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Biomedical subjects

T Fujiwara

Publications and source records attributed to T Fujiwara.

At least 649 records · Page 36Linked to original sources
PubMed1994

Molecular cloning and sequence analysis of a human 372-kDA protein localized in the Golgi complex.

Autoantibodies from a patient with chronic rheumatoid arthritis recognized an antigen localized in the Golgi complex of various cells from different tissues and species. The autoantibodies were used as a probe for screening human QGP-1 cDNA library, resulting in identification of a 10.3-kb cDNA. The cDNA insert contained an open reading frame which encodes a 3225-residue protein with a calculated mass of 372 kDa. The predicted protein was found to have no NH2-terminal signal sequence but a single hydrophobic domain at the COOH terminus. These results indicate that the 372-kDa antigen is cytoplasmically disposed and anchored to the Golgi membrane by the COOH-terminal hydrophobic domain.

Amino Acid Sequence↗
PubMed1994

Inconsistency between the fimbrilin gene and the antigenicity of lipopolysaccharides in selected strains of Porphyromonas gingivalis.

Immunochemical specificity of lipopolysaccharide and the molecular property of the gene encoding the fimbrilin (fimA) of Porphyromonas gingivalis strains were examined using 'fimbriated' strains 381 and HG564 and 'non-fimbriated' strains 381FL and W50. Lipopolysaccharide from strains 381, 381FL and HG564 reacted with monoclonal antibody raised to lipopolysaccharide from strain 381 to give a fused precipitin band by the immunodiffusion test. However, silver staining and Western blotting of lipopolysaccharide clearly revealed a difference in profile of bands between strains 381 and 381FL. On the other hand, lipopolysaccharide from W50 formed another precipitin band and reacted with the antibody, but only at higher concentrations of lipopolysaccharide. The fimA genes in these strains were amplified by polymerase chain reaction and cloned. Sequencing of the fimA gene revealed that the fimA(W50) was almost identical to fimA(HG564), but a notable difference was observed at the start codon of the open reading frame, while the fimA(381FL) was considerably different from fimA of other strains and its open reading frame was found to be missing. These results indicate that the molecular structure of the fimA genes of these strains is not homologous, indicating that molecular modifications in the fimA gene should occur during in vitro passages and maintenance of strains of P. gingivalis in laboratories.

Antibodies, Monoclonal↗
PubMed1994

Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model.

BACKGROUND: Mutations in the p53 tumor suppressor gene (also known as TP53) are common in human lung cancers. The wild-type form of p53 is dominant over the mutant; thus, restoration of wild-type p53 function in lung cancer cells may suppress their growth as tumors. PURPOSE: We investigated the therapeutic efficacy of direct administration of a retroviral wild-type p53 (wt-p53) expression vector (LNp53B) in an orthotopic human lung cancer model in nu/nu mice. METHODS: Proliferation of H226Br cells was determined by cell counting after infection with LNp53B in vitro. Irradiated (350 cGy) female BALB/c nu/nu mice were inoculated intratracheally with 2 x 10(6) H226Br cells (whose p53 gene has a homozygous mutation at codon 254) and treated beginning 3 days later with an intratracheal instillation of LNp53B retroviral supernatant for 3 days. RESULTS: Infection with LNp53B inhibited proliferation of H226Br cells in vitro. Thirty days after tumor cell inoculation, 62%-80% of the control mice showed macroscopic tumors of the right main stem bronchus. LNp53B suppressed H226Br tumor formation in 62%-100% of mice, and the effect was abrogated by dilution of the retroviral supernatant with inactive vector. CONCLUSIONS: Direct administration of a retroviral vector expressing wt-p53 may inhibit local growth in vivo of human lung cancer cells with abnormal p53 expression. IMPLICATIONS: Development of gene-replacement treatment strategies based on the type of mutations found in target cancers is warranted and may lead to the development of new adjunctive therapies and gene-specific prevention strategies for lung cancer.

