Biomedical subjects

T Fujiwara

Publications and source records attributed to T Fujiwara.

At least 55 records · Page 3Linked to original sources

PubMed2001

Combination of tumor necrosis factor alpha and interferon alpha induces apoptotic cell death through a c-myc-dependent pathway in p53 mutant H226br non-small-cell lung cancer cell line.

We investigated the role of wild-type p53 and c-myc activity in apoptosis induced by a combination of natural human tumor necrosis factor alpha (TNF-alpha) and natural human interferon alpha (IFN-alpha). Studies were performed with two human non-small-cell lung cancer cell lines, H226b, which has wild-type p53, and H226br, which has a mutant p53. The combination of IFN-alpha and TNF-alpha significantly inhibited cell growth and induced apoptotic cell death of both H226b and H226br, compared with IFN-alpha or TNF-alpha alone. Treatment with one or both cytokines did not affect the expression level of p53 in both cell lines. These results suggest that the combination of IFN-alpha/TNF-alpha induces apoptotic cell death through a p53- independent pathway. The c-myc oncogene is known to be involved in apoptosis induced by TNF. Antisense c-myc oligonucleotides have been reported to modulate cell growth or apoptosis in several cell lines. Antisense oligodeoxynucleotides were added to the culture of H226br cells before the addition of IFN-alpha/TNF-alpha. Antisense c-myc inhibited IFN-alpha/TNF-alpha cytotoxicity and apoptotic cell death. In conclusion, this study provides support for the speculation that TNF-alpha/IFN-alpha induce apoptosis through a c-myc-dependent pathway rather than a p53-dependent pathway. (c)2001 Elsevier Science.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2001

Optical filters inhibiting television-induced photosensitive seizures.

OBJECTIVE: Televised images are the most common stimulus for provoking photosensitive seizures in photosensitive persons. To inhibit photosensitive seizures in photosensitive persons who do or do not have epilepsy, the authors sought nonpharmacologic methods for reducing the levels of photic stimulation of televised images. BACKGROUND: The authors found two types of pathophysiologic mechanisms (wavelength-dependent and quantity of light-dependent mechanisms) for photoparoxysmal responses (PPR). METHODS: The authors tested two different types of optical filters, one reflecting long-wavelength red light selectively, which stimulates a wavelength-dependent mechanism, and the other absorbing light in the visible spectrum evenly (neutral density filters). Inhibiting effects of optical filters were studied by conventional intermittent photic stimulation (IPS) using strobe light and novel photic stimulation using flashing cathode ray tubes (CRT). RESULTS: Both filters individually inhibited PPR insufficiently (less than 50%). Compound optical filters, composed of both types of filters, can inhibit the PPR, approximately 90% for IPS and 95% for photic stimulation with CRT. These compound optical filters do not destroy chromaticity of emissions from the television's CRT. CONCLUSIONS: These compound filters may be useful to prevent seizures induced by television in photosensitive persons.

Adolescent↗

PubMed2001

Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse.

Evidence suggests that the specification of primordial germ cells (PGCs) in the mammalian embryo does not depend on maternal determinants. Rather, previous genetic analysis in the mouse has shown that bone morphogenetic protein 4 (Bmp4) is required for the formation of both PGCs and allantois. Bmp4 is expressed in both the trophoblast-derived extraembryonic ectoderm (ExE) and in the epiblast-derived extraembryonic mesoderm (ExM), in which the PGCs, allantois primordium, and angioblasts are first detected. We have shown that Bmp4 made in the ExE functions to induce precursors of PGCs and allantois in the adjacent epiblast, resulting in complete lack of both cell types in homozygous null mutants. However, the function of Bmp4 in the ExM is totally unknown. To address this question, we generated tetraploid (4N) chimeras by aggregating Bmp4 null ES cells with wild-type tetraploid embryos. In this combination, wild-type tetraploid cells contribute to the extraembryonic trophoblast and primitive endoderm lineages but are excluded from the epiblast and its derivatives, including the ExM. Our results clearly demonstrate that Bmp4 made in the ExM does not affect the establishment of either PGC or allantois lineages, but is required for PGC localization and survival and for the differentiation of the allantois. These findings suggest that Bmp4 expressed in epiblast-derived tissues plays vital roles in reproduction by regulating both the development of the germ line and the vascular connection between the embryo and the placenta.

