Itaru Yamamoto

Formation of a stable l-ascorbic acid α-glucoside by mammalian α-glucosidase-catalyzed transglucosylation

Abstract Enzymatic transglucosylation from maltose to l -ascorbic acid (AA) with mammalian tissue homogenates was determined by a high-performance liquid chromatography method and compared with the reaction catalyzed by α-glucosidase from Aspergillus niger. The homogenates of small intestine and kidney had a high transglucosylase activity to form a new type of glucosylated AA, which was associated with α-glucosidase activity. The new compound was demonstrated to be an equimolar conjugate of AA and glucose by the spectral and quantitative analyses. In particular, it showed a high stability in a neutral solution and no reducing activity toward cytochrome c and a dye. These properties were very different from those of AA and l -ascorbic acid α-glucoside formed with α-glucosidase form A. niger, but they were consistent with those of l -ascorbic acid 2-O-phosphate and l -ascorbic acid 2-O-sulfate. Moreover, it exhibited a reducing power associated with AA after mild acid hydrolysis or treatment with rat intestinal α-glucosidase. These results indicate that it should be assigned the 2-O-α-glucoside structure. Consequently, i should be assigned the 2-O-α-glucoside structure. Consequently, it is concluded that mammalian α-glucosidase is able to form a very stable and nonreducing form of glucosylated AA through a specific transglucosylation reaction distinct from that of microbial α-glucosidase.

Induction of hepatocyte growth factor in human skin fibroblasts by epidermal growth factor, platelet-derived growth factor and fibroblast growth factor.

Hepatocyte growth factor (HGF) is a potent mitogen for rat and human hepatocytes in primary culture and appears to be the physiological hepatotrophic factor that triggers or modulates liver regeneration. Regulation of HGF gene expression and the protein production in human skin fibroblasts was examined. Addition of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF) and transforming growth factor-alpha (TGF-alpha) to confluent cultures of the cells markedly stimulated HGF secretion from the cells. The stimulating effect of EGF, PDGF and bFGF was further investigated. The effect of all three growth factors was maximal at 3-30 ng/ml and was accompanied by an increase in HGF mRNA levels. The mRNA levels were not elevated at 5 h but were at 10 h or more after addition of EGF. The levels of HGF mRNA in fibroblasts treated with the optimal doses of EGF, PDGF, bFGF, aFGF and TGF-alpha for 24 h were 6, 4, 5, 4 and 5 times that of control cultures incubated in medium only, respectively. The growth factor-induced HGF mRNA expression and HGF secretion was inhibited by addition of TGF-beta 1 or dexamethasone. Pretreatment with a high dose of phorbol 12-myristate 13-acetate (PMA), which causes down-regulation in protein kinase C (PKC) activity and PMA-induced HGF secretion, did not reduce the effects of the growth factors on HGF mRNA expression and HGF secretion, but rather enhanced them.(ABSTRACT TRUNCATED AT 250 WORDS)

TGF-β is a potent inhibitor of hepatocyte growth factor secretion by human fibroblasts

Transforming growth factor-beta 1 (TGF-beta 1) inhibited secretion of human hepatocyte growth factor (hHGF), which is also known as scatter factor or fibroblast-derived tumor cytotoxic factor, by MRC-5 cells. The effect was detectable at as little as 10 pg/ml and was more potent than that of dexamethasone. Complete inhibition was observed after 12 h in the presence of 5 ng/ml of TGF-beta 1. Phorbol 12-myristate 13-acetate-induced secretion of hHGF from human skin fibroblasts was also suppressed by TGF-beta 1. TGF-beta 2 inhibited hHGF secretion by MRC-5 cells to the same extent as TGF-beta 1, but other growth factors such as epidermal growth factor and acidic and basic fibroblast growth factors had only a slight or null inhibitory effect.

