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Fumio Nomura

Publications and source records attributed to Fumio Nomura.

At least 19 recordsLinked to original sources
PubMed2007

Rapid detection of metastasis of gastric cancer using reverse transcription loop-mediated isothermal amplification.

Tailor-made surgeries for patients with solid malignancies have been under consideration on the basis of the development of new approaches for minor metastatic foci of malignant tumors. Accurate and reliable methods to detect metastases in biopsy specimens with certain rapidity are essential for the performance of these surgeries. The aim of this study was to develop a rapid and practical method to detect metastasis in specimens from patients with gastric carcinoma with the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction, a novel technique for detecting mRNA expressions of targeted sequences with high sensitivity, specificity and rapidity under isothermal conditions. RT-LAMP primers to detect cytokeratin19 (CK19) mRNA were generated and 92 lymph nodes (LNs) obtained from 9 patients with gastric cancer were tested for tumor metastases with this technique. Among 92 LNs, 15 were metastasis-positive by routine histopathological examination. RT-LAMP reaction detected CK19 expression in all of the pathologically positive LNs and in 16 of 77 negative LNs. Nested RT-PCR assay for CK19 expression was also performed on 2 of the 9 cases including 32 LNs. The agreement rate of CK19 expression detection by RT-LAMP and RT-PCR analysis was 31/32 (97%). The RT-LAMP technique showed similar sensitivity to detect metastases as nested RT-PCR assay, with a rapidity comparable to that of intraoperative histopathological examination with frozen sectioning and hematoxylin and eosin staining. This method is expected to play an essential role in the performance of tailor-made surgeries in the near future.

Aged↗
PubMed2007

Serum anti-myomegalin antibodies in patients with esophageal squamous cell carcinoma.

In order to identify new serum markers of esophageal squamous cell carcinoma (SCC), we performed serological identification of antigens by recombinant cDNA expression cloning (SEREX). E. coli was transformed with a lambdaZAPII phage cDNA library prepared from mRNA of an esophageal cancer cell line (T.Tn), and IPTG-induced cDNA products were screened for interaction with antibodies in allogeneic sera of patients with esophageal SCC. We identified myomegalin (MMGL, phosphodiesterase 4D interacting protein/PDE4DIP) as a new SEREX antigen for esophageal SCC. Western blot analysis revealed that serum anti-myomegalin antibodies (s-MMGL-Abs) were present in 43 (47%) of 91 patients, but in only one (2.2%) of 45 healthy controls. Of the 21 patients with stage I disease, 8 (38%) were sero-positive. The positive rate of s-MMGL-Abs was greater than those of other conventional tumor markers. Reverse transcription-PCR analysis suggested that alternative splicing from myomegalin variant 1 to variant 5 may explain, in part, the development of s-MMGL-Abs. Although the presence of s-MMGL-Abs was not related to any clinicopathological features of the patients, multivariate analysis indicated that the presence of s-MMGL-Abs was significantly associated with a favorable prognosis. Consequently, s-MMGL-Abs may be a useful tumor marker to diagnose and establish a prognosis in patients with esophageal SCC.

Aged↗
PubMed2006

Miliary brain metastases from adenocarcinoma of the lung: MR imaging findings with clinical and post-mortem histopathologic correlation.

INTRODUCTION: Miliary dissemination is a rare form of brain metastasis. The clinical and pathologic features of this form are unclear. METHODS: We report a 66-year-old man with miliary brain metastases from adenocarcinoma of the lung, describing MRI and neuropathologic findings in the context of previously reported cases. RESULTS: Initial disorientation progressed to an apallic state within 6 months. Although, CT with administration of contrast agent failed to demonstrate any lesions, MRI with Gd-DTPA administration showed multiple enhancing miliary nodules in the cerebral cortex, basal ganglia, thalamus, cerebellum, and brainstem. Some of those nodules also could be seen on T2-weighted imaging without Gd-DTPA, but were difficult to identify conclusively. A histopathologic examination at autopsy disclosed diffusely distributed miliary tumor nodules in a perivascular distribution without surrounding focal edema or reactive gliosis. Notably, this patient with miliary brain metastases developed disorientation followed by unconsciousness, which overshadowed other focal neurologic signs at that time. CONCLUSION: We should consider this pattern of brain dissemination when a cancer is associated with unexplained disturbance of consciousness.

Adenocarcinoma, Papillary↗
PubMed2006

Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli.

