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Biomedical subjects

A Iwamoto

Publications and source records attributed to A Iwamoto.

At least 127 records · Page 7Linked to original sources
PubMed1995

Design and anti-HIV-1 activity of ribozymes that cleave HIV-1 LTR.

A hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5'C(UUCG)G3' loop were designed to cleave the long terminal repeat (LTR) of HIV-1. When the ribozyme catalyzed RNA cleavage reaction for a chemically synthesized 19 mer (LTR 19) was measured, the t 1/2 value of LTR 19 mediated by RZ115 was smaller than that of the RZ119 case. Moreover, the transformed CEM cells harboring the gene encoding these ribozymes were challenged with a HIV-1IIIB strain, two ribozymes, HR112 and RZ119 possessed strong anti-HIV-1 activity. However, the anti-HIV-1 activity displayed by RZ115 was weak. On the basis of secondary structure predictions of the RNA transcribed with the gene encoding ribozymes, the secondary structure of the transcribed RNA with RZ115 sequences was observed to be different from those with the other ribozymes. It has been demonstrated that the secondary structures of transcribed RNAs can possibly influence the anti-HIV-1 activity.

Antiviral Agents↗
PubMed1994

Purification and characterization of a novel substrate for plasma kallikrein (PK-120) in human plasma.

A 120 kDa plasma protein, which is susceptible to plasma kallikrein, was purified from human plasma by polyethylene glycol fractionation followed by ion exchange chromatography using Q-Sepharose, S-Sepharose, and hydroxyapatite and gel filtration on Sephacryl S-200. The 120 kDa protein, termed PK-120 in this paper, was a single polypeptide chain containing about 20% sugar by weight and its concentration in plasma was estimated to be 80 micrograms/ml by ELISA. At least three fragments, 100, 70, and 35 kDa, were produced from PK-120 by plasma kallikrein. The N-terminal sequence and Western blot demonstrated that PK-120 was first cleaved to yield the 100 and 35 kDa fragments, then the 100 kDa fragment was cleaved into the 70 kDa fragment. N-Terminal sequence analyses of PK-120 and its fragments demonstrated that it is a novel plasma protein, distinct from high molecular weight kininogen, a natural substrate for plasma kallikrein.

Amino Acid Sequence↗
PubMed1994

N-ethylmaleimide-sensitive mutant (beta Val-153-->Cys) Escherichia coli F1-ATPase: cross-linking of the mutant beta subunit with the alpha subunit.

A beta subunit mutation, beta Val-153-->Cys, in the glycine-rich sequence (phosphate-binding loop) of Escherichia coli F1 was constructed. Like vacuolar-type ATPase, the mutant enzyme was inhibited by N-ethylmaleimide (NEM) and labeled with [14C]NEM. The inhibition and labeling were prevented by ATP. m-Maleimidobenzoyl-N-hydroxysuccinimide (MBS) (3 microM) almost completely inhibited the mutant enzyme, and cross-linked one pair of alpha and beta subunits. These results suggest that the interaction of the domain near beta Val-153 with the alpha subunit is essential for catalytic cooperativity of the enzyme and that beta Val-153 is within 10 A of the alpha subunit.

Adenosine Triphosphate↗
PubMed1994

Catalysis and energy coupling of H(+)-ATPase (ATP synthase): molecular biological approaches.

The molecular biological approach has provided important information for understanding the F0F1 H(+)-ATPase. This article focuses on our recent results on the catalytic site in the beta subunit, and the roles of alpha/beta subunit interaction and amino/carboxyl terminal interaction of the gamma subunit in energy coupling. Extensive mutagenesis of the beta subunit revealed that beta Lys-155, beta Thr-156, beta Glu-181 and beta Arg-182 are essential catalytic residues. beta Glu-185 is not absolutely essential, but a carboxyl residue may be necessary at this position. A pseudo-revertant analysis positioned beta Gly-172, beta Ser-174, beta Glu-192 and beta Val-198 in the proximity of beta Gly-149. The finding of the roles of beta Gly-149, beta Lys-155, and beta Thr-156 emphasized the importance of the glycine-rich sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser, E. coli beta residues between beta Gly-149 and beta Thr-156) conserved in many nucleotide binding proteins. The A subunits of vacuolar type ATPases may have a similar catalytic mechanism because they have conserved glycine-rich and Gly-Glu-Arg (corresponding to beta Gly-180-beta Arg-182) sequences. The results of these mutational studies are consistent with the labeling of beta Lys-155 and beta Lys-201 with AP3-PL, and of beta Glu-192 with DCCD [15]. The DCCD-binding residue of a thermophilic Bacillus corresponds to beta Glu-181, an essential catalytic residue discussed above. The defective coupling of the beta Ser-174-->Phe mutant was suppressed by the second mutation alpha Arg-296-->Cys, indicating the importance of alpha/beta interaction in energy coupling. The gamma subunit, especially its amino/carboxyl interaction, seems to be essential for energy coupling between catalysis and transport judging from studies on gamma Met-23-->Lys or Arg mutation and second-site mutations which suppressed the gamma Lys-23 mutation. Thus the conserved gamma Met-23 is not absolutely essential but is located in the important region for amino/carboxyl interaction for energy coupling.

