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Biomedical subjects

H Itoh

Publications and source records attributed to H Itoh.

At least 667 records · Page 37Linked to original sources
PubMed1996

Immunohistochemical detection of K-sam protein in stomach cancer.

The K-sam gene, originally isolated as an amplified gene from the stomach cancer cell line KATO-III, is characterized by its preferential amplification in the undifferentiated type (diffuse type) of stomach cancer and encodes one of the receptors for heparin-binding growth factors or fibroblast growth factors. The K-sam gene has been isolated by different methods and has been designated BEK, TK14, and Cek2. The receptor for keratinocyte growth factor was also found to be encoded by the same gene. To examine the expression of the K-sam protein in stomach cancer, polyclonal antibody pK1-2 was raised against the extracellular domain of the gene product. This antibody detected K-sam proteins by Western blot and flow cytometry analyses in stomach cancer cell lines KATO-III and HSC39, in which the K-sam gene is amplified and overexpressed. By immunohistochemical analysis, 20 of 38 cases of the undifferentiated type of advanced stomach cancer were K-sam positive, whereas none of 11 cases of the differentiated or intestinal type revealed K-sam staining. The K-sam product was observed predominantly in diffusely infiltrative lesions. In one autopsy case, the K-sam protein was detected only focally in the primary tumor, whereas markedly increased staining for the K-sam product was detected diffusely in the metastasized tumor in the lymph node and liver. These results suggest that K-sam overexpression is associated with the malignant phenotype of the undifferentiated type of stomach cancer, such as infiltrative growth and metastasis.

Amino Acid Sequence↗
PubMed1995

Cloning of the cDNA encoding a novel rat mast-cell proteinase, rMCP-3, and its expression in comparison with other rat mast-cell proteinases.

A cDNA encoding a novel rat mast-cell proteinase (MCP) named rMCP-3 was successfully cloned and sequenced from the peritoneal cells of Lewis rats infected with the intestinal nematode Nippostrongylus brasiliensis by using the combination of reverse transcription-PCR and rapid-amplification-of-cDNA-ends ('RACE') methods. The cDNA was 979 bp long and included a 741 bp open reading frame. When the deduced amino acid sequence was compared with those of other known mast-cell proteinases, rMCP-3 was considered to be translated as a preproenzyme with a 19-amino-acid signal peptide, a two-amino-acid activation peptide and a 226-amino-acid mature enzyme. The amino acid identity in the mature enzyme was 52.9% and 55.1% with rMCP-1 and rMCP-2 respectively. The rMCP-3 mRNA was not detected in the peritoneal cells of mast-cell-deficient Ws/Ws rats, though it was strongly detected in those of littermate +/+ and Lewis rats, indicating the mast-cell origin of rMCP-3 In addition to being present in peritoneal mast cells, the rMCP-3 mRNA was strongly detected in the skin, tongue, and RBL2H3 rat basophilic leukaemia cells and weakly in the jejunum of N. brasiliensis-infected rats by RNA blot analysis using a rMCP-3 gene-specific probe. By reverse transcription-PCR, the rMCP-3 mRNA was also detected in the lung. While the expression of rMCP-1 and rMCP-2 are clearly restricted in connective-tissue mast cells and mucosal mast cells respectively, rMCP-3 was widely expressed in both types of mast cells with a predominance in connective-tissue mast cells.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of mouse intestinal trefoil factor and its expression during goblet cell changes.

A cDNA encoding mouse intestinal trefoil factor (mITF) was successfully cloned and sequenced from the small intestine of C57BL/6 mouse by using the combination of reverse transcription-PCR and rapid amplification of cDNA ends methods. The gene was, similar to rat and human ITFs, mainly expressed in the small and large intestine. The mITF expression was up-regulated during the recovery phase after depletion of goblet cells in acetic acid-induced colitis. On the other hand, the expression in the jejunum was not altered, while goblet cell hyperplasia was induced by Nippostrongylus brasiliensis infection. These results suggest that the mITF expression did not simply correlate with the number of goblet cells. The mITF may play an important role in the maintenance and repair of mucosal function of the rectum. Additionally, the mITF in the jejunum may play a role in alteration of the physicochemical nature of goblet cell mucins, thereby affecting the establishment of intestinal helminths.

Acetates↗
PubMed1995

E-cadherin and urokinase-type plasminogen activator tissue status in gastric carcinoma.