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes: II. A major role of the mixed disulfide between 2-mercaptoethanol and cysteine

Five thiol compounds including 2-mercaptoethanol (2-ME) were examined for their augmenting effects on in vitro antibody response to sheep erythrocytes. Three compounds were effective with the following order of activity; 2-ME greater than dithiothreitol greater than cysteamine. Glutathione and thioglycollate failed to enhance the response. The same order or effectiveness was seen in the stimulation of [35S]cystine uptake by murine lymphocytes by these thiols. Murine lymphocytes took up cysteine five to six times more rapidly than cystine. It is, however, unlikely that 2-ME stimulation of cystine uptake is solely due to the reduction of cystine into cysteine, because 2-ME was still stimulatory after free thiol groups had disappeared in the medium containing 2-ME and [35S]cystine. The mixed disulfide of cysteine with 2-ME (Cys-2-ME) was found to be an only product after free thiols had been oxidized. [35S]Cys-2-ME was taken up by the lymphocytes with a comparable rate to cysteine via a transport system common to that of leucine and phenylalanine. Cysteine was, however, transported via a different route. It was observed that Cys-2-ME was readily metabolized to cysteine and glutathione after the uptake. Cys-2-ME added to cystine-free RPMI 1640 medium could support the antibody response as efficiently as cystine plus 2-ME. These observations strongly suggest that 2-Me stimulates cystine uptake and, therefore, enhances the antibody response through the formation of the mixed disulfide with cysteine.

Phorbol ester-induced secretion of human hepatocyte growth factor by human skin fibroblasts and its inhibition by dexamethasone

Human skin fibroblasts secreted a certain amount of human hepatocyte growth factor (hHGF), as determined by an enzyme‐linked immunosorbent assay for hHGF. This hHGF secretion was remarkably stimulated by protein kinase C (PKC)‐activating phorbol esters, which was inhibited by the simultaneous addition of dexamethasone. Pretreatment with phorbol 12‐myristate 13‐acetate (PMA) caused a down‐regulation in hHGF secretion. hHGF secreted by the PMA‐treated cells showed a potent hepatocyle growth‐promoting activity which was neutralized by an anti‐hHGF antiserum. These results indicate both that PMA‐treated human skin fibroblasts produce biologically active hHGF and the possible involvement of PKC activation in this process.

Enzyme immunoassay for clenbuterol, an beta 2-adrenergic stimulant.

A sensitive double antibody and heterologous enzyme immunoassay for the quantitation of clenbuterol is established. Specific antiserum to this agent was raised in rabbits by immunization with diazotized clenbuterol and human serum albumin conjugate. For competitive reactions, antibody was incubated with a mixture of diazotized clenbuterol analog (NA 1141) labelled with beta-D-galactosidase and unlabelled standard or sample clenbuterol. The antibody-bound enzyme hapten was separated from free hapten by anti-rabbit IgG immobilized to a polystyrene ball. Activity of the enzyme on the solid phase was fluorometrically determined. The assay system made it possible to ascertain values as low as 0.5 pg /tube of clenbuterol. By use of this assay method, the time course of plasma levels of clenbuterol was examined after a single oral administration (20 micrograms) to 3 healthy volunteers. It was shown that the maximum level was achieved after 2-3 hr with approximately 10 ng clenbuterol/dl of plasma.

Biological and Immunological Properties of Human Hepatocyte Growth Factor from Plasma of Patients with Fulminant Hepatic Failure.

We have recently purified human hepatocyte growth factor (hHGF), a heterodimer with molecular weight of about 83,000, from plasma of patients with fulminant hepatic failure (Gohda, E. et al., J. Clin. Invest. 81, 414-419, 1988). Biological and immunological properties of hHGF were examined. Out of the well-known growth factors tested, only epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulated DNA synthesis of adult rat hepatocytes in primary culture. hHGF enhanced the DNA synthesis at less than one-tenth of the molar concentrations of EGF and TGF-alpha. Half-maximal stimulations by hHGF, EGF and TGF-alpha were observed at 30, 400 and 900 pM, respectively. Maximal stimulation by TGF-alpha, however, was greater than those caused by hHGF and EGF. The effect of hHGF was additive with the maximal effects of EGF and TGF-alpha. Anti-hHGF antiserum was prepared in a rabbit by injecting with purified hHGF. This antiserum recognized nonreduced hHGF, but not reduced hHGF. The antiserum for hHGF did not inhibit growth-promoting activity of EGF, that was neutralized by incubation with anti-EGF antiserum. The activity of hHGF was completely inhibited by anti-hHGF antiserum, but not by anti-EGF antiserum. hHGF did not show any cross-reactivity to anti-EGF antiserum as measured by enzyme immunoassay for EGF. Thus, biological and immunological properties of hHGF are different from those of EGF and TGF-alpha.