Animals↗
PubMed1994

A patient with malignant paraganglioma that simultaneously produces adrenocorticotropic hormone and interleukin-6.

BACKGROUND: To the authors' knowledge, there have been no reports describing a case of malignant paraganglioma that produces both adrenocorticotropic hormone (ACTH) and interleukin-6 (IL-6). METHODS: The clinical course and pathophysiology of a patient with Cushing's syndrome induced by ectopic ACTH syndrome caused by a cervical malignant paraganglioma with elevated plasma levels of IL-6 was investigated. RESULTS: Ultrasonography, computed tomography, and magnetic resonance imaging of the neck revealed the presence of a tumor around the area from which the blood sample that showed the highest levels of ACTH and IL-6 was obtained by selective catheterization. Immunohistochemical staining of the removed tissue demonstrated the presence of ACTH and IL-6 proteins in the tumor cells. Reverse transcriptase-polymerase chain reaction also revealed the existence of IL-6 mRNA in those cells. CONCLUSION: The malignant paraganglioma of ectopic ACTH syndrome may produce IL-6. The present investigation provides new observations concerning ectopic ACTH syndrome.

ACTH Syndrome, Ectopic↗
PubMed1994

Mechanisms of augmented vascular responses to histamine in atherosclerotic rabbit common carotid arteries.

The vascular responsiveness to histamine, 2-pyridylethylamine and dimaprit was investigated in isolated and perfused atherosclerotic rabbit common carotid arteries enclosed by a hollow silastic collar. The constrictor effects of histamine were significantly enhanced in atherosclerotic arteries, although those of 2-pyridylethylamine were not changed. The dilator effects of histamine and dimaprit, which were completely inhibited after pretreatment with cimetidine, were not influenced by removal of the endothelium but significantly depressed in atherosclerotic arteries. The dose-response curves for histamine in the control group pretreated with cimetidine were shifted to the left and responses reached almost the same level as those of the atherosclerotic group. From these results, it is concluded that histamine-induced vasoconstrictions are enhanced in atherosclerotic arteries, and that the mechanism of the hyperreactivity to histamine might be an attenuation of the vasodilatations mediated via histamine H2 receptors in vascular smooth muscle.

Animals↗
PubMed1994

Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene.

Recombinant adenovirus-mediated transfer of the wild-type p53 gene into monolayer cultures or multicellular tumor spheroids of human non-small cell lung cancer cell line H358, which has a homozygous deletion of p53, markedly increased the cellular sensitivity of these cells to the chemotherapeutic drug cisplatin. Treated cells underwent apoptosis with specific DNA fragmentation. Direct injection of the p53-adenovirus construct into H358 tumors s.c. implanted into nu/nu mice, followed by i.p. administration of cisplatin, induced massive apoptotic destruction of the tumors. These results support the clinical application of a regimen combining gene replacement using replication-deficient wild-type p53 adenovirus and DNA-damaging drugs for treatment of human cancer.

Animals↗
PubMed1994

Molecular cloning and expression of a novel human gene that is highly homologous to human FK506-binding protein 12kDa (hFKBP-12) and characterization of two alternatively spliced transcripts.

We isolated a novel gene encoding a protein highly homologous to human FK506-binding protein 12kDa (hFKBP-12) from a human fetal brain cDNA library and determined the full-length cDNA sequence. The cDNA clone contained the open reading frame of 324 nucleotides encoding 108 amino acid and revealed 76% identity in DNA sequence and 88% identity in predicted amino acid sequence with hFKBP-12. The DNA and amino-acid sequence of this gene, designated OTK4, also had homology with other FKBPs in species ranging from humans to prokaryotes. Recombinant protein, produced in E.coli transformed by a pGEX2T expression vector containing the OTK4 cDNA and purified, showed peptidyl-prolyl cis-trans isomerase activity like other FKBP proteins. An alternatively spliced form of the transcript found in the cDNA library contained a 45-bp insertion which included a stop codon. Although the biological function of the truncated version of OTK4 is unknown, both transcripts were ubiquitously expressed in human tissues examined by the reverse-transcriptase PCR (RT-PCR) method.

