PHYSIOLOGICAL ACTIVITIES OF HELMINTHOSPOROL AND HELMINTHOSPORIC ACID: II. EFFECTS ON EXCISED PLANT PARTS

Effects of H-ol and H-acid were observed using excised partsof several plant species. Both H-ol and H-acid were active inelongation of oat coleoptile and mesocotyl, expansion of Raphanusleaf disk, but were inactive in elongation of wheat coleoptileand of green stem of pea. They gave also negative results inthe standard Avena curvature test and in the split pea test.In expansion of lettuce cotyledon, H-ol was inactive while H-acidwas active. In excised plant parts, as in intact plants, theactivity of H-ol and H-acid resembles rather gibberellins thanauxins and cytokinins. (Received August 20, 1966; )     

PROMOTING ACTIVITY OF TERPENIC LACTONES IN PHASEOLUS ROOTING AND THEIR REACTIVITY TOWARD CYSTEINE

Sesquiterpenoids which contain exomethylenes conjugated to -lactonecarbonyl-heliangine, helianginol, pyrethrosin and cyclopyrethrosinacetate—promoted the adventitious root formation on hypocotylsof cuttings taken from light-grown (1900 lux) 6-day old Phaseolusmungo seedlings, but their derivatives in which the methylene-lactone systems are reduced-to the saturated lactones—dihydroheliangine,hexahydro-heliangine, dihydrohelianginol, dihydrocyclopyrethrosinacetate and tetrahydrocyclopyrethrosin acetate—showedno effect on the root formation. As far as the present experiment is concerned, every substancewhich showed promoting activity in Phaseolus rooting reactedwith cysteine and formed an adduct, but any of the substancesincapable of promoting root formation did not react with cysteine.The correlation of promoting activity of terpenic lactones inPhaseolus rooting with their reactivity toward SH groups maythus be demonstrated. ¹ Contribution No. 14 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo.     

Species Specificity of the Insect Prothoracicotropic Hormone (PITH): the Presence of Bombyx-and Samia-Specific PTTHs in the Brain of Bombyx mori

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.     

PURIFICATION OF GONAD-STIMULATING SUBSTANCE OBTAINED FROM RADIAL NERVES OF THE STARFISH, ASTERIAS AMURENSIS

Purification of starfish gonad-stimulating substance (GSS), which induces shedding of gametes and oocyte maturation, was carried out using lyophilized radial nerves of Asterias amurensis as source material. In the first series of experiments, 1.3 mg of the purified GSS, which induced spawning at a concentration of 0.0096 μg/ml, was isolated from acetone powder of lyophilized radial nerves of 7,360 starfish through several steps of purification procedures consisting of gel-filtrations on Sephadex G–50 and G–25 columns of various sizes and ion-exchange chromatography on DEAE-Sephadex columns by gradient as well as step-wise elution. With this sample, the molecular weight and amino acid composition of GSS were estimated. Another series of experiments, conducted on a similar amount of material with purification procedures which were essentially the same as those of the first series except for employing 2 steps of partition chromatography instead of extensive gel-filtration, gave about 0.1 mg of purified sample which served as material for studies of the amino acid composition and electrophoretic properties of GSS.
The molecular weight of Asterias GSS was found to be about 2,100, as determined with the sedimentation equilibrium method. GSS seemed to consist of the following 22 amino acid residues: aspartic acid (2), threonine (1), serine (6), glutamic acid (1), proline (1), glycine (4), alanine (2), valine (1), isoleucine (1), leucine (1), histidine (1), and ornithine (1). The isoelectric point of GSS was found to be at about pH 4.5 as determined by the isoelectric focusing method.     

