Biomedical subjects

K Ozaki

Publications and source records attributed to K Ozaki.

At least 37 records · Page 2Linked to original sources

PubMed2002

Mast cell tumors of the gastrointestinal tract in 39 dogs.

Mast cell tumors (MCTs) of gastrointestinal origin that had been surgically removed from 39 dogs were examined to evaluate their pathologic features. Miniature breeds, especially Maltese, were most frequently affected. The average age of affected dogs was 9.7 +/- 2.6 years. No sex difference was apparent. The most frequently affected sites were in the upper digestive tract, and the prognosis was very poor. Grossly, the gastrointestinal wall was prominently thickened, and the lumen of the affected gut was usually narrowed. Microscopically, there was diffuse transmural invasion of round to pleomorphic tumor cells. Tumor cells had moderate to abundant cytoplasm, round to ovoid nuclei with scattered chromatin, and mitotic figures. Fibrous stroma was observed in about half of the tumors. There was variable infiltration of eosinophils. In all tumors, cytoplasmic granules showed weak metachromasia, but the number of granules was very small. Immunohistochemical staining for c-kit and mast cell tryptase was positive in 77% and 62% of tumors, respectively. All tumors were positive for at least two of these markers. Immunohistochemical staining for p53 was positive in 13% of the tumors. Reactivity for staining markers and p53 was unrelated to cell pleomorphism, vessel invasion, or survival time. Gastrointestinal MCTs have histologic and immunohistochemical features completely different from those of other primary or metastatic gastrointestinal tumors. The combination of immunostaining for mast cell tryptase and c-kit and histochemical staining for metachromasia appears to be a powerful tool for the diagnosis of gastrointestinal MCTs.

Animals↗

PubMed2001

ERK pathway positively regulates the expression of Sprouty genes.

Sprouty was originally identified as an inhibitor of Drosophila development-associated receptor tyrosine kinase (RTK) signaling. Although RTK signaling has been shown to induce Sprouty gene expression, the precise induction pathway downstream of RTK remains unclear. As RTK signaling pathway includes activation of extracellular signal-regulated kinases (ERKs), we have examined a correlation between activation of ERKs and induction of Sprouty gene expression. All reagents which induce the activation of ERKs induce Sprouty gene expression; these agents include not only growth factors which bind to RTK but also phorbol 12-myristate-13-acetate and active Raf-1 kinase. Furthermore, the Sprouty gene expression induced by all those agents is totally suppressed when the cells are pretreated with specific inhibitors of ERK kinase (MEK). Human tumor cells which exhibit constitutive activation of ERKs show elevated expression of Sprouty genes, which is abolished by treatment of these cells with MEK inhibitors. All these findings clearly indicate that Sprouty gene expression is positively regulated by the ERK pathway downstream of RTK.

Animals↗

PubMed2001

Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme.

The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.

Adaptation, Physiological↗

PubMed2001

Impaired FHIT expression characterizes serous ovarian carcinoma.

The FHIT (fragile histidine triad) gene on chromosome 3p14.2 is a candidate tumour suppressor gene. To define the role of the FHIT gene in the development of ovarian cancer, we have examined 33 ovarian carcinomas, 2 borderline tumours and 10 benign adenomas for the presence of FHIT gene alterations. FHIT transcripts were analysed by RT-PCR and sequencing. Aberrant FHIT transcripts were observed in 5/33 carcinomas (15%) and in 1 of 2 borderline tumours. Loss of normal FHIT transcript was observed in 5/33 carcinomas (15%) but not in 2 borderline tumours or 10 benign adenomas. Allelic losses at D3S1300 and D3S4103, both located within intron 5 of FHIT, were detected in 5/24 (21%) and 5/25 (20%) informative ovarian carcinomas, respectively. Expression of Fhit protein was analysed by immunohistochemistry in 44 carcinomas, 19 borderline tumours and 16 benign adenomas. Loss or significantly reduced expression of Fhit protein was observed in 6/44 (14%) ovarian carcinomas but not in any of 19 borderline tumours or 16 benign adenomas. The impaired Fhit protein expression was significantly correlated with the loss of normal FHIT transcription. Most notably, loss of normal FHIT transcript and impaired expression of Fhit protein occurred only in serous adenocarcinomas of grade 2 and 3 (5/15; 33% and 6/19; 32%, respectively). The present data suggest that inactivation of the FHIT gene by loss of expression is one of the important molecular events associated with the genesis of ovarian carcinoma, especially of high-grade serous carcinoma.

Acid Anhydride Hydrolases↗

PubMed2001

CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk.

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Isolation of a novel human lung-specific gene, LUNX, a potential molecular marker for detection of micrometastasis in non-small-cell lung cancer.

