Effect of Cycloheximide on the Encystment and Ultrastructure of the Ciliate, Histriculus

SYNOPSIS The effect of cycloheximide on the experimentally induced synchronous encystment of a hypotrich ciliate, Histriculus , was examined and the cytoplasmic ultrastructures of the control and the inhibitor-treated organisms were compared. Encystment was divided into the following 5 stages based on observations of living cells: stage 1, cells with brown cytoplasm; stage 2, cells with smooth, transparent cytoplasm: stage 3, spindle-shaped cells; stage 4, spherical cells without cyst walls; and stage 5, young cysts with cyst walls. When cycloheximide treatment was preceded by stage 2, encystment and transformation into the next stages were totally blocked, whereas even in the presence of the inhibitor, stage 4 and stage 5 cells encysted normally, and stage 3 cells transformed into stage 4 although they did not form cyst walls. On the basis of ultrastructural studies it is suggested that the formation and excretion of ectocyst precursors are severely inhibited by cycloheximide and that polysome formation, active in stages 1 and 2, might be almost finished by stage 3. The role of lysosomal enzymes in the early stages of encystment are discussed.     

Differences in cellulose digestive systems among castes in two termite lineages

Abstract.  Termites (Isoptera) are eusocial insects and express polyphenism. Soldiers have specialized morphology for colony defense, but their feeding activity is dependent on other colony members. To determine differences in cellulose degradation between soldier and worker termites, enzymatic activity and cellulase gene expression, as well digestive tract histology, are examined in two phylogenetically distant species. In Hodotermopsis sjostesti (family Termopsidae) , endo-β-1,4-glucanase activity is identified in the salivary glands, whereas β-glucosidase activity is identified in salivary glands and hindgut. The relative expression levels of endo-β-1,4-glucanase genes in soldiers are significantly lower than in workers. Thin sections of salivary gland of workers and soldiers are different in H. sjostedti . In Nasutitermes takasagoensis (family Termitidae), the endo-β-1,4-glucanase activity is restricted to the midgut in four tested castes (i.e. three types of workers and soldier). Examination of activity per termite reveals the highest activity in minor workers and the lowest activity in major workers and soldiers. The β-glucosidase activity is also concentrated on the midgut in all four castes. The relative expression level of the endo-β-1,4-glucanase gene does not correspond with its activity in the midgut. In thin sections prepared from N. takasagoensis , the folds and pulvillus in the gizzards, and cuticle structure of soldiers are less developed compared with the other three worker castes. The differences in digestive system among termite castes in terms of caste development in each species are discussed.     

Inductive Capacities for the Dorsal Mesoderm of the Dorsal Marginal Zone and Pharyngeal Endoderm in the Very Early Gastrula of the Newt,and Presumptive Pharyngeal Endoderm as an Initiator of the Organization Center*

The organization center of Cynops pyrrhogaster was divided into Parts 1, 2 and 3 of equal size (0.3×0.4 mm²) with presumptive fates as pharyngeal, pharyngeal+prechordal+trunk notochord, and trunk-tail notochord, respectively. Movements and changes in size and shape of each part were followed through gastrulation. Differentiation tendencies of each part were examined under three conditions: I, isolated; II, sandwiched with presumptive ectoderm; 111, sandwiched with presumptive ectoderm after preculture in isolation for various times. In I, Parts 2 and 3 differentiated into dorsal mesoderm. In II, each part induced dorsal mesoderm and neural tissues, the frequency being highest in Part 2 and lowest in Part 3. In III, Parts 1 and 2 realized their presumptive fates, through changes in inductive capacities from trunk-tail to head. This change progressed rapidly in Part 1, and slowly in Part 2. Part 3 required induction by neighbouring Part 2 to realize its presumptive fate. Changes of inductive capacity of Parts 1 and 2 respectively, were chronologically similar in normal development and in preculture experiments. Lastly, the primary presumptive pharyngeal zone at blastula was proposed to act as an initiator of the organization center, its programmed information being transmitted to Part 2, and then to Part 3.     

ENZYME ACTIVITIES ASSOCIATED WITH RIBOSOMES FROM SOYBEAN SEEDLINGS

Ribosomes from cotyledons of soybean seeds soaked overnightin water (resting-cotyledon ribosomes) and respective ribosomesfrom cotyledons (working-cotyledon ribosomes) and hypocotyls(hypocotyl ribosomes) of 5 day-old seedlings were prepared.These ribosomes mainly consisted of 80 S particles. However,hypocotyls contained 115 S particles in a small amount and working-cotyledonscontained 115 and 150 S particles which may correspond to thepolymers of ribosomes or polysomes. The abundant 80 S ribosomeswere fractionated by the sucrose density gradient centrifugation,and enzyme activities in the fractionated ribosomes were estimated.RNase, PDase, acid phosphatase, 5'-nucleotidase, XTPase, peroxidaseand ß-glucosidase were found in the ribosomes. Theenzyme activities were lower in the resting-cotyledon ribosomesand higher in the hypocotyl ribosomes. Working-cotyledon ribosomesshowed the middle of them. All these enzymes were also foundin the cytoplasmic solution (supernatant) of cotyledon and hypocotylcells. However, RNase, PDase, 5'-nucleotidase and peroxidasewere concentrated in ribosomes, and the specific activitiesof 5'-nucleotidase and ß-glucosidase were increasedby washing the ribosomes. The status of the enzymes found inthe ribosomes was discussed. (Received May 12, 1966; )     

