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Biomedical subjects

S Inouye

Publications and source records attributed to S Inouye.

At least 163 records · Page 9Linked to original sources
PubMed1995

Gene regulation by antisense DNA produced in vivo.

Antisense technology has been widely used for regulating gene expression. Single-stranded RNA or DNA complementary to a target mRNA can inhibit the translation of the mRNA. Antisense RNA is produced in vivo, while antisense DNA is chemically synthesized as an oligonucleotide, which is extracellularly added to the cells. To maintain the effect of antisense DNA, a synthetic oligonucleotide has to be constantly added to the system. An advantage of antisense DNA over antisense RNA is that the target mRNA hybridized with the antisense DNA can be specifically digested by ribonuclease H. Here, we attempted to produce in vivo short single-stranded DNAs complementary to a specific mRNA. We demonstrate that such antisense oligodeoxyribonucleotide of a desired sequence can be produced in Escherichia coli using a retron, a bacterial retroelement, as a vector and that the antisense DNA thus produced in vivo can effectively inhibit the expression of a specific E. coli gene, such as the gene for the major outer membrane lipoprotein.

Bacterial Outer Membrane Proteins↗
PubMed1995

Cloning, DNA sequencing, and amino acid sequencing of catechol 1,2-dioxygenases (pyrocatechase) from Pseudomonas putida mt-2 and Pseudomonas arvilla C-1.

Catechol 1,2-dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis-muconic acid and is encoded by a catA gene. We have cloned a catA gene from Pseudomonas putida mt-2 using a PCR product of amino acid sequence-based primers as a probe. The amino acid sequence deduced from the 930 nucleotides was in complete agreement with the chemically determined sequence of the protein. Crude extracts of Escherichia coli cells carrying the catA gene downstream from the lac promoter showed the enzyme activity. By using the same probe, we also cloned and sequenced the catA beta gene for catechol 1,2-dioxygenase isozyme beta beta from Pseudomonas arvilla C-1, which has three isozymes, alpha alpha, alpha beta, and beta beta (C. Nakai, H. Horiike, S. Kuramitsu, H. Kagamiyama, and M. Nozaki, 1990, J. Biol. Chem. 265, 660-665). There was very high homology between isozyme beta beta of the C-1 strain and the enzyme of the mt-2 strain in both the amino acid (98%) and the DNA sequences (97%). A preference for the use of codons terminating in C and G was found in the coding region of both the enzymes, which contributed to the high G + C content (65-66%) of the genes. A comparison of the DNA sequences of various catA genes from other sources revealed their common ancestry, whereas a comparison of the amino acid sequences of the enzymes revealed clear reflection of substrate specificity. Tyrosyl and histidyl residues for proposed ligands of ferric ion are conserved in all catechol 1,2-dioxygenases.

Amino Acid Sequence↗
PubMed1995

Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation.

A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279 residues show 37% identity to catalytic domain of Pkn1, another protein serine/threonine kinase expressed during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of 18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coli under a T7 promoter was phosphorylated at both serine and threonine residues. TEM-beta-lactamase produced in E. coli was found to serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent molecular mass from 29 to 44 kD. The phosphorylated beta-lactamase was unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity of endogenous beta-lactamase or related enzymes in response to an external signal yet to be identified.

Amino Acid Sequence↗
PubMed1995

The heparin binding site of follistatin is involved in its interaction with activin.

Whether the heparin-binding site of follistatin would interact with activin has been examined. When a mixture of recombinant human follistatin-288 (rhFS-288) and -315 (rhFS-315) was applied to an activin-coupled affinity column, followed by stepwise elution of the column using 4M urea, 8M urea, 1M guanidine-HCl and 2M guanidine-HCl, rhFS-315 was eluted with 4M urea, while rhFS-288 was eluted with 2M guanidine-HCl. This finding implies that the carboxylterminal 27 amino acid extension of rhFS-315, which is not present in rhFS-288, affects the binding of follistatin with activin. Addition of heparin (50 micrograms/ml) to the elution solvent caused rhFS-288 to elute with 4M urea, whereas rhFS-315 was not affected. These data suggest for the first time that these two structurally related follistatin molecules interact with activin by different modes of binding and, in the presence of heparin, the interaction of rhFS-288 with activin is indistinguishable from that of rhFS-315. Two analogs of rhFS-288 mutated at the heparin binding site were eluted with 8M urea or 1M guanidine-HCl, distinct from the elution profile of the intact rhFS-288. These results indicated that mutation at the heparin binding site alters the activin binding affinity. In addition, bioassay of the two mutants showed that they were less potent than the rhFS-288. These findings suggest that the heparin binding site of follistatin also contributes to its binding for activin, and heparin may play an important role in the bioactivity of follistatin.

