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Biomedical subjects

K Takai

Publications and source records attributed to K Takai.

At least 145 records · Page 8Linked to original sources

[Distribution of EIA reactive values and serum antibody titers of Chlamydia trachomatis urethritis and cervicitis at the first visit].

Among 120 non-gonococcal male urethritis, 83 were found to be Chlamydia trachomatis (CT) positive by Chlamydiazyme with 2 to 5 times repeated urethral swab collection during pre-treatment period. Among 97 female partner of male CT urethritis, 76 were CT positive by the same repeated specimen collection from cervix. In the 83 male CT urethritis and the 76 female CT cervicitis, EIA reactive values by Chlamydiazyme and serum CT antibody titer by FA at the first visit were investigated. The EIA reactive values of cervicitis were lower than those of urethritis. There was no case of "CT negative at the first visit and positive at repeated detection" in male urethritis. 3 case of "CT negative at the first visit and CT positive at repeated detection" were experienced among females who were the partner of male CT urethritis. The sensitivity of Chlamydiazyme was found to be enough to decide presence or absence of CT by single specimen collection in male urethritis but not enough in female cervicitis. It could be assumed that by the improved sensitivity of CT detection, CT detection rate would be raised among female cervicitis but not in male urethritis. Positive rate CT serum antibody were 63.9% in male urethritis and 100% in female cervicitis. The clinical value of CT antibody detection might be not as detection method of CT infection in progress, but as non-invasive screening for CT infection up to the present, namely the risk factor of STD, especially in females in whom detection of CT is not complete.

Antibodies, Bacterial↗

Phosphorothioate oligonucleotides block reverse transcription by the Rnase H activity associated with the HIV-1 polymerase.

We demonstrate the degradation of RNA bound to an antisense oligonucleotide by a reverse transcriptase enzyme-associated RNase H activity. We found that phosphorothioate oligonucleotides inhibit the RNase H activity by binding to AMV RT, rather than to the template RNA, whereas the RNase H activity of HIV-1 RT is not affected by the antisense phosphorothioate oligonucleotide. Selective inhibition of HIV-1 gene expression involves the degradation of the template RNA bound to the antisense phosphorothioate oligonucleotide by the RNase H activity associated with the HIV-1 polymerase.

Avian Myeloblastosis Virus↗

Properties of base-pairing in the stem region of hairpin antisense oligonucleotides containing 2'-methoxynucleosides.

We have designed a new type of antisense oligodeoxyribonucleotide. These oligonucleotides are able to form hairpin loop structures at the 3'-ends. The stability to nuclease degradation was observed by incubation of these hairpin oligonucleotides with snake venom phosphodiesterase, DNA polymerase, and fetal bovine serum. Of particular interest is the hairpin antisense oligonucleotide containing 2'-methoxynucleosides with base-pairing in the stem region at the 3'-end, which has increased nuclease resistance.

Base Composition↗

Lack of the expression of EBNA-2 and LMP-1 in T-cell neoplasms possessing Epstein-Barr virus.

We investigated 34 cases of T-cell neoplasm [15 cases of T-cell granular lymphocytic leukemia (T-GLL), 10 cases of T-cell non-Hodgkin's lymphoma (T-NHL), six cases of T-cell chronic lymphocytic leukemia (T-CLL), and three cases of cutaneous T-cell lymphoma] to study their association with Epstein-Barr virus (EBV). In 4 (three T-NHL and one T-GLL) of 34 cases, EBV genome was detected in a single episomal form, while polyclonal EBV-DNA was detected in one (T-NHL) of the remaining cases. All three cases of T-NHL having monoclonal EBV episome showed histologically diffuse large-cell lymphoma and developed leukemic conversion. Phenotypic analysis showed that two of these four cases were CD4+, CD8-, and the remaining two cases were CD4-, CD8+. The cells from all four cases were confirmed to be in T-cell lineage by detecting the rearrangement of T-cell receptor (TCR) beta or gamma chain gene. By reverse transcription-polymerase chain reaction (RT-PCR), EBNA-1 was detected at low levels, and neither EBNA-2 nor LMP-1 were found in any of the three cases examined. Lack of the expression of EBNA-2 and LMP-1 was also confirmed by immunocytochemical staining. The cells of these four cases did not show rearrangement or overexpression of c-myc and bcl-2 genes by Southern and Northern blots, and the mutation of p53 gene was detected in only one patient. These results suggest that other latent gene products of EBV or other cellular oncogenes are involved in the development of Japanese T-cell neoplasm after EBV infection.

