Biomedical subjects

Y Saeki

Publications and source records attributed to Y Saeki.

At least 73 records · Page 4Linked to original sources

PubMed1999

B-myb promoter retargeting of herpes simplex virus gamma34.5 gene-mediated virulence toward tumor and cycling cells.

Deletion of the gamma34.5 gene coding for virulence markedly reduces cytotoxicity mediated by herpes simplex virus type 1 (HSV-1) (J. M. Markert et al., Neurosurgery 32:597-603, 1993; N. S. Markovitz et al. , J. Virol. 71:5560-5569, 1997). To target lytic virulence to tumors, we have created a novel HSV-1 mutant, designated Myb34.5. This viral mutant is characterized by a deletion of the gene for infected cell polypeptide 6 (ICP6; also known as UL39 or ribonucleotide reductase) and of the two endogenous copies of the gamma34.5 gene (RL1) and by reintroduction of one copy of gamma34.5 under control of the E2F-responsive, cellular B-myb promoter. On direct intracerebral inoculation in BALB/c mice, the 50% lethal dose (LD(50)) for Myb34.5 was 2.7 x 10(7) PFU while that for HSVs with mutations in the gamma34.5 gene could not be technically achieved with available viral stocks and it was estimated as >1 x 10(7) PFU. The LD(50) for an HSV with a single defect in ICP6 function was 1.3 x 10(6) PFU. Conversely, Myb34.5's oncolytic efficacy against a variety of human glioma cells in culture and in vivo was enhanced compared to that of HSVs with gamma34.5 mutations, and in fact, it was comparable to that of the wild-type F strain and of viral mutants that possess a wild-type gamma34.5 gene. The characteristic shutoff of host protein synthesis, occurring after infection of human SK-N-SH neuroblastoma cells by gamma34.5 mutant viruses (J. Chou and B. Roizman, Proc. Natl. Acad. Sci. USA 89:3266-3270, 1992), was not present after infection with Myb34.5. There was an increase of almost 3 logarithmic units in the production of progeny virus in arrested fibroblasts compared to that in cycling fibroblasts infected with Myb34.5. These results suggest that transcriptional regulation of gamma34.5 by cell cycle-regulated promoters can be used to target HSV-1 virulence toward tumors while maintaining the desirable neuroattenuated phenotype of a gamma34.5 mutant.

Animals↗

PubMed1999

Skin toxicity of propranolol in guinea pigs.

The skin toxicities of propranolol were studied in guinea pigs. In the primary and cumulative skin irritation studies, the skin reactions and the histopathological changes were observed in all animals treated with propranolol, and those tended to increase with the increase of propranolol dosage. The skin reactions increased with the application times of propranolol up to 7 days in the cumulative skin irritation study. In the skin sensitization, the phototoxicity and the skin photosensitization studies, no skin reactions were observed in any animals used in the studies. These results indicate that propranolol caused skin irritation, but was negative for skin sensitization, phototoxicity and skin photosensitization in guinea pigs.

Adrenergic beta-Antagonists↗

PubMed1999

Inhibition of NF2-negative and NF2-positive primary human meningioma cell proliferation by overexpression of merlin due to vector-mediated gene transfer.

OBJECT: The absence of in vitro models of neurofibromatosis Type 2 (NF2)-defective meningiomas has limited investigative efforts to study the biological effects of this gene in the pathogenesis of these tumors. The goals of this report are to show that gene transfer vectors can efficiently express the wild-type NF2 transgene into primary meningioma cells and to determine effects on cellular proliferation. METHODS: In this study, the authors have compared the transducing capacities of a retrovirus, an adenovirus, and a herpes simplex virus amplicon vector for use in primary human meningioma cells harvested from human tumors excised from patients with and without NF2. Transduction efficiencies with the latter vector approached 100% and it was selected to transfer the wild-type NF2 transgene into these cells. Western blot analysis confirmed that vector-mediated gene transfer mediated the expression of the NF2-encoded polypeptide merlin. Overexpression of merlin significantly inhibited the proliferation of both NF2-negative and NF2-positive human meningioma cells when compared to the proliferation of cells transduced with a control vector. CONCLUSIONS: This study demonstrates the feasibility of using vector-mediated gene transfer to study wild-type NF2 gene function in short-term cultures of primary human meningioma cells.

Apoptosis↗

PubMed1998

LBP-p40 binds DNA tightly through associations with histones H2A, H2B, and H4.

