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Biomedical subjects

Y Saeki

Publications and source records attributed to Y Saeki.

At least 19 recordsLinked to original sources
PubMed2001

Flow cytometric assessment of transduction efficiency and cytotoxicity of herpes simplex virus type 1-based amplicon vectors.

BACKGROUND: In this study, we compared herpes simplex virus type 1 (HSV-1) amplicon vector stocks prepared by transient cotransfection with two different BAC-cloned packaging-defective HSV-1 helper genomes, fHSVDeltapacDelta27 and fHSVDeltapac, with respect to transduction efficiency and cytotoxicity. Both fHSVDeltapacDelta27 and fHSVDeltapac are packaging defective because the pac signals have been deleted; fHSVDeltapacDelta27 contains an additional deletion in the HSV-1 ICP27 gene, which increases the safety of the system. METHODS: HSV-1 amplicon pHSVGFP under the control of the HSV-1 immediate-early (IE) 4/5 promotor was packaged into virus particles by transient cotransfection with either fHSVDeltapacDelta27 or fHSVDeltapac DNA. Cultures were infected with the two different vector stocks and examined under the fluorescence microscope and analyzed by flow cytometry over a 5-day period to assess transduction efficiency and cytotoxicity. RESULTS: Both vector stocks, pHSVGFP[fHSVDeltapacDelta27] and pHSVGFP[fHSVDeltapac], efficiently transduced the target cells. Interestingly, the highest mean fluorescence intensities were measured at 1 day after infection, whereas the number of GFP-fluorescent cells reached a peak at day 3 after infection. At day 3 after infection, a slight increase in the number of dead cells was observed in those cultures transduced with high doses of vector stock. Between days 3 and 4 after infection, the number of dead cells increased dramatically in all the cultures, transduced and nontransduced. Only the cultures infected with a high dose of pHSVGFP[fHSVDeltapac] displayed a significant further increase in the number of dead cells between days 4 and 5 postinfection. CONCLUSIONS: Flow cytometry allowed comparison of transduction efficiency and cytotoxicity mediated by the two different amplicon vector stocks. Cultures infected with pHSVGFP[fHSVDeltapacDelta27] were more viable than those infected with pHSVGFP[fHSVDeltapac](P < 0.05). The practical implications of this study are at the level of vector design. Flow cytometry has proven a fast and reliable approach to assess the quality of potential gene transfer vectors prior to their use in (pre) clinical trials.

Animals↗
PubMed2001

Enhancement of T helper2 response in the absence of interleukin (IL-)6; an inhibition of IL-4-mediated T helper2 cell differentiation by IL-6.

Functional roles of interleukin (IL-)6 in T cell response were investigated. Mice deficient in IL-6 and wild mice were immunized with antigens (myelin oligodendrocyte glycoprotein or methylated BSA) and production of IL-4 and interferon (IFN)-gamma by regional lymph nodes was measured. IL-6 deficiency led to an enhancement of IL-4 and an inhibition of IFN-gamma production. Moreover, polyclonal stimulation of spleen T cells from unimmunized IL-6-deficient mice with anti-CD3 plus anti-CD28 antibodies (Abs) demonstrated an enhancement of T helper (Th)(2)responses. The presence of IL-6, however, augmented IL-4 production but it inhibited IFN-gamma expression by spleen T cells in response to polyclonal stimulation and by antigen-primed spleen T cells in response to re-challenge with the antigen. In contrast, the induction of spleen CD4-positive T cells into Th(2)cells in vitro by the anti-CD3 plus IL-4 was completely suppressed by exogenously added IL-6, whereas Th(1)differentiation of T cells by the anti-CD3 plus IL-12 was not inhibited by the presence of IL-6. Thus, these results indicate that IL-6 physiologically could modulate qualitative T cell response and suggest that it augments Th(1)responses partly through its inhibitory capability of IL-4-induced Th(2)differentiation of naive T cells.

Adjuvants, Immunologic↗
PubMed2001

Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.

