Biomedical subjects

F Nishikawa

Publications and source records attributed to F Nishikawa.

At least 37 records · Page 2Linked to original sources

PubMed1995

Conversion of Salmonella typhimurium to L-forms contributes to the maintenance of acquired immunity against murine typhoid.

Conversion of Salmonella typhimurium to L-forms, both in vitro and in vivo, resulted in the expression of proteins cross-reacting to the mycobacterial 65,000 MW heat-shock protein (hsp). Immunization of C3H/HeJ mice with a protective dose of stable L-form S. typhimurium induced gamma delta T cells in the liver, in accordance with the multiplication of L-form Salmonella in Kupffer cells. The number of gamma delta T cells decreased after the intracellular growth of L-form Salmonella plateaued. Persistance of the L-forms in Kupffer cells, however, allowed hepatic gamma delta T cells to increase within 48 hr of infection with virulent S. typhimurium. Thus, the intrahepatic colonization of L-form Salmonella seems to keep gamma delta T cells on standby, but the emergence of these T cells does not correlate with the expression of L-form hsp. In addition, Kupffer cells colonized by L-forms constitutively synthesized mRNA for interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha). These results suggest that conversion of S. typhimurium to L-forms in phagocytic cells builds up and maintains acquired resistance, conferred by live-cell vaccines of S. typhimurium, against murine typhoid.

Animals↗

PubMed1995

In vitro selection analysis of trans-acting HDV ribozyme.

In order to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. After 10 generations, a randomized pool of trans-acting ribozymes accumulated in which the secondary structure of each ribozyme confirmed to the pseudoknot model and important bases in single-stranded regions were all conserved. We were surprised that mutated ribozymes derived from genomic sequence were changed to anti-genomic-like sequences. Further investigations of the most active variant confirmed that each mutated base was the most appropriate nucleotide at every position of HDV ribozyme.

Base Sequence↗

PubMed1995

Magnesium is essential for formation of an active complex of a hammerhead ribozyme with its substrate: an investigation by NMR spectroscopy.

The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high-resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson Crick three-base-paired duplex and a four-base-paired duplex that included a wobble base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate-recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substrate interaction. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme-substrate complex.

Base Sequence↗

PubMed1994

Reinfection with influenza B virus in children: analysis of the reinfection influenza B viruses.

Influenza B virus reinfection in Japanese children was studied epidemiologically during 1979-91 and virologically during 1985-91. During this investigation, there were four epidemics caused by influenza B viruses, each of which accompanied antigenic drift. Between the epidemics in 1987/88 and 1989/90, the viruses changed drastically, both genetically and antigenically. The minimum rate of reinfection with influenza B virus during the whole period was 3-25% depending on the influenza seasons. The antigens of primary and reinfection strains of influenza B virus isolated from 18 children during 1985-90, which covered three epidemic periods, were studied by haemagglutination inhibition tests. The results showed that the viruses isolated in the 1984/85 and 1987/88 influenza seasons, which belonged to the same lineage, were antigenically close, and reinfection occurred with these viruses. The results of amino-acid analysis of the HA1 polypeptide of these viruses corresponded with those of antigenic analysis. There were no specific amino-acid changes shared by the primary infection and reinfection influenza B viruses; the patients were infected with the viruses epidemic at that time.

Adolescent↗

PubMed1994

Protective capacity of L-form Salmonella typhimurium against murine typhoid in C3H/HeJ mice.

L forms of Salmonella typhimurium LT2 conferred strong protection to a lethal challenge with its parental bacterium on innately hypersusceptible C3H/HeJ mice, and its minimal protective dose was approximately 150 L-forming units. Although L-form S. typhimurium was avirulent for C3H/HeJ mice, it multiplied slowly in both the liver and spleen with the maximal growth 2-3 weeks after immunization and thereafter it persisted in the liver until 24 weeks. Protective immunity began to work between 4 and 6 weeks after immunization, and it remained active as long as the L forms colonized the liver (until 24 weeks after immunization). Vaccination with the L form induced a population of T cells responding to L-form whole-cell lysate (WCL), while delayed-type hypersensitivity (DTH) to the extract of S. typhimurium was induced after the establishment of solid immunity. Moreover, neither T-cell responses nor DTH to heat-killed S. typhimurium was generated. In addition, antibody responses were elicited to WCL but not to heat-killed S. typhimurium. These results indicate that protection conferred by the L forms is attributable to the persistent colonization of the L forms rather than the presence of DTH, and also that Salmonella cytoplasmic antigens are involved in induction of immunological responses by vaccination with the L forms.