Subtilase cytotoxin (SubAB) is a recently identified AB5 subunit toxin produced by Shiga-toxigenic Escherichia coli. The A subunit is thought to be a subtilase-like, serine protease, whereas the B subunit binds to the toxin receptor on the cell surface. We cloned the genes from a clinical isolate; the toxin was produced as His-tagged proteins. SubAB induced vacuolation at concentrations greater than 1 microg/ml after 8 h, in addition to the reported cytotoxicity induced at a ng/ml level after 48 h. Vacuolation was induced with the B, but not the A, subunit and was dependent on V-type ATPase. The cytotoxicity of SubAB at low concentrations was associated with the inhibition of protein synthesis; the 50% inhibitory dose was approximately 1 ng/ml. The A subunit, containing serine 272, which is thought to be a part of the catalytic triad of a subtilase-like serine protease, plus the B subunit was necessary for this activity, both in vivo and in vitro. SubAB did not cleave azocasein, bovine serum albumin, ovalbumin, or synthetic peptides. These data suggest that SubAB is a unique AB toxin: first, the B subunit alone can induce vacuolation; second, the A subunit containing serine 272 plus the B subunit inhibited protein synthesis, both in vivo and in vitro; and third, the A subunit proteolytic activity may have a strict range of substrate specificity.

Amino Acid Sequence↗
PubMed2006

Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin.

Some shiga toxin-producing Escherichia coli secrete a novel AB5 cytotoxin, named subtilase cytotoxin (SubAB), which induces vacuole formation in addition to cytotoxicity in susceptible cells. By immunoprecipitation with SubAB from Vero cells, we discovered proteins of 100 kDa, 135 kDa and 155 kDa as potential candidates for its receptor. These proteins were N-glycosylated in their extracellular domains, a modification that was necessary for interaction with SubAB. Biotinylated receptors were partially purified by Datura stramonium agglutinin affinity chromatography and avidin-agarose and analysed by TOF mass spectroscopy. The peptide sequences of p135 were identical to beta1 integrin, and its identification was confirmed with anti-integrin beta1 antibody. The p155 protein was identified as alpha2 integrin using anti-integrin alpha2 antibody. In addition, treatment of Vero cells with beta1 integrin RNAi before exposure to SubAB prevented vacuolating activity. These results suggested that SubAB recognizes alpha2beta1 integrin as a functional receptor; this first interaction may be an important key step leading to the SubAB-induced morphological changes in Vero cells.

Animals↗
PubMed2006

Development of a novel fragments absorbed immunocapture enzyme assay system for tartrate-resistant acid phosphatase 5b.

BACKGROUND: Osteoclastic activity is mainly assessed by measuring urinary markers. To correct for differences in renal clearance, the levels of urinary markers are usually corrected by the urine creatinine concentration. Therefore, alternative serum markers to evaluate osteoclastic activity are required. We developed a novel system for the determination of serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity to evaluate osteoclastic activity. METHODS: Two unique monoclonal antibodies were generated and the specificity was tested using a surface enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI TOF-MS). A novel fragments absorbed immunocapture enzymatic assay (FAICEA) method was developed using 2 monoclonal antibodies. RESULTS: FAICEA gave a sensitivity 0.1 U/l, linearity of 0.1-28 U/l, recovery 92-103%, inter-assay CV 2.95% and intra-assay CV 2.15%. Unlike other TRACP5b assay systems, FAICEA avoided interference from TRACP 5a. CONCLUSIONS: According to the FAICEA, postmenopausal women had higher TRACP5b concentrations than younger women. The results show that TRACP5b is a novel bone resorption marker in serum.

Acid Phosphatase↗
PubMed2006

Critical roles of muscle-secreted angiogenic factors in therapeutic neovascularization.

The discovery of bone marrow-derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 "no-option" patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1beta (IL-1beta). We then examined an experimental model of limb ischemia using IL-1beta-deficient mice. Implantation of IL-1beta-deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1beta-deficient mononuclear cells increased expression of IL-1beta and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1beta markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.

Aged↗
PubMed2006

An essential role of alternative splicing of c-myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis.

Elevated expression of c-myc has been detected in a broad range of human cancers, indicating a key role for this oncogene in tumor development. Recently, an interaction between FUSE-binding protein-interacting repressor (FIR) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and might be important for suppressing tumor formation. In this study, we showed that enforced expression of FIR induced apoptosis. Deletion of the NH(2)-terminal repression domain of FIR rescued the cells from apoptosis as did coexpression of c-Myc with FIR; thus, repression of Myc mediates FIR-driven apoptosis. Surprisingly, a splicing variant of FIR unable to repress c-myc or to drive apoptosis was frequently discovered in human primary colorectal cancers but not in the adjacent normal tissues. Coexpression of this splicing variant with repressor-competent FIR, either in HeLa cells or in the colon cancer cell line SW480, not only abrogated c-Myc suppression but also inhibited apoptosis. These results strongly suggest the expression of this splicing variant promotes tumor development by disabling FIR repression and sustaining high levels of c-Myc and opposing apoptosis in colorectal cancer.