BACKGROUND: E-cadherin (ECD) is known to be an invasion suppressor gene, and urokinase-type plasminogen activator (uPA) plays a central role in infiltration of solid cancers. METHODS: To elucidate the relationship between expression of these factors and metastasis in patients with gastric cancer, the authors examined immunohistochemically a combination analysis of uPA and E-cadherin expression in 98 primary tumors, and the results were correlated with several parameters related to metastasis. RESULTS: Among 125 tumors, 42 (34%) were evaluated as having E-cadherin expression (E-cadherin-positive), and the other 83 (66%) were defined as having reduced E-cadherin expression (E-cadherin-negative). uPA immunoreactivity was observed in 82 tumors (66%). There were four subtypes of patterns of uPA and E-cadherin expression: 22 uPA-negative/E-cadherin-positive, 17 uPA-negative/E-cadherin-negative, 21 uPA-positive/E-cadherin-positive, and 65 uPA-positive/E-cadherin-negative, uPA overexpression and reduced E-cadherin expression were associated with lymph node metastasis, vessel invasion, serosal involvement, and poor prognosis. In addition, uPA-positive/E-cadherin-negative tumors were associated significantly with large tumors, positive serosal invasion, lymph node involvement, and poor prognosis. Patients with uPA-positive/E-cadherin-negative expression had the poorest prognoses, compared with the three other groups of patients uPA-positive/E-cadherin-negative tumors had a fourfold relative risk of death when compared with uPA-negative/E-cadherin-positive tumors. A Cox proportional hazard model projected lymph node status as the strongest of the prognostic variables followed by DNA ploidy patterns and uPA/E-cadherin tissue status. CONCLUSIONS: These results indicate that immunohistochemical combination analysis of uPA and E-cadherin expression may be a powerful aid in evaluating metastatic potential or the prognosis of patients with gastric cancer.

Adult↗
PubMed1995

Isoenzymes of cyclic nucleotide phosphodiesterase in the human aorta: characterization and the effects of E4021.

In extracts of the human aorta, five isoenzymes of cyclic nucleotide phosphodiesterase, namely, phosphodiesterase I, phosphodiesterase II, phosphodiesterase III, phosphodiesterase IV and phosphodiesterase V, were identified exclusively in the cytosolic fraction, and no phosphodiesterase activity was detected in the particulate fraction. Phosphodiesterase V and phosphodiesterase I were the major cGMP-hydrolyzing enzymes in the human aorta. A novel vasorelaxant, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl ]piperidine-4- carboxylate sesquihydrate (E4021), relaxed prostaglandin F2 alpha-precontracted strips of human pulmonary artery with an ED50 value of 0.5 microM. E4021 potently and highly selectively inhibited the activity of phosphodiesterase V from human aorta with a Ki value of 2.4 nM. These results suggest that there is a unique distribution of phosphodiesterase isoenzymes in the human aorta and that inhibitors of phosphodiesterase V might be useful as a new type of vasodilator in the treatment of clinical disorders.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction.

BACKGROUND: We have demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone predominantly synthesized in and secreted from the ventricle. We have also reported that, compared with atrial natriuretic peptide (ANP), the plasma concentration of BNP is increased to a greater degree in patients with congestive heart failure and more rapidly in patients with acute myocardial infarction (AMI). METHODS AND RESULTS: To investigate ventricular gene expression of BNP in AMI, we analyzed plasma and ventricular BNP concentrations along with ventricular BNP mRNA in rats with AMI produced by coronary artery ligation. The BNP concentration in the left ventricle increased about 2-fold as early as 12 hours postinfarction and 5-fold 1 day postinfarction compared with sham-operated rats, whereas left ventricular ANP concentration remained unchanged within 1 day. The tissue concentration of BNP increased in the noninfarcted region as well as in the infarcted region. The surviving myocytes in and around the necrotic tissues in the infarcted region were intensely stained with the anti-BNP antiserum, indicating augmented production in the remaining myocytes in the infarcts. The BNP concentration in the right ventricle also increased about 10-fold 12 hours postinfarction, whereas the ANP concentration remained unchanged within 12 hours. Northern blot analysis revealed that BNP mRNA expression was augmented 3-fold in the left ventricle as early as 4 hours postinfarction. In contrast, ANP mRNA expression was unchanged. Reflecting the rapid induction of ventricular BNP production, the plasma BNP concentration rose to about 100 pg/mL 12 hours postinfarction (sham-operated rats, < 70 pg/mL). CONCLUSIONS: These results demonstrate the rapid induction of ventricular BNP gene expression in rats with AMI compared with ANP and suggest that BNP gene expression in the ventricle is regulated distinctively from ANP gene expression against acute ventricular overload. They also suggest that the BNP gene can be one of the acutely responsive cardiac genes for the ventricular overload and suggest a possible pathophysiological role of BNP distinct from ANP in AMI.

