Biomedical subjects
Publications and source records attributed to Katsuyoshi Matsunami.
The chemical investigation of leaves of Bridelia glauca f. balansae afforded six megastigmane glucosides, named bridelionosides A-F, along with seven known megastigmane glucosides. Their structures were determined by a combination of spectroscopic analyses and by application of the modified Mosher's method.
From the leaves of Helicia cochinchinensis, collected on Okinawa Island, seven phenolic glucosides and two terpenic glucosides were isolated. Five of the phenolic glucosides were previously known, being identified with p-coumaric and ferulic acids glucosyl esters, rhodioloside, helicidiol, and naringenin 5-O-beta-D-glucopyranoside. The structures of two other phenolic glucosides, named heliciosides A and B, were elucidated to be 5-O-beta-D-glucosides of 3-hydroxyflavanone, namely aromadendrin and taxifolin, by means of spectroscopic analyses. The two terpenic glucosides were identified with ampelopsisionoside and icariside C1.
The pig cDNA encoding C1 esterase inhibitor (C1-INH) was isolated and the homology of the sequence was compared with that from other animals. The structure of pig C1-INH contains a two disulfide bridge pattern identical to the human C1-INH. In the amino acid sequence of the first Cys-91 to the C-terminal end, the pigC1-INH has a 76.2% homology with the human protein, and the sequence of the reactive site is close to the human. A surface-bound form of pig and human C1-INH, pC1-INH-PI and hC1-INH, respectively, were next constructed. Stable Chinese hamster ovarian tumor (CHO) cell lines and pig endothelial cell (PEC) lines expressing these C1-INH-PI were prepared by transfection. The basic function and the species specificity of pCI-INH were then investigated using these transfectants. pC1-INH and hC1-INH have almost the same suppressive effect on pig, human, dog and rabbit sera in complement-dependent cell lysis, indicating little species specificity.
HLA-E functions as an inhibitory signaling molecule of natural killer (NK) cell-mediated cytolysis. However, the cell surface expression of HLA-E molecules is quite restricted because of the limited repertoire of binding peptide sequences, such as signal peptides of other HLA molecules, especially on xenogeneic cells. In this study, we successfully determined that position-147 is an important amino acid position for cell surface expression by producing point substitutions. For further studies concerning transplantation therapy, the point substitution, Ser147Cys, that resulted in a single atom change, oxygen to sulfur, designated as HLA-Ev(147), led to a much higher expression on the human and pig cell surface and a greater inhibitory function against human NK cells than wild type HLA-E in an in vitro model system of pig to human xenotransplantation. Consequently, HLA-Ev(147) might be a promising alternative gene tool for future transplantation therapy such as xenotransplantation.
Xenotransplantation is one of the most attractive solutions for the current worldwide shortage of organs. The knocking out of alpha1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, more recent studies indicate that other xeno-antigens, so-called non-Gal antigens, will also need to be downregulated. In this study, pig N-acetylglucosaminyltransferase I (GnT-I), a key enzyme that initiates the biosynthesis of hybrid- and complex-type N-linked sugar chains, was isolated and the pigGnT-I.2 specific for the O-linked sugar chain was also isolated. Point mutants, pigGnT-I(123) and pigGnT-I(320), were subsequently constructed. While pigGnT-I(123) shows an indistinct dominant negative effect for endogenous GnT-I in pig cells, pigGnT-I(320) had a drastic effect. In addition, in the case of pig cell transfectants with pigGnT-I(320), cell surface carbohydrate structures were significantly altered and its antigenicity to human serum was reduced. Consequently, pigGnT-I(320) appears to be potentially useful in xenotransplantation by remodeling the carbohydrate structures on pig cells.