Alternative Splicing↗
PubMed2006

An assay for separating and quantifying four bilirubin fractions in untreated human serum using isocratic high-performance liquid chromatography.

BACKGROUND: The quantification of serum bilirubin fractions has been widely performed with both the diazo-method and an enzymatic method; however, the accuracy of these methods has not been evaluated because quantitative fractional high-performance liquid chromatography (HPLC) reference methods have yet to be established. METHODS: Samples were analyzed using HPLC and Shodex Asahipak GS-320HQ columns. Human serum was subjected to HPLC using direct injection, then eluted with acetonitrile: 0.3 mol/l phosphate buffer (pH 6.5) containing 1% Brij 35 and 0.08% sodium ascorbate (30:70, v/v). RESULTS: Serum bilirubin was separated into 4 fractions; retention times of 9.24, 19.92, 24.07, 35.75 min were identified as delta bilirubin, bilirubin diglucuronide, bilirubin monoglucuronide, and unconjugated bilirubin, respectively. Mean recovery was 93.0%-99.2%. Total precision of peak retention time, height and area exhibited <4.26% variation. Detection range was 3.1 to 348 mg/l. Hemoglobin (6 g/l) and immunoglobins produced a small positive interference. beta-carotene (20 mg/l), vitamin-B2 (370 microg/l) and B(12) (9.5 microg/l) did not interfere with this analysis. Results (n=30) with this method were closely correlated to those by Adachi's HPLC method as r=0.9941 to 0.9960, slope=0.88 to 1.27, intercept=-3.2 to +4.9, for each fraction. CONCLUSIONS: Since this method was a precise quantitative HPLC method for serum bilirubin fractionation, it might be used to evaluate the accuracy and the characteristics of various routine methods for bilirubin measurement.

Bilirubin↗
PubMed2006

Bladder sensation in peripheral nerve lesions.

OBJECTIVE: To present bladder sensory data of three common peripheral nerve lesions (e.g., distal, intermediate/focal, and proximal). METHODS: We measured first sensation (FS) and bladder capacity (BC) (not exceeding 600 ml) in 71 patients with peripheral nerve lesions: 35 diabetic neuropathy (D group), 6 post-pelvic surgery (S), and 27 cauda equina syndrome due to lumbar spondylosis (L). We excluded those with detrusor overactivity or low compliance that might affect bladder sensation. RESULTS: The mean FS was 301.7 ml (D), 271.3 ml (S), and 189.4 ml (L), with the largest being in the D group (P < 0.05); the mean BC was 495.2, 475.4, and 391.4 ml, with the largest being in the D group (P < 0.05); who commonly had less frequent toileting. The mean post-void residual volume was 106.5, 29.0, and 42.0 ml; the values tended to increase along with BC. In the D group, the mean FS in patients with skin hypoalgesia as detected by pin prick and in those without it was 407.8 and 210.0 ml. The percentage of patients with FS < 100 ml was 5.7%, 0%, and 7.4%, respectively, who commonly had urinary urgency and frequency. CONCLUSIONS: Bladder sensation is affected in diabetic neuropathy more severely than in intermediate/proximal lesions, together with somatic sensory disturbance. Bladder sensory disturbance leads to less frequent toileting, resulting in bladder over-distension and large post-void residuals. A small proportion of patients with peripheral nerve lesions develop urinary urgency, presumably reflecting irritation of the afferent nerve fibers or the urothelium.

Aged↗
PubMed2006

Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin.

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.

Aged↗
PubMed2006

Development and characterization of novel monoclonal antibodies against tartrate-resistant acid phosphatase 5.

Serum band 5 tartrate-resistant acid phosphatase (TRACP 5; EC 3.1.3.2) is a glycoprotein that exists as two very similar isoforms, TRACP 5a and TRACP 5b. The similarity of these two isoforms has made it difficult to establish monoclonal antibodies specific for either isoform. We report here the development of a monoclonal antibody with high specificity for TRACP 5b. We prepared TRACP 5b antigens from four sources: TRACP 5b purified from human bone, recombinant TRACP 5 from Escherichia coli, recombinant TRACP 5 from insect cells, and a synthetic TRACP 5b peptide. Thirty-seven mice were each immunized with 1 of the 4 different TRACP antigens to generate 473 antibody-producing clones. Three of these clones, Trk27, Trk49, and Trk62, reacted with TRACP 5b. These three clones were all established from mice exposed to native bone TRACP 5b antigen. In fact, none of the other antigens were able to generate anti-TRACP 5b monoclonal antibodies in mice. Furthermore, Trk62 interacted more strongly with TRACP 5b than with TRACP 5a. These results suggested that although recombinant proteins can be effective antigens, the native TRACP 5 protein might be more effective at generating monoclonal antibodies of greater specificity due to its more faithful representation of the native three-dimensional structure of the protein.