Animals↗

PubMed1994

Transfer of protection to murine typhoid conferred by L-form Salmonella typhimurium in dependence of cooperation between L form-adopted macrophages and L form-induced Lyt-2+ T cells.

The effector cells responsible for protection to Salmonella typhimurium in C3H/HeJ mice, conferred by L-form S. typhimurium, were determined by cell transfer test. Nonfractionated spleen cells from 6-week immune mice but not from 24-week immune animals transferred anti-S. typhimurium immunity. Treatment with anti-macrophage antiserum and complement most effectively abolished protective capacity in 6-week immune cells, while anti-T cell monoclonal antibody plus complement reduced it to a lesser extent. However, adoptive protection was achieved only by transfer of immune macrophages along with Lyt-2+ T cells selected from 6-week immune spleen cells. These Lyt-2+ T cells were cytotoxic to Kupffer cells from C3H/HeJ mice which had been infected 48 hr previously and from the mice which had been immunized 1 week previously, but not to the cells from 6-week immune mice and from normal animals. Moreover, protective capacity in immune macrophages seemed to be correlated to the degree of colonization by the L forms, and the inability to transfer immunity of 24-week immune spleen cells may be due to the decrease in the L form-colonization. These results suggest that cooperation between the L form-colonized macrophages and L form-induced cytotoxic Lyt-2+ T cells contributes to anti-S. typhimurium immunity, and might imply the immunological difference between the 6-week immune phagocytes and the cells at an early stage of infection or immunization.

Animals↗

PubMed1993

High-resolution NMR study of a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein, ricin.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Its substrate domain forms a double helical stem and a 17-base loop that includes the sequence GAGA, the second adenosine of which corresponds to A4324. Recently, studies of mutant RNAs have shown that the four-nucleotide loop, GAGA, can function as a substrate for ricin. To investigate the structure that is recognized by ricin, we studied the properties of a short synthetic substrate, the dodecaribonucleotide r-CUCAGAGAUGAG, which forms a RNA hairpin structure with a GABA loop and a stem of four base pairs. The results of NMR spectroscopy allowed us to construct the solution structure of this oligonucleotide by restrained molecular-dynamic calculations. We found that the stem region exists as an A-form duplex. 5G and 8A in the loop region form an unusual G:A base pair, and the phosphodiester backbone has a turn between 5G and 6A. This turn seems to help ricin to gain access to 6A which is the only site of depurination in the entire structure. The overall structure of the GAGA loop is similar to those of the GAAA and GCAA loops that have been described but that are not recognized by ricin. Therefore, in addition to the adenosine at the depurination site, the neighboring guanosine on the 3' side (7G) may also play a role in the recognition mechanism together with 5G and 8A.

Base Composition↗

PubMed1993

Identification of important bases in a single-stranded region (SSrC) of the hepatitis delta (delta) virus ribozyme.

Models for the secondary structure of genomic and antigenomic self-cleaving RNAs of human hepatitis delta (delta) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single-stranded regions [nucleotides 726-731 (SSrA) and nucleotides 762-766 (SSrB)]. For the identification of the important residues in the remaining single-stranded region, nucleotides 708-715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single-base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in-vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild-type SSrC (HDV86). By analyzing the self-cleavage activities of various mutants, we determined the base requirements for SSrC as 5'-(U/C/G)-C-N-N-(C/A/G)-(G/A/U)-N-N-3'.

Base Sequence↗

PubMed1993

Systematic substitution of individual bases in two important single-stranded regions of the HDV ribozyme for evaluation of the role of specific bases.

