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Biomedical subjects

T Kojima

Publications and source records attributed to T Kojima.

At least 19 recordsLinked to original sources
PubMed2006

A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.

Retrospective analysis has shown that activating mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene are a predictor of response to gefitinib. We conducted a phase II trial to evaluate the efficacy and safety of gefitinib as first-line therapy for advanced non-small cell lung cancer (NSCLC) with EGFR mutations. Patients with stage IIIB or IV chemotherapy-naïve NSCLC with EGFR mutation were treated with 250 mg gefitinib daily. For mutational analysis, DNA was extracted from paraffin-embedded tissues and EGFR mutations were analysed by direct sequence of PCR products. Twenty (24%) of the 82 patients analysed had EGFR mutations (deletions in or near E746-A750, n=16; L858R, n=4). Sixteen patients were enrolled and treated with gefitinib. Twelve patients had objective response and response rate was 75% (95% CI, 48-93%). After a median follow-up of 12.7 months (range, 3.1-16.8 months), 10 patients demonstrated disease progression, with median progression-free survival of 8.9 months (95% CI, 6.7-11.1 months). The median overall survival time has not yet been reached. Most of the toxicities were mild. This study showed that gefitinib is very active and well tolerated as first-line therapy for advanced NSCLC with EGFR mutations.

Administration, Oral↗
PubMed2006

Molecular cloning and characterization of a novel salt-inducible gene encoding an acidic isoform of PR-5 protein in soybean (Glycine max [L.] Merr.).

We identified a novel salt-inducible soybean gene encoding an acidic-isoform of pathogenesis-related protein group 5 (PR-5 protein). The soybean PR-5-homologous gene, designated as Glycine max osmotin-like protein, acidic isoform (GmOLPa)), encodes a putative polypeptide having an N-terminal signal peptide. The mature GmOLPa protein without the signal peptide has a calculated molecular mass of 21.5 kDa and a pI value of 4.4, and was distinguishable from a known PR-5-homologous gene of soybean (namely P21 protein) through examination of the structural features. A comparison with two intracellular salt-inducible PR-5 proteins, tobacco osmotin and tomato NP24, revealed that GmOLPa did not have a C-terminal extension sequence functioning as a vacuole-targeting motif. The GmOLPa gene was transcribed constitutively in the soybean root and was induced almost exclusively in the root during 24 h of high-salt stress (300 mM NaCl). Interestingly, GmOLPa gene expression in the stem and leaf, not observed until 24 h, was markedly induced at 48 and 72 h after commencement of the high-salt stress. Abscisic acid (ABA) and dehydration also induced expression of the GmOLPa gene in the root; additionally, dehydration slightly induced expression in the stem and leaf. In fact, the 5'-upstream sequence of the GmOLPa gene contained several putative cis-elements known to be involved in responsiveness to ABA and dehydration, e.g. ABA-responsive element (ABRE), MYB/MYC, and low temperature-responsive element (LTRE). These results suggested that GmOLPa may function as a protective PR-5 protein in the extracellular space of the soybean root in response to high-salt stress and dehydration.

Amino Acid Sequence↗
PubMed2006

Ewing's sarcoma fusion protein, EWS/Fli-1 and Fli-1 protein induce PLD2 but not PLD1 gene expression by binding to an ETS domain of 5' promoter.

It was reported that short interfering RNA (siRNA) of EWS/Fli-1 downregulated phospholipase D (PLD)2 in Ewing's sarcoma (EWS) cell line, suggesting that PLD2 is the target of aberrant transcription factor, EWS/Fli-1. Here, we further investigated the regulation of PLD2 gene expression by EWS/Fli-1 and Fli-1 in another EWS cell line, and also in EWS/Fli-1- or Fli-1-transfected cell line. EWS/Fli-1- or Fli-1-overexpressed cells showed higher PLD2 but not PLD1 protein expression and enhanced cell proliferation as compared to mock transfectant. The treatment of these cells with 1-butanol or siRNA of PLD2 inhibited cell growth, suggesting the pivotal role of PLD in cell growth promotion. PLD2 but not PLD1 mRNA level was also increased in EWS/Fli-1 or Fli-1-transfectants. After determining the transcription initiation points, we cloned the 5' promoter of both PLD1 and PLD2 and analysed promoter activities. Results showed that EWS/Fli-1 and Fli-1 increase PLD2 gene expression by binding to an erythroblast transformation-specific domain (-126 to -120 bp from the transcription initiation site) of PLD2 promoter, which is the minimal and most powerful region. Electrophoresis mobility shift assay using truncated proteins showed that both DNA-binding domain and trans-activating domain were necessary for the enhanced gene expression of PLD2.