Amino Acid Sequence↗
PubMed1994

Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses.

We developed an in vitro assay for antibodies to hepatitis C virus (HCV) that bind to virions and prevent initiation of the replication cycle in susceptible cells in vitro. These antibodies therefore appear to be capable of neutralizing the virus. Using this assay and a standard inoculum of HCV of known infectivity, we have measured the antibody in serial serum samples obtained from the same chronically infected patient over 14 years following onset of his hepatitis. Such antibody was found in sera collected within 5 years of onset of hepatitis but not in later sera. In double immunoprecipitation experiments with anti-human immunoglobulin, the same sera that contained neutralizing antibody were found to contain antibody that bound to HCV to form antigen-antibody complexes immunoprecipitable with anti-human globulin. Similarly, plasma collected from this patient in 1990, 13 years after onset of hepatitis, and which contained HCV that had diverged genetically from the 1977 strain, did not contain antibody capable of neutralizing either the 1977 or the 1990 strain of HCV. However, plasma collected a year later (1991, 14 years after onset of hepatitis) contained neutralizing antibody to the 1990, but not the 1977, strain of HCV. These results suggest that HCV does induce antivirion antibody, as measured by blocking of initiation of the replication cycle of virus in cells and by the formation of immunoprecipitable antigen-antibody complexes but that these antibodies are isolate specific and change over time. Thus, these antivirion antibodies function as neutralizing antibodies and are probably in vitro correlates of the attempt of the host to contain the emergence of neutralization-resistant variants of HCV over time.

Cell Line↗
PubMed1994

Interaction between the dominant negative mutant and the wild-type envelope proteins of Friend murine leukemia virus.

Interaction between the previously obtained dominant negative mutant, referred to as fcr (T. Matano, T. Odawara, M. Ohshima, H. Yoshikura, and A. Iwamoto, J. Virol. 67:2026-2033, 1993), and the wild-type envelope proteins (Env) of Friend murine leukemia virus was examined. The wild-type Env was bound to the fcr mutant Env and trapped in the endoplasmic reticulum. The virus receptor was not involved in this interaction.

Cell Line↗
PubMed1993

Domains near ATP gamma phosphate in the catalytic site of H+-ATPase. Model proposed from mutagenesis and inhibitor studies.

The beta Gly-149 residue is in a glycine-rich sequence (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr; residues 149-156) of the Escherichia coli H(+)-ATPase (ATP synthase) beta subunit. Substitution of beta Gly-149 by Ser suppressed the effect of the beta Ser-174-->Phe mutation (Iwamoto, A., Omote, H., Hanada, H., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1991) J. Biol. Chem. 266, 16350-16355), suggesting that beta Gly-149 is located near beta Ser-174. In this study, we introduced different residues at position 149 and found that a single mutant beta Cys-149 was defective. The effect of beta Cys-149 mutation was suppressed by beta Gly-172-->Glu, beta Ser-174-->Phe, beta Glu-192-->Val, or beta Val-198-->Ala replacement. These results suggest that beta Gly-149, beta Gly-172, beta Ser-174, beta Glu-192, and beta Val-198 residues are located close together in the catalytic site. From these findings we propose a model of the catalytic site of the enzyme near the gamma phosphate moiety of ATP. F1 enzymes with the double mutations beta Cys-149/beta Glu-172, beta Cys-149/beta Phe-174, beta Cys-149/beta Val-192, and beta Cys-149/beta Ala-198 were less sensitive than wild-type F1 to dicyclohexylcarbodiimide and adenosine triphosphopyridoxal (an affinity analogue of ATP forming a Schiff base with the epsilon-amino group of beta Lys-155 or beta Lys-201), and became sensitive to N-ethylmaleimide in an ATP-protected manner. These results of inhibitor studies are consistent with the proposed model.

Adenosine Triphosphate↗
PubMed1993

Endoscopic local injection of a new drug-delivery format of peplomycin for superficial esophageal cancer: a pilot study.