Antigens, Viral↗

Antiviral effect of phosphorothioate oligodeoxyribonucleotides complementary to human immunodeficiency virus.

Modifications of oligodeoxyribonucleotides include the replacement of the backbone phosphodiester groups with phosphorothioate (S-ODNs) groups and the substitution of phosphorothioate (SO-ODNs) groups at both the 3'- and 5'-ends. In assays for HIV, oligomers (S-ODNs) were more active at the micromolar range than were SO-ODNs of the same sequence. Furthermore, the abilities of antisense-, sense-, random-, and mismatched-oligomers, or homo-oligomers containing internucleotidic phosphorothioate linkages to inhibit HIV-1 replication were examined. Antisense oligonucleotides inhibit the replication and the expression of HIV-1 more efficiently than random-, sense-, mismatched-, and homo-oligomers of the same length or with the same internucleotide modification. Five different target sites (gag, pol, rev, tat, and tar) within the HIV genes were also studied with regard to the inhibition of HIV replication by antisense oligonucleotides. Antisense oligomers complementary to the sites of initiation sequences and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting viral replication was also investigated. Of particular interest was the S-ODNs-rev 15 mer, which possessed higher anti-HIV activity than the sense-, random-, mismatched-, and homo-20 mers.

Antiviral Agents↗

[Comparative study on minimum inhibitory concentration of gonococcal strains according to their auxogroups and serovars].

Auxotypes, serovars and minimum inhibitory concentration (MIC) of various antibiotics are investigated on 85 gonococcal strains isolated from the patient with gonorrhea from 1984 to 1986 in Japanese Red Cross Medical Center. As to the auxotypes 33 strains were identified to be Proto, 33 to be Pro, 10 to be Arg and 9 to be PAU. No AHU strain was identified. Serovars were divided into 1A and 1B. MIC of 22 kinds of antibiotics including penicillins, cephems, spectinomycin, tetracycline and new quinolones were determined. Cumulative MIC curves were found to be shifted to the right according to the order of auxotypes Arg, PAU, Proto, Pro in the most antibiotics. Serovar 1A was found to have lower MIC as compared to 1B, though only 5 strain of 1A were in the study. Seventy-eight % of isolates were Proto or Pro, so-called Asian auxotype which were revealed to have higher MIC not only of penicillin but also of the other antibiotics. Nine strains were identified as PAU which was lately identified in Canada. This might suggest that gonococcal strains are spreading over the world in spite of the local predominance of auxotype distribution.

Adult↗

[Inhibition of gene expression by antisense DNA].

We demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV, MMLV, or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment, bound to the antisense oligonucleotide, by reverse transcriptase associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization, by binding to the AMV- and MMLV-RTs, rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription. Furthermore, the RNase H activity of HIV-RT was only slightly affected by the phosphorothioate oligonucleotide. The anti-HIV activities of phosphorothioate- or 5'-linked lipid-oligonucleotides are also described and some of the problems that still need to be solved are pointed out.

Avian Myeloblastosis Virus↗

Saturable uptake of a recombinant human granulocyte colony-stimulating factor derivative, nartograstim, by the bone marrow and spleen of rats in vivo.