BACKGROUND: Porcine endogenous retrovirus (PERV) released from pig cells is a main problem associated with clinical xenotransplantation. In a previous study, we demonstrated that the high mannose type of N-glycan of the envelope glycoprotein is closely related to PERV infectivity with respect to human cells. In this study, we addressed the effects of reducing the high mannose type of N-glycan on PERV infectivity. METHODS: Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection to produce PEC(Z). The PEC(Z)s were then further infected with PERV subtype B (PERV-B) to produce PEC(Z)/PB. The PEC(Z)/PBs were next transfected with the alpha 1,2 mannosidase Ib (Man Ib), N-acetylglucosaminyltransferase I (GnT-I) or alpha-mannosidase II (Man II) gene in order to reduce the levels of high mannose type of N-glycan. HEK293 cells were inoculated with the PERV in each of the culture supernatants. The inoculated cells were histochemically stained and the LacZ-positive cells were counted. RESULTS: In experiment I, PERV transmission from the PEC(Z)/PB with GnT-I or Man II to HEK 293 cells was significantly reduced in comparison with control PEC(Z)/PB, while the PEC(Z)/PB with Man Ib was not. However, in experiment II, PERV transmission from the PEC(Z)/PB with ManIb to HEK 293 cells was also significantly reduced in comparison with control PEC(Z)/PB. CONCLUSION: The transfection of these genes to pig cells is effective in reducing the susceptibility of human cells to PERV infection. The results suggest that this represents a potentially useful strategy for further decreasing the likelihood of PERV infections.
Two new diarylheptanoids, designated muricarpones A and B, together with three known diarylheptanoids, 1,7-di-(3',4'-dihydroxyphenyl)-4-hepten-3-one, 1-(3',4'-dihydroxyphenyl)-7-(4''-hydroxyphenyl)-4-hepten-3-one, and 1,7-bis(p-hydroxyphenyl)-4-hepten-3-one, were isolated from the rhizomes of Amomum muricarpum ELMER (Zingiberaceae). Their structures were determined using spectroscopic analyses.
Four new megastigmane glucosides, named macarangiosides A-D (2-5), together with mallophenol B, lauroside E, methyl brevifolin carboxylate, and hyperin and isoquercitrin as a mixture were isolated from the leaves of Macaranga tanarius (L.) MULL.-ARG. (Euphorbiaceae). Their structures were elucidated by spectroscopic and chemical analyses. Macarangioside A-C (2-4) and mallophenol B were galloylated on glucose moiety and possessed the potent 2,2-diphenyl-picrylhydrazyl (DPPH) radical-scavenging activity.
Four new natural neolignans and lignans, which were given the trivial names odoratisols A-D (1-4), together with (-)-licarin A, kachirachirol B, obovatifol, and machilin-I were isolated from the air-dried bark of the Vietnamese medicinal plant Machilius odoratissima NEES (Lauraceae). Their absolute structures were determined on the basis of spectroscopic analyses including circular dichroism spectra.
From the aerial parts of Scoparia dulcis L. (Scrophulariaceae) grown in Vietnam, four scopadulane-type diterpenoids (4-7), of which 7 is new and was given the trivial name scopadulcic acid C, together with nine known compounds were isolated. Their structures were elucidated by spectroscopic analyses. The absolute configurations of 4-7 were ascertained by applying the modified Mosher's method to iso-dulcinol (6). The isolation of the lignans nirtetralin and niranthin for the first time from S. dulcis is also of chemotaxonomic interest. The cytotoxic activity in KB cells, inhibitory effect on LPS/IFNgamma-induced NO production, inhibition of multidrug resistance (MDR), and antibacterial and antifungal activities of the scopadulane-type diterpenoids 4-7 were examined in this study.
Eight ent-kaurane glucosides, named tricalysiosides H-O, were isolated from Tricalysia dubia. Tricalysioside H possessed a hydroxyl group at the 1-position, to which the glucose moiety was attached. The structure was first elucidated by means of spectroscopic data analysis and finally confirmed by X-ray crystallography. Since acid hydrolysis of 1 gave D-glucose, the aglycone was proved to have an enantio-kaurane type skeleton. The structures of tricalysiosides I-O were mainly elucidated from analysis of spectroscopic evidence.
