Search PubMed⌕ Search

Biomedical subjects

S Inouye

Publications and source records attributed to S Inouye.

At least 271 records · Page 15Linked to original sources
PubMed1991

Protein U, a late-developmental spore coat protein of Myxococcus xanthus, is a secretory protein.

Protein U is a spore coat protein produced at the late stage of development of Myxococcus xanthus. This protein was isolated from developmental cells, and its amino-terminal sequence was determined. On the basis of this sequence, the gene for protein U (pru) was cloned and its DNA sequence was determined, revealing an open reading frame of 179 codons. The product from this open reading frame has a typical signal peptide of 25 amino acid residues at the amino terminal end, followed by protein U of 154 residues. This result indicates that protein U is produced as a secretory precursor, pro-protein U, which is then secreted across the membrane to assemble on the spore surface. This is in sharp contrast to protein S, a major spore coat protein produced early in development, which has no signal peptide, indicating that there are two distinct pathways for trafficking of spore coat proteins during the differentiation of M. xanthus.

Amino Acid Sequence↗
PubMed1991

Association of a retroelement with a P4-like cryptic prophage (retronphage phi R73) integrated into the selenocystyl tRNA gene of Escherichia coli.

A new multicopy single-stranded DNA (msDNA-Ec73) was found in a clinical strain of Escherichia coli. Retron-Ec73, consisting of an msDNA-coding region and the gene for reverse transcriptase (RT), was found to be a part of a 12.7-kb foreign DNA fragment flanked by 29-bp direct repeats and integrated into the gene for selenocystyl-tRNA (selC) at 82 min on the E. coli chromosome. Except for the 2.4-kb retron region, the integrated DNA fragment showed remarkable homology to most of the bacteriophage P4 genome. Among the phage genes found in this element, however, the integrase gene had very low identity (40%) to P4 integrase, indicating that the cryptic prophage associated with the retroelement has its own unique site-specific integrase different from P4 integrase. Recently, we have shown that P2 phage can act as a helper to excise the cryptic prophage and to package its genome into an infectious virion. The newly formed phage (retronphage phi R73) can also lysogenize a new host strain, reintegrating its genome into the selC gene and enabling the newly formed lysogen to produce msDNA-Ec73 (S. Inouye, M. G. Sunshine, E. W. Six, and M. Inouye, Science 252:969-971, 1991).

Amino Acid Sequence↗
PubMed1991

Survey of multicopy single-stranded DNAs and reverse transcriptase genes among natural isolates of Myxococcus xanthus.

Twenty different isolates of the soil bacterium Myxococcus xanthus were examined for the presence of multicopy single-stranded DNA (msDNA)-producing retroelements, or retrons. Each strain was analyzed by ethidium bromide staining for msDNA, 32P labeling of the msDNA molecule by the reverse transcriptase (RT) extension method, and DNA hybridization experiments with probes derived from two retrons, Mx162 and Mx65, previously cloned from M. xanthus DZF1. These analyses revealed that all M. xanthus strains contain an msDNA very similar to Mx162 msDNA, and 13 strains also contain a second smaller msDNA very similar to Mx65 msDNA. In addition, the strains contained retron-encoded genes msr and msd, which code for msDNA, and a gene for RT responsible for the synthesis of msDNA. These genes show greater than 80% nucleotide sequence similarity to retrons Mx162 or Mx65. The near-ubiquitous occurrence of msDNA retrons among M. xanthus strains and their homogeneous nature are in marked contrast to the highly diverse but rarely occurring msDNA-producing elements of Escherichia coli. The possible origin and evolution of RT and retron elements is discussed in view of these findings.

Bacteria, Aerobic↗
PubMed1991

A unique repetitive DNA sequence in the Myxococcus xanthus genome.

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.

Base Sequence↗
PubMed1991

Follistatin binds to both activin and inhibin through the common subunit.