Acid Phosphatase↗
PubMed2006

Nocardia exalbida sp. nov., isolated from Japanese patients with nocardiosis.

Two bacterial strains isolated from different hospitals in Japan were subjected to a polyphasic analysis. Strains IFM 0803(T) and IFM 10383 were found to have morphological, biochemical, physiological and chemotaxonomic properties consistent with their classification in the genus Nocardia. Strains IFM 0803(T) and IFM 10383 clustered with the type strain of Nocardia xishanensis, showing 16S rRNA gene sequence similarities of 98.6-98.9 % with this species. The novel strains could be distinguished from N. xishanensis by a range of phenotypic properties. Based on their phenotypic and phylogenetic characteristics, the two isolates are proposed as members of a novel species of the genus Nocardia, Nocardia exalbida sp. nov., with the type strain IFM 0803(T) (=NBRC 100660(T) = JCM 12667(T) = DSM 44883(T)).

Humans↗
PubMed2006

Presence of serum tripartite motif-containing 21 antibodies in patients with esophageal squamous cell carcinoma.

SEREX has been applied to esophageal SCC, and the TRIM21 gene was identified as a novel SEREX antigen of esophageal SCC. The presence of s-TRIM21-Abs was confirmed by Western blotting using bacterially expressed TRIM21 gene product and was evaluated for clinicopathological significance in patients with esophageal SCC. s-TRIM21-Abs were detected in 18 (20%) of 91 patients with esophageal SCC but not in 42 healthy donors. The presence of s-TRIM21-Abs was partly associated with tumor size (P = 0.063) and poor survival (P = 0.067). To measure serum antibody levels, ELISA using purified recombinant TRIM21 protein was developed. The levels of s-TRIM21-Abs were significantly higher in patients with esophageal SCC than in healthy donors (P = 0.013). s-TRIM21-Abs may be a useful tumor marker to diagnose and predict disease progression in patients with esophageal SCC.

Adult↗
PubMed2006

Comprehensive analysis of the ICEN (Interphase Centromere Complex) components enriched in the CENP-A chromatin of human cells.

The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.

Aneuploidy↗
PubMed2006

Identification of a novel SEREX antigen, SLC2A1/GLUT1, in esophageal squamous cell carcinoma.

We have carried out SEREX (serological identification of antigens by recombinant cDNA expression cloning), and identified SLC2A1 (solute carrier family 2/facilitated glucose transporter, member 1) as an antigen recognized by serum IgG antibodies in patients with esophageal squamous cell carcinoma (SCC). The levels of serum anti-SLC2A1 antibodies (s-SLC2A1-Abs), examined by enzyme-linked immunosorbent assay using bacterially expressed glutathione-S-transferase-SLC2A1 fusion protein, were significantly higher in patients with esophageal SCC than in healthy donors. When using a cut-off level as the mean + 2x standard deviations of healthy donors, a total of 12 (21%) out of 57 SCC patients were revealed as positive for s-SLC2A1-Abs. The presence of s-SLC2A1-Abs was not associated with either clinicopathological factors or survival. Because s-SLC2A1-Abs were not associated with the positivity of other conventional serum markers, a combination assay of s-SLC2A1-Abs with these conventional serum markers may be useful for the diagnosis and monitoring of esophageal SCC.

Adult↗
PubMed2006

[Clinical proteomics in laboratory medicine].

During the past few years, the emphasis on genomics has shifted via transcriptomics to proteomics, the science of understanding how the whole set of proteins are expressed and function at the cellular level. Recent progress in clinical proteomics is mostly thanks to sophisticated new methodologies for proteome analyses. In search for novel biomarkers for cancers and other disorders, we have taken advantage of the surface-enhanced laser/desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) and the MALDI-TOF/TOF. Since personal and verbal reporting of alcohol use is not necessarily accurate, objective markers to assess alcohol consumption are required. We used the ProteinChip SELDI technology to generate comparative protein profiles of the consecutive serum samples obtained during abstinence and found that a 5.9kD peptide detected and identified by this technology could be a novel marker of heavy alcohol consumption. Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We could identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. A number of proteins with altered expression between cancer and adjacent non-cancer tissues were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting and immunohistochemistry. Clinical proteomics consists of the discovery proteomics and the measurement proteomics. Although we still stand at the discovery proteomics, undoubtedly, the clinical proteomics will be a major contributor in laboratory medicine in the near future.

Clinical Laboratory Techniques↗