of the proenzyme besides the activation process. From the results of kinetic experiments, a close relationship between the production of the activator and that of tyrosinase is suggested.

Glucose 6-phosphate dehydrogenase (E.C. 1.1.1.49) was purified almost homogeneously from the eggs of silkworm, Bombyx mori. Apparent Km values for glucose 6-phosphate (G-6-P) and nicotinamide adenine dinucleotide phosphate (NADP) were estimated to be 1.3 × 10−4M and 1.8 × 10−6M respectively. The optimum pH of the reaction was observed at 9.5–10.0. The apparent molecular weight of the enzyme was determined to be 5.6 × 104 by Sephadex G-150 gel filtration.

Extract from silkworm eggs contains two kinds of nicotinamide adenine dinucleotide phosphate (NADP)-specific polyol dehydrogenases, which catalyse the dehydrogenation of various polyols as well as the reduction of a number of sugars to the corresponding polyol. These are designated as polyol dehydrogenase-I and -II. The purified polyol dehydrogenase-I catalyses the reduction of glucose, xylose, erythrose and d,l -glyceraldehyde but does not catalyse the reduction of the phosphate esters of sugars (except ribose-5-phosphate, R-5-p). Polyol dehydrogenase-II catalyses the reduction of glucose-6-phosphate (G-6-P) as well as xylose, erythrose and d,l -glyceraldehyde, but does not catalyse the reduction of hexoses (except galactose). The molecular weights of polyol dehydrogenase-I and -II were determined to be 56,000 and 76,000 respectively by gel filtration. The roles of these dehydrogenases are discussed in relation to the metabolic pathway which leads to the accumulation of sorbitol and glycerol in the diapausing silkworm eggs.

From the silkworm eggs two acid phosphatases have been partially purified and their properties are characterized. One of the enzymes, designated as ‘acid phosphotase I’, has p H optimum at 3·5 and hydrolyses αGP, βGP, and AMP. The other enzyme, designated as ‘acid phosphatase II’, has p H optimum at 6·5, and hydrolyses αGP, S6P, F6P, G6P, and R 5 P. The latter enzyme requires divalent metals, e.g., Mg 2+ , Mn 2+ , Co 2+ , Ni 2+ , and Fe 2+ for its activity, and is inhibited almost completely by 10 m M ATP. These characteristics are discussed in relation to the sorbitol and glycerol formation in the diapausing eggs.

Investigations were undertaken to elucidate the mechanism of the accumulation of 3-hydroxykynurenine (3-OHK) in pupal ovarian follicles of the silkworm, Bombyx mori L. In diapause-egg-producer pupa (D-pupa), 3-OHK is actively transferred into and accumulated in the follicles from hemolymph, whereas no such phenomenon is observed in non-diapause-egg-producer pupa (ND-pupa). In D-pupa, the following facts indicate that an active transport is one of the major factors involved in the accumulation of 3-OHK; namely 3-OHK accumulation in follicles exceeds that of hemolymph by about 18-fold and the transport is inhibited by DNP and sodium azide. Autoradiography revealed that accumulated 3-OHK localized specifically on the surface of the yolk spheres of the oocytes. On the other hand, in ND-pupa, 3-OHK is considered to be transferred into the follicles by a simple diffusion, since 3-OHK concentration in a follicle nearly coincides with that of hemolymph. The accumulation of 3-OHK could be arrested by the removal of suboesophageal ganglion (Sg) in D-pupa. These results are discussed in relation to the action of the “diapause factor” released from Sg.

Depending upon the light conditions during a certain period prior to pupation, pupae of the cabbage white butterfly, Pieris rapae crucivora, are either green or greyish brown: The colouration of the green type is due to a combination of yellow cuticle and underlying bluish green epidermis. The extractable yellow pigment in the cuticle was shown to be a carotenoid, lutein, in terms of its absorption spectra and its behaviour in thin-layer chromatography. The blue component is a bile pigment, probably mesobiliverdin, as judged by its absorption spectra and other chemical properties. The colour of the brown pupae is due to the presence of black spots (melanin) throughout the cuticle in combination with a reddish-brown pigment in the epidermis. The reddish-brown pigment was shown to be the ommochrome, xanthommatine, by paper chromatography and by its behaviour before and after reduction by SO2 or ascorbic acid.

Carotenoids in integument, haemolymph, and fat bodies of orange pupae of the swallowtail, Papilio xuthus, were analysed qualitatively. The pigment described as ‘astaxanthin ester-like pigment’ in a previous paper is identical to canthaxanthin. Papilioerythrin, one of the main pigments of the integument, is assumed to be 4-ketolutein and identical to α-doradexanthin. The carotenoids present in all 3 sites are α-carotene, β-carotene, and free lutein. Besides these 3 carotenoids, the integument contains large amounts of 2 ketonic carotenoids, canthaxanthin and papilioerythrin, and a small amount of astaxanthin. The carotenoids typical of the fat bodies are mono- and di-esters of lutein.