Activins↗
PubMed1995

The formation of the 2',5'-phosphodiester linkage in the cDNA priming reaction by bacterial reverse transcriptase in a cell-free system.

Bacterial reverse transcriptase (RT) is responsible for synthesis of multicopy single-stranded DNA (msDNA) consisting of single-stranded DNA linked to an internal guanosine residue of RNA by an unusual 2',5'-phosphodiester linkage. Here we purified a bacterial RT to homogeneity from Escherichia coli harboring the RT gene from retron-Ec73. The purified RT-Ec73 was able to synthesize msDNA in a cell-free system using an RNA template produced in vitro by T7 RNA polymerase. The in vitro synthesized msDNA was released from the template RNA only when treated with yeast debranching enzyme DBR1, a specific nuclease for a 2',5'-phosphodiester linkage. The position of the branching G residue in the template RNA and the DNA sequence of the cell-free product were identical to those of msDNA-Ec73 synthesized in vivo. These results clearly demonstrate that the formation of the 2',5'-phosphodiester linkage in msDNA synthesis is carried out by RT itself.

Base Sequence↗
PubMed1995

Structure, function, and evolution of bacterial reverse transcriptase.

The discovery of retroelements in the prokaryotes raises intriguing questions concerning their roles in bacteria and the origin and evolution of reverse transcriptases. We first discuss a possible structure of bacterial reverse transcriptases on the basis of the known three-dimensional structure of HIV-1 reverse transcriptase, and how such a putative three-dimensional structure is able to recognize a single primer-template RNA molecule to initiate DNA chain elongation from the 2'-OH group of an internal G residue. This reaction leads to the production of a unique RNA-DNA complex called msDNA (multicopy single-stranded DNA) in which a single-stranded DNA branches out from an RNA molecule via a 2',5'-phosphodiester linkage. Second, the mobility of the bacterial retroelements called retrons, responsible for the production of msDNA, are discussed and compared with the mobility of group I and group II introns. Third, the original and evolution of bacterial reverse transcriptases are discussed in light of the question of whether the bacterial reverse transcriptases are older than eukaryotic reverse transcriptases.

Amino Acid Sequence↗
PubMed1995

Inhibition of pulmonary metastases and tumor cell invasion in experimental tumors by sodium D-glucaro-delta-lactam (ND2001).

Sodium D-glucaro-delta-lactam (ND2001) inhibited spontaneous pulmonary metastases of the highly metastatic B16 melanoma variant with a maximal inhibition rate of 99.5%, and 6 of 7 animals remained metastasis-free. Likewise, ND2001 inhibited the spontaneous pulmonary metastases of both Lewis lung carcinoma (3LL) with a rate of 98.0% (3 of 5 animals remaining metastasis-free) and rat KDH-8 liver carcinoma with a rate of 82.5% (3 of 7 animals remaining metastasis-free), although it was unable to inhibit the metastases of mouse BMT-11 fibrosarcoma and rat SST-2 breast carcinoma. Pretreatment with ND2001 in vitro inhibited the pulmonary metastases of the B16 variant and 3LL cells, which indicates direct action upon the cancer cells. When the invasive activity of cancer cells was measured by the Boyden chamber method, the number of invading B16 variant or 3LL cells was reduced with maximal inhibition rates of 93.0% or 89.9%, respectively, but pretreatment with ND2001 failed to reduce the invasive activity of BMT-11 or SST-2 cells. ND2001 showed neither cytocidal nor antitumor activity. These results suggest that ND2001 inhibited pulmonary metastases at the invasive step into the basement membrane by directly changing some property of the tumor cells.

Animals↗
PubMed1995

Inhibition of tumor cell haptotaxis by sodium D-glucaro-delta-lactam (ND2001).