Alternative Splicing↗
PubMed1994

Retroviral-mediated transduction of p53 gene increases TGF-beta expression in a human glioblastoma cell line.

Transforming growth factor-beta (TGF-beta) has been implicated as a potent growth regulator; the degree of responses to it, whether positive or negative, generally correlates with the stage of cell differentiation in various cell types. We examined the effect of the p53 gene, which participates in the control of cell-cycle progression, on the expression of human TGF-beta. The human glioblastoma cell line SNB-19, which expresses the latent form of TGF-beta, was transfected with a retroviral vector containing wild-type p53 (wt-p53) or p53 with a mutation (mut-p53) at codon 273. Stable G418-resistant SNB-19 clones were isolated. The growth kinetics of wt-p53 transfectants were suppressed compared with those of parental cells, vector transfectants, or mut-p53 transfectants, as assayed by growth-curve measurements and 3H-thymidine incorporation; however, RNA dot blot and Western blot analyses demonstrated that wt-p53 and mut-p53 transfectants expressed higher amounts of TGF-beta 1 and TGF-beta 2 mRNA and intracellular TGF-beta isoform proteins, respectively, than parental cells. By means of the biological assay for active TGF-beta (Mv1Lu cell-growth-inhibition assay), we observed that both transfectants produced active TGF-beta, whereas the parental cells produced only the latent form. These results suggest that, while only the wt-p53 gene inhibits tumor-cell progression, both wt-p53 and codon 273-mutated p53 can cause increased TGF-beta expression.

Base Sequence↗
PubMed1994

Developmental regulation of rat serine dehydratase gene expression: evidence for the presence of a repressor in fetal hepatocytes.

To determine why the rat serine dehydratase gene becomes transcriptionally activated just after birth, we examined the interactions of DNA binding proteins of fetal and adult rat livers with the serine dehydratase gene promoter by DNase I protection analysis and gel mobility shift assay. Several binding regions of nuclear proteins were found to be common to fetal and adult livers and interaction of factors with the characteristics of Sp1 or NF-Y was suggested. Two additional regions, named regions B and I, were specific to fetal liver. These regions contain GATA-like sequences and competition experiments by gel mobility shift assay suggested that the fetal liver-enriched factor binds to the GATA-like sequences. The function of the regions B and I in transcription regulation was investigated in fetal and adult hepatocytes by transient DNA transfer experiments with serine dehydratase-chloramphenicol acetyltransferase fusions. These experiments showed that these regions functioned as negative cis-acting elements in fetal hepatocytes, but not in adult hepatocytes.

Animals↗
PubMed1994

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Tissue-specific and temporal regulation of a beta-conglycinin gene: roles of the RY repeat and other cis-acting elements.

Upstream regulatory sequences (URS) of the gene that encodes the alpha' subunit of beta-conglycinin, the 7S soybean seed storage protein, includes two RY repeat elements. The role of RY elements and sequences that bind soybean embryo factors 3 and 4 (SEF3 and SEF4; Allen et al., Plant Cell 1 (1989) 623-631) in regulating expression of the promoter was studied following site directed mutagenesis. Specific mutations introduced into these sequences abolished the in vitro binding activities of SEF3 and SEF4. The biological activities resulting from the mutations were determined in transgenic plants using two chimeric promoters comprising sequences from the CaMV 35S promoter and the alpha' subunit promoter. The uidA reporter gene was used to assess the levels of gene expression in transgenic plants. The mutations in the RY element and SEF3 and SEF4 binding sites had little effect on expression of the alpha' promoter. By contrast, mutations in the RY element had significant effect on gene expression when the URS from the alpha' promoter was ligated upstream of the core 35S promoter. Mutations in the RY element abolished the seed specific enhancing activity of the alpha' URS and caused expression of the chimeric promoter in leaves. These results indicate that the RY element plays a key role in seed-specific gene regulation in coordination with other cis-acting elements.