Inhibin, activin, and follistatin are three families of polypeptides originally isolated and characterized from ovarian follicular fluid based on their modulation of FSH release from pituitary cell culture. In addition to their effects on FSH synthesis and secretion, inhibin and activin have other biological functions. By contrast, the physiological significance of follistatin was obscure, until it was discovered that follistatin is a binding protein to activin. Since activin binds to follistatin, it is imperative to determine the nature of the activin/follistatin binding complex. Moreover, because inhibin contains a beta-subunit derived from activin, it is important to determine whether inhibin will also bind follistatin. Using a double-ligand blotting technique, we have determined that activin-A has two binding sites for follistatin, whereas inhibin-A has only one binding site for follistatin. Therefore, these results suggest that follistatin binds to both activin and inhibin through the common beta-subunit.

Activins↗
PubMed1991

Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin.

Follistatin is a glycosylated monomeric protein originally isolated from ovarian follicular fluid based on its ability to specifically inhibit pituitary FSH release. To further explore the physiological role of follistatin, we have expressed recombinant human follistatins with 315 (rhFS-315) and 288 (rhFS-288) amino acids in Chinese hamster ovary cells under the control of the simian virus-40 promoter. The two types of FS originated from alternatively spliced mRNAs and rhFS-315 differed from rhFS-288 by having an extra 27-amino acid sequence at the carboxyl-terminal. The yield of the purified rhFS-315 and rhFS-288 after a single step of affinity chromatography on an activin-coupled Affi-Gel column was 3-5 mg/liter conditioned medium. Using the rhFS-315 and rhFS-288 as molecular mass markers, Western blotting with FS carboxyl-terminal-specific antibodies demonstrated that the majority of native FS isolated from porcine ovarian follicular fluid was neither FS-315 nor FS-288, but was composed of 300 amino acids in various forms of glycosylation. This finding is consistent with our earlier results obtained from tryptic peptide fragment analysis of native FS. Only a very small percentage (less than 1%) of native porcine FS was FS-288. In cultures of rat anterior pituitary cells, rhFS-315 (ED50, 115.2 +/- 16.2 pM) is equipotent to native porcine FS (ED50, 86.7 +/- 14.1 pM) on the suppression of FSH release, but, surprisingly, rhFS-288 (ED50, 9.6 +/- 2.2 pM) is 8-10 times more potent than the native protein, similar to the potency of inhibin-A (ED50, 8.6 +/- 0.9 pM). Interestingly, when the in vivo FSH-suppressing activity of rhFS-288 was compared to that of inhibin-A in 1-week ovariectomized adult rats, it was found that rhFS-288 was more potent and longer acting than inhibin-A. Hence, these results indicate that FS-288 is probably one of the most potent natural FSH suppressors.

Amino Acid Sequence↗
PubMed1991

Isolation of new minor benanomicins.

Four minor benanomicins, dexylosylbenanomicins A and B, 2'-demethylbenanomicin A and 7-methoxybenanomicinone have been isolated from the culture filtrate of Actinomadura sp. MH193-16F4. Their structures were confirmed by spectral analyses. Dexylosylbenanomicins A and B were derived chemically from benanomicins A and B, respectively.

Anthracyclines↗
PubMed1991

In vitro and in vivo antibacterial activities of ME1220 and ME1221, novel cephalosporins.

The antibacterial activities and serum pharmacokinetic properties of ME1220 and ME1221, new aminothiazolylalkoxyiminoacetylcephalosporins having an N-alkylpyridiniumthiomethyl side chain at C-3, were compared with those of cefpirome and ceftazidime. ME1220 and ME1221 exhibited broad antibacterial activity against Gram-positive and Gram-negative bacteria. The in vitro and in vivo activities of ME1220 were similar to cefpirome, while ME1221 was superior to ceftazidime against almost all test organisms except pseudomonas. On intravenous injection in rats, ME1220 and ME1221 were excreted mainly in the urine. ME1221 was excreted moderately in the bile and showed higher serum concentration and AUC than those of ME1220.

Animals↗
PubMed1991

[Kinetics of the falling of airborne mite allergens (Der I and Der II)].

A futon (Japanese quilt) was beaten to disperse mite allergens into the air in a closed room, and the airborne allergens were collected both by Andersen air sampler for particle size analysis and by slit air sampler for kinetic analysis of the clearing of the allergens from the air. After extraction of the allergens from the agar plates in the samplers, two kinds of major mite allergens (Der I and Der II) were immunochemically quantitated. We found that the aerodynamic diameters of both allergens were mainly above 5.5 microns, and that airborne allergen levels decreased to about 10% of the starting level in 30 minutes, indicating the rapidity of the falling of both allergens.