Kinetic analysis of the in vivo tissue distribution of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) derivative, nartograstim (NTG; nartograstim is an international nonproprietary name of KW-2228, Kyowa Hakko Kogyo, Tokyo, Japan) was studied in rats to determine the mechanism of the growth factor's nonlinear pharmacokinetic properties. The early-phase tissue uptake clearance (CL(uptake)) by each tissue was determined within 5 min after the i.v. administration of 125I-NTG. Coadministration of various amounts (0.6-150 microgram/kg) of unlabeled NTG did not produce any significant change in CL(uptake) by kidneys and liver. The hepatic and renal extraction ratios of NTG were low, 0.10 to 0.15, which suggests that there is no saturable uptake system in either of these organs. CL(uptake) by the kidneys was comparable to the glomerular filtration rate of unbound NTG. By contrast, a dose-dependent reduction in CL(uptake) by bone marrow and spleen was clearly observed with increasing doses of unlabeled NTG, i.e., at the maximal dose, these values were 14% and 20%, respectively, of those after a tracer dose. A saturable process was, therefore, involved in the tissue uptake of 125I-NTG by bone marrow and spleen. The intrinsic clearance (Vmax/Km) of this saturable uptake by bone marrow, 1.4 ml min-1 kg-1, was greater than that by the spleen, 0.09 ml min-1 kg-1. The sum of the intrinsic clearances of the saturable process was comparable with the value for the clearance of the saturable elimination process of NTG obtained previously by nonlinear pharmacokinetic analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Epstein-Barr virus associated natural killer cell leukemia: report of an autopsy case].

A 20-year-old female was admitted because of high fever, hepatosplenomegaly, severe hepatic dysfunction and coagulopathy. Peripheral blood showed pancytopenia and granular lymphocytes bearing the natural killer cell phenotype (CD2+CD3-CD16+CD56+CD57-TCR alpha beta-TCR gamma delta-) constituted 97% of leucocytes. Southern blot analysis of DNA obtained from peripheral blood mononuclear cells showed germ-line configuration of TCR beta, gamma and delta chain genes. EBV-DNA was detected in a single episomal form by using EBV-terminal repeat probe. Bone marrow findings were consistent with hemophagocytic syndrome and administration of VP-16 was effective transiently. After ten months she died from massive gastrointestinal bleeding. An in situ hybridization study identified EBV-RNA (EBER-1) in atypical lymphocytes infiltrating bone marrow, spleen and lymph nodes. Sections of liver showed steatosis and infiltration of T cells (CD3+ and EBER-1-negative) in the portal areas and few atypical lymphocytes in sinusoids. The patients developed an EBV-associated clonal proliferation of natural killer (NK) cells, but the clinical features were suggestive of chronic active EBV infection or virus-associated hemophagocytic syndrome (VAHS) rather than leukemia. Bone marrow transplantation for NK cell leukemia is an issue to be discussed.

Adult↗

[Consecutive annual changes in minimum inhibitory concentration in clinically isolated Neisseria gonorrhoeae strains].

Between 1983 and 1991, 465 gonococcal strains isolated in the urological department of Japanese Red Cross Medical Center. The minimum inhibitory concentration (MIC) of these isolates to 22 kinds of antibiotics including penicillins (PC), cephems, tetracyclins (TC) and new quinolons (NQ) were determined and the annual difference of MIC was studied. The annual incidence of penicillinase producing neisseria gonorrhoeae (PPNG) of these 9 years were distributed in 3 to 17% and increasing tendency was not observed. As for Penicillin G, the MIC 90 of PPNG was seven fold higher than that of non-PPNG. No remarked difference was observed between MIC 90 of Cephems, TC and NQ of PPNG and non-PPNG. No annual difference was observed in MIC of PC, Cephems and SPCM. However the rising tendency of MIC was observed in NQ.

Adolescent↗

Selective deuteration of RNA for NMR signal assignment.

For site-selective deuterium labeling of RNA, [5-2H]uridine phosphoramidite was prepared. The uridine at position 10 of a 25-mer RNA, GGACAGACUUCGGUCGGAGUACUCG, was labeled in two different manners for "positive" (U = [5-1H]U, U = [5-2H] U) and "negative"(U = [5-2H]U, U = [5-1H]U) observations. By comparison of NOESY spectra of the two labeled samples with that of the unlabeled RNA, we could unambiguously assign the H5-H6 signals of U10, and measure their NOE connectivities.

Base Sequence↗

Triple helix formation with oligodeoxyribonucleotides containing 8-oxo-2'-deoxyadenosine and 2'-modified nucleoside derivatives.