Malacosoma neustria testacea accumulates crystals of Ca oxalate monohydrate in the Malpighian tubes abundantly. The mode of accumulation in the fifth instar larvae was followed by estimating the calcium content of the Malpighian tubes. To determine the incorporation of the radioactivity into Ca oxalate, glucose-U- 14 C, Na citrate-1,5- 14 C, glycine-1,2- 14 C, Na oxalate-1,2- 14 C, and Ca oxalate-1,2- 14 C were administered by mouth or by injection into the larvae. The results revealed that most of the oxalic acid in the crystals comes from dietary oxalic acid. A minor pathway of oxalic acid formation from glycine is also indicated.

In silkworm eggs there are two forms of malate dehydrogenase, one in the cytosol and the other in the mitochondria. The cytosol form has been purified 607 times. Km values for malate, NAD, and oxaloacetate are estimated to be 952 μM, 238 μM, and 78.7 μM respectively. The molecular weight is 56,000, comprising two subunits. The enzymic activity is inhibited by N-ethylmaleimide and PCMB.

Activation by α-chymotrypsin of the prephenoloxidase, purified from the silkworm, Bombyx mori , was studied. The pre-enzyme was activated by the proteinase linearly with time without any lag period. The reaction velocity is proportional to the concentrations of α-chymotrypsin and of the pre-enzyme. The activation was suppressed by diisopropylfluorophosphate (DFP) and l -l-tosylamino-2-phenylethyl-chloro-methyl ketone (TPCK) and also by high concentrations of NaCl. The activated product seems to have a distinct molecular size as judged by gel filtration on Sephadex G-150, and showed a substrate specificity different from that of the activated enzyme produced by the action of the cuticular activator. Semialkaline proteinase of Aspergillus melleus showed an activating capacity nearly comparable to α-chymotrypsin. Neutral proteinase of Bacillus sublilis had slight activity, whereas, trypsin, leucine aminopeptidase, carboxypeptidase A, and alkaline proteinase of B. sublilis were inactive.

From an acetone-ethanol extract of the developing embryos of the silkworm, Bombyx mori, the presence of α-ecdysone, but not of β-ecdysone, was shown by high pressure liquid chromatography and bioassay. The amount of α-ecdysone was estimated to be 0.74 μg per gram of eggs. The absence of a hydroxylating system at C-20 in the embryos is suggested.

Two novel ecdysteroid conjugates, 2,22-dideoxy-20-hydroxyecdysone 3-phosphate (1) and bombycosterol 3-phosphate (2), as well as four known ecdysteroid 22-phosphate esters, have been isolated and characterized from the ovaries of the silkworm,Bombyx mori.

A novel ecdysteroid, for which the name bombycosterol is proposed, has been isolated from the ovaries of the silkworm Bombyx mori and its structure was determined as (20S)-Cholesta-7,14-diene-3β,5α,6α,20,25-pentaol (1) by spectroscopic means and 1H n.m.r. comparison with a reference compound (3).

Changes in the ecdysone titre of the silkworm, Bombyx mori , during pupal-adult development were estimated. The average value of the maximum titre, which was observed on the second day after pupal ecdysis, was about 0·8 μg equivalent of β-ecdysone/g of live weight in both sexes. There is a distinct sexual dimorphism in the pattern of the ecdysone titre. The male exhibited a single sharp peak on the second day whereas the female showed the second peak on the fifth day. When the female was ovariectomized, the ‘female type’ ecdysone pattern was converted to the ‘male type’. In the female pharate adult 7 days after pupal ecdysis, ecdysone activity accumulated in the ovaries. The relationship between the ecdysone titre and adult differentiation, especially during ovarian development, is discussed.

Metabolic pathways of estradiol-17 beta and other vertebrate steroid hormones of cultured silkworm pupal ovaries were examined using 14C-labeled steroids. The isolated ovaries showed significant uptake and metabolic activity of the 14C-labeled estradiol-17 beta added to the medium. Analysis of the metabolized compounds by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed extensive metabolic conversion of [14C]estradiol-17 beta and estrone; i.e., estrone was reduced to estradiol-17 beta and estradiol-17 beta was metabolized to conjugates, including estradiol-3-beta-D-glucoside and estradiol-17-alpha-D-glucoside. [14C]Testosterone was not transformed appreciably by the ovaries. Metabolic activity and physiological significance of the vertebrate steroid hormones in the silkworm ovaries are discussed.

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.