Base Sequence↗
PubMed2006

CSF1 gene associated with aggressive periodontitis in the Japanese population.

Aggressive periodontitis (AgP) is characterized by the early onset of the rapid and progressive destruction of the alveolar bone. We investigated the correlation of single nucleotide polymorphisms (SNPs) in candidate genes with AgP in the Japanese population in order to determine the genetic risk factors for this complex disease. Among 11 genes related to bone formation and resorption, 43 known SNPs were tested in 98 case and 88 control samples for association with AgP by using SNP genotyping techniques. Among these, three polymorphisms located in the colony stimulating factor 1 (CSF1) gene showed a positive association with AgP. This is the first case of an association between a CSF1 polymorphism and a human disease.

Adolescent↗
PubMed2006

A novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus.

The suprachiasmatic nucleus of the anterior hypothalamus is the center of an internal biological clock in mammals. Glutamate is the neurotransmitter of retino-hypothalamic tract responsible for mediating the circadian actions of light in rodents. N-methyl-d-aspartate receptors, particularly NR2B subunit are reported to be principally involved in photic resetting of the biological clock in vivo and in slice culture. But, the precise cellular mechanisms of the resetting are not elucidated, because no adequate neuronal cell lines derived from the suprachiasmatic nucleus have been established. We established a neuronal cell line, N14.5, derived from the suprachiasmatic nucleus of a transgenic rat harboring the temperature-sensitive simian virus 40 large T-antigen gene. When the cells were cultured at 39 degrees C, the morphological features were turned fibroblastic into neuronal round cell body with neurite extensions. These cells showed immunoreactivities for neuronal markers (betaIII-tubulin, microtubule-associated protein 2 and TAU2) and as well as for vasoactive intestinal peptide which is expressed in the ventrolateral region of the suprachiasmatic nucleus. The cells expressed N-methyl-d-aspartate receptors, particularly NR1 and NR2B subunits as revealed by quantitative PCR. N-methyl-d-aspartate activated phosphorylation of p44/42 mitogen-activated protein kinase and increased expression level of Per1 and Per2 mRNA. These results suggest that the N14.5 is a novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus, and that N-methyl-d-aspartate receptors expressed in the cells are a functional receptor. The N14.5 cells may be a useful tool to elucidate numerous chronobiological processes, especially resetting mechanism induced by an external light signal.

Animals↗
PubMed2006

Substance P stimulates release of RANKL via COX-2 expression in human dental pulp cells.

OBJECTIVE: Our previous study found that substance P (SP), a sensory neuropeptide, was expressed in the dental pulp of rats during experimental tooth movement. We examined the effects of SP on the production of prostaglandin (PG) E2 and the receptor activator of nuclear factor- B ligand (RANKL) by human dental pulp fi broblast-like (HDPF) cells. MATERIALS AND METHODS: SP was added to cultured HDPF cells at concentrations ranging from of 10(-4) to 10(-12) mol/L. PGE2 and soluble RANKL (sRANKL) levels were determined using enzyme-linked immunosorbent assay kits. Gene expression was confi rmed by RT-PCR analysis. Pit formation assays using dentin slices were carried out to examine the effect of SP on osteoclastogenesis. RESULTS: The levels of PGE2 and sRANKL increased in the presence of SP, though the increases were greater in the experimental groups in both a time- and concentration-dependent manner, and the increase of RANKL was partially mediated by PGE2. The gene expression of cyclooxygenase (COX)-2 and RANKL was up-regulated, and conditioned medium samples obtained from HDPF cells treated with SP induced bone resorption. CONCLUSIONS: SP stimulated the production of PGE2 and RANKL, and promoted bone resorption. Therefore, SP may be involved in pulpal inflammation and root resorption during orthodontic tooth movement.