The crystal structure and physicochemical properties of l-ascorbic acid 2-glucoside

The stable L-ascorbic acid glucoside produced by the action of the cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus stearothermophilus was crystallized from an aqueous solution. Determination of the molecular structure by single crystal X-ray analysis showed the compound to be 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G). The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell dimensions a = 11.929 A, b = 24.351 A, and c = 4.864 A. The D-glucopyranose residue has the 4C1 conformation. These conclusions are in good agreement with those based on the 13C-NMR spectrum. The general physicochemical properties of crystalline AA-2G are reported.

Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-α-glucoside formation

In this study, we characterized cyclodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus in L-ascorbic acid-2-O-alpha-D-glucoside (AA-2G) formation and compared its enzymological properties with those of rat intestinal and rice seed alpha-glucosidases which had the ability to form AA-2G. CGTase formed AA-2G efficiently using alpha-cyclodextrin (alpha-CD) as a substrate and ascorbic acid (AA) as an acceptor. Several AA-2-oligoglucosides were also formed in this reaction mixture, and they could be converted to AA-2G by the additional treatment of glucoamylase. The optimum temperature for AA-2G formation was 70 degrees C and its optimum pH was around 5.0. CGTase also utilized beta- and gamma-CDs, maltooligosaccharides, dextrin, amylose, glycogen and starch as substrates, but not any disaccharides except maltose. CGTase showed the same acceptor specificity as two alpha-glucosidases, whereas its hydrolyzing activity towards AA-2G was very low compared with those of alpha-glucosidases. Cleavage profiles of AA-2-oligoglucosides by CGTase present a possible mechanism for AA-2G formation that CGTase transfers a glucose-hexamer to an acceptor at the first step and then a glucose is stepwisely removed from the non-reducing end of the product through glucoamylase-like action of this enzyme. These results indicate that CGTase is able to synthesize AA-2G more efficiently than rat and rice alpha-glucosidases and utilization of this enzyme makes the mass production of AA-2G possible.

Effects of Rooibos Tea Extract on Antigen-specific Antibody Production and Cytokine Generation in Vitro and in Vivo

Rooibos tea contains a large amount of flavonoids and acts as a potent antioxidant. In this study, we examined the effects of Rooibos tea extract on antigenspecific antibody production and cytokine generation in vitro and in vivo. The primary in vitro anti-ovalbumin (anti-OVA) or sheep red blood cell (SRBC) antibody production in murine splenocytes was markedly stimulated by the addition of the tea extract at concentrations of 1-100 μg/ml. On the other hand, a nonspecific antibody response elicited with lipopolysaccharide (LPS) in purified splenic B-cells was not modified by the extract. Rooibos tea extract caused an increase in the generation of interleukin 2 (IL-2) both in OVA- and anti-CD3-primed splenocytes at concentrations ranging from 10 μg/ml to 1000 μg/ml. In contrast, this tea extract suppressed the generation of interleukin 4 (IL-4) in OVA-primed splenocytes. Moreover, the reduction of OVA-induced antibody production in serum of the cyclosporin A (CyA)-treated rats can be significantly restored and the IL-2 generation in murine splenocytes was stimulated, following oral administrations of Rooibos tea extract. Thus, our findings suggested that Rooibos tea extract may facilitate the antigen-specific antibody production through selective augmentation of IL-2 generation both in vitro and in vivo. Collectively, Rooibos tea intake may be of value in prophylaxis of the diseases involving a severe defect in Th1 immune response such as cancer, allergy, AIDS, and other infections.