PHYSIOLOGICAL RESPONSES OF LIGHT-GROWN CUCUMBER HYPOCOTYLS TO HELMINTHOSPOROL AND ITS DERIVATIVES

Helminthosporol, helminthosporic acid and dihydrohelminthosporicacid stimulated hypocotyl elongation of light-grown cucumberseedlings. The relative activities of the three compounds werein the order H₂-H-acid> H-acid> H-ol. The higher the dosage,the longer the promotion of the hypocotyl elongation lasted.The response of the hypocotyl to H-acid depended on the presenceof cotyledons. As the amount of the cotyledon attached to thehypocotyl was reduced, the response decreased. When IAA-oxidase was estimated as IAA metabolized per dry weightof the hypocotyl, there was an inverse relationship betweenIAAoxidase activity and H-ol-induced elongation of the hypocotyl.However, there was no difference between control and H-ol treatedmaterials when IAA metabolized per plant was compared. It isunlikely that the mechanism of H-ol action is closely relatedto IAA-oxidase activity of the plant. (Received April 5, 1967; )     

PHYSIOLOGICAL ACTIVITIES OF HELMINTHOSPOROL AND HELMINTHOSPORIC ACID: I. EFFECTS ON GROWTH OF INTACT PLANTS

Helminthosporol (H-ol) and helminthosporic acid (H-acid) wereeffective in promoting elongation of leaf sheaths of rice, Japanesebarnyard grass and dwarf maize (d-2 and d-5) and of hypocotylsof taisai (Brassica chinensis), but inactive in leaf sheathsof oat and wheat, hypocotyls of sesame and morning glory (Pharbitisnil) and epicotyls of Pharbitis and dwarf and tall peas. Onthe elongation of the leaf sheath of maize d-1, H-ol was promotivebut the activity of H-acid was doubtful. On hypocotyls of lettuceand daikon (Raphanus sativus), only H-acid was active. Multiplicationrate and size of fronds of Lemna perpusila were not affectedby either of the substances. Compared with gibberellic acid for the effect on the shoot growth,H-ol and H-acid were weak in activity and narrower in the scopeof plants that responded. H-ol and H-acid characteristicallypromoted the elongation of the primary root. Comparative effectivenessof H-ol and H-acid varied with plant species or parts examined. ¹ This study was supported in part by grant-in aid of the Ministryof Education (No. 0417). The results reported here were presentedat the Annual Meeting of the Botanical Society of Japan at Kanazawain 1964 (S).     

GLYCOSIDE HYDROLASES OF SEA URCHIN SPERMATOZOA AND THEIR POSSIBLE INVOLVEMENT IN SPERM ISOAGGLUTINATION BY EGG WATER*

The whole sperm of sea urchins, Anthocidaris crassispin and Hemicentrotus pulcherrimus, were found to contain neuraminidase and 4-methylumbelliferyl α-mannosidase. The maximal activity was attained after a lag of a few minutes. The sperm hydrolyzed 8 kinds of 4-methylumbelliferyl glycosides including hexosides, hexosaminides and a fucoside. The enzymes were found to be membrane-bound and solubilized by KCl, sodium taurocholate or Triton X-100. When the enzymes were solubilized, the pH optima were at about 5.5, although the optima were at about 7.5 in the whole sperm. Divalent cation requirement was not detectable. On gel filtration, neuraminidase was eluted as a low molecular weight form, whereas α-mannosidase was eluted near the void volume as a high molecular weight form. The activity hydrolyzing other 4-methylumbelliferyl glycosides was found to be concentrated in the fraction showing α-mannosidase activity. Our results suggest that when the sperm are mixed with egg water, glycosidases on the sperm is fixed at sugar residues in the heterosaccharide chains of the jelly network and hydrolyze the glycosidic bonding after a lag period. The sequence of reactions is supposedly responsible for the reversible agglutination of the sperm by egg water.     

PURIFICATION AND PROPERTIES OF THE PROTHORACICOTROPIC HORMONE OF THE SILKWORM, BOMBYX MORI

A simple and highly reproducible procedure for partial purification of prothoracicotropic hormone (PTTH) was established starting with 96,000 male adult Bombyx heads. Approximately 28,500-fold purification 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.