Laminin binding protein precursor p40 (LBP-p40) was long believed to be located exclusively in the cytoplasm. We recently reported localization of epitope-tagged LBP-p40 to the nucleus tightly associated with nuclear structure as well as on ribosomes. In this paper, we analyze the interaction of LBP-p40 with DNA and nuclear proteins in vitro. LBP-p40 was found to bind to a double-stranded DNA cellulose column at moderate salt. However, when mixed with a high salt nuclear extract, LBP-p40 was eluted from the DNA cellulose column only at higher salt. An LBP-p40 affinity column indicated that both histone H1 and in particular the core histones associate with LBP-p40. Using recombinant core histone molecules fused with glutathione S-transferase (GST), we demonstrate that histones H2A, H2B, and H4 are capable of interacting with LBP-p40, whereas H3 is not. These results suggest that association of LBP-p40 with histones H2A, H2B, and H4 confers tight binding of LBP-p40 to chromatin DNA in the nucleus.

Animals↗

PubMed1998

Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors.

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.

Animals↗

PubMed1998

Growth-dependent change of the 26S proteasome in budding yeast.

The 26S proteasome is assembled from the 20S proteasome and the regulatory subunit complex in an ATP-dependent manner. In the present study, we found that the ATP-dependent activity and the protein amount of the 26S proteasome change during growth of the budding yeast Saccharomyces cerevisiae. Both levels in the stationary phase are higher than those in the exponentially growing phase. On the other hand, the levels of the 20S proteasome appear to remain unchanged during growth. These results suggest that the 26S proteasome undergoes a growth-dependent change and that the 26S proteasome plays a role in the survival of yeast cells under starvation conditions.

Adenosine Triphosphate↗

PubMed1998

Interleukin 6 plays a key role in the development of antigen-induced arthritis.

To investigate the direct role of interleukin (IL) 6 in the development of rheumatoid arthritis, IL-6-deficient (IL-6 -/-) mice were backcrossed for eight generations into C57BL/6 mice, a strain of mice with a genetic background of susceptibility for antigen-induced arthritis (AIA). Both histological and immunological comparisons were made between IL-6-deficient (IL-6 -/-) mice and wild-type (IL-6 +/+) littermates after the induction of AIA. Although all IL-6 +/+ mice developed severe arthritis, only mild arthritis was observed in IL-6 -/- mice. Safranin O staining demonstrated that articular cartilage was well preserved in IL-6 -/- mice, whereas it was destroyed completely in IL-6 +/+ mice. In addition, comparable mRNA expression for both IL-1beta and tumor necrosis factor alpha, but not for IL-6, was detected in the inflamed joints of IL-6 -/- mice, suggesting that IL-6 may play a more crucial role in cartilage destruction than either IL-1beta or tumor necrosis factor alpha. In immunological comparisons, both antigen-specific in vitro proliferative response in lymph node cells and in vivo antibody production were elicited in IL-6 -/- mice, but they were reduced to less than half of that found in IL-6 +/+ mice. Lymph node cells of IL-6 -/- mice produced many more Th2 cytokines than did IL-6 +/+ mice with either antigen-specific or nonspecific stimulation in in vitro culture. Taken together, these results indicate that IL-6 may play a key role in the development of AIA at the inductive as well as the effector phase, and the blockade of IL-6 is possibly beneficial in the treatment of rheumatoid arthritis.

Animals↗

PubMed1998

Role of microtubules in the viscoelastic properties of isolated cardiac muscle.

Myocardial viscoelastic properties are determined by both interstitial collagens and intramocyte structures, including sarcolemma, contractile proteins and the cytoskeleton. It is not known whether myocyte microtubules are significant constituents that contribute to the viscoelastic properties of cardiac muscle. We examined the passive properties of isolated right-ventricular papillary muscles before and after altering the polymerization states of microtubules. The muscles were subjected to sinusoidal changes in length (strain) and the resultant changes in resting tension (stress) were measured. The elastic constant was determined by the slope of the stress-strain relation during the slow increase in muscle length (duration 60 s). The viscous constant was determined by the loop area between the stress-strain relation obtained during the rapid increase and decrease in muscle length (duration 1 s). Colchicine (1 micromol/l, 1 h), which depolymerized microtubules, had little effect on either the elastic constant or viscous constant. In contrast, taxol (10 micromol/l), which hyperpolymerized and stabilized microtubules, exerted a time-dependent increase in the viscous constant (133+/-9% of control; n=9, P<0.05), but did not affect the elastic constant (18. 9+/-2.2 to 17.7+/-2.1; n=7, P=n.s.). The increase of viscosity by taxol closely paralleled the increase in the strain rate. The specificity of each pharmacological intervention for the microtubule polymerization state was confirmed by both a Western blot analysis and the immunofluorescence micrographs of myocyte tubulin. Like other cytoskeleton and extracellular collagens, the increase in the myocyte microtubule density was able to modify the viscous component of the passive properties of the isolated cardiac muscle.