Herpes simplex virus type 1 (HSV-1) amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 "helper" functions, amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 "stuffer" sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU amplicon vectors), and (iii) the resulting amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 amplicon vector stocks further improves safety in gene transfer applications.

Animals↗
PubMed2001

Remission of the renal involvement in a patient with primary Sjögren's syndrome (SS) after pulse high-dose corticosteroid infusion therapy.

We report the case of a young female patient with primary Sjögren's syndrome (SS). In addition to sicca symptoms she also suffered from progressive renal insufficiency and renal tubular acidosis (RTA). She was treated with three sets of pulse high-dose corticosteroid infusion and subsequent low-dose corticosteroid oral administration. When the efficacy was evaluated about 6 months after the start of the therapy, dramatic improvements were seen with no adverse effects, not only in laboratory tests but also histopathologically, as indicated by the repeat kidney biopsy. This suggests that renal involvements of SS might be reversible in some cases, and that there might be a clinical benefit of pulse high-dose corticosteroid infusion therapy in SS with progressive renal involvement.

Acidosis, Renal Tubular↗
PubMed2001

Two configurations of static magnetic fields for treating rheumatoid arthritis of the knee: a double-blind clinical trial.

OBJECTIVE: To assess the efficacy of a nonpharmacologic, noninvasive static magnetic device as adjunctive therapy for knee pain in patients with rheumatoid arthritis (RA). DESIGN: Randomized, double-blind, controlled, multisite clinical trial. SETTING: An American and a Japanese academic medical center as well as 4 community rheumatology and orthopedics practices. PATIENTS: Cohort of 64 patients over age 18 years with rheumatoid arthritis and persistent knee pain, rated greater than 40/100mm, despite appropriate use of medications. INTERVENTION: Four blinded MagnaBloc (with 4 steep field gradients) or control devices (with 1 steep field gradient) were taped to a knee of each subject for 1 week. MAIN OUTCOME MEASURES: The American College of Rheumatology recommended core set of disease activity measures for RA clinical trials and subjects' assessment of treatment outcome. RESULTS: Subjects randomly assigned to the MagnaBloc (n = 38) and control treatment groups (n = 26) reported baseline pain levels of 63/100mm and 61/100mm, respectively. A greater reduction in reported pain in the MagnaBloc group was sustained through the 1-week follow-up (40.4% vs 25.9%) and corroborated by twice daily pain diary results (p < .0001 for each vs baseline). However, comparison between the 2 groups demonstrated a statistically insignificant difference (p < .23). Subjects in the MagnaBloc group reported an average decrease in their global assessment of disease activity of 33% over 1 week, as compared with a 2% decline in the control group (p < .01). After 1 week, 68% of the MagnaBloc treatment group reported feeling better or much better, compared with 27% of the control group, and 29% and 65%, respectively, reported feeling the same as before treatment (p < .01). CONCLUSIONS: Both devices demonstrated statistically significant pain reduction in comparison to baseline, with concordance across multiple indices. However, a significant difference was not observed between the 2 treatment groups (p < .23). In future studies, the MagnaBloc treatment should be compared with a nonmagnetic placebo treatment to characterize further its therapeutic potential for treating RA. This study did elucidate methods for conducting clinical trials with magnetic devices.

Arthralgia↗
PubMed2001

A novel method to study contraction characteristics of a single cardiac myocyte using carbon fibers.

To facilitate cardiac muscle research, we developed a novel method by which the force and length of a single ventricular myocyte can be recorded with a pair of carbon graphite fibers attached firmly to both ends. One fiber was stiff, whereas the other fiber was compliant to allow the recording of force and shortening during twitch contractions. The image of the compliant carbon fiber was projected onto a pair of photodiodes, and their output was fed to a piezoelectric transducer after variable amplifications to alter the effective compliance of the carbon fiber. Thus contraction of the myocyte was induced under virtually isometric conditions as well as under auxotonic conditions. We obtained a bell-shaped relation between the compliance under an auxotonic load and the work output of the myocyte, which was directly related to myocyte performance in the heart. Because it is easy to attach myocytes to the experimental apparatus, the present method would allow us to study cardiac muscle mechanics at the cellular and molecular levels.