To elucidate the role of specific bases in the self-cleavage activity of the human hepatitis delta virus (HDV) ribozyme, systematic substitutions of individual bases in two important single-stranded regions [between nucleotides 726-731 (SSrA region) and 762-766 (SSrB region)] were carried out by oligonucleotide-directed point mutagenesis. Among the mutants obtained, 12 mutants (G726 variants, G727A, G727C, G728C, G762A, G762C, C763 variants and A766C) could not tolerate the respective base-substitutions and self-cleavage activities were reduced to very low levels (10%), suggesting a requirement of the respective bases. In particular, G726 in the SSrA region and C763 in the SSrB region were found to be essential for the ribozyme activity. We could determine the preferred sequences, 5'-G-G-(G/A/U)-N-(A/U/G)-Pu-3' for SSrA and 5'-(G/U)-C-N-(A/G/U)-A-3' for SSrB regions, respectively.

Base Sequence↗

PubMed1993

Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages.

Hammerhead ribozymes are considered to be potential therapeutic agents for HIV virus because of their site-specific RNA cleavage activities. In order to elucidate structure--function relationship and also to hopefully endow ribozymes with resistance to ribonucleases, we firstly synthesized chimeric DNA/RNA ribozymes in which deoxyribonucleotides were substituted for ribonucleotides at noncatalytic residues (stems I, II, and III). Kinetic analysis revealed that (i) DNA in the hybridizing arms (stems I and III) enhanced the chemical cleavage step. (ii) stem II and its loop do not affect its enzymatic activity. Secondly, we introduced deoxyribonucleotides with phosphorothioate linkages to the same regions (stems I, II, and III) in order to test whether such thio-linkages further improve their resistance to nucleases. Kinetic measurements revealed that this chimeric thio-DNA/RNA ribozyme had seven-fold higher cleavage activity (kcat = 27 min-1) than that of the all-RNA ribozyme. In terms of stability in serum, DNA-armed ribozymes gained about 10-fold higher stability in human serum but no increase in stability was recognized in bovine serum, probably because the latter serum mainly contained endoribonucleases that attacked unmodified catalytic-loop regions of these ribozymes. Thirdly, in order to protect them from endoribonucleases, three additional modifications were made at positions U7, U4 and C3 within the internal catalytic-loop region, that succeeded in gaining more than a hundred times greater resistance to nucleases in both serums. More importantly, these catalytic-loop modified ribozymes had the comparable cleavage activity (kcat) to the wild-type ribozyme. Since these chimeric thio-DNA/RNA ribozymes are more resistant to attack by both exonucleases and endoribonucleases than the wild-type all-RNA ribozymes in vivo and since their cleavage activities are not sacrificed, they appear to be better candidates than the wild type for antiviral therapeutic agents.

Animals↗

PubMed1993

Antigenic and molecular properties of Murayama virus isolated from cynomolgus monkeys: the virus is closely related to avian paramyxovirus type 2.

A new virus that belonged to a member of paramyxovirus was isolated from cynomolgus monkeys showing respiratory disorders about 20 years ago and was named Murayama virus (MrV). Interestingly, it showed no serological relationship with the mammalian paramyxoviruses tested. On the other hand, it was related to Yucaipa (YuV) and Bangor virus (BaV) belonging to avian paramyxovirus type 2 (PMV2). In analysis using anti-MrV monoclonal antibodies, MrV showed the closest relationship with YuV, and furthermore some conserved epitopes were found among avian paramyxovirus, MrV, YuV, BaV, and Newcastle disease virus (NDV). Subsequently, the nucleotide sequences of the F and HN genes of MrV were determined. In comparison with the deduced amino acid sequence of MrV and other paramyxoviruses, MrV showed the highest homology with NDV and higher similarity to HPIV2 group rather than to HPIV1 group. The present study clearly indicates that MrV belongs to PMV2 adapting to monkeys.

Amino Acid Sequence↗

PubMed1993

Serum level of carbohydrate-deficient transferrin as a marker of alcoholic liver disease.

