Yoshinari Shoyama

Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography

HIV-1 protease is a dimeric aspartic protease that plays an essential role in viral replication. To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp-25 and Asp-125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9-Å resolution and X-ray crystallography at 1.4-Å resolution. The resulting structural data show that the catalytic residue Asp-25 is protonated and that Asp-125 (the catalytic residue from the corresponding diad-related molecule) is deprotonated. The proton on Asp-25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine (Apns) group in KNI-272. The deprotonated Asp-125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease and can therefore contribute substantially to the development of specific inhibitors for therapeutic application.

Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa.

Cannabidiolic‐acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic‐acid into CBDA, the dominant cannabinoid constituent of the fiber‐type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene‐specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic‐acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug‐type Cannabis plants.

Micropropagation ofPanax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets

Somatic embryogenesis was induced in callus tissues derived from young flower buds ofPanax notoginseng via callus within 18 weeks of culture. The mature somatic embryos were germinated on half-strength Murashige and Skoogs (MS) medium supplemented with gibberellic acid A3(GA) and 6-benzyladenine (BA). The most suitable medium for optimal root formation proved to be MS medium supplemented with 1-naphthaleneacetic acid (NAA). Total DNA was extracted from the leaves of the regenerated plantlets ofP. notoginseng. Analysis of random-amplified polymorphic DNA (RAPD) using 21 arbitrary oligonucleotide 10-mers, showed the genetic homogeneity ofP. notoginseng. The amplification products were monomorphic for all of the plantlets ofP. notoginseng regenerated by embryogenesis, suggesting that somatic embryogenesis can be used for clonal micropropagation of this plant.

Crocin and crocetin derivatives inhibit skin tumour promotion in mice

The addition of 100 μg/mL of a 50% EtOH extract of Crocus sativus (ECS) exhibited significant inhibitory effects on EBV‐EA activation and preserved the high viability of Raji cells. The attenuating effect of ECS was dose‐dependent. The oral administration of ECS demonstrated an inhibitory effect on two‐stage carcinogenesis of mouse skin papillomas, using DMBA as an initiator and TPA as a promoter. Crocetin gentiobiose glucose ester and crocetin di‐glucose ester were less potent than crocin in inhibiting the EBV‐EA induction effect. When crocin was applied before each TPA treatment, it delayed the formation of papillomas; only about 10% of mice bore papillomas at 9 weeks of promotion and after 13 weeks of promotion 20% of mice still bore no papilloma. The effect of crocin was not mimicked by gentiobiose or glucose alone.

Identification and Characterization of Cannabinoids That Induce Cell Death through Mitochondrial Permeability Transition in Cannabis Leaf Cells

Cannabinoids are secondary metabolites stored in capitate-sessile glands on leaves of Cannabis sativa. We discovered that cell death is induced in the leaf tissues exposed to cannabinoid resin secreted from the glands, and identified cannabichromenic acid (CBCA) and Δ1-tetrahydrocannabinolic acid (THCA) as unique cell death mediators from the resin. These cannabinoids effectively induced cell death in the leaf cells or suspension-cultured cells of C. sativa, whereas pretreatment with the mitochondrial permeability transition (MPT) inhibitor cyclosporin A suppressed this cell death response. Examinations using isolated mitochondria demonstrated that CBCA and THCA mediate opening of MPT pores without requiring Ca2+ and other cytosolic factors, resulting in high amplitude mitochondrial swelling, release of mitochondrial proteins (cytochrome c and nuclease), and irreversible loss of mitochondrial membrane potential. Therefore, CBCA and THCA are considered to cause serious damage to mitochondria through MPT. The mitochondrial damage was also confirmed by a marked decrease of ATP level in cannabinoid-treated suspension cells. These features are in good accord with those of necrotic cell death, whereas DNA degradation was also observed in cannabinoid-mediated cell death. However, the DNA degradation was catalyzed by nuclease(s) released from mitochondria during MPT, indicating that this reaction was not induced via a caspase-dependent apoptotic pathway. Furthermore, the inhibition of the DNA degradation only slightly blocked the cell death induced by cannabinoids. Based on these results, we conclude that CBCA and THCA have the ability to induce necrotic cell death via mitochondrial dysfunction in the leaf cells of C. sativa.

Biosynthesis of propyl cannabinoid acid and its biosynthetic relationship with pentyl and methyl cannabinoid acids

Abstract Biosynthesis of propyl cannabinoid acid has been determined by in vitro incubation with a crude enzyme solution from three strains of Cannabis sativa using 14 C-labelled cannabinoid acid. Biosynthetic relationships between methyl, propyl and pentyl cannabinoid acids have been demonstrated.

Somatic embryogenesis and plant regeneration from the anther of Aconitum carmichaeli Debx.

Anthers of Aconitum carmichaeli Debx. were used for callus induction. After the addition of 5 ppm 2,4-D and 1 ppm kinetin callus induction occurred over a period of 15 weeks. When calluses were subcultured on a medium containing 1 ppm 2,4-D for 12 weeks embryogenesis occurred. Mature somatic embryos developed normal shoots when transferred to basal medium inoculated with 1 ppm GA and 5 ppm BAP. Rooting occurred after the transfer of shoots to a new medium containing 0.5 ppm IAA and plantlets formed. The transplantation of these was successful and all plants matured during 5 months subsequent cultivation.

Crystallization of Δ1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

Delta1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure-function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A. The calculated Matthews coefficient was approximately 4.1 or 2.0 A3 Da(-1) assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

Somatic Embryogenesis in Ginseng (Panax Species)

Almost all Panax spp. (family Araliaceae) have been used in folk medicine. The most famous variety is P. ginseng, which was recorded in Chinese Materia Medica 2000 years ago. Ginseng is one of the most important Chinese medicines used in tonics to combat stress and cancer, disturbances of the central nervous system, and hypothersmia, and for radio-protection. It contains many dammarane and oleanane saponins of which the biological activity has been studied widely. P. pseudoginseng var.notoginseng is cultivated in Kumming province in China. It is also an important Chinese crude drug used as an astringent and tonic. P. japonicus grows mainly in Japan and China, and is used as a stomachic and a hair-growing tonic in Japan.

Elucidations of the catalytic cycle of NADH-cytochrome b5 reductase by X-ray crystallography: new insights into regulation of efficient electron transfer

NADH-Cytochrome b5 reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome b5 (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68Å resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD(+). Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78Å resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.