Animals↗
PubMed2001

Cross-bridge and calcium behavior in ferret papillary muscle in different thyroid states.

X-ray diffraction studies were made using synchrotron radiation on ferret right ventricular papillary muscle under three different thyroid states: euthyroidism, hyperthyroidism, and hypothyroidism. The latter two states were induced by treatment with L-thyroxine and methimazole, respectively. The X-ray equatorial reflections were recorded at a time resolution of 10 ms to study the mass movement of myosin cross-bridges from thick to thin filaments. The myosin isomer content was measured by gel electrophoresis which showed that V3 isomer was predominant in euthyroid muscle and 27% of myosin was V1 isomer in hyperthyroid muscle. The intracellular free Ca concentration was measured by using the aequorin method. The peak Ca concentration was similar in all three states, but in the hypothyroid state the time-to-peak was longer and the decay was slower. The time-to-peak of twitch tension was shorter in hyperthyroidism and longer in hypothyroidism than in euthyroidism. The different time courses of twitch tension in different thyroid states accompanied a cross-bridge movement which closely followed the tension development. In hyperthyroidism, the cross-bridge movement significantly preceded tension development, suggesting that hyperthyroid myosin (V1) has a longer latency period between the shift to the vicinity of the thin filament and force development.

Animals↗
PubMed2001

Protein kinase A increases the rate of relaxation but not the rate of tension development in skinned rat cardiac muscle.

To clarify the contribution of cross-bridge kinetics to the contraction profile of cardiac twitch during beta-adrenergic stimulation, we studied the rate of tension development and relaxation following laser flash photolysis of caged compounds in rat-skinned ventricular trabeculae before and after treatment with the catalytic subunit of protein kinase A (PKA, 0.5 U/microl, 40 min). Tension development following nitrophenyl (NP)-EGTA photolysis was fitted with a single exponential function. The rate constant increased with an increase in postphotolysis steady tension, and the relation between the rate constant and the tension was not influenced by PKA. The rate of relaxation following diazo-2 photolysis was fitted with a double exponential function. The rate of both initial rapid and subsequent slow relaxation was independent of the extent of relaxation. PKA increased the rate of initial rapid relaxation by about twofold, but showed no significant effect on the rate of subsequent slow relaxation. These results suggest that in beta-receptor stimulated rat cardiac muscle, the increased rate of tension development and the facilitated relaxation rate during twitch can be partly explained as being due to the combined effects of decreased Ca(2+) affinity of troponin C and increased cycling rate of cross-bridges (subtractive combination for tension development and additive combination for tension relaxation).

Animals↗
PubMed2000

Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes.

The 26S proteasome is a multisubunit protein- destroying machinery that degrades ubiquitin-tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain-binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [(125)I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B-type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo-specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development.

Alternative Splicing↗
PubMed2000

Rapid isolation and characterization of the yeast proteasome regulatory complex.

The 26S proteasome, which catalyzes degradation of ubiquitinated proteins, is composed of the 20S proteasome and the 19S complex. Recently, it has been reported that the 26S complex can be dissociated into the lid complex and the 20S-proteasome-base complex in a mutant yeast and that the lid complex is required for ubiquitin-dependent proteolysis. In the present study, we established methods for rapid isolation of the 19S complex, the lid complex, and the base complex from wild-type yeast. The isolated 19S complex was capable of binding to the 20S proteasome to reconstitute the 26S proteasome. In contrast with the previously reported result showing that Rpn10, a multiubiquitin chain binding subunit, is a component of the base complex, we present evidence that the lid complex isolated from wild-type yeast contains Rpn10.

Amino Acid Sequence↗
PubMed2000

Enhancement of Th2 response in IL-6-deficient mice immunized with myelin oligodendrocyte glycoprotein.