Serum levels of carbohydrate-deficient transferrin (CDT) were assayed in 87 patients with alcoholic liver disease, 25 alcoholics without liver disease, 25 cases with viral liver disease and 37 healthy subjects, by two different methods (Pharmacia CDT RIA kit and Axis % CDT kit). The serum level of Pharmacia-CDT was significantly higher in the patients with alcoholic liver disease (38.9 +/- 2.8 U/l) compared to the normal subjects (18.9 +/- 0.2 U/l), alcoholics without liver disease (21.7 +/- 1.5 U/l) and non-alcoholic liver disease (viral liver disease) (23.4 +/- 1.6 U/l) (P < 0.001). The serum level of Axis-CDT was also significantly higher in the patients with alcoholic liver disease (4.22 +/- 0.48%) compared to the normal subjects (0.84 +/- 0.14%), alcoholics without liver disease (1.14 +/- 0.23%) and non-alcoholic liver disease (1.84 +/- 0.29%) (P < 0.001). A significant correlation was found between serum levels of CDT determined by the two kits (r = 0.718, P < 0.001). The serum level of Axis-CDT was significantly higher in patients with alcoholic hepatitis compared to the normal subjects (P < 0.005), while the serum level of Pharmacia-CDT was not increased in the patients with alcoholic hepatitis. These results indicate that determination of serum CDT levels is a useful marker of alcoholic liver disease, not a marker for alcohol consumption. Axis-CDT is more useful than Pharmacia-CDT for assaying the serum level of CDT in patients with alcoholic liver disease.

Adult↗

PubMed1993

Prevalence of hepatocellular carcinoma in patients with alcoholic cirrhosis and prior exposure to hepatitis C.

Sixty-three patients with alcoholic cirrhosis were retrospectively studied for the prevalence of antibodies to core (P22) and nonstructural (C100) region of hepatitis C virus (HCV). The prevalence rate of anti-P22 antibodies in patients with alcoholic cirrhosis was higher than that of anti-C100 antibodies (63.5% vs. 54.9%). The positivity rate of anti-C100 and/or anti-P22 antibodies was 73.0% (46/63) in alcoholic cirrhosis. We performed a multivariate analysis on the effects of age, sex, cumulative alcohol intake, anti-HCV antibodies, indocyanine green excretion test, and serum albumin on the development of hepatocellular carcinoma HCC in patients with cirrhosis, using Cox's proportional-hazards model, which revealed that anti-HCV positivity was the only independent prognostic variable for HCC in patients with alcoholic cirrhosis. The probability of HCC was significantly higher in the anti-HCV-positive patients than in the negative patients with alcoholic cirrhosis (p < 0.05). The 3-, 5- and 10-yr cumulative occurrence rate of HCC was, respectively, 13.3%, 41.3%, and 80.7% for anti-HCV-positive patients with alcoholic cirrhosis, compared with 0%, 8.3%, and 18.5% for anti-HCV-negative patients. In nonalcoholic patients with type C cirrhosis, the 3-, 5-, and 10-yr cumulative occurrence rate of HCC was 7.3%, 23.1%, and 56.5%, respectively. The follow-up studies indicate that hepatocarcinogenesis is hastened significantly in patients with alcoholic cirrhosis if they are positive for anti-HCV antibody, and that heavy alcohol consumption also is a risk factor for the development of HCC in patients with type C cirrhosis.

Adult↗

PubMed1993

Chemical probing studies of the hepatitis delta virus (HDV) genomic ribozyme.

We have investigated the higher-order structure of HDV genomic ribozyme using various base-and phosphate-specific chemical probes under native, semi-denaturing and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethylsulfate and carbodiimide. In general, our results are consistent with the proposed pseudoknot model of secondary structure. The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea.

Base Sequence↗

PubMed1992

Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme.

In elucidating function of two important single-stranded regions [SSrA (726-731 nt) and SSrB (762-766 nt)] derived mainly from three secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several random mutants at those two regions on the HDV88 molecule (683-770 nt) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among mutants, at the region SSrA, G726 was found to play an important role during cleavage reaction since substitutions of the base to A (mutant A20) or C (mutant A16) or U (mutant A23), reduced the ribozyme activity to very low levels suggesting the importance of G726 position. C763 at SSrB region was found to play a more significant role during catalysis than G726 (at region SSrA) since any substitutions at C763 completely inactivated the ribozyme. Other bases located in these two regions could be substituted to other bases at the expense of some self-cleavage activity. The results presented here together with our previous deletion analysis indicate that these two regions may play an important role during cleavage process.