The ability of homopyrimidine oligonucleotides containing 8-oxo-2'-deoxyadenosine (dAOH) and/or 2'-methoxyuridine (Um) or 2'-fluorouridine (Uf) to form stable, triple helical structures with the sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-oxo-2'-deoxyadenosine substituted oligomers were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise specificity. On the other hand, the oligonucleotides containing 2'-modified nucleosides (Uf ,Um, dAOH-Um, and dAOH-Uf) bind in a pH-dependent mannerto the target duplex.

Binding Sites↗

Inhibition of influenza virus RNA polymerase and nucleoprotein of gene expression by antisense oligonucleotides.

We demonstrated that unmodified and modified (phosphorothioate) antisense oligonucleotides inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to dexamethasone. Antisense oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for the their inhibitory effects by a CAT-ELISA assay. Antisense phosphorothioate oligonucleotides (S-ODNs) complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed a high inhibitory effect. On the other hand, the inhibitory effect of the S-ODNs targeted to PB1 was considerably decreased in comparison with the other three target sites.

Base Sequence↗

Properties of nicked and circular dumbbell RNA-DNA chimeric oligonucleotides.

We have designed a new type of antisense oligodeoxyribonucleotide. These oligonucleotides form two hairpin loop structures with RNA-DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem. The nicked and circular dumbbell RNA/DNA chimeric oligonucleotides are molecules, with or without free ends, that are more resistant to exonuclease attack. Of particular interest, antisense DNA is liberated by RNase H treatment of the dumbbell RNA/DNA chimeric oligonucleotides.

Base Sequence↗

Codon recognition by tRNA molecules with a modified or unmodified uridine at the first position of the anticodon.

Effects of a single nucleoside modification at the first position of the anticodon of a transfer RNA molecule on its codon reading properties were investigated by use of a cell-free protein synthesis. We prepared two artificial tRNA molecules that differ only in the nucleotide at the first position of the anticodon. One has an unmodified uridine and the other has a 5-methoxyuridine (mo5U). These molecules were charged with labeled serine and introduced into a cell-free protein synthesis directed by a designed mRNA, and the relative codon reading efficiencies were calculated. The results showed that the modification of U into mo5U elevates the reading efficiencies of the UCU and UCG codons but reduces that of the UCA codon.

Anticodon↗

pH-independent inhibition of restriction endonuclease cleavage via triple helix formation by oligonucleotides containing 8-oxo-2'-deoxyadenosine.

The ability of homopyrimidine oligonucleotides containing 8-oxo-2'-deoxyadenosine to form stable, triple helical structures with the sequence containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-oxo-2'-deoxyadenosine-substituted oligomers were shown to inhibit enzymatic cleavage and to bind within the physiological pH range in a pH-independent fashion without compromising specificity.

Base Sequence↗

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis.

Nonnatural amino acids with photofunctional groups were incorporated site-specifically into a polypeptide by using in vitro protein synthesizing system. The nonnatural amino acids were attached to tRNA(CCU) through chemical misacylation method, and added to the in vitro system with a mRNA containing a single AGG codon. L-p-Phenylazophenylalanine, L-2-anthrylalanine, L-1-naphthylalanine, L-2-naphthylalanine and L-p-biphenylalanine were successfully incorporated into a polypeptide, but l-1-pyrenylalanine was not. The polypeptides containing the nonnatural amino acids showed photofunctionalities.

Acylation↗

Recognition of UUN codons by two leucine tRNA species from Escherichia coli.

Codon recognition by Escherichia coli tRNA(Leu)4 and tRNA(Leu)5 was investigated by analysis of the competition between two aminoacyl-tRNA species in an in vitro protein synthesis. Both tRNA species strictly obey the wobble rule when they are in competition with other tRNA species. This is probably due to the post-transcriptional modifications at the first position of the anticodon of these tRNA(Leu) species, supporting the proposal that the conformational rigidity of post-transcriptionally modified pyrimidine nucleotides guarantees the correct codon recognition.

Amino Acid Sequence↗