Adolescent↗
PubMed2006

A case of coagulation factor V deficiency caused by compound heterozygous mutations in the factor V gene.

We investigated the molecular basis of a severe factor V (FV) deficiency in a Japanese female, and identified two distinct mutations in the FV gene, a novel cytosine insertion (1943insC) and a previously reported point mutation (A5279G). We expected the patient to be a compound heterozygote for those mutations, as a 1943insC, but not an A5279G, was found in the mother and a sibling. The 1943insC will cause a frame-shift after 590Gln, resulting in amino acid substitutions with two abnormal residues followed by a stop codon in the FV A2 domain (FS592X). The A5279G will cause an amino acid alteration in the FV A3 domain (Y1702C), which has been observed in several ethnic groups. We found that both mutant mRNAs were detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the patient's platelets, whereas no FV antigen and activity were detected in plasma. On the one hand, the RT-PCR signal from the FS592X-FV mutant mRNA was markedly reduced, suggesting that the RNA surveillance system would eliminate most of the abnormal FS592X-FV transcripts with a premature termination. On the other hand, expression analyses revealed that only small amounts of Y1702C-FV with a low specific activity were secreted, and that the FS592X-FV was not detected in cultured media. These data indicated that both mutant FV molecules would be impaired, at least in part, during the post-transcriptional process of protein synthesis and/or in secretion. Taken together, it seems to suggest that each gene mutation could be separately responsible for severe FV deficiency, while this phenotype is due to the in-trans combination of the two defects.

Adult↗
PubMed2006

Fatal thrombosis of antithrombin-deficient mice is rescued differently in the heart and liver by intercrossing with low tissue factor mice.

BACKGROUND: We previously reported that the targeted disruption of murine antithrombin (AT) gene resulted in embryonic lethality before 16.5 gestational days (gd) because of severe cardiac and hepatic thrombosis. OBJECTIVE AND METHODS: To investigate the influences of lowered tissue factor (TF) activity upon hypercoagulation of AT-/- embryos, we crossed AT+/- with low TF (mTF-/- hTF+) mice to yield homozygous AT-deficient mice with the extremely low TF activity, that is expressed from the inserted human TF mini gene. RESULTS: AT-/- embryos either with 50% TF (AT-/- mTF+/- hTF+) or with low (approximately 1% TF, AT-/- mTF-/- hTF+) were not born, although the survival was prolonged until 18.5 gd. In both genotypes, histological examination showed disseminated thrombosis in hepatic sinusoidal space or in the portal veins, suggesting that the thrombogenesis caused loss of hepatic blood flow. As in original AT-/-, AT-/- mTF+/- hTF+ showed subcutaneous (s.c.) bleeding and also suffered from the myocardial degeneration apparently because of coronary thrombus formation. However, AT-/- mTF-/- hTF+ had no skin hemorrhage and the thrombosis and degeneration were completely abolished in the heart. Myocardium of adult low TF mice had exhibited fibrosis secondary to hemorrhage; however, it was significantly decreased in low TF mice with AT+/-. CONCLUSIONS: Our current model suggests that, in the heart, TF plays an important role in the thrombogenesis and it counterbalances AT-dependent anticoagulation. AT may be a potent anticoagulant during mice development and the activation and subsequent regulation of TF-procoagulant activity take place differently between the liver and the heart. These differences appear to point to local regulatory mechanisms in murine hemostasis.

Animals↗
PubMed2006

In vitro characterization of missense mutations associated with quantitative protein S deficiency.

