Toshio Hara

A novel cell-free translation/glycosylation system prepared from insect cells

A cell-free translation/glycosylation system derived from lepidopteran (Sf21) cells, which are widely used to express high yields of foreign active proteins that have post-translational modifications, was constructed. The insect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-translational components. The gp120 mRNA was transcribed from the human immunodeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polymerase. When the gp120 mRNA was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, and they both had reduced mobility compared with gp120 secreted by recombinant baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molecules expressed on the surface of quail cells. These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.

A Study on the Mechanism of DNA Excretion from P. aeruginosa KYU-1—Effect of Mitomycin C on Extracellular DNA Production—

The mechanism of excretion of DNA was investigated using strain KYU-1. This strain produced a considerable amount of DNA extracellularly (7.8mg/ml), and the amount of extracellular DNA per ml of the culture was scores of times more compared to that of intracellular DNA per ml of the culture. DNA synthesis was repressed by the addition of inhibitors (nalidixic acid, 5-fluoro uracil or mitomycin C), their inhibition rates were 35 to 54%. With the addition of 100 μg/ml of mitomycin C at the middle of the logarithmic growth phase, only 0.89 mg of extracellular DNA per ml of the culture was obtained, the inhibition rate was 95%. However, the amount of organic phosphate synthesized in the culture was almost the same no matter whether with or without an inhibitor.Furthermore, the excretion of DNA from the cell surface of strain KYU-1 was observed with on electron microscope and the DNA molecules excreted were found to be double-stranded.

The DNA sequence of γ-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1

SummaryThe γ-glutamyltranspeptidase (γ-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined. The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49356. Putative -35 and -10 sequences, TTCAAA and TATTAT, were observed as the consensus sequence for the promoter recognized by the σ43 RNA polymerase of B. subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The amino acid sequence of the gene with the segment of putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian γ-GTPs does not show any similarity at all.

Coinfection of Spodoptera exigua and Spodoptera frugiperda cell lines with the nuclear polyhedrosis viruses of Autographa californica and Spodoptera exigua

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) has a broad host range among Lepidoptera. In contrast, the Spodoptera exigua MNPV (SeMNPV) can replicate efficiently only in S. exigua larvae or S. exigua-derived cell lines. In this study, we examined the coinfection of S. exigua Se301 and Spodoptera frugiperda IPLB-SF21AEII (Sf21) cell lines with SeMNPV and AcMNPV recombinant (Ac360-501β-gal) which was constructed for expression of β-galactosidase under control of the polyhedrin promoter. Coinfection led to the restriction as the level of late gene expression, nonoccluded virus production, and DNA replication of Ac360-501β-gal in both Se301 and Sf21 cell lines. In contrast, Ac360-501β-gal supported the SeMNPV replication in Sf21 cells. Occurrence of recombinants, between Ac360-501β-gal and SeMNPV, with expanded host range was not observed in coinfected Sf21 cells. This suggests that Ac360-501β-gal supports the SeMNPV replication through trans-activation.

Colorimetric detection of DNA-DNA hybridization in microdilution wells for taxonomic application on bacterial strains

Abstract Colorimetric detection of hybridization in microdilution wells without radioisotope was designed and established for rapid and routine identification of taxonomic relatedness among bacterial species. Total chromosomal DNA for hybridization reactions was labeled with photoreactive biotin (photobiotin). The biotinylated DNA was hybridized with unlabeled single-stranded DNAs which were immobilized on the surfaces of microdilution wells. After hybridization, the biotinylated DNA was quantitatively detected by using an enzyme, streptavidin-conjugated peroxidase, and a dye, 3,3′,5,5′-tetramethylbenzidine, and analyzed with a microplate reader. The homology values among Bacillus spp. with biotinylated DNA of B. subtilis (natto) were in good agreement with those obtained by the membrane filter method with radioisotope. The method presented here is simple, rapid, and safe, and may be applicable routinely for the identification of bacterial species.

Differential Level in Co‐Down‐Modulation of CD4 and CXCR4 Primed by HIV‐1 gp120 in Response to Phorbol Ester, PMA, among HIV‐1 Isolates