Allantois↗

PubMed2001

Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells.

Heparan sulfate proteoglycans is a major component of the cell surface and extracellular matrix and functions as a barrier against cationic molecules and macromolecules. Heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate, and its activity is associated with the metastatic potential of tumor cells. To inhibit human heparanase expression in human cancer cells, we constructed an adenoviral vector carrying a full-length human heparanase cDNA in an antisense orientation (Ad-AS/hep). Increased heparanase expression in T.Tn human esophageal cancer cells and A549 human lung cancer cells after infection with an adenovirus vector expressing the human heparanase gene (Ad-S/hep) was specifically inhibited by simultaneous infection with Ad-AS/hep in a dose-dependent manner. A modified Boyden chamber assay demonstrated that infection with Ad-AS/hep significantly inhibited in vitro invasion of A549 cells after Ad-S/hep infection. Moreover, intrathoracic administration of Ad-AS/hep reduced the number and size of heparanase-expressing A549 tumors implanted intrathoracically into BALB/c-nu/nu mice. Our results suggest that heparanase contributes to the invasive phenotype of tumor cells, and that antisense-mediated inhibition of heparanase activity may be efficacious in the prevention of pleural dissemination.

Adenoviridae↗

PubMed2001

Hybridization mechanism for cohesion of Cd-based quasicrystals.

The cohesion mechanism of cubic approximant crystals in newly discovered binary quasicrystals, Cd(6)M ( M = Yb or Ca), is studied theoretically. It is found that stabilization due to alloying is obtained if M is an element with low-lying unoccupied d states. This leads to the conclusion that the cohesion of Cd-based compounds is due to hybridization of the d states of Yb and Ca with a wide sp band. The Hume-Rothery mechanism does not play a principal role in the stability because neither a distinct pseudogap nor stabilization due to alloying is obtained for isostructural Cd-Mg. In addition to the electronic origin, matching of the atomic size is very crucial for quasicrystal formation in Cd-based compounds.

Journal Article↗

PubMed2001

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus.

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.

Amino Acid Sequence↗

PubMed2001

Influence of anesthesia on brain distribution of [(11)C]methamphetamine in monkeys in positron emission tomography (PET) study.

We investigated the influence of anesthesia on the brain distribution of [11C]methamphetamine (MAP) obtained by the positron emission tomography (PET) using the normal rhesus monkeys. We clarified that the brain uptake of [11C]MAP under halothane anesthesia was faster and higher than that under pentobarbital. The difference of the effect of anesthesia is an important problem in pharmacokinetic study in PET with experimental animals.

Adjuvants, Anesthesia↗

PubMed2001

Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells.

Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cancer treatments. This cellular suicide program is known to be regulated by many different signals from both intracellular and extracellular stimuli. Here we report that p53 suppressed expression of the cellular FLICE-inhibitory protein (FLIP) that potentially blocks apoptotic signaling in human colon cancer cell lines expressing mutated and wild-type p53. In contrast, the expression of the death receptor KILLER/DR5 (TRAIL-R2) had no effect on FLIP expression, although exogenous p53 is known to induce KILLER/DR5 expression. In line with these observations, FLIP-negative cancer cells were sensitive to both p53- and KILLER/DR5-mediated apoptosis, whereas cells containing high levels of FLIP underwent apoptotic cell death when triggered by ectopic p53 expression but not by KILLER/DR5 expression. Treating the cells with a specific inhibitor of the proteasome inhibited the decrease of FLIP by p53, suggesting that p53 enhances the degradation of FLIP via a ubiquitin-proteasome pathway. Thus, the data indicate that p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer.

Adenoviridae↗

PubMed2001

Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides.

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.

Base Pairing↗

PubMed2001

Anti-influenza virus activities of 2-alkoxyimino-n-(2-isoxazolin-3-ylmethyl)acetamides.

