Hitoshi Kakidani

Chemo-enzymatic synthesis of 3-(2-naphthyl)- L-alanine by an aminotransferase from the extreme thermophile, Thermococcus profundus

Hyper-thermostable aminotransferase from Thermococcus profundus (MsAT) was used to synthesize 3-(2-naphthyl)-l-alanine (Nal) by transamination between its corresponding α-keto acid, 3-(2-naphthyl)pyruvate (NPA) and l-glutamate (Glu) at 70 °C. Equilibrium of this reaction was shifted toward Nal production due to its low solubility, giving rise to Nal precipitate. Optically pure Nal (>99% ee) was synthesized with 93% (mol mol−1) yield from 180 mM NPA and 360 mM Glu.

Oxidation of both termini of p- and m-xylene by Escherichia coli transformed with xylene monooxygenase gene

Abstract Xylene monooxygenase (XMO) from Pseudomonas putida mt-2 catalyzes oxidation of methyl group of toluene and xylenes. While it has been postulated that this enzyme oxidizes one methyl group of xylene, we observed that both methyl groups in p - and m -xylene were oxidized to alcohol and aldehyde when the relevant genes ( xylM and xylA ) were co-expressed in Escherichia coli C600 and MC4100. When p -xylene was used as a substrate, p -hydroxymethylbenzaldehyde and p -xylyleneglycol were identified, in addition to p -methylbenzylalcohol and p -tolualdehyde. When m -xylene was used as a substrate, m -hydroxymethylbenzaldehyde and m -xylyleneglycol were identified, in addition to m -methylbenzylalcohol and m -tolualdehyde. Ratio of the products varied significantly according to the reaction condition and host strain, presumably reflecting the relative activity of XMO and host-derived dehydrogenase(s). Using various oxidized compounds as substrates, it was indicated that dialcohol ( p - or m -xylyleneglycol) was formed via p - or m -hydroxymethylbenzaldehyde, respectively, rather than directly from corresponding monoalcohol ( p - or m -methybenzylalcohol).

Suppressive Effect of Liposomes Containing DNA Coding for Diphtheria Toxin A-Chain on Cells Transformed with Bovine Leukemia Virus

A recombinant plasmid which contained a gene for diphtheria toxin A‐chain (DT‐A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV‐LTR) was constructed to test a novel application of liposomes as antiviral agents. The promoter activity of BLV‐LTR was estimated by the chloramphenicol acetyltransferase (CAT) assay using a plasmid which contains the coding sequence of CAT under the control of BLV‐LTR (pBLVCAT). When BLV‐infected cells were transfected with pBLVCAT, CAT activity was detected. BLV‐uninfected cell lines, however, showed no detectable CAT activity. The plasmid DNA entrapped in liposomes was added to BLV‐infected cells in culture. Syncytium formation induced by BLV‐infected cells was effectively suppressed by the liposomes containing the gene for DT‐A under the control of BLV‐LTR. Conversely, liposomes containing the gene for DT‐A without a promoter showed no such effect. DT‐A gene‐containing liposomes with BLV‐LTR did not affect formation of syncytium induced by bovine immunodeficiency virus. These observations indicate that BLV‐infected cells were readily targeted on the level of gene expression. This strategy could be applied to the treatment of BLV‐induced B‐cell proliferation of cattle, and further to other viral/neoplastic diseases where specific gene expression is exerted.

Preparative-scale Enzyme-catalyzed Synthesis of (R)-α-Fluorophenylacetic Acid

A preparative-scale asymmetric synthesis of (R)-α-fluorophenylacetic acid, a useful chiral derivatizing reagent, is described. Starting from ethyl α-bromophenylacetate, α-fluorophenylmalonic acid dipotassium salt was prepared in three steps (54% yield), including nucleophilic substitution by the fluoride ion as the keystep. Both the purified form and crude preparation of arylmalonate decarboxylase in E. coli worked well on this substrate, and (R)-α-flurophenylacetic acid (>99% e.e.) was prepared in a quantitative yield.

Antitumor Effect of Diphtheria Toxin A-Chain Gene-containing Cationic Liposomes Conjugated with Monoclonal Antibody Directed to Tumor-associated Antigen of Bovine Leukemia Cells

Monoclonal antibody c143 against tumor‐associated antigen (TAA) expressed on bovine leukemia cells was conjugated to cationic liposomes carrying a plasmid pLTR‐DT which contained a gene for diphtheria toxin A‐chain (DT‐A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) in the multicloning site of pUC‐18. The specificity and antitumor effects of the conjugates were examined in vitro and in vivo using TAA‐positive bovine B‐cell lymphoma line as the target tumor. In vitro studies with the TAA‐positive cell line indicated that luciferase genecontaining cationic liposomes associated with the c143 anti‐TAA monoclonal antibody caused about 2‐fold increase in luciferase activity compared with cationic liposomes having no antibody, and also that the c143‐conjugated cationic liposomes containing pLTR‐DT exerted selective growth‐inhibitory effects on the TAA‐positive B‐cell line. Three injections of pLTR‐DT‐containing cationic liposomes coupled with c143 into tumor‐bearing nude mice resulted in significant inhibition of the tumor growth. The antitumor potency of the c143‐conjugated cationic liposomes containing pLTR‐DT was far greater than that of normal mouse IgG‐coupled cationic liposomes containing pLTR‐DT as assessed in terms of tumor size. These results suggest that cationic liposomes bearing c143 are an efficient transfection reagent for BLV‐infected B‐cell lymphoma cells, and that the delivery of the pLTR‐DT gene into BLV‐infected B‐cells by the use of such liposomes may become a useful technique for gene therapy of bovine leukosis.

Growth inhibition of cancer cells by Co-transfection of diphtheria toxin A-chain gene plasmid with bovine leukemia virus-tax expression vector

We constructed a plasmid containing bovine leukemia virus (BLV)‐tax gene driven by SRα promoter, designated as pME‐BLVtax, to activate the promoter of the long terminal repeat (LTR) of BLV in various tumor cells. Activation of the promoter of BLV‐LTR by pME‐BLVtax was confirmed by luciferase assay. When the cells, such as COS‐1, C8, and KU‐1, were transfected with a plasmid pBLV‐LUC1, which contained the luciferase gene under the control of BLV‐LTR, and pME‐BLVtax, luciferase was expressed in these cells, whereas no luciferase gene expression was observed when only pBLV‐LUC1 was introduced into the cells. Activation of the BLV‐LTR promoter was regulated by pME‐BLVtax and 0.5 μg of pME‐BLVtax was sufficient for the expression of the gene under the control of BLV‐LTR. Furthermore, pME‐BLVtax was used to direct the cell expression of the gene for diphtheria toxin A‐chain under the control of BLV‐LTR (pLTR‐DT) to various tumor cell lines, KU‐1, C8, COS‐1, BL2M3, and HeLa cells. The transfection was carried out with cationic liposomes. In this experiment, co‐transfection of pLTR‐DT with pME‐BLVtax exerted selective growth inhibitory effects on the tumor cell lines. Moreover, three co‐introductions of pLTR‐DT with pME‐BLVtax into the cell lines resulted in significant inhibition of the cell growth. This result suggests that the delivery of the pLTR‐DT and pME‐BLVtax genes into tumor cells by the use of cationic liposomes may be potentially useful as a novel approach for the treatment of tumor cells.

Improved Chromatographic Method for the Assay of Retroviral Proteases

The retroviral protease is essential for the maturation of infectious virus, and is therefore an attractive target for the design of antiviral drugs. To facilitate the screening of inhibitors of retroviral proteases, an improved chromatographic assay for the protease activity was developed.