Biomedical subjects
Publications and source records attributed to T Nakaya.
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A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.
BACKGROUND AND OBJECTIVES: In order to evaluate the clinical relevance of angiogenesis in patients with gastric cancer, we investigated the microvessel count in gastric cancer tissues and compared the results with several clinicopathologic factors and prognosis. METHODS: A total of 55 patients with gastric cancer were included in this study. Microvessel count was determined by immunohistochemical staining using antifactor VIII-related antigen antibody. Histologically recognizable blood vessels within tissue sections served as internal control for immunostaining. Normal mouse IgG diluted to an equivalent protein concentration was used as a negative control in place of the primary antibody in each experiment. RESULT: The microvessel count ranged from 4.4 to 39.8 and the median count was 15.6. Microvessel count was significantly higher in patients with hepatic metastasis, lymph node metastasis, adjacent organ invasion, and lymphatic invasion. In patients who had undergone a curative operation, survival time in the hypervascular group was significantly shorter than that in the hypovascular group. CONCLUSIONS: Microvessel count correlated well with tumor progression and may serve as a useful prognostic factor in patients with gastric cancer.
Cytoplasmic membranes were isolated from the cells of a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F and membrane-bound cytochromes were characterized. Redox difference spectra at 77 K revealed the presence of cytochromes with the alpha peaks at 552 and 556 nm while CO-binding difference spectra showed the presence of o-type cytochrome(s). Partial purification of the cytochromes demonstrated that the membranes contain cytochromes c550, c551, c556 and possibly d1 besides high molecular mass cytochrome c and cytochrome c3. It turned out that two kinds of novel CO-binding c-type cytochromes are present in the membrane. The membranes and a partially purified fraction showed weak ubiquinol-1 oxidase activity but no cytochrome c oxidase activity. Results suggest that D. vulgaris does not express the heme-copper terminal oxidase under our growth conditions in spite of the presence of the col gene, which is homologous to the gene of subunit I of the aa3-type oxidase.
Sero- and molecular-epidemiological studies on Borna disease virus (BDV) infection show that BDV RNA is not always detected in the peripheral blood mononuclear cells (PBMCs) from serum anti-BDV antibody-positive individuals such as horses, sheep, cattle, cats, and humans. In this study we demonstrated BDV RNA signals by polymerase chain reaction only in restricted regions of the brain from horses with locomotor disease. Four of six horses examined showed apparently positive reactions for anti-BDV antibodies. Specific regions of the brain of these four horses were positive for BDV RNA but the internal organs, lymph nodes, and PBMCs were negative. Histological studies of their brains revealed no apparent histological abnormalities such as inflammatory reactions. These results suggest that BDV chronically infects certain restricted regions of brain in seropositive horses.
A significant level of superoxide (O2-) generation was observed in a U937-derived subclone following infection with human cytomegalovirus (HCMV). Although there were no detectable levels of viral mRNA and/or protein expression, HCMV DNA content transiently increased immediately before O2- generation. Similarly, O2- generation was also observed in peripheral blood monocytes derived from healthy donors.
In asymptomatic carriers, the vast majority of human immunodeficiency virus type 1 (HIV-1) infection is non-productive whilst the clinical stage of disease is associated with significant virus expression. Virus-specific CD8+ T-cell functions are believed to play a major role in the generation of heterogeneous virus populations and in subsequent disease progression. Here, we prepared two types of HIV-1 isolate by culturing whole and CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) from five asymptomatic carriers. The former is expected to be escape variant populations, whereas the latter would be mixed populations including the former viruses. The analyses of Nef and Env V3 sequence variations of viruses in a total of 77 and 44 DNA clones, respectively, allowed a direct comparison to be made of the differences between the paired isolates. Comparison of Nef sequences between the paired isolates showed them to be more distinct in two carriers with a relatively stable CD4/CD8 ratio (Nos 68 and 69), than in two other carriers with similar CD4/CD8 ratios (Nos 53 and 57), or in carrier No. 67, which had an extremely lower CD4/CD8 ratio. By contrast, a distinction between the paired isolates by use of the Env V3 sequences was only apparent in the latter three carriers. These results indicate that the predominant populations of HIV-1 in Nos 68 and 69 were sensitive to selective pressure from Nef-specific CD8+ T-cells, while those in Nos 53, 57, and 67 were sensitive to pressure from V3-specific CD8+ T-cells. It is noteworthy that Nos 53 and 57 progressed to an AIDS-related complex shortly and several years after this examination. These data suggest that HIV-1-induced pathogenesis is more strongly associated with the generation of variant nef alleles than with env V3 variants, and that these arise by CD8+ T-cell pressure.