HIV‐1 enters cells through interacting with cell surface molecules such as CD4 and chemokine receptors. We generated recombinant soluble gp120s derived from T‐cell line‐tropic (T‐tropic) and macrophage‐tropic (M‐tropic) HIV‐1 strains using a baculovirus expression system and investigated the association of CD4‐gp120 complex with the chemokine receptor and/or other surface molecule(s). For monitoring the co‐down‐modulations of the CD4‐gp120 complex, a cytoplasmic domain deletion mutant (tailless CD4), which is not capable of undergoing down‐modulation by itself in response to phorbol ester PMA, was used. Our studies revealed both cell‐type and HIV‐1 strain‐specific differences. We found that T‐tropic gp120s were capable of priming co‐down‐modulation with tailless CD4 by interacting with CXCR4, whereas M‐tropic SF162 gp120 could not after PMA treatment even in the presence of CCR5. Among the T‐tropic HIV‐1 envelopes, IIIB gp120 was the most potent. Furthermore, the ability of gp120 to prime the PMA induced co‐down‐modulation of tailless CD4 appeared to be dependent on the concentration of the principal coreceptor CXCR4. Nevertheless, the observation that IIIB gp120 strongly primed tailless CD4 co‐down‐modulation on human osteosarcoma HOS cells that express undetectable levels of surface CXCR4 raised the possibility that membrane component(s) other than those recently identified can be involved in down‐modulation of the CD4/gp120 complexes.

Screening of compounds stimulating spore formation and mycelial growth of pock-forming plasmid-carrying strains in Streptomyces azureus

SummarySporulation-stimulating compounds were screened by using mutant PK100a of Streptomyces azureus ATCC 14921, in which spore formation was markedly inhibited by the pock-forming plasmid pSA1.1. On agar media, cysteine, bacitracin, glutathione and β-NAD induced the formation of coloured spores or spore mass of strain PK100a. These compounds also stimulated the spore formation of the wild-type and its plasmid-cured (good spore-forming) strains and the growth of aerial mycelia of these three strains. This screening method appears to be an effective method for the screening of substances that stimulate spore formation and mycelial growth of streptomycetes or that overcome the inhibitory effect of pock-forming plasmids.

Comparison and Characterization of Aroma Components of Chinese Oolong Tea and Japanese Semi Fermented Tea

中国産ウーロン茶と,わが国で試作したウーロン茶の香気成分を分析し,次の結果を得た。(1) 中国産ウーロン茶は日本産のものより,ネロリドール,ロンギフォレン,ファルネッセン,トリエンー3―オール,シスージャスモン,ジャスミンラクトン,ベンジルシアニド,インドールなどが多く含まれていた。これに対し,日本産ウーロン茶にはトランスー2―ヘキセナール,シスー3―ヘキセンー1オール,ベンジルアルコールなどが多く含まれていた。この結果は,中国産ウーロン茶に特有の花のような香りが強く,日本産ウーロン茶は青臭いにおいが強いという官能検査の結果とよく一致していた。(2) 中国産ウーロン茶に多く含まれるセスキテルペンとして,茶から初めてロンギフォレンを同定した。(3) 日本産ウーロン茶と中国産のものの香気形成の差異を明らかにするため,ウーロン茶に多く含まれるネロリドールと,紅茶に多く含まれるリナロールおよびシスー3―ヘキセン―1オールの含量比を調べた。その結果,日本産ウーロン茶は,中国産ウーロン茶と紅茶の中間型の香気形成を示した。この研究を行うにあたり,標品のロンギフォレンを分与していただき,同定についてご教示を賜ったお茶の水女子大学教授小林彰夫博士に厚くお礼申し上げます。また,供試材料のウーロン茶を提供していただいた,三井農林株式会社三浦宣安氏,静岡県茶業試験場高橋宇生氏,三重県茶業センター木下 〓氏に深く感謝いたします。

Off-flavor components in stored packaged green tea.

Green tea off-flavor components were analyzed in two different types of tea storage pouches by gas chromatography. In a moisture proof pouch, the reversion flavor with green notes was found in sensory tests, and off-flavor components, such as 1-penten-3-ol, (Z)-2-penten-l-ol, (E, Z)-2, 4-heptadienal and (E, E)-2, 4-heptadienal, increased remarkably in storage of over three months. The amount of firing tea aroma components, such as 2-methylpyrazine, 2, 5-dimethylpyrazine and 1-ethyl-2-formylpyrrole, remained unchanged in four months storage. In an ordinary cellophane-polyethylene laminated pouch, a slightly acidic flavor was organoleptically detected, and acetic acid increased markedly in storage over three months. The off-flavor components did not increase in ordinary packaged tea as in the moisture proof package.

Changes in Aroma Components during Roasting of Green Tea

Volatile components were obtained from raw tea (Ban-cha) and roasted green tea by vacuum distillation followed by ether extraction for gas liquid chromatography using capillary column.The production of pyrazines, furans and pyrroles was remarkable during roasting of green tea indicating they have important contributions to the aroma of roasted green tea.Two isomeric 2, 4-heptadienals were newlyidentified from volatile components of green tea before roasting. These compounds were remarkably decreased during roasting.