We have isolated a novel human lung-specific gene, LUNX (lung-specific X protein), by differential-display mRNA analysis. The full-length cDNA contained 1,015 nucleotides including an open reading frame of 768 nucleotides encoding 256 amino acids. We localized the gene to chromosomal region 20p11.1-q12 by radiation hybrid mapping. Using an RT-PCR assay specific for LUNX mRNA, 35 non-small-cell lung-cancer (NSCLC) tumors and 0 of 16 normal lymph nodes were positive. Furthermore, LUNX mRNA expression was enhanced in 26 (84%) of 31 NSCLC tumors vs. corresponding cancer-free lung tissues by semi-quantitative analyses with multiplex RT-PCR. We assessed the possibility of LUNX mRNA as a molecular marker for detection of micrometastasis in dissected lymph nodes obtained from 20 patients with NSCLC tumors. LUNX mRNA was detected in 16 (80%) of 20 histologically positive lymph nodes and 21 (25%) of 84 histologically negative lymph nodes. Comparative analyses of the conventional histological examination and the RT-PCR detection assay for LUNX mRNA showed that the detection rate of metastases in lymph nodes by the RT-PCR assay was higher in 12 and consistent in 6 of the total 20 NSCLC patients. We demonstrate that the LUNX RT-PCR assay is a potential diagnostic method for detection of micrometastases in lymph nodes of NSCLC patients.

Amino Acid Sequence↗

PubMed2001

Nucleotide and deduced amino acid sequences of an alkaline pullulanase from the alkaliphilic bacterium Bacillus sp. KSM-1876.

The nucleotide sequence of an alkaline pullulanase-encoding gene from alkaliphilic Bacillus sp. strain KSM-1876 was determined. The open reading frame of the gene encoded 1142 amino acids with a calculated molecular mass of 128739 Da. The alkaline pullulanase showed very limited homology (<32% identity) to previously reported debranching enzymes from prokaryotes and eukaryotes. It contained unique tandem repeats in both the N-terminal and the C-terminal regions.

Amino Acid Sequence↗

PubMed2001

Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis.

An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.

Alkalies↗

PubMed2001

Evaluation of resting brain conditions measured by two different methods (i.v. and oral administration) with 18F-FDG-PET.

Our aim was to evaluate regional differences between brain activity in two resting control conditions measured by 3D PET after administration of FDG through either the intravenous (i.v.) or the oral route. Ten healthy male volunteers engaged in the study as the i.v. group (mean age, 26 +/- 9.3 years, +/- S.D.) who received FDG intravenously and another 10 volunteers as the oral group (mean age, 27.9 +/- 11.3 years, +/- S.D.) who received FDG per os. A set of 3D-PET scans (emission and transmission scans) were performed in both groups. To explore possible functional differences between the brains of the two groups, the SPM-96 software was used for statistical analysis. The results revealed that glucose metabolism was significantly higher in the superior frontal gyrus, superior parietal lobule, lingual gyrus and left cerebellar hemisphere in the i.v. group than in the oral group. Metabolically active areas were found in the superior, middle and inferior temporal gyrus, parahippocampal gyrus, amygdaloid nucleus, pons and cerebellum in the oral group when compared with the i.v. group. These differences were presumably induced by differences between FDG kinetics and/or time-weighted behavioral effects in the two studies. This study suggests the need for extreme caution when selecting a pooled control population for designated activation studies.

Administration, Oral↗

PubMed2001

Advantage of delayed whole-body FDG-PET imaging for tumour detection.

Delayed imaging that coincides with the highest uptake of fluorine-18 fluorodeoxyglucose (FDG) by tumour may be advantageous in oncological positron emission tomography (PET), where delineation of metastasis from normal tissue background is important. In order to identify the better imaging protocol for tumour detection, whole-body FDG-PET images acquired at 1 h and 2 h after injection were evaluated in 22 subjects, with a post-injection transmission scan at 90 min for attenuation correction. After visual interpretation, tumour uptake [tumour standardised uptake ratio (SUR)], normal tissue uptake (normal SUR) and tumour to background contrast (tumour SUR/normal tissue SUR) were evaluated in the images acquired at 1 h and at 2 h. Most malignant lesions, including primary lung cancer, metastatic mediastinal lymph nodes and lymphoma lesions, showed higher FDG uptake at 2 h than at 1 h. By contrast, benign lesions, with the exception of sarcoidosis, showed lower uptake of FDG at 2 h than at 1 h. Among normal tissues, the kidney, liver, mediastinum, lung, upper abdomen and left abdomen showed significant falls in FDG uptake from 1 h to 2 h. The lower abdomen, right abdomen and muscles (shoulder and thigh) showed no significant changes. Consequently, malignant lesions of the lung, mediastinum and upper abdomen showed significant increases in tumour to background contrast from 1 to 2 h. Three lesions (two lung cancers and a malignant lymphoma) that were equivocal on 1-h images became evident on 2-h images, changing the results of interpretation. All other malignant lesions were detected on 1-h images, but were clearer, with higher contrast, on 2-h images. Lesion-based sensitivity was improved from 92% (49/53) to 98% (52/53), and patient-based sensitivity from 78% (14/18) to 94% (17/18). It is concluded that delayed whole-body FDG-PET imaging is a better and more reliable imaging protocol for tumour detection.