Animals↗

PubMed1998

Influences of protein kinase A and D-cAMP on actin-myosin interaction and energy consumption of cardiac muscles.

To address controversies concerning the effects of beta-adrenergic stimulation on the rate of myocardial cross-bridge cycling, we measured three mechanical variables, isometric tension development, transient tension response to a step stretch in length (< 1% of muscle length), maximum velocity of shortening, and a chemical variable, ATPase activity before and after treatment with the catalytic subunit of protein kinase A (PKA) in demembranated rat right ventricular trabeculae, and also measured three mechanical variables before and after treatment with D-cAMP in intact ryanodine-induced tetanized preparations. PKA treatment (I U/microliter, 40 min) shifted the pCa-tension relation to the right from 5.41 to 5.26 at pCa50 (the [Ca2+] required for half maximal steady tension) without changing the steepness of the pCa-tension relation and the maximum tension. The rate of the transient tension changes was significantly increased after either PKA or D-cAMP treatment (5 mM, 15 min), regardless of the level of isometric tension. Vmax was increased for a given Ca2+ concentration after either the PKA or D-cAMP treatment, despite the reduced level of isometric tension. The PKA treatment also shifted the pCa-ATPase activity to the right slightly from 5.47 to 5.40 at pCa50, but increased the ATPase activity during a given level of steady isometric tension generation, resulting in an increased tension cost (ATPase activity/tension). These results suggest that, in rat right ventricular trabeculae, beta-adrenergic stimulation may increase the rate of cross-bridge cycling by increasing the rate of crossbridge detachment from actin through a PKA-mediated mechanism, although PKA reduces the Ca(2+)-sensitivity of the contractile system.

Adenosine Monophosphate↗

PubMed1998

Characterization of a newly established human chondrosarcoma cell line, CS-OKB.

A clonal cell line, CS-OKB, was derived from a human chondrosarcoma and characterized by cytogenetic study, immunocytochemical staining, and reverse transcriptase polymerase chain reaction (RT-PCR). Chromosomal abnormalities characteristic of malignant cartilaginous neoplasms were identified. CS-OKB cells were intensely stained with anti-type II collagen and anti-keratan sulphate antibodies. RT-PCR indicated that CS-OKB transcribes cartilage-specific genes such as type II, X procollagen, and aggrecan. This human chondrosarcoma cell line is stable and expresses well-differentiated chondrocyte-specific genes. It synthesizes well-differentiated chondrocyte-specific molecules in uncoated plastic dishes. CS-OKB may be useful for studies of human chondrocytes and in characterizing human chondrosarcomas.

Aged↗

PubMed1998

The induction of apoptosis in HeLa cells by the loss of LBP-p40.

To analyze the function of the laminin-binding protein precursor p40 (LBP-p40) in higher eukaryotic cells, plasmid DNA expressing antisense or sense cDNA for p40 under the control of the LacSwitch system was introduced into HeLa cells. Stable transformants were isolated, and the expression of p40 was assayed by Western and Northern blotting. The expression level of p40 was not affected in HeLa cell transformants cultured in 10% serum-supplemented media with the induction of antisense (AS)-p40 with 5 mM IPTG. However, both the protein and message for endogenous p40 in serum-depleted media with 5 mM IPTG were reduced to about 30 - 10% of the expression level in serum-free media without 5 mM IPTG. Colony formation was inhibited with the suppression of p40. AS-p40 clones died in 7 days when cultured in serum-depleted media with 5 mM IPTG, while clones without 5 mM IPTG AS-p40 clones never died, even in serum-depleted media. Additionally, sense (S)-p40 clones and control CAT clones survived more than 2 weeks in serum-free media with 5 mM IPTG. DNA fragmentation assay revealed that cell death induced by the reduction of AS-p40 resulted from apoptosis. Both the inhibition of cell growth and apoptotic cell death were partially rescued by the transfer of the p40 cDNA expression vector to AS-p40 clones. Moreover, the introduction of a synthetic hammerhead ribozyme for LBP-p40 using a fusigenic viral liposome suppressed the message for LBP-p40 even in the presence of 10% serum, and it also induced apoptosis.

Apoptosis↗

PubMed1998

Sustained transgene expression in vitro and in vivo using an Epstein-Barr virus replicon vector system combined with HVJ liposomes.