Air Pollutants↗
PubMed1991

[Evaluation of different treatments of Japanese bed quilts for reducing mite allergens].

We evaluated the effectiveness of different treatments of Japanese bedquilt (futon) in reducing mite allergens: vacuum-cleaning, beating plus vacuum-cleaning and washing of the whole futon in water. Before and after these treatments, small amounts of cotton were taken out of the futon and mite allergens were extracted from the cotton into water. The absolute contents of two kinds of major allergens of two Dermatophagoides species were immunochemically quantitated. We found that beating and vacuum-cleaning reduced the allergen contents by only about 40%, whereas washing reduced the allergens by more than 90%. Therefore, for reducing airborne mite allergens generated from futon, we think that washing the whole futon in water is the most effective method.

Allergens↗
PubMed1990

Photoprotein aequorin: use as a reporter enzyme in studying gene expression in mammalian cells.

Aequorin is a luminescent protein present in the jellyfish Aequoria victoria which emits light (at 460 nm) in the presence of Ca2+. We report here that aequorin can be used as a reporter enzyme to monitor gene expression in eukaryotic cells. A cDNA encoding apoaequorin was fused to several eukaryotic promoters, including those of SV40, RSV and the HSV-1 tk gene, and introduced into several cell lines such as CV-1, COS and HeLa. At appropriate times after transfection, the aequorin activities in cell extracts were measured by monitoring the intensity of light emitted at 460 nm when triggered by Ca2+ by the use of a photomultiplier photometer. The aequorin assay was shown to be as sensitive as the conventional CAT assay, and the relative activities of various promoters estimated by the aequorin assay were in general agreement with those obtained by the CAT assay. The aequorin assay can be done within 6-7 h from the preparation of extract to the measurement of activity without using radioactive compounds.

Aequorin↗
PubMed1990

Upstream regulatory sequence for transcriptional activator XylR in the first operon of xylene metabolism on the TOL plasmid.

Transcription of the first operon coding for m-xylene-degrading enzymes on the TOL plasmid of Pseudomonas putida is activated by the xylR gene product in the presence of m-xylene. The operon has the consensus sequence of the ntr/nif promoters at -24 and -12 regions, and the transcription is dependent on an RNA polymerase containing a sigma factor NtrA (RpoN or sigma 54). Deletion derivatives of the upstream sequence of the operon promoter were made in vitro and connected with the xylE gene on a plasmid. Their promoter activities were analyzed in Escherichia coli by monitoring catechol 2,3-dioxygenase activity, the xylE gene product. A cis-acting DNA element was identified, which is required for activation of the operon promoter by XylR protein in the presence of the inducer. This regulatory sequence of about 40 base-pairs in length was located 150 base-pairs upstream from the transcription start site. Analysis of the mutants containing insertions between the upstream regulatory sequence and the promoter sequence demonstrated strong dependence of the activation upon helical periodicity of DNA. The regulatory sequence functioned in the inverse orientation or at a distance of more than 1 x 10(3) base-pairs upstream from the promoter though less efficient. These results indicated that this upstream regulatory sequence might be the binding site for XylR protein. DNA-loop formation through protein-protein interaction between XylR protein attached to the upstream sequence and the NtrA-containing RNA polymerase bound by the promoter sequence was suggested for activation of the operon transcription. A sequence similar to the regulatory sequence of the first operon of xylene metabolism was found in the upstream region of the xylS gene, which is also activated by XylR protein in the presence of m-xylene.

Base Sequence↗
PubMed1990

Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.

Aequorin↗
PubMed1990

Bioluminescent immunoassay using a fusion protein of protein A and the photoprotein aequorin.

Aequorin is a photoprotein that emits light in the presence of Ca2+ ions. To develop a bioluminescent immunoassay based on the light emission property of aequorin, we have expressed the apoaequorin fusion protein with S. aureus protein A in E. coli by recombinant DNA techniques. The fusion protein expressed was purified by IgG-Sepharose affinity chromatography, gel filtration and HPLC procedures. The purified protein A-apoaequorin fusion protein has both the luminescent activity of aequorin and the IgG-binding ability of protein A. We compared results obtained using the protein A-aequorin fusion protein with those obtained using a protein A conjugated horseradish peroxidase based immunoassay, and found them to yield similar results.