Fluorodeoxyglucose F18↗

PubMed2001

Histochemical and morphometrical analysis of skeletal muscle in spontaneous diabetic WBN/Kob rat.

Spontaneous diabetic WBN/Kob rats develop diabetic peripheral neuropathy characterized by primary segmental demyelination and secondary axonal degeneration. The objective of this study was to evaluate the histochemical and morphometric characteristics of the lesions of skeletal muscles innervated by the affected nerves in diabetic rats. The following groups of rats were investigated: 24-month-old males that had been diabetic for less than 12 months, 10-month-old pre-diabetic males, 24-month-old non-diabetic females, and 10-month-old nondiabetic females. The soleus (SOL), extensor digitorum longus (EDL) and biceps femoris (BF) muscles were studied by light and electron microscopy, including histochemical and morphometric analyses. Muscle weight was reduced with age to a remarkable degree in diabetic BF and EDL. Dispersed atrophy of muscle fiber was observed in type 2a fibers of BF and EDL, and type 2c fibers of SOL, and the incidence was higher in diabetic rats. Multi-core, myofibrillar disorientation and an increased number of central nucleus of SOL, along with connective tissue proliferation of BF perimysium were noted in diabetic rats. The fiber population and type of composition varied with age, but no remarkable changes attributable to diabetic conditions were observed. Electron microscopically, an abnormal arrangement of myofibrils, a number of myelin figures, mitochondrial swelling and lysis of mitochondrial cristae were seen in diabetic rats. However, the neuromuscular junction and capillaries were intact. These findings indicate that the diabetic skeletal muscle lesion in WBN/Kob rats was mainly myogenic in nature, and was aggravated by the age-related change.

Aging↗

PubMed2001

Cronkhite-Canada syndrome associated with advanced rectal cancer treated by a subtotal colectomy: report of a case.

A 41-year-old man with Cronkhite-Canada syndrome presented with multiple juvenile polyps with hyperplastic and adenomatous changes throughout his stomach and entire colorectum. Dysgeusia was recognized and the degree of hypoproteinemia was remarkable. A barium enema study and colonofiberscopy also revealed an advanced cancer in the rectum. Chronic hepatitis B and membranous glomerulonephritis were also present. It was difficult to design a conservative protocol using steroids for the treatment of protein-loosing enteropathy because the patient was a hepatitis B virus carrier. As a result, a subtotal colectomy while preserving the cecum with cecorectal anastomosis was performed. Pathologically, the ulcerated rectal tumor was a moderately differentiated adenocarcinoma with invasion into the muscularis propria. Most polyps showed cystically dilated glands without dysplasia or edematous stroma with inflammatory cell infiltration. A few polyps were juvenile-type polyps with adenoma components. Although no remarkable improvement was observed in the hypoproteinemia postoperatively, an alpha1-antitrypsin clearance test showed a significant decrease in protein loss from the gastrointestinal tract, which was only about one third of the loss seen preoperatively. These findings lead us to conclude that when improvement using conservative treatment can be neither obtained nor is expected, then the use of surgery should be considered when treating patients with Cronkhite-Canada syndrome.

Adenocarcinoma↗

PubMed2001

Characterization of a new citrus viroid species tentatively termed Citrus viroid OS.

A new viroid was obtained from a viroid sample, named 'OS', collected from a citrus plant. The viroid consists of 330-331 nucleotides, contains the central conserved region (CCR) characteristic of the genus Apscaviroid, and has the highest sequence similarity (only 68%) with Citrus III viroid (CVd-III) among known viroids. This viroid, by itself, caused only mild petiole necrosis and characteristically very mild leaf bending in Arizona 861-S1 Etrog citrons (Citrus medica L.), the degree of which differed from that induced by other citrus viroids. This viroid could be a new citrus viroid species belonging to the genus Apscaviroid; for convenience, it was tentatively named Citrus viroid OS (CVd-OS) after the original sample. CVd-OS has chimeric features related to other viroids. In particular, CVd-OS has high sequence similarity with CVd-III and Apple dimple fruit viroid in the putative central and terminal left domains, including a duplicative sequence from the lower CCR of the genus Pospiviroid in the left terminus. Further, CVd-OS shares high sequence similarity with Citrus exocortis viroid (CEVd) in the lower strand of the putative variable and terminal right domains, including the sequence identical to that of the right termini of CEVd and of Citrus IV viroid.

