Verbascoside production by plant cell cultures

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium.     

Differentiation of extracutaneous melanocytes in embryos of the turtle, Trionyx sinensis japonicus.

The present study investigates the mode of differentiation of neural crest-derived melanocytes in the embryos of the soft-shell turtle, Trionyx sinensis japonicus. DOPA reaction-positive melanoblasts were first detected in 10-day-old embryos. Melanocyte differentiation in terms of pigmentation takes place from the day 16 of development. Melanin pigments were found in the dorsal integument as well as in various extracutaneous tissues such as skeletal muscle, dorsal aorta, peritoneum, blood vessels, choroid, lung, bone marrow, fat tissues and in the connective tissue of the nose. These results suggest the presence of a specific environmental regulation of the melanoblast differentiation in the soft-shell turtle.     

A new DNA profiling system for cell line identification for use in cell banks in Japan

Summary Using the polymorphic DNA probes, ChdTC-15, ChdTC-114, pYNH24, and λTM-18, a DNA profiling system was developed that verified identities of individual cultured cell lines collected in the Japanese cell banks, JCRB, RCB, and IFO. These highly polymorphic DNA probes include both VNTR (Variable Number of Tandem Repeats) sequences and substantial lengths of unique regions. In the mixed probe system, several distinct bands from four to eight can be used for cell line identification. These bands were widely spread in a range of molecular sizes, and were stable and reproducible under stringent conditions of Southern blot hybridization. Because the DNA profile was specific for each individual human cell line, it is useful not only to authenticate many existing cultured cell lines but also to monitor their identity during propagation in a laboratory, and to confirm newly established lines as unique.     

Formation of Hassall's corpuscles in vitro by the thymic epithelial cell line IT-26 R 21 of the rat

Summary In the monolayer of an established epithelial cell line from the rat thymus, IT-26R21, characteristic cell aggregates quite similar to Hassall's corpuscles were formed. These aggregates were examined by light and electron microscopy, and immunohistochemically. Their interpretation as Hassall's corpuscles is based on the following observations: (1) The aggregates are formed in the monolayer of cells that greatly resemble medullary epithelial cells of the thymus. (2) They consist of flattened epithelial cells in a concentric pattern with one or more degenerating cells in the center. (3) Loss of microvilli suggests that these cells are keratinizing. (4) The aggregates show strongly positive reactions in immunofluorescent staining with antikeratin and antiprekeratin.When Hassall's corpuscles increase in size, cellular proliferation is somewhat suppressed. Both in vivo and in vitro, they may be interpreted as an expression of a changing growth pattern in confined spaces and thus seem to have little immunological function.     

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B₂ were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF_1α reached the maximum at 1 hr after carrageenin, then PGE₂ and TXB₂ showed peaks at 3 hr and waned off before 9 hr. he PGF_2α level was kept low, but PGD₂, PGE₁ and PGF_1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB₂ levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE₂, definitely play an important role in the exudation during the first 5 hr.     

Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene- 7,8-dihydrodiol-9,10-oxide in Escherichia coli

Plasmid-mediated transformation and mutagenesis induced by (±)-trans- benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E. coli) have been studied. Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells. Plasmid pK0482 DNA, which has two dominant genes, β-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E. coli N99, AB1157, AB2463(recA^?) and AB1886(uvrA^?). Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene. (1) Approx. 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA. (2) In AB1886(uvrA^?) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157. (3) In AB2463(recA^?), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed. (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.     

Effects of IAA, Zeatin, Ammonium Nitrate and Sucrose on the Initiation and Development of Floral Buds in Torenia Stem Segments Cultured In Vitro

In Torenia stem segments cultured on a defined medium from whichammonium nitrate and growth regulators were omitted, adventitiousbuds were readily formed from epidermal tissue, with subsequentdifferentiation of floral buds. Using this plant material, thecorrelation between the time of application of various chemicalsand the time-course of floral bud differentiation was investigated.Histological examination showed that adventitious buds werevegetative during the first two weeks of the culture, and floralprimordia appeared after about three to four weeks of culture.We divided the flowering process in Torenia stem segments intothe following 3 phases: the first phase (first 2 weeks) duringwhich adventitious buds are formed, the second phase (3rd and4th weeks) during which floral buds are initiated and the thirdphase (5th to 12th weeks) during which floral buds develop.Then we added IAA, zeatin, ammonium nitrate or a high concentrationof sucrose to the medium during one, two or three of these phases.Ammonium nitrate added during the third phase suppressed floralbud development, but the high concentration of sucrose givenduring this phase stimulated it. These two chemicals influencedonly the development of floral buds previously initiated. Theapplication of IAA during the first phase promoted both theinitiation and development of floral buds. However, its applicationafter 2 weeks of culture failed to promote floral bud formation.Zeatin inhibited floral bud formation in a manner similar toammonium nitrate, but if it was added to the medium only duringthe first phase, it slightly promoted the initiation and developmentof floral buds. (Received July 7, 1981; Accepted October 12, 1981)     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Pumpkin (Cucurbita sp.) seed globulin VI. Proteolytic activities appearing in germinating cotyledons

N--benzoyl D,L-arginine p-nitroanilide (BAPA), leucine p-nitroanilide(LPA) and casein hydrolytic activities were assayed in germinatingcotyledons. BAPA hydrolytic activity was not detected in dryseeds, but increased rapidly from 1 to 4 days of germinationand then decreased. LPA and casein hydrolytic activities weredetected in dry seeds and increased from 2 to 4 days. Caseinhydrolytic activity decreased faster than the other two activities. BAPA hydrolytic enzyme was partially purified. It was inhibitedby p-chloromercuribenzoate and activated by ß-mercaptoethanoland dithiothreitol, but was not affected by EDTA, phenylmethylsulfonylfluoride, pumpkin trypsin inhibitor and several divalent cations.It had no ability to hydrolyze globulin or the chain to produce F_ß or smaller polypeptides,respectively, which was referred to as proteolytic activityI in the preceding paper (14), but released small peptides andamino acids from the chain and F_ß. However, it wasdifferent from proteolytic enzyme II which was present in dryseeds and inhibited by EDTA (14). Pumpkin trypsin inhibitor was purified. Its molecular weightwas estimated to be 10,500 by gel filtration. It did not inhibitthe BAPA hydrolytic enzyme. Both proteolytic activities I andII were also not reduced by the inhibitor (14). The inhibitoryactivity decreased gradually during germination. (Received November 9, 1979; )