Antigens, Plant↗
PubMed1994

Intra-abdominal desmoplastic small cell tumour in a patient with Peutz-Jeghers syndrome.

A surgical case of intra-abdominal desmoplastic small cell tumour with divergent differentiation (IDS-CT) in a patient with Peutz-Jeghers syndrome (PJS) is reported. The patient was a 23-year-old woman who underwent surgery for a tumour in the left paraovarian region. Widespread peritoneal metastases were noted. Histopathological examination revealed solid cell nests composed of medium-sized cells demarcated by desmoplastic stroma. Gland-like spaces were found within many cell nests. Tumour cells were positive for cytokeratin and smooth muscle actin, but negative for desmin. Both ovaries contained minute foci of a sex cord tumour with annular tubules. This is the first documentation of the association of IDSCT with PJS. Since patients with PJS have an increased risk of cancer in various organs, the occurrence of IDSCT in the present patient might not be fortuitous.

Adult↗
PubMed1994

Transformation of fibroblasts into endothelial cells during angiogenesis.

Light- and electron-microscopic autoradiography have been used to study fibroblast transformation into endothelial cells in the formation of new blood vessels during wound healing in rabbit ear chambers. When cultured fibroblasts labeled with tritium thymidine were transplanted autologously into the chambers, newly formed blood vessels contained endothelial cells labeled with tritium thymidine. This result suggests that fibroblasts play a pivotal role in angiogenesis, as progenitors of endothelial cells in newly formed blood vessels.

Animals↗
PubMed1994

Intra-arterial chemotherapy for brain stem glioma: report of four cases.

Four patients with pontine gliomas were treated with radiation and intra-arterial chemotherapy using ACNU, cisplatin, and/or carboplatin. All underwent biopsy and were histologically proven to have gliomas. Even though three were benign histologically, all appeared malignant on neuroimaging. Two patients had complete remissions; the others had partial responses; three patients died, 36, 14, and 15 months after the primary diagnosis. One patient, now in complete remission, has been alive for 24 months. There was no neurotoxicity except for transient nausea and vomiting. Intra-arterial chemotherapy is effective against brain stem gliomas, producing remission with minimum side effects.

Adolescent↗
PubMed1994

Plasmodesmal-mediated cell-to-cell transport in wheat roots is modulated by anaerobic stress.

Cell-to-cell transport of small molecules and ions occurs in plants through plasmodesmata. Plant roots are frequently subjected to localized anaerobic stress, with a resultant decrease in ATP. In order to determine the effect of this stress on plasmodesmal transport, fluorescent dyes of increasing molecular weight (0.46 to 1OkDa) were injected into epidermal and cortical cells of 3-day-old wheat roots, and their movement into neighboring cells was determined by fluorescence microscopy. Anaerobiosis was generated by N2 gas or simulated by the presence of sodium azide, both of which reduced the ATP levels in the tissue by over 80%. In the absence of such stress, the upper limit for movement, or size exclusion limit (SEL), of cortical plasmodesmata was <1 kDa. The ATP analogue TNP-ADP (mw 681) moved across the plasmodesmata of unstressed roots, indicating that plasmodesmata may be conduits for nucleotide (ATP and ADP) exchange between cells. Upon imposition of stress, the SEL rose to between 5 and 10 kDa. This response of plasmodesmata to a decrease in the level of ATP suggests that they are constricted by an ATP-dependent process so as to maintain a restricted SEL. When roots are subjected to anaerobic stress, an increase in SEL may permit enhanced delivery of sugars to the affected cells of the root where anaerobic respiration could regenerate the needed ATP.

Adenosine Triphosphate↗