The possibility of preventing the transmission of porcine endogenous retrovirus (PERV) to human cells using short interfering RNAs (siRNA) was investigated. The siRNA for the p30 of PERV gag region was cloned into pSUPER, the polymerase-III H1-RNA gene promoter. A green fluorescence protein (GFP) was also cloned into pSUPER to establish pSXGH. Pig endothelial cells (PEC) were transduced with the LacZ gene by pseudotype infection, and infected with PERV subtype B, resulting in the formation of PEC(LacZ)/PB. The PEC(LacZ)/PB was next transfected with pSXGH-siRNA. The expression of siRNA was provisionally checked by determining the level of expression of GFP. Culture supernatants of infected cells were then inoculated into HEK293 cells. The siRNA clearly destroyed the PERV infectivity of PEC(LacZ)/PB in both transient cell lines and stable clones. Moreover, the decreased levels of mRNA and gag protein were evidenced in the stable clones by real-time PCR and Western blotting, respectively. The final goal of our study was to establish a transgenic pig expressing the siRNA for PERV. The results suggest that siRNA represents a novel approach for controlling PERV infections in clinical xenotransplantation.
Three new diarylheptanoids, called alpinnanins A-C (1-3), together with two known chalcones, 2',4'-dihydroxy-6'-methoxychalcone and 4',6'-dimethylchalconaringenin, two known flavanones, alpinetin and naringenin 5-O-methyl ether, a known diarylheptanoid, (3S,5S)-trans-3,5-dihydroxy-1,7-diphenyl-1-heptene, stigmasterol and beta-sitosterol as a mixture, and beta-sitosterol 3-O-beta-D-glucopyranoside were isolated from the rhizomes of Alpinia pinnanensis T. L. WU et SENJEN (Zingiberaceae). Their structures were elucidated by spectroscopic analyses.
Twelve natural bis-spirolabdane-type diterpenoids, including eight new, named leoheteronones A-E, 15-epileoheteronones B, D, and E, and four known leopersin B, 15-epileopersin B, leopersin C, and 15-epileopersin C, together with hispanone and galeopsin were isolated from the aerial parts of the medicinal plant Leonurus heterophyllus SW. (Lamiaceae) grown in Vietnam. Their structures were determined by spectroscopic analyses. The current study emphasized the accumulation of C-15 oxygenated bis-spirolabdane-type diterpenoids of both 13R and 13S configurations in L. heterophyllus.
Two new megastigmane glucosides, called excoecariosides A and B were isolated from the leaves of a medicinal Vietnamese plant, Excoecaria cochinchinensis LOUR. var. cochinchinensis (Euphorbiaceae) together with seven known compounds. Their structures were elucidated by spectroscopic analyses and chemical reactions including the modified Mosher's method.
Chemical investigation of leaves of Staphylea bumalda DC., collected in the suburbs of Hiroshima City, afforded 11 new megastigmane glucosides, named staphylionosides A-K (3-13), along with two known megastigmane glucosides (1, 2). The relative structures were elucidated from spectroscopic evidence, and the absolute structures of the aglycones were determined by means of the combination of beta-D-glucosylation-induced shift-trends and the modified Mosher's method.
Five new natural labdane-type diterpenoids (5-9), designated leoheteronins A-E, together with four known diterpenoids (1-4), two phytosterols as a mixture of beta-sitosterol and stigmasterol, and the flavone genkwanin (10) were isolated from the aerial parts of Leonurus heterophyllus SW. (Lamiaceae) collected in northern Vietnam. Compound 1 was isolated for the first time from a Leonurus species, and 10 is considered to be a chemotaxonomic marker of the Leonurus genus. Their structures were determined using spectroscopic analyses.
NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.