OBJECTIVE: To elucidate the molecular consequences of hereditary protein S (PS) deficiency, we investigated the in vitro synthesis of the PS missense mutants in COS-1 cells and their activated protein C (APC) cofactor activities. PATIENTS: Four patients with quantitative PS deficiency suffering from venous thrombosis were examined. RESULTS: We identified three distinct novel missense mutations, R275C, P375Q and D455Y, and two previously reported missense mutations, C80Y and R314H. The P375Q and D455Y mutations were found in one patient and observed to be in linkage on the same allele. The R314H mutant showed the lowest level of expression (32.7%), and the C80Y, P375Q + D455Y, and R275C mutants exhibited a moderate impairment of expression, that is, 43.8%, 49.5%, and 72.3% of the wild type, respectively. Furthermore, pulse-chase experiments demonstrated that all mutants showed impaired secretion and longer half-lives in the cells than the wild type PS. In the APC cofactor assays, the C80Y mutant showed no cofactor activity, and the R275C mutant showed reduced activity, 62.3% of the wild type PS, whereas the R314H and P375Q + D455Y mutants exhibited normal cofactor activity. CONCLUSION: These data indicate that the C80Y and R275C mutations affect the secretion and function of the PS molecule, and that the R314H and P375Q + D455Y mutations are responsible for only secretion defects, causing the phenotype of quantitative PS deficiency observed in the patients.

Adult↗
PubMed2006

Preliminary laboratory based diagnostic criteria for immune thrombocytopenic purpura: evaluation by multi-center prospective study.

BACKGROUND: We proposed diagnostic criteria for immune thrombocytopenic purpura (ITP) by modifying the existing guidelines for diagnosis of ITP and by incorporating laboratory tests found useful for predicting its diagnosis, for example erythrocyte count, leukocyte count, anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, percentage of reticulated platelets, and plasma thrombopoietin. OBJECTIVE AND METHODS: To validate our criteria, we conducted a multi-center prospective study involving 112 patients with thrombocytopenia and a morphologically normal peripheral blood film at the first visit. Each patient underwent a physical examination, routine laboratory tests, and specialized tests for the anti-GPIIb/IIIa antibody response and platelet turnover. RESULTS: Ninety-one patients (81%) satisfied the proposed criteria at first visit. Clinical diagnosis was made by skilled hematologists > 6 months after the first visit; ITP was diagnosed in 88 patients and non-ITP disorders in 24. The proposed criteria had 98% sensitivity, 79% specificity, a 95% positive predictive value, and a 90% negative predictive value. A relatively low specificity appears to be attributed to a few patients who had both ITP and aplastic anemia or myelodysplastic syndrome. CONCLUSIONS: Our preliminary diagnostic criteria based on ITP-associated laboratory findings were useful for the differential diagnosis of ITP, but additional evaluations and modifications will be necessary to develop criteria that can be used routinely.

Adolescent↗
PubMed2006

Evaluation of RANK/RANKL/OPG gene polymorphisms in aggressive periodontitis.

BACKGROUND AND OBJECTIVE: Aggressive periodontitis (AgP) is a specific type of periodontal disease that is characterized by rapid attachment loss and bone destruction. While attempting to identify genetic polymorphisms associated with AgP, previous research has focused on candidate genes that may be involved in immune responses to microbial infections. In this study, the focus was on single nucleotide polymorphisms (SNPs) in the key mediators of osteoclast differentiation and activation, which involve receptor activator of nuclear factor-kappaB (RANK), RANK ligand (RANKL) and osteoprotegrin (OPG), in the Japanese population. The aim of this study was to evaluate the association of RANK/RANKL/OPG gene polymorphisms with AgP in the Japanese population. MATERIAL AND METHODS: We examined 99 patients with AgP and 89 controls from the Japanese population to explore the possibility of RANK/RANKL/OPG loci as candidate regions associated with the disease. All exons and relevant exon-intron boundaries of these three candidate genes were amplified by polymerase chain reaction (PCR) using 19 primers, followed by direct sequencing. The polymorphisms were identified by comparing the sequences obtained from 48 subjects. RESULTS: We identified 27 SNPs in RANK, including 10 novel SNPs and seven SNPs each in both RANKL and OPG. A pairwise linkage disequilibrium analysis using the r2 statistic showed that some SNP pairs from the three loci are in tight linkage disequilibrium. CONCLUSION: An association analysis with allelotypes showed that SNPs identified in the RANK/RANKL/OPG genes have no significant association with AgP in the Japanese population.