To investigate the mechanism for the resistance of IL-6-deficient mice to experimental autoimmune encephalomyelitis (EAE), we examined the production of cytokines in lymph nodes (LNs) of wild-type and IL-6-deficient mice immunized with myelin oligodendrocyte glycoprotein (MOG) by reverse transcriptase-polymerase chain reaction analysis. Significant up-regulation of IL-4 production and down-regulation of IFN-gamma production were found in LNs from IL-6-deficient mice as compared to LNs from wild-type mice. Administration of IL-6, which caused typical EAE in IL-6-deficient mice immunized with MOG, reduced IL-4 production but did not restore IFN-gamma production in LNs of IL-6-deficient mice. These results implied that the resistance of IL-6-deficient mice to EAE might be mainly due to enhancement of Th2 response.

Animals↗
PubMed2000

Expression of prostaglandin E synthase mRNA is induced in beta-amyloid treated rat astrocytes.

Deposition of beta-amyloid (A beta) in the brain is considered to be one of the most critical events in the onset of Alzheimer's disease (AD). In order to identify factors involved in the exacerbation of AD, we investigated transcriptionally A beta-induced genes using a cDNA subtraction technique in rat astrocytes. One gene obtained was rat prostaglandin (PG) E synthase. In this report, we present the deduced sequence for rat PGE synthase for the first time and demonstrate the induction of PGE synthase mRNA by treatment of cells with A beta. Our results suggest a possibly significant role of this enzyme in the progression of AD.

Alzheimer Disease↗
PubMed2000

Enhancement of transgene expression by cotransfection of oriP plasmid with EBNA-1 expression vector.

We have attempted to develop a system for specific enhancement of transgene expression, which has been one of the most important issues in human gene therapy. When an Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) expression vector, pCMV-trEBNA-1, was cotransfected with an origin of latent viral DNA replication (oriP)-harboring plasmid, poriP-CMV-luciferase, luciferase gene expression was up to 20 times greater than in the absence of EBNA-1. This enhancement was regulated mainly at the transcriptional level and was dependent on the oriP sequence and the amount of EBNA-1. However, cointroduction of poriP-CMV-luciferase with purified recombinant EBNA-1 inhibited luciferase gene expression whereas no inhibition was observed when pCMV-luciferase was cointroduced with recombinant EBNA-1. We also introduced poriP-CMV-luciferase into mouse liver via the use of HVJ (hemagglutinating virus of Japan)-liposomes. By 10 days after transfer, luciferase gene expression was decreased to low levels. We then introduced pCMV-trEBNA-1 to mouse liver via HVJ-liposomes on day 10. Luciferase gene expression was reactivated, whereas no reactivation was detected by the injection of EBNA-1 expression plasmid into liver injected with pCMV-luciferase lacking the oriP sequence. Thus, cotransfection of oriP-harboring expression vector with EBNA-1 expression plasmid should be promising for human gene therapy, although the safety of the system must be investigated thoroughly.

Animals↗
PubMed2000

Enhanced expression and DNA binding activity of two CCAAT/enhancer-binding protein isoforms, C/EBPbeta and C/EBPdelta, in rheumatoid synovium.

OBJECTIVE: To investigate the activation and expression of CCAAT/enhancer-binding proteins (C/EBP), especially C/EBPbeta and -delta, in rheumatoid synovium, and their pathogenic implications in rheumatoid arthritis (RA). METHODS: The activation of C/EBPbeta and -delta was assessed in synovial tissues from patients with RA by electrophoretic mobility shift assay (EMSA); DNA binding activity of C/EBPs was evaluated by measuring EMSA band density. The expression and distribution of C/EBPbeta and -delta in synovial tissues were examined by immunohistochemistry analysis. As a control, synovial tissues from patients with osteoarthritis (OA) were studied. RESULTS: Enhanced DNA binding activity of C/EBPbeta and -delta, 2 major members of the C/EBP family, was detected in synovial tissues from RA patients, while synovial tissues from the patients with OA showed only faint or marginal activity (mean +/- SEM arbitrary units [AU] RA 23.3 +/- 11.7 in RA versus 4.5 +/- 1.3 in OA; P < 0.05). Moreover, the binding activities of the C/EBP proteins were correlated with both serum C-reactive protein levels (r = 0.62, P < 0.05) and synovial interleukin-6 messenger RNA levels (r = 0.60, P < 0.05). In immunohistochemistry studies, C/EBPbeta and -delta were detected predominantly in the rheumatoid synovial lining cells (both CD14+ and CD14- cells). CONCLUSION: C/EBPbeta and -delta may contribute to the pathology of rheumatoid synovitis.