ELECTRON MICROSCOPIC DEMONSTRATION OF TYROSINASE IN PTERINOSOMES OF THE FROG XANTHOPHORE,AND THE ORIGIN OF PTERINOSOMES*

In the frog, Rana japonica, the successive appearance of types I, II and III pterinosomes, which were defined according to the degree of lamellar structure, is in keeping with the xanthophore differentiation at the larval stage, but these three types coexist in a single xanthophore in the adult. An intense tyrosinase reaction was found in type I–II intermediate form in the larval and adult xanthophores, but it was rarely observed in types I and III. A tyrosinase reaction was always found in the GERL (Golgi-associated Endoplasmic Reticulum) of larval and adult xanthophores, and it was similarly evident in small Golgi vesicles which were separated from the GERL and dispersed in the cytoplasm. The above findings suggest that tyrosinase and pterinosome originate from different parts of the cytoplasm. The hypothesis that small Golgi vesicles are transported to the tyrosinase-negative premelanosomes involved in the origin of the melanosome is also applicable to the origin of pterinosomes.     

Cytochemical and Electrophoretic Studies on the Cyst Wall of a Ciliate, Histriculus muscorum Kahl

The localization of proteins and polysaccharides in the cyst wall of a ciliate, Histriculus muscorum, was examined by light and electron microscope cytochemistry and by using an enzyme digestion test on thin sections. The endocyst and ectocyst were digested by treatment with pepsin or protease VI. The endocyst was intensely PAS-positive and alcian blue-positive. The ectocyst was also PAS- and alcian blue-positive, but the reaction was weaker than that of the endocyst. At the electron microscope level, an intensely positive reaction to methenamine silver was observed in the endocyst and a weak reaction in the ectocyst of the mature cyst. In the ectocyst of the encysting organism, however, the reaction to methenamine silver was more intense than that of the mature cyst. These results demonstrate the possible presence of glycoproteins in the ectocyst and endocyst. The mesocyst was negative to all cytochemical and enzyme digestion tests examined. The cyst wall, isolated by sonication, was analyzed by SDS-polyacrylamide gel electrophoresis. Two bands, 190 and 140 kilodaltons, were specific for the cyst wall. The 190 kilodalton band was the only PAS-positive band and its localization in the cyst was was discussed.     

CHARACTERIZATION OF FORCED SUSPENSION CULTURE OF SPONTANEOUSLY TRANSFORMED MOUSE FIBROBLASTS (B-6)

Mouse fibroblasts (B-6) were cultured on agar-coated dishes. After cells grew for 2–3 generations relatively rapidly in suspension, they began to grow very slowly (stationary phase). Electron microscopic studies showed that cells in a stationary phase developed intracellular organella: membranous structures (endoplasmic reticulum and Golgi apparatus) became manifest and the number of mitochondria increased. The specific activities of succinic-cytochrome c reductase and 5′-nucleotidase were three and five times higher, respectively, than those of cells on the dish.     

CELL ASSOCIATION PATTERN IN AGGREGATES CONTROLLED BY MULTIPLE CELL-CELL ADHESION MECHANISMS

We have previously assumed the presence of two mechanisms for the aggregation of Chinese hamster V79 cells, the Ca²⁺-dependent one and the Ca²⁺-independent one. In order to examine if each of these mechanisms contributed differently to the various aspects of cell aggregation, the morphology of V79 cell aggregates, pretreated so that they were provided with only one of the two adhesion mechanisms, was compared by light and electron microscopy. The adhesion among cells with only the Ca²⁺-dependent mechanism was very tight, with the formation of gap and intermediate junctions. Cells were arranged in a rod or dendric shape in aggregates. In aggregates of cells with only the Ca²⁺-independent mechanism, cells were loosely attached to each other without the formation of specialized junctions and the aggregates were of globular shape. In aggregates of cells with both mechanisms, both characteristics of the above two aggregates were found. Four clones of V79 cells, which formed colonies with different morphology when they were grown in soft agar, were isolated. It was found that such differences were due to the different activity of the Ca²⁺-independent mechanism among these clones. These results suggested that the two adhesion mechanisms play different roles in the cell arrangement in aggregates.     

PRODUCTION OF GERMLINE CHIMERIC CHICKENS BY TRANSFER OF CULTURED PRIMORDIAL GERM CELLS

Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3–3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.