Shigeyasu Nabeshima

Regeneration of horseradish hairy roots incited by Agrobacterium rhizogenes infection

Surface-sterilized leaf disks of horse-radish (Armoracia lapathifolia) were immersed in a suspension of Agrobacterium rhizogenes harboring the root-inducing plasmid (pRi) and cultured on a solid medium. Within about 10 days after inoculation, adventitious roots (hairy roots) emerged from the leaf disks. No roots emerged from the uninoculated leaf disks. The excised hairy roots grew vigorously in the dark and exhibited extensive lateral branches in the absence of phytohormones. When the hairy roots were moved into the light, numerous adventitious buds thrust out of the roots within about 10 days, and they developed into complete plants (R0 generation). R0 plants revealed leaf wrinkle. Root masses of cultured R0 plants were of two types. One had fibrous roots only and the other had both fibrous and tuberous roots Leaf disks of the R0 plants proliferated adventitious roots (R1 generation) on a solid medium after 1–2 weeks of culture. Phenotypical characters of the R1 roots were the same as those observed with the initial hairy roots. The T-DNA sequences of pRi were detected within DNA isolated from the hairy roots and their regenerants.

Production of Tropane Alkaloids by Hairy Root Cultures of Scopolia japonica

Hairy root clones of Scopolia japonica were established by selection of adventitious roots formed on the root segments inoculated with Agrobacterium rhizogenes strain 1 5834. Twenty-nine isolated hairy root clones displayed various phenotypes characterized by growth rate, opine production and tropane alkaloid production. Of these, two highly alkaloid productive clones SI and S22 were examined for their growth rate and alkaloid productivity under various cultural conditions. Whenthe most scopolamine-productive clone SI was cultured for 4 weeks at 25°C in the dark, the weight of the root tissue was increased by 40 times and the content of scopolamine reached a level of 0.5% on a dry weight basis in each optimum medium. On culture of the most hyoscyamine-productive clone S22under the same conditions as with SI , the weight was increased by 102 times and the content of hyoscyamine was 1.3% on a dry weight basis in each optimum medium.

Studies on enantioselective hydrolysis of the acetic ester of a secondary alcohol with Arthrobacter lipase

SummaryCharacteristics of the enantioselective hydrolysis of the acetic ester of 4-hydroxy-3-methyl-2-(2-propynyl)-2-cyclopentenone (HMPC) by Arthrobacter lipase were investigated in a water/oil biphasic reaction mixture. Kinetic studies revealed that the strict enantioselectivity was entirely due to a difference in the catalytic constants for the enantiomeric substrates and that (S)-HMPC acetate acted as a competitive inhibitor. The comparison of enantioselectivity for the acetates of HMPC analogues indicated that hydrophobic substituents in the HMPC molecule were essential for the strict enantioselectivity.

Characterization of recombinant DNA-derived human granulocyte macrophage colony stimulating factor by fast atom bombardment mass spectrometry

Abstract Proteolytic digests of biologically active fractions of recombinant DNA-derived human granulocyte macrophage colony stimulating factor (rhGM-CSF) expressed in large quantities in Escherichia coli have been analyzed by fast atom bombardment mass spectrometry (FAB-MS) and high-performance liquid chromatography (HPLC). The major fraction fractionated by reversed-phase HPLC (RP-HPLC) was N-terminal methionine additional type of human GM-CSF. Disulfide bond linkages were confirmed between Cys-54 and Cys-96, Cys-88 and Cys-121. Furthermore five minor fractions were isolated, and characterized by FAB-MS and HPLC. One of them was a rhGM-CSF in which two amino acids at the N-terminal (Met-Ala) were deleted. Other minor fractions were rhGM-CSFs of which one of four methionine residues was sulfoxidated, respectively. These findings suggest that the removal of amino acids occurs post-translationally, and that the sulfoxidation of methionine residues occurs during oxidative renaturation and/or purification. FAB-MS combined with HPLC provides a very useful method for the characterization of recombinant proteins, because the method is very convenient and simple.

Selective Killing of HIV-Infected Cells by Liposomes Composed of dimyristoylphosphatidylcholine/phosphatidylserine/cholesterol

We have previously shown that liposomes containing fragment A of diphtheria toxin, which were prepared by the detergent-dialysis method with egg phosphatidylcholine, phosphatidylserine and cholesterol, possess a selective killing activity against human immunodeficiency virus (HIV)-1-infected cells, but not against uninfected cells (Ikuta et al., 1987). Since the liposomes were found to be unstable in human plasma in vitro, we prepared improved liposomes by the extrusion method with dimyristoylphosphatidylcholine instead of egg phosphatidylcholine. These liposomes were found to be very stable in human plasma, and also possessed the selective killing activity against HIV-1-infected cells. In addition, it was found that the fragment A in the liposomes was not necessary for the selective cell killing activity. The cell killing activity and selectivity of HIV-1-infected cells of the liposomes were remarkably affected by cholesterol content and the acyl chain length of the saturated fatty acid of phosphatidylcholines. These data suggest that membranes of liposomes can interact specifically with HIV-1-infected cells, but not with uninfected cells, resulting in the inhibition of cell proliferation.