For long-term gene expression in tissues, we constructed an Epstein-Barr virus (EBV) replicon-based plasmid, pEB, containing the latent viral DNA replication origin (oriP) and EBV nuclear antigen-1 (EBNA-1). When pEB was transferred to human cells (HeLa-S3, HEK 293 and FS 3) and rodent cells (BHK-21) using HVJ-cationic liposomes, luciferase expression was observed in those cells for at least 10 days. Luciferase activity was two to 10 times higher in those cell lines on and after day 3 post-transfection of pEBActLuc compared with plasmids without the EBV replicon sequence. Southern blot analysis showed that the pEB vector luciferase gene was maintained extrachromosomally in BHK-21 cells. In human cells, transformation was five to 20 times more efficient with pEBc than with pcDNA3, and 18-35% of the introduced EBV replicon plasmid was replicated autonomously. The luciferase gene or lacZ gene was introduced into mouse liver using HVJ-AVE liposomes. Luciferase gene expression was observed for at least 35 days in cells transfected with pEBActLuc, whereas it was not detected on day 14 in cells transfected with pActLuc, which lacks the EBV sequence. By the transfer of pEBActNlacF, the lacZ gene expression rate in hepatocytes was approximately 35 and 12% on days 7 and 35, respectively.

Animals↗

PubMed1998

Suboptimal clinical response to anti-tumor necrosis factor alpha (TNFalpha) antibody therapy in a patient with severe rheumatoid arthritis and lymphadenopathy.

This concerns a patient with severe rheumatoid arthritis (RA) and lymphadenopathy (LA) who showed suboptimal clinical response to antitumor necrosis factor alpha (TNFalpha) antibody (Ab), cA2 therapy. The assessment of TNFalpha and IL-6 mRNA expression in the swollen lymph-node (LN) of the patient by reverse transcription, polymerase chain reaction (RT-PCR) before cA2 treatment, showed only enhanced IL-6 production, but not TNFalpha. Moreover, cA2 failed to inhibit in-vitro spontaneous IL-6 production in the LN block culture from the patient. Taken together, these results indicate that IL-6 production in the swollen LNs of the patient might not depend on TNFalpha. This might partly cause suboptimal clinical response to anti-TNFalpha Ab therapy in the patient.

Antibodies, Monoclonal↗

PubMed1998

Impairment of autonomic function induced by posture change in postmenopausal women.

This study was designed to determine if there is a difference in autonomic regulation induced by posture change between postmenopausal and young women. To evaluate autonomic nervous system function, spectral analysis of heart rate variability (HRV) was done in postmenopausal women (n = 13, 46-59 years of age), age-matched men (n = 8, 45-55 years of age), and young women (n = 10, 20-37 years of age) for 3-min periods of controlled frequency breathing (15 breaths/min) in supine followed by sitting positions. In the supine position, the R-R interval variation in older persons decreased significantly compared with that during the follicular phase in young women. Furthermore, the high-frequency (HF) components of HRV, which reflect only parasympathetic activity, were lower in older subjects than in young women. Following a change of position from supine to sitting, the HF component did not change significantly in the postmenopausal women or the men, but the low/high frequency (LF/HF) component ratio, which reflects the balance of autonomic nerve activities, increased significantly in the men. These results suggest that cardiac parasympathetic tone may be reduced in older persons in comparison with young women. Furthermore, arterial baroreflex control of parasympathetic nerve activity caused by posture changes is impaired in the postmenopausal women and aged-matched men. The baroreflex control of the sympathetic component is maintained in the men but not in the postmenopausal women. These differences might result in part from changes in the level of female hormones.

Adult↗

PubMed1998

IL-6-deficient mice are resistant to the induction of experimental autoimmune encephalomyelitis provoked by myelin oligodendrocyte glycoprotein.

The role of IL-6 in experimental autoimmune encephalomyelitis (EAE) provoked by myelin oligodendrocyte glycoprotein (MOG) was investigated using IL-6-deficient mice. We show here that IL-6-deficient mice were resistant to the MOG-induced EAE as compared to wild-type mice (one out of 18 versus 17 out of 20). The delayed-type hypersensitivity response, lymphocyte proliferation response and antibody reactivity to MOG in IL-6-deficient mice were significantly lower than those in wild-type mice. Furthermore, the histological examination revealed that no infiltration of inflammatory cells was observed in the central nervous system of IL-6-deficient mice. These results indicate that IL-6 may play a crucial role in the induction phase of EAE. Given the potential relevance of this animal model for multiple sclerosis (MS), it is possible that anti-IL-6 therapy may be useful in the prevention of relapses of MS.

