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Biomedical subjects

J Nagai

Publications and source records attributed to J Nagai.

At least 91 records · Page 5Linked to original sources
PubMed1987

Construction of a new plasmid vector that can express cloned cDNA in all translational reading frames.

Construction of a bacterial expression vector, pSI4001, is described. The vector contains the lac promoter-operator and three sets of ribosome-binding sites (RBSs) tandemly arranged in all possible reading frames. cDNA can be directly cloned downstream from these translational start points in the fixed and proper orientation by using the method of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289]. The open reading frame of any cDNA inserted may be automatically aligned in phase with either of the three ATG start codons, thus enabling its expression with a maximum theoretical probability of unity. Fusion with the lacZ gene (coding for beta-galactosidase) has shown that at least two of the three translation initiation sites exhibit high expression capacities and the remaining one can also function at a lower but significant rate. We used the vector to construct a bovine pituitary cDNA library, from which clones coding for prolactin were detected by immunological screening with an efficiency as high as two in three clones. The construction with triple RBSs should also provide a unique experimental model to study the regulation of overlapping translations.

Animals↗
PubMed1987

Characterization of nystatin-resistant mutants of Saccharomyces cerevisiae and preparation of sterol intermediates using the mutants.

Analysis of sterols of Saccharomyces cerevisiae mutants N3, N15, N26, and N3H, defective in sterol biosynthesis, was performed. Strains N3, N15, and N26 were isolated from their mother strain, M10, by screening with nystatin (Nagai et al. (1980) Mie Med. J. 30, 215-224), and strain N3H was isolated from N3 as a doubly-mutated strain. The main sterols of N3, N15, N26, and N3H were ergosta-7,22-dienol, ergost-8-enol, cholesta-5,7,24-trienol, and ergosta-7,22,24(28)-trienol, respectively. The former three strains were characterized as defective in delta 5-desaturation, delta 8--delta 7 isomerization, and C-24 transmethylation. Strain N3H was found to be defective in delta 5-desaturation as well as in delta 24(28)-reduction. However, the defect of N26 and N3H was suggested to be leaky, since small amounts of ergosterol and ergosta-7,22-dienol were found in these mutants, respectively. In N15, an accumulation (2% in total sterols) of the compound likely to be hydroxylated sterol was found. By aerobic adaptation of these strains, the accumulation of these strains, the accumulations of ergosta-7,22-dienol (22 mg/g dry cells), ergosta-7,22,24(28)-trienol (24 mg), ergosta-8,24(28)-dienol (18 mg), and cholesta-8,24-dienol (22 mg) reached a maximum in N3, N3H, N15, and N26 after 20, 20, 30, and 30 h, respectively. These strains appear to be useful for making 14C-labeled and non-labeled preparations of the above sterols.

Drug Resistance, Microbial↗
PubMed1985

Intra uterine survival and growth of Wistar and Wistar x Brown Norway rat embryos.

Embryos of the Wistar strain and its F(1) cross (Wistar females mated with Brown Norway males) of rats were transferred nonsurgically to 48 Wistar, 17 F(1) cross and 20 Wistar-Imamichi recipients. The two types of embryos were transferred together to each recipient to compare the viability of the embryos. Pregnancy rate was 78.8% (67 85 ). The survival rate of fetuses to term was 11.5% (20 174 ) and 25.1% (42 168 ) for the Wistar and F(1) embryos, respectively. Placental weight differed significantly (P<0.05) between embryo types and among recipient types while fetus weight differed (P<0.01) only among recipient types, with a significant interaction between recipient and embryo types (P<0.01). It was concluded that the F(1) embryos (Wistar x Brown Norway) were twice as viable as Wistar embryos under the conditions provided.

Journal Article↗
PubMed1984

Base-pair formation between noncapped influenza virus in vitro transcripts and 18S rRNA in rabbit reticulocyte 80S ribosome-mRNA complexes detected by psoralen photoreaction.

RNA-RNA interactions between 18S ribosomal RNA and noncapped influenza cRNA were detected by psoralen photochemical cross-linking in reticulocyte 80S ribosome-cRNA complexes. In vitro transcripts of type A influenza virus synthesized by endogenous RNA polymerase with adenylyl-(3'----5')-guanosine primer formed 80S complexes with rabbit reticulocyte ribosomes. The extent of the complex formation by these noncapped cRNAs was less than that by the m7G-capped reovirus in vitro transcripts, but the former RNAs in the 80S complexes were cross-linked to 18S rRNA as efficiently as the latter RNAs by photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen. These results suggested that mRNA with or without the cap structure on the 5'-terminal can form complexes with the ribosomes in a eukaryotic cell-free translation system by base-pair formation with 18S rRNA. Correspondingly, sequences capable of forming extensive base-pairs including four- to five-base complementarities were found between the 3'-terminal of rabbit reticulocyte 18S ribosomal RNA and the 5'-noncoding regions of either influenza virus transcripts or reovirus mRNA.