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We have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (EIAV), designated V70. Five viruses (F1V, F2V, F3V, F4V and F5V) were recovered during periodic febrile episodes. Cross-neutralization tests revealed that all of these variants and the parental V70 were antigenically distinct. Sequencing of their full-length env gp90 genes and gp45 5' sequences revealed novel mutations at a limited number of nucleotide positions, consisting of insertions and duplications in the gp90 principal neutralizing domain (PND) in F1V, F3V and F5V. Parts or all of small units (6, 9 and 12 nucleotides) located just before the insertion site were used for the duplications. Furthermore, amino acid substitutions in the env PND and hypervariable region were also observed in all five viruses. These mutations may contribute to the generation of serial variants. Consequently, the full-length gp90 sequences showed close relationships between V70, F2V and F4V, and between F1V, F3V and F5V. In addition to the two domains (PND and hypervariable region), a comparison of these viruses with the reported env gp90 sequences revealed four additional variable domains, although these four domains were highly conserved among the five variants.
The relationship between Borna disease virus (BDV) infection and schizophrenia in the clinical time course was investigated. By nested reverse-transcribed polymerase chain reaction (RT-PCR) and Western blotting, BDV-specific RNA and anti-BDV antibodies were examined in the EDTA-treated blood from 67 schizophrenic patients (according to DSM-III-R) in Japan. A significantly higher proportion (45%) of anti-BDV antibody and/or BDV RNA carriers were found among these 67 schizophrenic patients than in 26 controls (0%). There were no apparent associations of BDV infection with age, age at onset, period of hospitalization, accompanying somatic diseases, a past history of tuberculosis, a history of transfusion, a family history, or doses of psychotropic drugs. It is possible that, at least, BDV infection in schizophrenic patients may not be a nosocomial (hospital-acquired) infection, although the route of BDV infection in humans remains unidentified. More studies on the relationship between BDV infection and clinical psychosomatic features should be performed in order to elucidate the pathogenesis of schizophrenia.
Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev, that bind to target RNA sequences. These are the trans-activation responsive (TAR) RNA and the Rev-responsive element (RRE), respectively. The Rev protein shifts RNA synthesis to viral transcripts by binding to the RRE within the env gene. In the present study we prepared a RNA-DNA chimera consisting of 29 or 31 nucleotides to inhibit the Rev regulatory function by means of the decoy approach. The chimera oligonucleotides (anti-Rev oligonucleotides [AROs]) contained an RNA "bubble" structure (13 oligonucleotides; the Rev-binding element in RRE) that bound Rev with a high affinity in an in vitro assay. The controls were RNA-DNA chimera oligonucleotides (negative control oligonucleotides [NCOs]) similar to ARO, but without the bubble structure, that bound with considerably less affinity to Rev. When the inhibitory effects of these decoys on HIV-1 replication were examined, we found that AROs, but no NCOs, reduced more than 90% of the HIV-1 production generated by productively infected human T-cell lines. The production of primary HIV-1 isolates in healthy donor-derived peripheral blood mononuclear cells was also similarly inhibited by AROs. In addition, the induction of viral mRNAs and antigens in latently HIV-1-infected ACH-2 cells by tumor necrosis factor alpha was specifically inhibited by AROs, but not by NCOs. No apparent cytotoxicity was caused by either decoy. Thus, the use of a Rev-binding element-based decoy, the RNA-DNA chimera oligonucleotide, may represent a safer approach to gene therapy for reducing the virus load in HIV-1-infected individuals.
Previous seroepidemiological and molecular epidemiological studies of Borna disease virus (BDV) showed considerably high rates of infection in horses, cattle, cats, and humans in Hokkaido, Japan. Here, we further demonstrate high rates of specific antibodies to BDV and BDV RNA in peripheral blood mononuclear cells (PBMCs) from healthy sheep bred on the same island. The BDV prevalences by immunoblotting and/or reverse transcriptase PCR were 0% (0 of 19) in newborns (<1 month old), 51.7% (15 of 29) in lambs (1 to 6 months old), and 36.7% (11 of 30) in adults (>2 years old). Among animals positive for BDV, 60% of lambs and 45.5% of adults contained BDV RNA in PBMCs while 46.7% of lambs and 90.9% of adults contained specific antibodies to BDV. Thus, it is suggested that virus replication in the blood, as observed in lambs, is usually reduced in adulthood by raising immune responses to BDV.
We developed a simple and sensitive microplate hybridization procedure with which to identify Borna disease virus cDNA in amplified products from human peripheral blood mononuclear cells. The mean values for the positive PCR products were significant compared with those for any of the negative products, indicating that this method can be applied to rapidly diagnose a large number of clinical specimens.
