Biomedical subjects
Publications and source records attributed to S Inouye.
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A new aminosugar named nojirimycin B (1) has been isolated as its bisulfite adduct from the culture broth of Streptomyces lavendulae SF-425, together with nojirimycin. Microbiological oxidation of 1 with Gluconobacter suboxydans IAM 1829 gave a delta-lactam (2). The structures of 1 and 2 were determined to be 5-amino-5-deoxy-D-mannopyranose and D-mannonic-delta-lactam, respectively, on the basis of 1H NMR spectroscopy and X-ray structural analysis. Both 1 and 2 exhibited powerful inhibitory activity against rat epididymal alpha-mannosidase and apricot beta-glucosidase.
Oligonucleotide-directed site-specific mutagenesis was applied to alter the cleavage site in the signal peptide of the major outer membrane lipoprotein of Escherichia coli. Replacing the glycine residue at the cleavage site with an alanine residue did not affect the processing of the signal peptide. However, when the same cleavage site was constructed by the deletion of the glycine residue, the signal peptide was no longer cleaved. These results indicate that stringent structural integrity at the cleavage site in the lipoprotein signal sequence is required for correct processing of prolipoprotein.
We have examined the importance of the positively charged NH2 terminus of the major outer membrane lipoprotein precursor, prolipoprotein, in the early steps of secretion in Escherichia coli. For this purpose, we have generated three mutants using oligonucleotide-directed mutagenesis in which the charge at the NH2-terminal region was changed from +2 to +1, 0, and -2. The results indicate that the synthesis of prolipoprotein is facilitated by the presence of a positively charged NH2 terminus. In addition, the translocation of prolipoprotein across the cytoplasmic membrane does not absolutely require any basic amino acids at its NH2 terminus. However, the presence of a net negatively charged NH2 terminus causes an initial cytoplasmic accumulation of prolipoprotein which is slowly, post-translationally translocated across the cytoplasmic membrane at a rate which is dependent on the number of positive charges present in this region. The analysis of these mutants clearly demonstrates the importance of the NH2 terminus of the lipoprotein signal peptide in initiating the secretion of this protein in E. coli.
Two improvements that greatly enhance the rate of phosphotriester oligonucleotide synthesis are described: 1) use of hindered primary amines, e.g. t-butyl amine for decyanoethylation of oligonucleotide triester intermediates, and 2) a simplified isolation procedure that eliminates the tedious bicarbonate extraction after each condensing reaction. Using the improved procedures, oligonucleotide fragments can be synthesized as rapidly as using solid phase chemistry. The final products are purer than those obtained by solid phase chemistry since each intermediate block is purified by chromatography. The technique has been used to synthesize five oligonucleotide fragments (size 15 to 20) for the purpose of performing guided site-specific mutagenesis on a cloned E. coli lipoprotein gene.
Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2. We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene. The fragment included only one open translational frame of sufficient length to accommodate the enzyme. The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M. (1983) J. Biol. Chem. 258, 2916-2922). A mutant plasmid was isolated which did not direct the synthesis of the active enzyme. This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide. From the deduced amino acid sequence, the secondary structure was predicted. Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa. A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene.
Protein S, a development-specific protein of Myxococcus xanthus is produced only during fruiting body formation. More than 15% of total protein synthesis during this period is accounted for by the production of protein S. The genes for protein S were identified and cloned with the use of mixed probes consisting of eight synthetic oligodeoxyribonucleotides (tetradecamers) which correspond to a carboxyl-terminal portion of protein S. The two genes are oriented in the same direction and are separated approximately 1.2 kilobases. The DNA sequences of the carboxyl-terminal portions of the two genes reveal that both can code for the identical eleven amino acid sequence which corresponds to the carboxyl-terminal end of protein S. However, there are a few base substitutions upstream of these regions. This duplication of genes in M. xanthus may facilitate the extremely rapid synthesis of protein S during fruiting body formation.
A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.
Numerous blebs were observed in contact with smooth muscle cells (SMC) by light microscopy in the myometrium of the rat uterus after parturition. Electron-microscopically the cell surface of SMC showed bulbous protrusions, which often lacked a basement membrane and were less electron-dense than the surrounding cytoplasm or sometimes nearly electron-lucent. Many bulbous protrusions were separated from SMC and became the isolated structures which we called cytoplasmic blebs. These bulbous protrusions and cytoplasmic blebs were often found to be phagocytosed by fibroblast-like cells and macrophages. A series of these tissue changes in the uterine myometrium after delivery, possibly due to hypoxic conditions, contribute to a rapid involution of SMC which have enlarged during pregnancy.
In the post-partum rat endometrium, ultrastructural distinction could be made between stromal cells (fibroblast-like cells) and macrophages, especially by the freeze-fracture technic. The stromal cells were characterized by a well-developed rough-surfaced endoplasmic reticulum (RER) and intercellular junctions, while the macrophages had many vacuoles and vesicles, but no intercellular contact with each other. The freeze-fracture image showed that the stromal cells had many low linear elevations and gap junctions on the cleaved plane of the cell membranes, while the macrophages had no linear elevations or intercellular junctions. The cell membranes of the stromal cells had more intramembranous particles (IMP) (P-face 697 +/- 63/micrometers 2, E-face 303 +/- 52/micrometers 2) than those of the macrophages (P-face 467 +/- 50/micrometers 2, E-face 217 +/- 35/micrometers 2). It was confirmed that these two types of cell phagocytosed collagen fibrils.