Aged↗
PubMed2000

Tumour necrosis factor alpha (TNF-alpha) interferes with Fas-mediated apoptotic cell death on rheumatoid arthritis (RA) synovial cells: a possible mechanism of rheumatoid synovial hyperplasia and a clinical benefit of anti-TNF-alpha therapy for RA.

To investigate the mechanism of rheumatoid synovial hyperplasia (RASH), the influence of tumour necrosis factor alpha (TNF-alpha) on Fas-mediated apoptotic cell death (Fas-ACD) was examined on cultured rheumatoid synovial cells (RASCs). RASCs were obtained from the synovial tissues of eight patients with rheumatoid arthritis (RA) and SCs from eight patients with osteoarthritis (OA) were used as a control. To examine the influence of TNF-alpha on Fas-ACD, SCs were cultured with anti-Fas antibody (CH11) for 16 h in the absence or presence of different doses of recombinant TNF-alpha. ACD was determined by electron microscopic analysis and the percentage of apoptotic cells was calculated by trypan blue staining. In addition, the expression of Fas and Bcl-2 on RASCs was examined by flow cytometry. As a result, RASCs were more susceptible to Fas-ACD in vitro than OASCs. TNF-alpha interfered with Fas-ACD on RASCs in a dose-dependent manner. Moreover, removal of TNF-alpha activity by a neutralizing anti-TNF-alpha antibody (cA2) restored Fas-ACD. Flow cytometric analysis showed no significant changes in either Fas or Bcl-2 expression on RASCs after the culture with TNFalpha. These results suggest the following: (1) Fas-ACD might be diminished in vivo by local excessive TNF-alpha and this might contribute in part to RASH. (2) The inhibition of Fas-ACD on RASCs by TNF-alpha might not be associated with changes in the expression of Fas or Bcl-2. (3) In addition, considering a magnetic resonance imaging (MRI) finding of marked reduction in the RASH after cA2 treatment, blockade of TNF-alpha activity could restore Fas-ACD in RA synovium, implicating a clinical benefit of anti-TNF-alpha therapy for RA.

Antibodies, Monoclonal↗
PubMed2000

Quantitative changes in the mRNA for contractile proteins and metabolic enzymes in masseter muscle of bite-opened rats.

To study the effects of bite opening on the fibre phenotypes of rat masseter, the mRNAs of four predominant myosin heavy-chain isoforms (MHC I, IIa, IId/x and IIb) and two alkali light-chain isoforms (LC1f and 3f) as well as those of two metabolic enzymes, carbonic anhydrase III (CAIII, oxidative enzyme) and glucose-phosphate isomerase (GPI, glycolytic enzyme), were measured in relation to the total RNA of masseter muscle by competitive, reverse transcriptase-polymerase chain reaction in control and bite-opened rats. Bite opening (2.8 mm increase in the vertical dimension for 1 week) significantly (P<0.05) increased the amount of MHC IIa mRNA but decreased (P<0.001) the amount of MHC IIb mRNA without changing the amount of MHC IId/x mRNA. No MHC I mRNA was found in any masseter studied. A significant (P<0.01) increase in the mRNA of LC1f associated with a decrease (P<0.05) in that of LC3f was observed after the bite opening. The CAIII mRNA increased significantly (P<0.001), while the GPI mRNA decreased (P<0.05) in association with the bite opening. These results strongly suggest that in 1 week of bite opening changes the rat masseter muscle from a glycolytic, MHC IIb-LC3f-dominant fibre to an oxidative, MHC IIa-LC1f-dominant fibre.

Adaptation, Physiological↗