Animals↗

PubMed1998

Molecular cloning, expression, and characterization of a novel mouse liver SULT1B1 sulfotransferase.

A mouse liver homogenate was shown to contain enzymatic activities catalyzing the sulfation of 3,4-dihydroxyphenylalanine (Dopa) and tyrosine isomers with a pH optimum of 8.25. Western blot analysis revealed a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against rat liver SULT1B1 sulfotransferase. By employing the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, a 910-base pair product encoding the putative mouse liver SULT1B1 sulfotransferase was obtained. Using this PCR product as a probe, a cDNA containing the entire open reading frame of the mouse liver SULT1B1 sulfotransferase was cloned from a mouse liver Lambda ZAP cDNA library. The nucleotide sequence indicated it is a new enzyme. The deduced amino acid sequence exhibited 87.6, 72.3, 55.9, 54.2, 52.8, 51.1, and 49.4% identity to the amino acid sequences of the rat liver SULT1B1 sulfotransferase, human thyroid hormone sulfotransferase, mouse phenol sulfotransferase, rat liver phenol sulfotransferase, rat liver hydroxyarylamine sulfotransferase, mouse estrogen sulfotransferase, and rat estrogen sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pcDNA3) harboring the cDNA encoding this new enzyme, a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against the rat liver SULT1B1 sulfotransferase was expressed. The recombinant sulfotransferase exhibited enzymatic activities toward Dopa and tyrosine isomers, as well as dopamine and 3,3',5-triiodo-L-thyronine. Northern blot analyses indicated the SULT1B1 sulfotransferase was predominantly expressed in liver, but not in the other ten mouse organs examined. Furthermore, the enzyme was found to be expressed in a developmental stage-dependent manner, being at a very low level in liver samples from 1-day-old mice and then gradually increasing to the maximum level in liver samples from 4-week-old mice.

Amino Acid Sequence↗

PubMed1998

Involvement of phosphoinositide 3-kinase in regulation of adhesive activity of highly metastatic hepatoma cells.

We have established hepatoma clones from benzopyrene-treated liver cells, one of which (G-5) shows extensive metastasis to the lung when injected subcutaneously into mice [Tanigaki, Y. et al. (1995) Invasion Metastasis 15, 70-80]. In the present study, we performed in vitro assays suitable for examination of the adhesive and invasive properties of the highly metastatic cells. G-5 cells efficiently entered the pores of fibronectin-coated filters. Treatment of the cells with an inhibitor of phosphoinositide 3-kinase (PI 3-kinase), wortmannin, significantly impaired the invasive activity. A structurally unrelated inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) also prevented invasion. Both inhibitors suppressed cell adhesion to fibronectin-coated dishes. G-5 cells were next transfected with a mutant regulatory subunit (Deltap85) of PI 3-kinase, which was expected to impair the function of PI 3-kinase. The transfectants showed suppressed adhesion to the dishes and did not efficiently migrate into the filters. The lower adhesive ability of the transfected cells was not further affected by inhibitors of PI 3-kinase. Thus, PI 3-kinase activity contributes significantly to the adhesive and invasive properties of G-5 cells.

Androstadienes↗

PubMed1998

Dynamic relations among length, tension, and intracellular Ca2+ in activated ferret papillary muscles.

To study the effects of mechanical constraints on the Ca2+ affinity of cardiac troponin C, we analyzed the tension and aequorin light (AL) responses to sinusoidal length changes (5-10% of the initial muscle length) in aequorin-injected, tetanized cardiac muscles. The amplitude of the quasi-sinusoidal tension and AL responses decreased with increasing length-perturbation frequency from 0.5 to 1 Hz at 24 degreesC and from 1 to 3 Hz at 30 degreesC. The increase in AL corresponded well to the decrease in tension; likewise, the decrease in AL to the increase in tension and the tension response lagged behind the length change. A further increase in frequency (>1 Hz at 24 degreesC and >3 Hz at 30 degreesC) markedly increased the amplitude of the tension responses but decreased the amplitude of the AL responses. The increase in AL lagged behind the decrease in tension; likewise, the decrease in AL lagged behind the increase in tension, and the tension response led the length change. From previous mechanistic interpretations of the frequency dependence of the amplitude of tension response, we argue that the Ca2+ affinity of cardiac troponin C changes in parallel with the active tension (i.e., the number of active cross bridges) but not with the passive tension produced by the length perturbation-induced cross-bridge strain.

Aequorin↗