A series of 2-alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides and related compounds were synthesized and their antiviral activities against human influenza A virus were assessed. Studies of the structure-activity relationships revealed the strongest antiviral activity when position-5 of the isoxazoline ring was substituted with a tert-butyl group. When the alkoxyimino moiety was substituted with a methyl, ethyl, isopropyl or allyl group, good antiviral activity was obtained. Among the geometrical isomers at the oxime moiety, the E-isomers were more active than the Z-isomers. Among the compounds examined, (E)-2-allyloxyimino-2-cyano-N-(5-tert-butyl-2-isoxazolin-3-ylmethyl)acetamide (1j) was the most active inhibitor with an EC(50) of 3 microg/mL in vitro.

Antiviral Agents↗

PubMed2001

Differential effects of isoflurane and halothane on the induction of heat shock proteins.

Isoflurane is considered to be a less hepatotoxic volatile anesthetic than halothane since it not only undergoes quantitatively much less metabolism to form toxic reactive intermediates, but also preserves better hepatic blood flow. However, the biochemical basis for the reduced hepatotoxicity has not been elucidated. In this study, we examined the induction of two heat shock proteins, heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1), in the livers of rats pretreated with or without phenobarbital, followed by exposure to isoflurane or halothane under hypoxic conditions. In the phenobarbital-pretreated rats, the maximal induction of HSP70 was observed by halothane-hypoxia treatment, followed by a half-maximal induction by isoflurane-hypoxia treatment, and less than 30% induction by hypoxia treatment alone. Serum alanine aminotransferase (ALT) activity, an indicator of hepatic dysfunction, which correlated well with the extent of centrilobular necrosis, showed similar changes with increases in HSP70 mRNA. In contrast, HO-1 mRNA was induced only by treatment with halothane-hypoxia. In addition, changes in the expression of HSP70 and HO-1 mRNAs were correlated with their protein expression in the liver. In non-pretreated rats, neither isoflurane-hypoxia exposure nor halothane-hypoxia exposure caused apparent hepatic injury. There was also no induction of HSP70 or HO-1 mRNA by these treatments in non-pretreated animals. These findings demonstrate that there is a significant difference in hepatic injury, and in the induction of HO-1 and HSP70 between halothane-hypoxia and isoflurane-hypoxia treatments. Isoflurane is known to be safer than halothane, which may, in part, be accounted for by the generation of less oxidative stress in the presence of isoflurane, as assessed by reduced induction of heat shock proteins compared with halothane treatment.

Anesthetics, Inhalation↗

PubMed2001

Crystallization and preliminary X-ray analysis of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui.

Catalase-peroxidases are bifunctional enzymes found in many microorganisms. Crystals of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui were obtained using the hanging-drop vapour-diffusion method. The rhombic plate-shaped crystals were grown from purified protein solution using (NH(4))(2)SO(4) as precipitant at 293 K. The crystal belongs to the monoclinic system, space group C2, and diffracted beyond 2.0 A resolution.

Archaeal Proteins↗

PubMed2001

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein.

It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.

Animals↗

PubMed2001

Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen.

In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.

Amino Acid Sequence↗

PubMed2001

Characterization of ultrastructure and its relation with DNA fragmentation in Fas-induced apoptosis of cultured cardiac myocytes.

The purposes of the present study were to define precisely the ultrastructural features of apoptosis in cultured cardiomyocytes and to determine whether DNA fragmentation is essential for the apoptotic morphology. When cultured neonatal murine cardiomyocytes were incubated with an agonistic anti-Fas antibody in the presence of a non-toxic amount of actinomycin D or cycloheximide, approximately 70% of them had lost their viability after 24 h. The dead cardiomyocytes showed the typical ultrastructural changes of apoptosis on transmission and scanning electron microscopy, as well as by positive in situ nick end-labelling (TUNEL), positive Taq polymerase-based in situ ligation, a DNA ladder pattern on gel electrophoresis, and an increase in the active fragment of caspase-3. According to TUNEL at the electron microscopic level, apoptotic nuclear change, cytoplasmic shrinkage, and DNA fragmentation always occurred simultaneously in apoptotic cardiomyocytes. Other ultrastructural features of apoptosis were the appearance of abundant lipid-like structures in the cytoplasm of cardiomyocytes at the early phase, and a high incidence of plasma membrane rupture and formation of apoptotic bodies at the later phase. When zinc, an inhibitor of Ca2+/Mg2+-dependent endonuclease, was added to the present model, activation of caspase-3 and an apoptotic ultrastructure were still observed in spite of the lack of DNA fragmentation, indicating that this type of myocyte death is also apoptosis. In conclusion, the typical apoptotic ultrastructure and DNA fragmentation occur simultaneously in association with caspase-3 activation in Fas-stimulated cultured cardiomyocytes. Apoptotic morphology can, however, be observed even without DNA fragmentation.