We used the Boyden chamber system to investigate the mechanism by which the antimetastatic agent sodium D-glucaro-delta-lactam (ND2001) inhibits tumor cell invasion, and by establishing what ND2001 did not achieve, we were able to pinpoint the areas in which it was successful as an inhibitor. ND2001 did not inhibit cell adhesion of a highly metastatic B16 melanoma variant (the B16 variant) to the reconstituted basal membrane Matrigel, nor did it affect the production or activity of basal membrane-degrading type i.v. collagenase, but, in the Boyden chamber, ND2001 inhibited cell migration of the B16 variant toward a chemoattractant, laminin, on the lower surface of a Matrigel-free filter set (haptotaxis). Lewis lung carcinoma (3LL) cells that had been treated with ND2001 also exhibited hardly any haptotaxis, although the cells showed no alteration in behavior during cell adhesion to Matrigel. Since ND2001 did succeed in inhibiting the pulmonary metastases of the B16 variant and 3LL, we infer that inhibition of the metastases by ND2001 in these tumors is likely to be due to the inhibition of haptotactic migration.

Adenocarcinoma↗
PubMed1995

Sensitivity to two major allergens (Cry j I and Cry j II) in patients with Japanese cedar (Cryptomeria japonica) pollinosis.

BACKGROUND: Japanese cedar (Cryptmeria japonica: CJ) pollinosis is one of the most important allergic diseases in Japan. Recently, the second major allergen (Cry j II) was isolated from CJ pollen. There have been no prevalence studies of sensitivity to Cry j I and Cry j II among a large number of patients with pollinosis. OBJECTIVE: This study was conducted to evaluate the prevalence of sensitivity to Cry j I and Cry j II. We measured specific IgE antibodies to these allergens in the sera of 145 patients. Furthermore, comparison of the sensitivity to Cry j I and Cry j II was examined by the histamine release assay. METHODS: Specific IgE antibodies to Cry j I and Cry j II were assayed by a fluorometric ELISA. Allergen-specific histamine release was measured by a radioimmunoassay kit. RESULTS: More than 90% of 145 patients had specific IgE antibodies to both allergens, the remainder had specific IgE to either one or the other. There were seasonal changes in the level of specific IgE. The changes in the levels of anti-Cry j II IgE antibodies were parallel to those of anti-Cry j I IgE. The histamine release assay with leucocytes from the patients demonstrated that the allergenic potency of the two allergens is almost the same. CONCLUSION: Cry j II is an as important a major allergen as Cry j I.

Adult↗
PubMed1995

Molecular evolution of the Ca(2+)-binding photoproteins of the Hydrozoa.

Alignment of the primary structures of the hydrozoan photoproteins, aequorin, mitrocomin, clytin and obelin showed very strong amino acid sequence identities. The Ca(2+)-binding sites of the proteins were found to be highly conserved. The Ca(2+)-binding sites were also homologous to the Ca(2+)-binding sites of other Ca(2+)-binding proteins. However, aequorin, mitrocomin, clytin and obelin differed from other Ca(2+)-binding proteins in that they contained a relatively large number of cysteine, tryptophan, histidine, proline and tyrosine residues, suggesting that these residues may have evolved as part of the light-emitting mechanism. Construction of a phylogenetic tree showed that aequorin, mitrocomin, clytin and obelin form a closely related group of proteins.

Aequorin↗
PubMed1995

[Type-specificity of serum antibodies from genital herpes patients as determined by an enzyme-linked immunosorbent assay using HSV-infected cells as antigens].

Type-specificity of serum antibodies from genital herpes patients was determined by an enzyme-linked immunosorbent assay (ELISA) using antigens extracted from herpes simplex virus (HSV) type 1- and 2-infected cells. Sixty-three of HSV type-known panel sera, which had been typed by HSV glycoprotein G-specific immunodot analysis, consisted of 3 groups; group I (25 sera; gG-1 antibody- and HSV-1 isolation-positive), group II (19 sera; gG-2 antibody- and HSV-2 isolation-positive) and group III (19 sera; gG-1 and gG-2 antibodies- and HSV-2 isolation-positive), were assayed for IgM, IgA, IgG1 and IgG3 antibody activities (optical densities) against HSV-1- as well as HSV-2-infected cell antigens. IgG antibodies of these 3 groups showed 2 different patterns of reactivities. The group II sera reacted with the two antigens to the same extent and could be differentiated from other 2 groups. The latter 2 groups were difficult to differentiate because of similar reaction patterns showing higher reactivities to HSV-1 antigen. In contrast, type-specificity was not observed in IgM antibody activities. The higher reactivities of IgG antibody to HSV-1 antigen than to HSV-2 antigen in the group III sera indicate the "original antigenic sin" phenomenon, i.e.; memory B cells produced in prior infection with HSV-1 were activated by cross-reactive antigens of HSV-2 which infected secondarily. To presume the type of infected HSV from serum antibody reactivities was difficult as long as HSV-infected cells were used as antigens in ELISA.

