Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

The activity of cutin-esterase, cutinase, was detected in themycelial homogenate of Botrytis cinerea cultured in a peptone-sucrosemedium at 25°C for 7 days. The crude enzyme solution wasprepared from the homogenate by centrifugation at 106,600xg,treatment with (NH₄)₂SO₄ at 70% saturation, and dialysis against0.01 M phosphate buffer. The optimum pH, temperature and assayduration for enzyme activity were 5.0, 25°C and 18 hr, respectively.Specific activity was 255 mµmoles/mg protein/18 hr aspalmitate under optimum conditions. 83% of the activity waslost by heating the enzyme solution (pH 7.6) for 4 min at 95°C.Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic,dihydroxyeicosanoic and octadecanedioic acids were recognizedin the enzymic hydrolysate of tomato-cutin using gas-liquidchromatography. Among these fatty acids, palmitic, oleic andoctadecanedioic acids were readily liberated by the enzyme,but dihydroxyeicosanoic acid, the major component of tomato-cutin,was isolated only in small amounts. The enzyme is, therefore,an exo-type cutinase which hydrolyses minor side chains of fattyacids bound to the major structure of cutin. Cutinesterase mayfacilitate cuticular invasion by fungi as a result of reductionin mechanical strength of the cuticle by the enzyme ¹Biological Laboratory, Research Department, Nihon Noyaku Co.,Ltd., Kawachinagano, Osaka, Japan (Received June 16, 1970; )     

FORMATION OF GERM CELL-LIKE CELLS IN THE PERITONEAL EPITHELIUM OF THE TESTES OF ESTROGEN-TREATED ADULT MALES OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER

Columnar cells of the peritoneal epithelium in slender cords of the testes were examined in normal and estradiol benzoate-treated Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, by light and electron microscopy. In normal newts, the peritoneal epithelium covering the slender cord consists of columnar cells, which contain extraordinarily large, oval or spindle-shaped nuclei with conspicuous indentations. The nucleus contains chromatin granules and the cytoplasm is filled with numerous tonofilaments. The primordial germ cells are scattered throughout the slender cord, and each cell is surrounded by a few follicle cells. Between the germ cells and follicle cells there are microvilli-like processes. The nucleus of primordial germ cells is multilobate and has electron lucent areas, dispersed chromatin and several electron-dense nucleoli. In the lighter cytoplasm, the nuage material is found very near to nuclear pores, and is frequently seen among the mitochondria. The nucleolus-like body is not associated with other organelles. The primary spermatogonia have bilobate nuclei. It is remarkable that most of the cytoplasmic organelles are found in the deep nuclear indentations. The nuage material and nucleolus-like body are well developed in the cytoplasm. After treatment of newts with estradiol benzoate for one year, four types of cells can be distinguished in the peritoneal epithelium. One type is quite different from the columnar cells. These newly appeared cells are large and light in appearance. Their nucleus is highly lobate, and contains dispersed chromatin and several nucleoli with compact electron dense material in its periphery. The cells are characterized by the presence of nuage material and nucleolus-like bodies in the cytoplasm. There are microvilli-like processes between these cells and adjacent elongated cells. These ultrastructural characteristics of the light cells are very similar to those of primordial germ cells and/or primary spermatogonia in normal testes. These findings suggest that the light cells which appear in the peritoneal epithelium of the testes on administration of estrogen may be germ line cells.     

BEHAVIOR OF SECONDARY SPERMATOGONIA OF THE NEWT, CYNOPS PYRRHOGASTER, IN IN VITRO CULTURE

Several cell types migrated cut from small pieces of newt testes cultivated in vitro. Flat fibroblastic cells migrated out within a few days. Then, secondary spermatogonia, identified by the presence of germ cell-specific substances and by the shape and appearance of their nucleus and subcellular organelles, migrated out over the sheet of fibroblastic cells. Sertoli cells co-migrated with secondary spermatogonia, maintaining a similar cellular arrangement to that of testicular cells in vivo. Mitosis of secondary spermatogonia both in clusters and as single cells was frequent from the third day until about 2 weeks after inoculation. During mitosis, active and periodic rotation of chromosomes was observed. Identification of the cell types and studies on their behavior were performed by electron microscopy and phase contrast microscopy.     

HISTOCHEMICAL AND ELECTRON MICROSCOPIC OBSERVATIONS ON THE STEROID HORMONE-SECRETING CELLS IN THE TESTIS OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER

The testis of the Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, caught in the middle of May was investigated in order to demonstrate the presence of steroid hormone (SH)-secreting cells, by means of histochemical and electron microscopic techniques. The newt testis is divided into two main parts of spermatogenetic stages. The anterior part is immature and consists of many seminal cysts containing spermatogonia and spermatocytes. The posterior part is mature and made up of many cysts containing mature spermatozoa and glandular tissue. Histochemical studies revealed Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSD) activity in the pericystic cells of the mature part, weak in those before spermiation, then gradually becoming more intense in the pericystic cells just after spermiation. Activity was most pronounced in the outer layer of cells of the glandular tissue which originate from the pericystic cells. In contrast to this, the Δ⁵-3β-HSD reaction was entirely negative in the pericystic cells of the immature part, the Sertoli cells, and the inner cells of the glandular tissue originating from the Sertoli cells. Electron microscopic observation of the pericystic cells and glandular tissue gave further information on the histochemical features. The pericystic cells in the immature part are fibroblast-like cells. The cell organelles of these cells are poorly developed except for rough-surfaced endoplasmic reticulum and rod-shaped mitochondria. In the mature part, the fibroblast-like cells are transformed into cells characteristic of SH-secreting cells. In the cytoplasm, the amount of smooth-surfaced endoplasmic reticulum increases and typical globular mitochondria with tubular cristae take the place of the rod-shaped mitochondria. In addition, a number of lipid droplets and lipofuscin granules appear. Most of the fine structures of the outer layer of the cells of the glandular tissue were found to be similar in appearance to those of the SH-secreting cells in mammals and other vertebrates. These results strongly suggest that, as in the other urodeles, the SH-secreting cells in the testis of the Japanese red-bellied newt are the pericystic cells in the mature part and in the outer layer of cells of the glandular tissue.     

Studies on cutin-esterase I. Preparation of cutin and its fatty acid component from tomato fruit peel

Examination was made of the fatty acid component of tomato cutinvia gas-liquid chromatography and thin layer chromatography.Dihydroxyeicosanoic acid was identified as a major componentof tomato cutinic acid in contrast with the results of BAKERand MARTIN (1) who recognized 10,16-dihydroxyhexadecanoic acidas the dominant acid of cutin in all plants tested. On the thinlayer chromatograms we found more than nine kinds of fatty acidsin the cutin hydrolysate which was saponified with ethanol-potashsolution. The gas-liquid chromatogram for trimethylsilyl etherderivatives of methyl cutinate showed somewhat different results,i.e., unsaturated decanoic, t_R 1.4, unsaturated stearic, t_R4.2 and unsaturated octadecanedioic acid, t_R 16.0 as unsaturatedfatty acids. Two more than C₂₂-hydroxyfatty acids were recognizedas minor components. Beside these components, octanoic, t_R 0.9,hydroxydecanoic, t_R 7.0 and cis-epoxy-hydroxyoctadecanoic acid,t_R 18.7 were identified. The biosynthesis of cutin is positednot to be fulfilled or to be delayed due to less lipoxidaseactivity in tomato fruit. ¹Biological Laboratory, Research Department, Nihon Noyaku Co.Ltd., Kawachinagano, Osaka, Japan (Received December 8, 1969; )