Animals↗
PubMed1984

Conformation and template activity of human reovirus genome RNA.

In vitro activation of human reovirus transcriptase by alpha-chymotrypsin digestion of viral outer shell proteins was completely dependent on the ionic size of the monovalent cation in the medium. Cations with nonhydrated ionic radii larger than 1.3 A showed full potency of activation of chymotrypsin digestion, and produced transcriptionally active virus cores. Smaller cations having ionic radii of 0.6 A or 0.95 A, on the other hand, promoted the chymotrypsin digestion to lesser extents, and yielded subviral particles showing latent or very low transcriptase activities. Differential conformational changes would be induced in viral outer shell proteins by these monovalent cations, resulting in the varied accessibility to chymotrypsin. Electron microscopic analyses under denaturing conditions of the cross-linked reovirus core genome RNAs with the AMT photoreaction revealed that they were almost evenly cross-linked by the psoralen adducts forming no reproducible size of "bubbles." This result suggests that the double helical reovirus genome may not be bound tightly by the inner viral proteins forming such nucleoprotein structures as nucleosomes in eukaryotic chromatin.

Chymotrypsin↗
PubMed1984

Identification of source of excessive ACTH production by improved selective venous sampling: simultaneous assay of PRL as a pituitary marker.

To identify an obscure site of excessive ACTH production, selective venous sampling was performed by Seldinger's method in a 32-year-old man. Differential diagnosis between an ectopic ACTH producing tumor and Nelson's syndrome was quite inconclusive in this patient in spite of extensive clinical and laboratory evaluations, including pituitary surgery. In this study, PRL was measured in every sample as a reliable marker for pituitary drainage. Significantly higher rations (2.6 to 5.3) of catheter to peripheral (C/P) concentration of PRL at sites closer to the pituitary indicated successful samplings of pituitary drainage. C/P ratios of ACTH correlated very closely (correlation coefficient = 0.906, p less than 0.01) with those of PRL at every sampling site. Thus, it was concluded that the site of excessive ACTH production in this patient was in the pituitary gland. The methods applied in this study appear to be useful in differentiating ectopic from eutopic production of other pituitary hormones as well.

Adolescent↗
PubMed1983

Characterization of a Saccharomyces cerevisiae mutant, N22, defective in ergosterol synthesis and preparation of [28-14C]ergosta-5,7-dien-3 beta-ol with the mutant.

Analysis of sterols was made with a Saccharomyces cerevisiae mutant, N22, which was resistant to nystatin and defective in ergosterol synthesis, and with its parent strain, M10 (haploid type, methionineless, petite). The main sterol of M10 was ergosterol, whereas that of N22 was ergosta-5,7-dien-3 beta-ol. A small amount of ergosterol was found also in N22. This indicates that the delta 22-desaturation reaction in N22 is blocked, though not completely. Ergosta-5,8-dien-3 beta-ol, an unusual sterol, was detected in N22 but not in M10. Variations in the amounts and compositions of free and esterified sterols of both strains were examined during cultivation and the subsequent aerobic adaptation. The contents of free sterols, which were mostly composed of the respective main sterols of both strains, did not change markedly. In contrast, the contents and compositions of esterified sterols of both strains varied depending on the growth conditions. When the cells of both strains were aerobically adapted, the total contents of esterified sterols increase, as did, as the intermediate sterols. Upon aerobic adaptation in the presence of [methyl-14C]methionine, [28-14C]ergosta-5,7-dien-3 beta-ol accumulated markedly in N22 cells. Using this mutant, we devised a convenient method for preparation of the radioactive sterol in high yield.

Chemical Phenomena↗
PubMed1983

Cross-linking of tobacco mosaic virus RNA and capped polyribonucleotides to 18S rRNA in wheat germ ribosome-mRNA complexes.

Tobacco mosaic virus RNA, forming 40S or 80S initiation complexes with wheat germ ribosomes, was covalently bound to 18S ribosomal RNA by the photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). Synthetic polyribonucleotide, poly(A, U), with the cap structure m7GpppGmC at the 5'-terminal was also cross-linked to 18S ribosomal RNA in 40S or 80S complexes with ribosomes by the AMT photoreaction. Polyuridylic acid with the same 5'-cap structure, forming 40S complexes but not 80S complexes with ribosomes, was most efficiently cross-linked to 18S ribosomal RNA by the psoralen photoreaction. These results suggest that the interactions between mRNA and 18S rRNA are not necessarily of strict complementarity but occur during formation of the complexes in eukaryotes. The 40S complexes would be then converted to 80S complexes in the presence of the AUG initiation codon or AUG-like triplets containing A and U on the polyribonucleotide chains which interact with 18S ribosomal RNA.

Cross-Linking Reagents↗