Thiamine requirement of two different cultured cell lines of soybean

When supplied with 6-benzyladenine (0.5–5 mg/liter) insteadof thiamine, thiamine-requiring soybean cells (strain TU) couldgrow successively. The effect of cytokinin was much more remarkableat a relatively higher concentration of 2,4-dichlorophenoxyaceticacid (4 mg/liter) than at a lower concentration (0.5 mg/liter).Among calli initiated from soybean hypocotyls on a medium withoutthiamine, the thiamine-nonrequiring variant (strain G) was obtainedincidentally. As this cell line became green in light, it couldbe visually separated from the other necrotic tissues. StrainG cells could grow successively not only without thiamine butalso without phytohormones, auxin and cytokinin. This cell linehad relatively higher amounts of chlorophyll and thiamine, andgrew in rigid, large cell aggregates which differed from cellaggregates of the strain TU cell line. The thiamine requirementof plant cultured cells seems to be associated with the degreeof dedifferentiation of the cells rather than the kind of plant.In general, the higher the degree of redifferentiation of thecells, the higher is their thiamine level and the less theyrequire externally supplied thiamine. ¹Present address: Section of Phytochemical Research, Eisai Co.,Ltd., Kawashima, Gifu 483, Japan. (Received December 15, 1978; )     

Immunohistochemical, autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein.

Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using 35S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for 35S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for 35S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

Hormonal Control of Elongation of Tobacco Cells Derived from Protoplasts

Spherical protoplasts (30–40 µm in diameter), isolatedfrom cultured tobacco cells, elongated rapidly during a cultureperiod of 3 to 8 days and finally assumed a long cylindricalform (300–400 µm in length). The optimum concentrationof a-naphthaleneacetic acid and benzyladenine for the elongationwas 0.1 mg/liter and 1 mg/liter respectively. (Received August 10, 1982; Accepted November 12, 1982)     

Polyclonal B-cell stimulative and immunosuppressive activities at different developmental stages of Trypanosoma gambiense

Bloodstream trypomastigote and cultured procyclic (insect midgut) forms of a monomophic strain of Trypanosoma gambiense were tested for their abilities to induce polyclonal B-cell activation (PBA) and immunosuppression (IS) in mice. Injection of a cell homogenate of bloodstream trypomastigotes induced both PBA and IS, while neither PBA nor IS was observed in mice injected with a cell homogenate of cultured procyclics. The results indicate that the substance(s) inducing PBA or IS is related to the developmental stage of the parasites.     

Biosynthesis of cytokinins by tobacco cell cultures

In synchronously cultured tobacco cells (Nicotiana tabacum cv.Xanthi), the incorporation of U-¹⁴C-adenosine into butanol-solublecytokinins in vivo was studied. The radioactivity was incorporatedinto zeatin, ribosylzeatin, isopentenyladenosine and glucosylzeatinafter 20 min. The radioactive cytokinins were identified bythin-layer chromatography and high performance liquid chromatography.From the short time course of the incorporation of ¹⁴C-adenosineinto butanol-soluble cytokinins, the presence of the followingbiosynthetic pathway in vivo was suggested: adenosine is converedinto isopentenyladenosine and then into zeatin via ribosylzeatin.The biosynthetic pathway of free cytokinins in vivo is comparedwith that in vitro. (Received June 20, 1980; )     

Immunohistochemical,autoradiographic and electron microscopic studies on the transformation of fibroblasts into chondrocytes in the mouse subfascia induced by bone morphogenetic protein

Summary Functional morphology on the transformation of fibroblasts into chondrocytes induced by bone morphogenetic protein (BMP) was studied by light and electron microscopy using ³⁵S autoradiography and immunohistochemistry for S-100 protein and type-II collagen. A pellet containing BMP obtained from a murine osteosarcoma was transplanted into the mouse subfascia. By 3 days after implantation, many typical fibroblasts, which were free of the silver grains for ³⁵S and devoid of both S-100 protein and type-II collagen, entered the pellet region. By 5 days, the fibroblasts in the pellet region became polygonal in shape, and cytoplasmic vesicles and vacuoles appeared, both containing a homogeneous substance of low electron density. At 5 days, autoradiography revealed many silver grains for ³⁵S over the Golgi apparatus and vesicles and vacuoles of the cells in the pellet region as well as over the surrounding extracellular matrix. Moreover, the cells at 5 days displayed immunoreactivity to both proteins. The extracellular matrix around the cell began to show clear metachromasia and increased in amount with time. At 9 days all the cells in the pellet region were round or oval in shape and surrounded by an abundant cartilaginous matrix. The rough endoplasmic reticulum and Golgi apparatus were extremely well developed, and a large number of vacuoles and vesicles were seen in the cytoplasm. These cells showed intense immunoreactivity to both proteins, and strong accumulation of sulfur was visualized in the extracellular matrix by autoradiography. These results suggest that the fibroblasts in the pellet region change into chondroblasts by 5 days, and become typical chondrocytes by 9 days.     

The exudation and characterization of iron-solubilizing compounds in a rice cell suspension culture

Some compounds capable of solubilizing ferric chloride as theiron source were released into the growth medium from culturedrice cells in the latter half of the growth cycle. This exudationof iron-solubilizing compounds was repressed by an additionof ammonium ion, which, of five cations, was taken up most rapidly.These iron-solubilizing compounds from cultured rice cells wereidentified by chromatography to be principally malic acid andsome citric acid. In the medium containing almost insolubleferric chloride as the iron source, the growth rate of the culturedcells decreased when cells were inoculated at a low populationdensity. This iron deficiency at the low initial populationwas rectified by an addition of a small amount of conditionedmedium or malic acid. (Received May 22, 1980; )     

A CHOLESTEROL-ESTERIFYING ENZYME IN RAT CENTRAL NERVOUS SYSTEM MYELIN1

Aontrary to our earlier finding (Eto & Suzuki , 1971), the myelin fraction purified from young adult rat brain consistently showed cholesterol-esterifying activity. The specific activity in myelin was the highest among subcellular fractions. Extensive washing wiih various aqueous salt solutions failed to remove the activity from myelin. The enzyme was evenly distributed among the arbitrarily defined light, medium and heavy myelin subfractions. The myelin-localized activity showed the pH optimum and heat stability identical to the microsome-bound activity. Although there were minor differences in the effect of detergents or exogenous lipids added to the reaction mixture, no firm evidence was obtained to indicate that the myelin-bound cholesterol-esterifying enzyme is different from that in other subcellular fractions. On the other hand, the distribution among the myelin subfractions and heat stability of the myelin-bound cholesterol-esterifying activity were different from those of the myelin-specific cholesterol ester hydrolase. Therefore, the esterification does not appear to be a mere reverse reaction catalyzed by the previously known myelin-specific hydrolase. The rat brain myelin, therefore, is capable of both synthesizing and hydrolyzing cholesterol esters.     

The exudation and characterization of iron-solubilizing compounds in a rice cell suspension culture

Some compounds capable of solubilizing ferric chloride as theiron source were released into the growth medium from culturedrice cells in the latter half of the growth cycle. This exudationof iron-solubilizing compounds was repressed by an additionof ammonium ion, which, of five cations, was taken up most rapidly.These iron-solubilizing compounds from cultured rice cells wereidentified by chromatography to be principally malic acid andsome citric acid. In the medium containing almost insolubleferric chloride as the iron source, the growth rate of the culturedcells decreased when cells were inoculated at a low populationdensity. This iron deficiency at the low initial populationwas rectified by an addition of a small amount of conditionedmedium or malic acid. (Received May 22, 1980; )