Acute Disease↗
PubMed2006

Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro.

OBJECTIVE: To determine the levels of the receptor activator of NFkB ligand (RANKL) and osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) during orthodontic tooth movement. A second objective was to investigate the effect of compression force on RANKL and OPG production from human periodontal ligament (hPDL) cells. DESIGN: Ten adolescent patients were included. GCF was collected at the distal cervical margins of the experimental and control teeth 0, 1, 24, and 168 h after the retracting force was applied. Thisin vitro study was performed to examine the secretion of RANKL and OPG from hPDL cells following a compression force (0, 0.5, 1.0, 2.0, or 3.0 g/cm(2) for 48 h). Enzyme-linked immunosorbent assay (ELISA) kits were used to determine RANKL and OPG levels in the GCF and the conditioned medium. RESULTS: GCF levels of RANKL were significantly higher, and the levels of OPG significantly lower, in the experimental canines than in the control teeth at 24 h, but there were no such significant differences at 0, 1, or 168 h. In vitro study indicated that the compression force significantly increased the secretion of RANKL and decreased that of OPG in hPDL cells in a time- and force magnitude-dependent manner. The compression-stimulated secretion of RANKL increased approximately 16.7-fold and that of OPG decreased 2.9-fold, as compared with the control. CONCLUSIONS: The results obtained suggest that the changes of amount of RANKL and OPG may be involved in bone resorption as a response to compression force.

Adolescent↗
PubMed2006

RANKL increase in compressed periodontal ligament cells from root resorption.

The ligand receptor activator of NFkappaB (RANKL) plays an important role in osteoclast formation. However, very little is known about the relationship between external apical root resorption during orthodontic treatment and RANKL. We hypothesized that compressive force is responsible for RANKL formation and up-regulation of osteoclastogenesis in periodontal ligament (PDL) cells from patients with severe orthodontically induced external apical root resorption. RANKL and osteoprotegerin (OPG) production, TRAP-positive cells, and resorptive pits were determined. The increase of RANKL and the decrease of OPG were greater in the severe root resorption group than in the non-resorption group. The numbers of TRAP-positive cells and resorptive pits were also increased in the severe root resorption group than in the non-resorption group. These results support the hypothesis that the compressed PDL cells obtained from tissues with severe external apical root resorption may produce a large amount of RANKL and up-regulate osteoclastogenesis.

Adolescent↗
PubMed2005

Transcription factor specificity protein 1 (Sp1) is the main regulator of nerve growth factor-induced sphingosine kinase 1 gene expression of the rat pheochromocytoma cell line, PC12.

Sphingosine kinase (SPHK) is known to exert an anti-apoptic role in various cells and cell lines. We previously reported that human brain is rich in SPHK1 (Murate et al. 2001). After showing a high expression of SPHK1 in rat brain, we examined the gene expression mechanism using nerve growth factor (NGF)-stimulated rat PC12 cells. With RT-PCR, we found that both rat brain and PC12 utilized exon 1d mostly out of eight untranslated first exons. NGF induced an increase in SPHK enzyme activity and protein about double those in PC12 cells, and NGF-induced SPHK1 mRNA was three times higher than in the control. The minimal 5' promoter was determined, and TrkA specific inhibitor K252a inhibited the NGF-induced promoter activity of SPHK1. The truncation or mutation of putative transcription factor-binding motifs revealed that one specificity protein 1 (Sp1) binding motif of the 5' region of exon 1d is prerequisite. Electrophoresis mobility shift assay confirmed the promoter analysis, indicating increased Sp1 protein binding to this motif after NGF treatment. Chromatin immunoprecipitation assay also showed the binding of Sp1 and the promoter region in vivo. These results suggest the signal transduction pathway from NGF receptor TrkA to transcription factor Sp1 protein binding to the promoter Sp1-like motif in NGF-induced rat SPHK1 gene expression.

Animals↗