Aequorin↗
PubMed1990

Reverse transcriptase from Escherichia coli exists as a complex with msDNA and is able to synthesize double-stranded DNA.

Reverse transcriptase required for the synthesis of msDNA.Ec67 in an Escherichia coli strain was purified as a large molecular weight complex with msDNA. The complex sedimented in a glycerol gradient at an s value greater than 19. The predominant protein species co-purifying with reverse transcriptase activity in the complex had a molecular weight estimated at 65,000 which is close to the expected size of 67,227 for the Ec67-reverse transcriptase. In addition, the large complex also contained msDNA.Ec67. The purified complex was able to synthesize cDNA using 5 S rRNA as a template (annealed to a synthetic DNA primer), and a double-stranded DNA using a synthetic DNA template (annealed to a synthetic DNA primer). When msDNA.Ec67 was used as a natural template:primer, the purified complex produced two major products: a 103-base single-stranded DNA by extending the 3' end of msDNA using msdRNA as a template, and a 60-base double-stranded DNA product resulting from the converse reaction in which the 3' end of msdRNA is extended using msDNA as a template. The results suggest that bacterial reverse transcriptase is capable of producing single-stranded cDNA and possibly double-stranded DNA as well. Possible implications of these findings on the biology of the msDNA-retron system are discussed.

Base Sequence↗
PubMed1990

Nucleoside diphosphate kinase from Myxococcus xanthus. I. Cloning and sequencing of the gene.

By photoaffinity labeling with a photolysable analog of GTP, 8-N3GTP, we were able to find at least five distinct GTP-binding proteins in Myxococcus xanthus; two of them located in the membrane and the other three in the soluble fraction. The amino-terminal sequence of the 16-kDa GTP-binding protein from the soluble fraction was determined, and the gene that encodes this protein was isolated and cloned using degenerate oligonucleotides as a probe. The DNA sequence of the gene was determined, which did not show similarity with other known proteins. The gene product was overexpressed in Escherichia coli, by using the lacZ promoter, to a level of 13% of the soluble protein. Attempts to isolate deletion mutants were unsuccessful, although double crossing-over events leading to a deletion mutation of the gene were detected by Southern blot hybridization. This result indicates that this gene is essential for cell growth. In the following paper (Muñoz-Dorado, J., Inouye, S., and Inouye, M. (1990) J. Biol. Chem. 265, 2707-2712), the gene product was biochemically characterized and identified to be a nucleoside diphosphate kinase.

Affinity Labels↗
PubMed1990

Nucleoside diphosphate kinase from Myxococcus xanthus. II. Biochemical characterization.

The gene that encodes the 16-kDa GTP-binding protein from Myxococcus xanthus has been cloned, and its DNA sequence has been determined. The gene has been expressed in Escherichia coli by using the lacZ promoter, and its gene product was overproduced (Muñoz-Dorado, J., Inouye, M., and Inouye, S. (1990) J. Biol. Chem. 265, 2702-2706). The gene product thus overproduced in E. coli was purified to homogeneity by a simple four-step procedure and crystallized. Gel filtration of the purified protein revealed that the protein forms a complex of an apparent molecular weight of 50,000, indicating that it exists as a trimer in the cell. It was found that the purified protein can bind not only GTP, but also equally well the other nucleoside diphosphates and triphosphates with no specificity for either the base or the sugar. Nucleoside monophosphates, Pi, and pyrophosphate do not bind to the protein. In the presence of Mg2+, the protein hydrolyzes nucleoside triphosphates to diphosphates and Pi. However, in the presence of EDTA, most of the phosphate remains bound to the protein. The phosphorylated protein can then transfer the phosphate group to a nucleoside diphosphate to form the corresponding nucleoside triphosphate in the presence of Mg2+. The reaction is reversible, and it is considered to occur by a two-step ping-pong mechanism. These results unambiguously demonstrate that the M. xanthus 16-kDa GTP-binding protein is a nucleoside diphosphate kinase.

Adenosine Triphosphate↗