Base Sequence↗

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↗

PubMed2001

High-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the entire human DiGeorge syndrome critical region 2 (DGCR2) gene at 22q11.2.

We constructed a high-density single-nucleotide polymorphism (SNP) map in the 96-kb region containing the DiGeorge syndrome critical region 2 (DGCR2) gene at chromosome 22q11.2, a human counterpart of mouse seizure-related gene SEZ-12. A total of 102 SNPs were isolated from the region by systematic screening among 48 Japanese individuals: 9 SNPs in the 5' flanking region, 3 in the 5' untranslated region, 2 in the coding regions, 77 in introns, 7 in the 3' untranslated region, and 4 in the 3' flanking region. By a comparison of our data with SNPs deposited in the dbSNP database in the National Center for Biotechnology Information, 80 SNPs (78.4%) were considered to be novel. The ratio of transition to transversion was 3.08:1. In addition, eight other types of genetic variations (one GA dinucleotide polymorphism and seven insertion/deletion polymorphisms) were discovered. The high-resolution map that we constructed will be a useful resource for analyzing gene scans of complex diseases mapped to this local segment on chromosome 22.

3' Untranslated Regions↗

PubMed2001

A high-throughput SNP typing system for genome-wide association studies.

One of the most difficult issues to be solved in genome-wide association studies is to reduce the amount of genomic DNA required for genotyping. Currently available technologies require too large a quantity of genomic DNA to genotype with hundreds or thousands of single-nucleotide polymorphisms (SNPs). To overcome this problem, we combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can reduce the required reaction volume. We amplified 100 genomic DNA fragments, each containing one SNP, in a single tube, and analyzed each SNP with the Invader assay. This procedure correctly genotyped 98 of the 100 SNP loci examined in PCR-amplified samples from ten individuals: the genotypes were confirmed by direct sequencing. The reproducibility and universality of the method were confirmed with two additional sets of 100 SNPs. Because we used 40 ng of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.4 ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 microg of genomic DNA is available. Our results indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5-10 ml.

Alleles↗

PubMed2001

Deduced amino-acid sequence of a calcium-free alpha-amylase from a strain of Bacillus: implications from molecular modeling of high oxidation stability and chelator resistance of the enzyme.

Alkaline alpha-amylase (AmyK38) from the alkaliphilic Bacillus sp. strain KSM-K38 is a unique enzyme in that it is highly chelator-resistant and oxidatively stable [Hagihara, H., Igarashi, K., Hayashi, Y., Endo, K., Ikawa-Kitayama, K., Ozaki, K., Kawai, S. & Ito, S. (2001) Appl. Environ. Microbiol. 67, 1744-1750]. This enzyme was found to contain no Ca and require Na (or monovalent cations) for manifestation of activity. The nucleotide sequence of the gene for the novel enzyme was determined, and it harbored an ORF of 1503 bp encoding the enzyme of 501 amino acids, including a 21-amino-acid signal peptide. The deduced amino-acid sequence of the mature enzyme (55 097 Da) showed moderate homology to those of alpha-amylases from Bacillus licheniformis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, with approximately 63% identity. A methionine residue, which is conserved and susceptible to chemical oxidation, was replaced with leucine in AmyK38. Moreover, many conserved residues that are crucial ligands for Ca were replaced with other amino acids, thereby leading to loss of the Ca coordination geometries. By building a molecular model, we showed the calcium-independent, oxidatively stable active-site topology and structural integrity of AmyK38.

Alkalies↗

PubMed2001

Clonal nature of seborrheic keratosis demonstrated by using the polymorphism of the human androgen receptor locus as a marker.

We evaluated the clonality of seborrheic keratoses using a polymorphism due to the random inactivation of one of two X chromosomes in females. Thirty-eight seborrheic keratoses obtained from the skin of females with polymorphism of the human androgen receptor (HUMARA) locus were examined by a fluorescent polymerase chain reaction procedure, which allowed accurate measurement of the peak intensities of each HUMARA allele. The epithelial portion of seborrheic keratosis and normal control epidermis adjacent to the seborrheic keratosis were removed by laser capture microdissection. As biopsied specimens of seborrheic keratoses contained small amounts of normal epidermis, the effect of digestion by a restriction enzyme (HhaI) recognizing the nonmethylated active sites was compared between seborrheic keratoses and normal control epidermis in only five seborrheic keratosis cases. Disappearance or significant reduction in intensity of one of two HUMARA alleles was observed after HhaI digestion in seborrheic keratoses, but not in the normal control epidermis. Although the skewing of the polymorphism was not corrected by the normal control epidermis in the remaining 33 seborrheic keratosis cases, one of two HUMARA peaks practically disappeared after HhaI digestion in 20 of 33 seborrheic keratosis cases. In total, 25 of 38 seborrheic keratoses were considered to be monoclonal. The histologic type of seborrheic keratoses did not affect clonality.

Adult↗