Antibodies, Viral↗
PubMed1995

The role of ribonuclease H in multicopy single-stranded DNA synthesis in retron-Ec73 and retron-Ec107 of Escherichia coli.

Bacterial reverse transcriptase is responsible for the synthesis of multicopy single-stranded DNA (msDNA). Reverse transcriptases from retron-Ec73 and retron-Ec107 do not contain an RNase H domain. Cellular RNase H is therefore considered to be required to make the mature form of msDNA. We found that RNase HI, but not RNase HII, is required for the production of the mature form of both msDNAs.

Base Sequence↗
PubMed1995

Two recA genes in Myxococcus xanthus.

Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined. On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E. coli RecA, respectively. Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid. Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated. Both the recA1 and recA2 genes expressed in E. coli complement the UV sensitivity of an E. coli recA strain.

Amino Acid Sequence↗
PubMed1995

Germination of myxospores from the fruiting bodies of Myxococcus xanthus.

Germination of myxospores from fruiting bodies of Myxococcus xanthus was examined under a light microscope as well as by analyzing the incorporation of [3H]uracil into the RNA fraction. Efficient germination was observed in 0.2% Casitone containing 8 mM MgSO4 and 1 mM CaCl2 at 30 degrees C. Under this condition, spherical myxospores were converted into rod-shaped vegetative cells within 5 to 6 h. The germination was severely inhibited in the presence of 1 mM phenylmethylsulfonyl fluoride, a protease inhibitor, indicating that a serine protease(s) is required for the myxospore germination. EGTA (1 mM) also completely blocked germination, indicating that Ca2+ plays an important role in myxospore germination. In 1% Casitone without added Mg2+ and Ca2+ or 0.2% Casamino Acids with 8 mM MgSO4 and 1 mM CaCl2, myxospores lost their refractility under a phase microscope, while no RNA synthesis took place within 6 h, as judged by the incorporation of [3H]uracil. A group of proteins were found to be specifically synthesized during an early stage of germination. In addition, a new major spore-associated protein with a size of 41.5 kDa became detectable in the spore shell fraction 3 h after germination. The present results demonstrate that myxospore germination occurs in at least two steps: the loss of myxospore refractility, followed by an outburst of metabolic activities. The first step can occur even in the absence of energy metabolism, while the second step was blocked by rifampin, EGTA, and protease inhibitors.

Amino Acids↗
PubMed1995

MlpA, a lipoprotein required for normal development of Myxococcus xanthus.

The mlpA gene encoding a 236-residue polypeptide has been identified immediately downstream of the oar gene of Myxococcus xanthus (M. Martinez-Canamero, J. Munoz-Dorado, E. Farez-Vidal, M. Inouye, and S. Inouye, J. Bacteriol. 175:4756-4763, 1993). The amino-terminal 21 residues of MlpA encode a typical prokaryotic signal sequence with a putative lipoprotein cleavage site. When expressed in Escherichia coli in the presence of [2-3H]glycerol, 3H-labeled MlpA had a molecular mass of 33 kDa and was found to be associated with the membrane fraction. Globomycin, an inhibitor of signal peptidase II, caused a shift in the mobility of E. coli-expressed MlpA to 35 kDa. Subsequently, a mlpA disruption strain (oar+) was constructed and found to have delayed fruiting body formation (by approximately 36 h), with significantly larger fruiting bodies being produced compared with those of the wild-type strain. Nevertheless, spore yields for the two strains were identical after 120 h of development. These data indicate that MlpA, the lipoprotein identified in M. xanthus, is required for normal fruiting body formation.

Anti-Bacterial Agents↗
PubMed1995

Hypersensitivity to Japanese cedar (Cryptomeria japonica) pollen in dogs.

Four of 51 dogs with chronic dermatitis were made to react to crude Japanese cedar (Cryptomeria japonica, CJ) pollen allergen by the intradermal skin test (IDST). These four dogs had specific IgE to CJ as determined by Prausnitz-Küstner test. In the provocation test, nasal discharge increased 5 to 20 min after introduction of the crude CJ pollen allergen into the nasal cavities, in two of three dogs. These results demonstrated that these dogs had hypersensitive to CJ pollen, which might be a cause of atopic disease in dogs.

Allergens↗