BACKGROUND: A new drug-delivery format comprising activated carbon particles adsorbing peplomycin (PEP-CH) was developed for the treatment of superficial esophageal cancer. METHODS: The drug distribution was measured in rats that received subcutaneous injections of PEP-CH or peplomycin aqueous solution. In 6 patients with superficial esophageal cancer, peplomycin as PEP-CH, 5-10 mg once a week for 4-10 weeks (total, 40-100 mg/patient) was injected endoscopically into primary lesions. RESULTS: Rats given PEP-CH had significantly higher peplomycin levels in the regional lymph nodes and the injection site than rats given aqueous solution. Five patients have survived to the present or died without cancer after 27-72 months. The remaining patient has survived without cancer for 8 months after a second course of PEP-CH against recurrence. CONCLUSIONS: PEP-CH therapy seems to have a good therapeutic effect on superficial esophageal cancer, although the present clinical study may have been biased by patient selection.

Aged↗
PubMed1993

Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of < or = 1.09 g/ml (principally approximately 1.06 g/ml), while that of strain F was found in fractions with densities of approximately 1.06 and approximately 1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.

Animals↗
PubMed1993

trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.

3T3 Cells↗
PubMed1992

Escherichia coli F0F1-ATPase. Residues involved in catalysis and coupling.

The molecular biological approach has provided important information toward understanding the complexities of the F0F1 ATPase. This article focuses on our recent results on the ATPase catalytic site contained in the beta subunit and the role of the gamma subunit in regulation of proton transport. We used a combination of affinity labeling and mutagenesis to locate several residues of the alpha and beta subunits in the catalytic site. Adenosine triphosphopyridoxal (AP3-PL) labeled beta Lys-155, beta Lys-201 and alpha Lys-201, suggesting that they are near the gamma-phosphate moiety of ATP. Turning to a mutagenesis approach we demonstrated that the two conserved residues, beta Lys-155 and beta Thr-156 in the glycine-rich sequence, are essential for catalysis. Finally, using pseudorevertant analysis, we positioned residue beta Gly-149 (also in the glycine-rich sequence) in proximity to beta Ser-174, beta Glu-192 (binding site for DCCD), and beta Val-198 (only three residues away from the AP3-PL binding site, beta Lys-201). Genetic studies suggested that the gamma subunit plays a role in regulation of catalysis and its coupling with proton conduction. We found that four mutations in the carboxyl-terminal region (gamma Gln-269-->Leu, gamma Gly-275-->Lys, gamma Thr-277-->end, or frameshift) had similar membrane ATPase activities but different ATP-dependent proton pumping and growth by oxidative phosphorylation. These results suggested a perturbation in the coupling between catalysis and proton translocation. We were able to clearly define the "uncoupling" by introducing mutations in the amino-terminal region of the gamma subunit. We were led to gamma Met-23-->Lys and Arg which resulted in an enzyme still regulated by delta microH+, but with profoundly inefficient coupling between ATPase catalytic sites and proton translocation in both ATP-dependent proton pumping and delta microH(+)-driven ATP synthesis. Second-site mutations in the carboxyl-terminal region of the gamma subunit reversed this effect.

Amino Acid Sequence↗
PubMed1992

Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line.

A human T-cell line, MOLT-4, either uninfected or infected with murine retroviruses, was tested for its susceptibility to hepatitis C virus (HCV) infection. The cell cultures were inoculated with a serum containing HCV and then examined for the presence of viral sequences by cDNA/PCR. In murine retrovirus-infected MOLT-4 (MOLT-4 Ma) cells, intracellular minus-strand viral RNA, a putative replication intermediate, was first detected 3 days after inoculation, and the maximum signal was seen on day 7. When the cells were continuously subcultured in fresh medium, HCV sequences were intermittently detected in cells over a period of 3 weeks. In MOLT-4 cells free of retroviruses, replication of minus-strand HCV RNA appeared less efficient than in MOLT-4 Ma cells. The presence of minus-strand viral RNA in MOLT-4 Ma cells inoculated with HCV was confirmed by in situ hybridization with a strand-specific RNA probe. Immunofluorescence tests with antibodies specific for HCV core and NS4 antigens showed that MOLT-4 Ma cells were positive for viral antigen 7 days after inoculation. Thus, it appears likely that the HCV genome can replicate in the human T-cell line MOLT-4.

Animals↗
PubMed1992

Similarity of the genomic structure between the two members in a new family of heparin-binding factors.

By differential hybridization of cDNA libraries from Fv-1 restrictive and non-restrictive somatic mutant mouse cells, we isolated a cDNA clone specifically present in the Fv-1 restrictive cells. Its sequence was found identical to that of heparin-binding growth-associated molecule or pleiotrophin (HB-GAM/PTN). We report here its genomic organization which has not been yet reported. Although the exon organization was similar to that of midkine, another member of the same family of heparin-binding factors, the sizes of introns were much larger and occupied more than a three-fold larger chromosomal region than midkine. As its introduction into Fv-1 non-restrictive cells failed to confer the Fv-1 restrictive character, HB-GAM/PTN is probably unrelated to Fv-1 restriction.

Animals↗