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During differentiation of Myxococcus xanthus, a large amount of protein S is produced and assembled on the surface of the myxospore by a process that specifically requires Ca2+. The gene for protein S has been cloned, and two tandemly repeated homologous genes have been found to be within a short distance of each other in the M. xanthus chromosome. We determined the DNA sequence of 3,692 bp encompassing both genes and deduced the amino acid sequences of the two gene products. The gene 1 (upstream) product and the gene 2 (downstream) product show extensive amino acid sequence homology (88%). However, from their structures, protein S was found to be produced from gene 2, indicating that gene 2 is specifically turned on during differentiation. The structure of protein S shows striking similarities with calmodulin: protein S is composed of four internally homologous domains. In particular, the first and the third domains, consisting of 38 residues each, show a high level of homology (79%), and the second and the fourth domains, consisting of 40 residues each, show homology of 65%. In the first and the third domains, there is a common sequence of nine residues, Glu (or Asp)-Asn-Asn-Thr-Ile-Ser-Ser-Val-Lys, which is highly homologous to one of the proposed Ca2+-binding sequences in bovine brain calmodulin, Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys.
After disruption of echovirus type 7 virions with urea and heat, VP1 and VP2 were separated by isoelectric focusing in urea-containing sucrose gradients. Antisera to these two polypeptides were produced in guinea pigs. In complement fixation, antiserum to VP1 reacted with native and heated virions (N and H antigens, respectively) of homologous virus, and also cross-reacted with heated virions of some other enteroviruses used. Antiserum to VP2 was reactive only with heated virions of homologous and heterologous viruses. Interestingly, the anti-VP2 serum reacted neither with native nor even with heated procapsids (naturally-occurring empty capsids). Antiserum to VP1, but not VP2, showed neutralizing and hemagglutination-inhibiting activities. These results suggest that 1) both VP1 and VP2 possess cross-reactive antigenic determinants which are exposed on the surface of heated virions, and 2) type-specific determinants of VP1 are located on the surface of native virions.
An outer capsid glycoprotein, VP 7, was purified from a bovine rotavirus, neonatal calf diarrhea virus, by isoelectric focusing in glycerol gradients after disruption of the outer capsid. Its isoelectric point was found to be about 4.5. Guinea pigs were immunized with this VP 7 preparation. The antisera possessed both neutralizing and hemagglutination-inhibiting activities as well as complement-fixing activity, suggesting that VP 7 is a protein involved in hemagglutination and initiation of infection. When these antisera were reacted with a simian rotavirus, their antibody titers were low by hemagglutination-inhibiting and complement-fixing assays, but one serum neutralized the simian rotavirus as efficiently as it did homologous neonatal calf diarrhea virus.
The regulatory gene xylR of the TOL plasmid, which functions positively on both xylABC and xylDEGF operons in the presence of m-xylene or m-methylbenzyl alcohol, was cloned onto an Escherichia coli vector, pACYC177. A fused operon consisting of the operator-promoter region of the xylABC operon and the xylE gene was cloned onto pBR322. The xylE product, catechol 2,3-dioxygenase, was induced by m-xylene or m-methylbenzyl alcohol in the cells containing the fused operon when a 2.8-kilobase segment of the TOL plasmid was provided in trans. Therefore, the segment appeared to contain the regulatory gene xylR. The xylR gene was mapped very close to the other regulatory gene, xylS, determined previously. The xylR gene was not effective on activation of the xylDEGF operon unless an additional region containing xylS was provided together with the inducer. These results indicate that both xylR and xylS are essential to the m-methylbenzyl alcohol-dependent induction of the xylDEGF operon. The map positions of xylR and xylS were precisely determined by subcloning or insertion inactivation. In addition, the operator-promoter regions of the xylABC and xylDEGF operons were mapped to the 0.6- and 0.4-kilobase regions of the TOL plasmid, respectively.
We studied the type specificity of the antisera which were produced in guinea pigs by immunization with heat-disrupted virions of human enteroviruses (echovirus types 7 and 11 and coxsackievirus type B5). When guinea pigs were immunized with heated virions (H particles), the antibodies produced reacted type specifically by complement fixation with the homologous H particles. By contrast, when guinea pigs were immunized with disrupted virions prepared by heating in the presence of sodium dodecyl sulfate, antibodies were produced which reacted broadly. Heterotypic complement-fixing antibody responses of humans to enterovirus infections are discussed.
5-O-Substituted derivatives of 5-hydroxypicolinic acids were synthesized from nojirimycin and studied for their antihypertensive activity in unanesthetized spontaneously hypertensive rats (SHRs) restrained in wire mesh cage and acute toxicity in mice. 5-n-Butoxy-picolinic acid (ND-186) was found to have antihypertensive activity comparable to fusaric acid and lesser acute toxicity. Introduction of halogeno group, in particular trifluoromethyl group to the omega-position of 5-n-butoxy group resulted in the enhancement of antihypertensive activity. The acute toxicity was also lowered 3-5 times compared to that of fusaric acid. Replacement of alkyl group with phenyl group resulted in a slight increment of activity. Some of ester derivatives of ND-186 potentiated the antihypertensive activity and reduced the acute toxicity. There was good correlation between partition coefficient (log P) of ester groups and their antihypertensive activities; compound with higher lipophilicity showed higher antihypertensive activity under the condition of oral administration. However, esterification of other compounds such as 5-halogenoalkoxy- and 5-(substituted) phenoxypicolinic acid was not accompanied with activity increment. Considering from the balance of the antihypertensive activity in SHRs and the acute toxicity in mice, 5-(5',5',5'-trifluoropentoxy)-picolinic acid was selected as a candidate for further evaluation.