Equine infectious anemia virus (EIAV) is a good model for studying mechanisms generating escaped retrovirus variants. We previously sequenced the entire gp90-encoding region of 22 cDNA clones obtained from five antigenically distinct isolates (F1V to F5V) recovered during febrile episodes in horse 493 experimentally infected with the Japanese virulent EIAV strain V70. The results showed that the mutations occurred in the principal neutralizing domain (PND) by insertions/duplications. In this study, we further characterized the PND of virus isolates sequentially recovered during 22 febrile episodes in seven horses newly infected with V70 or one of the V70-derived variants. Sequencing of 70 cDNA clones derived from the 22 episodes confirmed the generation of various new viral quasispecies with insertions/duplications in the PND. Although the insertion/duplication sequences in a total of 92 cDNA clones were extensively heterogeneous, we hypothesized that all the insertions/duplications occurred during reverse transcription from viral genomic RNA to minus strand DNA. The insertion/duplication regions were derived from a part of the PND sequence, which consisted of five small units. These small units, some with various substitutions and/or deletions, were also generated, especially in regions with insertions/duplications. Of particular note was that all these virus variants, except for two cDNA variants, were generated by essentially four different duplication pathways. Thus, these results extend the significance of insertions/duplications in the PND to the novel generation of EIAV in vivo during febrile episodes.
A new diol, bis[2-(2-hydroxyethyldimethylammonio)ethyl] butylenediphosphate, that contains two phosphatidylcholine analogous moieties in one diol molecule, was synthesized and characterized. The diol together with 1,4-butanediol as chain extender was further incorporated into the propolymer of poly(ethylene oxide) (Mn = 1000, 2000, 6000) and 4,4'-methylenediphenyl diisocyanate. The resulting phospholipid poly(ether urethane)s show viscosity behavior of common polyelectrolytes. The bulk and surface characteristics of the new phospholipid poly(ether urethane)s were investigated by IR, GPC, ATR-FTIR, ESCA and contact angle measurements. The new polymers possessed relatively hydrophilic surface revealed by contact angle measurements. The blood compatibilities of the polyurethanes were evaluated by platelet rich plasma contacting studies and scanning electron microscopy observation using medical grade PVC as the reference. No platelet adhesion was observed for all new phospholipid polyurethane casting films. This new type of phospholipid polyurethane is expected to have potential biomedical applications.
Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev. The Rev protein facilitates the transport of unspliced and singly-spliced RNA to the cytoplasm in infected host cells by binding to target RNA (Rev response element: RRE). A variety of approaches targeting Rev function, including gene therapy, have been developed that inhibit HIV-1 replication in cells cultured in vitro. This minireview summarizes the recent developments as well as our application of the Rev-binding element-based decoy approach using RNA-DNA chimera oligonucleotide modeling.
It is believed that Borna disease virus (BDV), an etiological agent of progressive polioencephalomyelitis in horses and sheep, is closely associated with psychiatric disorders in humans since the prevalence of BDV is higher in psychiatric patients than in blood donors. We investigated whether or not BDVs in humans are derived from infected domestic animals, by characterizing the BDVs in blood donors and horses derived from the same region of Hokkaido island, Japan. The seroprevalences (2.6 to 14.8%) of BDV were significantly higher in the blood donors from four regions where most horse farms are concentrated, compared with only 1% in the blood donors from Sapporo, the largest city in Hokkaido.BDV RNA was also detected in peripheral blood mononuclear cells from most of the seropositive horses and blood donors by nested reverse transcriptase-polymerase chain reaction. These findings support that BDV may be horizontally transmitted, at least in part, from infected horses to humans.
Pharmacokinetic parameters after intra-peritoneal administration of cis-platinum (CDDP) were evaluated and compared with those after intravenous administration. CDDP at a dose of 70 mg/m2 were administered intra-peritoneally in 5 patients with advanced gastric cancer (IP group). Pharmacokinetic parameters and toxicities were evaluated in these patients and compared with those in 4 esophageal cancer patients administered the same dose of CDDP intravenously (IV group). In IP group, Cmax of total- and free-Pt were 3.41 +/- 0.89 micrograms/ml and 1.10 +/- 0.30 micrograms/ml, and AUC of total- and free-Pt were 59.6 +/- 14.3 micrograms.hr/ml, 3.12 +/- 0.89 micrograms.hr/ml, respectively. On the other hand, Cmax and AUC of total- and free-Pt in IV group were 3.31 +/- 0.59 micrograms/ml. 1.13 +/- 0.21 micrograms/ml and 45.89 +/- 9.24 micrograms.hr/ml, 1.22 +/- 0.8 micrograms.hr/ml, respectively. AUC of free-Pt in IP group was significantly higher than in IV group. This result suggests that a more promising antitumor effect will be obtained systemically by intraperitoneal administration of CDDP. The incidence and grade of toxicities were similar in these two groups.