Base Sequence↗

PubMed1992

Deletion of internal sequence on the HDV-ribozyme: elucidation of functionally important single-stranded loop regions.

In elucidating functionally important single-stranded loop regions derived mainly from three models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several internal deletion variants of the HDV133 molecule (654-786 nt on genomic RNA) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among variants, the HDV133DI-1 (deletion of 701-718 nt) and HDV133DI-3 (deletion of 740-752 nt) ribozyme could maintain their self-cleavage activity, despite at reduced level. However, the activity could be regained in both mutants by some extent under partially denaturing conditions. These results suggest that the above two single-stranded RNA loop regions in HDV ribozyme are not part of the catalytic core but might be involved in the stability of the molecule. In contrast, deletion mutants such as HDV133DI-2 (deletion of 696-722 nt), HDV88DI-1 (deletion of 701-718 nt), HDV88DI-2 (deletion of 696-722 nt), and HDV88DI-4 (deletion of 733-760 nt) abolished catalytic activity. These results suggest that the remaining single-stranded regions of bases between 726-731 and 762-766 in the HDV88 ribozyme may be the potential regions to interact with Mg2+ ions.

Base Composition↗

PubMed1992

Contribution of interferon gamma and membrane-associated interleukin 1 to the resistance to murine typhoid of Ityr mice.

Resistance of mice to Salmonella typhimurium in the early phase of infection is known to be controlled by the expression of chromosome 1 locus Ity. To clarify the mechanism by which the genetically resistant (Ityr) mice can overcome the first phase of salmonellosis, the early response in DBA/2 (Ityr) and BALB/c (Itys) mice was compared after a subcutaneous injection of S. typhimurium. In both strains, the growth of S. typhimurium was controlled in livers and Kupffer cells until day 3, but thereafter the bacteria multiplied rapidly in BALB/c mice. Over the first 2 days nonspecific responses (changes in levels of blood leukocytes, plasma iron, and alpha 1-antitrypsin) were not significantly different between the strains, and the capacity of Kupffer cells isolated from infected mice of both strains to produce interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) was of the same degree. Thereafter, only DBA/2 Kupffer cells were able to produce membrane-associated IL-1 (ma IL-1) as well as TNF-alpha. Moreover, only DBA/2 splenocytes were able to produce interferon gamma (IFN-gamma) upon stimulation with Salmonella antigens, although concanavalin A-stimulated splenocytes of both strains produced the same level of interleukin 2. Furthermore, administration of recombinant murine IFN-gamma and DBA/2 Kupffer cells of day 6 to BALB/c mice 3 days after infection resulted in a significant level of protection, whereas neither of these materials alone induced protection. Injection of anti-TNF-alpha antibodies did not affect the resistance of DBA/2 mice. Thus, these findings suggest that the early resistance of Ityr mice is partly attributable to their capacity to produce IFN-gamma and ma IL-1 after infection.

Animals↗

PubMed1992

Comparison of the HA genes of type B influenza viruses in herald waves and later epidemic seasons.

From January 1985 to May 1991, herald strains of influenza B virus were isolated in 1987 and 1989 in Japan. In both cases, influenza epidemics caused by the same type followed in the next winter season. The HA gene sequences of the influenza B viruses isolated in Japan from 1987-91, which covers two herald waves of influenza B viruses, were analysed and located on the phylogenetic tree for influenza B viruses after the B/Singapore/64 strain. Co-circulation of at least two evolutionary lineages of the HA genes existed for influenza B viruses in Japan during the period of this study. The herald viruses in one wave (1987) were genetically close to the winter isolates and were considered to be the parental viruses for the following influenza season, while in the other wave (1989) winter isolates belonged to another lineage on which one of the herald viruses was located, but they were genetically and antigenically different from the herald viruses.

Antigens, Viral↗