Animals↗

PubMed2001

Transient spontaneous regression of aggressive non-Hodgkin's lymphoma confined to the adrenal glands.

A 72-year-old-man with night sweats and a low-grade fever was found to have bilateral adrenal enlargement associated with incipient adrenal insufficiency. Without any intervention, these adrenal lesions regressed spontaneously, accompanied by disappearance of clinical symptoms. Seven months later, however, the lesions became enlarged and exceeded their initial size while remaining confined to the adrenals, associated with reappearance of nights sweats and overt adrenal insufficiency. Upon unilateral laparoscopic adrenalectomy, the lesion proved histopathologically to be diffuse large B cell non-Hodgkin's lymphoma (NHL). After contralateral laparoscopic adrenalectomy and adjuvant chemotherapy, the patient remains alive with no recurrence at 26 months. Treatment with bilateral adrenalectomy and chemotherapy is effective for aggressive NHL confined to bilateral adrenal glands. A transient clinical improvement without treatment was considered to be due to a spontaneous regression of NHL, although we could not confirm the histological results before the regression. Such tumor behavior showing spontaneous regression in spite of aggressive histology may also be related to the favorable outcome, even though regression was transient.

Adrenal Gland Neoplasms↗

PubMed2001

A single amino acid change at Leu-188 in the reverse transcriptase of HIV-2 and SIV renders them sensitive to non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are selective for human immunodeficiency virus type 1 (HIV-1) and generally not effective on HIV-2 or simian immunodeficiency virus (SIV). Only SIVagm was found to be sensitive to NNRTIs. When the amino acid differences in RT between SIVmac and SIVagm were compared with the known amino acid substitutions of NNRTI-resistance variants of HIV-1, we came to consider that the amino acid residue Leu-188 of HIV-2 and SIVmac might be related to their resistance to NNRTIs. To test this hypothesis, we substituted Leu-188 to Cys or Tyr in HIV-2 and SIVmac, and examined sensitivity of the mutant molecular clones to NNRTIs. The L188Y mutant of HIV-2 became completely sensitive to delavirdine and efavirenz, while that of SIVmac was also significantly sensitive to these NNRTIs. We further isolated NNRTI-resistant variants from these mutant viruses and determined amino acid substitutions in RT. The roles of the observed substitutions in NNRTI-resistance were further confirmed by site-directed mutagenesis. Our study reveals the crucial role of L188 in the natural resistance of HIV-2 and SIVmac to NNRTIs. Furthermore, the observed substitutions in RT of HIV-2 and SIVmac support the common mechanism of action of NNRTIs against HIV-1, HIV-2 and SIV.

Alkynes↗

PubMed2001

Identification of GFAT1-L, a novel splice variant of human glutamine: fructose-6-phosphate amidotransferase (GFAT1) that is expressed abundantly in skeletal muscle.

Glutamine:fructose-6-phosphate amidotransferase (GFAT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway, which plays an important role in hyperglycemia-induced insulin resistance. To evaluate the role of GFAT1 expression, we analyzed the expression profiles of GFAT1 mRNA in various human tissues using reverse transcriptase-polymerase chain reaction. We report here the identification and cDNA cloning of a novel GFAT1 splice variant expressed abundantly in skeletal muscle and heart. This subtype, designated GFAT1-L, contains a 54-bp insertion within the GFAT1 coding sequence. Recombinant GFAT1-L protein possessed functional GFAT activities and biochemical characteristics similar to GFAT1. Previously, GFAT1 was considered a simplex enzyme. The identification of a novel GFAT1 subtype possessing functional enzymatic activity and tissue-specific expression should provide additional insight into the mechanism of skeletal muscle insulin resistance and diabetes complications.

Alternative Splicing↗