Animals↗
PubMed1995

Carboxyl terminal of G protein beta subunit is required for association with gamma subunit.

To investigate a complex formation of heterotrimeric G protein beta and gamma subunits, we constructed a deletion mutant which lacked the C-terminal WD40 repeating unit of beta 1 and cotransfected into HEK293 cells with gamma 2 which was tagged with FLAG-epitope. Co-immunoprecipitation assay indicated that the mutant beta 1 failed to associate with FLAG gamma 2. Furthermore, from the analysis of additional deletion mutants, the last 10 amino acids of C-terminal region of beta 1 were found to be necessary to associate with gamma 2. These results suggest that the C-terminal region, in addition to the N-terminal alpha-helical structure, of beta subunit may be involved in the beta gamma complex formation.

Amino Acid Sequence↗
PubMed1995

The complete amino acid sequence of chum salmon stanniocalcin, a calcium-regulating hormone in teleosts.

Stanniocalcin (STC) is a hypocalcemic hormone secreted from the corpuscles of Stannius of bony fish. Chum salmon STC was isolated in pure form by ion-exchange chromatography and reversed phase high performance liquid chromatography following ethanol-ammonium extraction of the tissues. A cDNA was cloned from cDNAs of the tissue by the PCR method using two primers corresponding to the N- and C-terminal amino acid sequence of the hormone. Sequence analysis of the protein and the cDNA revealed that chum salmon STC is a homodimer, and that the monomer consists of 179 amino acids including 11 half-Cys residues and one N-linked glycosylation site, which is 44 residues smaller at the C-terminal region than the sequence deduced from coho salmon STC cDNA.

Amino Acid Sequence↗
PubMed1995

Young borderline hypertensives are hyperreactive to mental arithmetic stress: spectral analysis of R-R intervals.

To investigate whether sympathetic tone and its reactivity to stress are increased in borderline hypertension, we compared pressor and autonomic nervous responses to mental arithmetic stress in male borderline hypertensives (BH) and normotensive volunteers (NT). Three age groups, 30, 40 and 50-year-old groups, which included 30 to 39, 40 to 49 and 50 to 59-year-old subjects, were studied. Fractional LF (%LF), fractional HF (%HF) and L/H, obtained from the power spectrum of R-R intervals, were used as indices of autonomic nervous function. Baseline autonomic nervous indices did not differ between NT and BH of any age group. Blood pressure rose higher during mental arithmetic stress in 50 than in 30-year-old NT but not in comparable age groups of BH. Pressor responses were augmented in BH compared to NT only in the 30-year-old group. However, the differences were not significant when pressor responses were expressed as percent increases in blood pressure. Both %L and L/H increased during arithmetic stress. The increase in %L did not differ between NT and BH but that in L/H (% delta L/H) was larger in 50 than in 30-year-old NT. % delta L/H was larger in BH than in NT only in the 30-year-old group. These findings suggest that both pressor and autonomic nervous responses to metal arithmetic stress were altered by aging and augmented in BH compared to NT in the 30-year-old group.

Adult↗
PubMed1995

Cloning of the cDNA encoding mast cell tryptase of Mongolian gerbil, Meriones unguiculatus, and its preferential expression in the intestinal mucosa.

By using the combination of reverse-transcription PCR and rapid amplification of cDNA ends methods, a cDNA encoding mast cell tryptase was successfully cloned from the small intestine of Mongolian gerbil, Meriones unguiculatus, infected with Nippostrongylus brasiliensis. The cDNA was 1219 bp long including 810 bp of an open reading frame. Based on the deduced amino acid sequences of known mast cell tryptases of other species, the gerbil mast cell tryptase (gMCT) was highly similar to mouse mast cell protease (mMCP)-7, and seems to be translated as a prepro-enzyme with 25 amino acids of signal and activation peptides and 245 amino acids of mature enzyme. The gMCT mRNA was preferentially transcribed in the intestinal mucosa and to a far lesser extent in the connective tissue such as skin and tongue. Moreover, kinetic study after infection revealed that the amount of gMCT mRNA in the small intestine correlated well with the degree of intestinal mastocytosis. Throughout the course of infection, enzyme-histochemically detectable tryptase activity was limited to mucosal mast cells. Since mucosal mast cells of other rodents, including mice and rats, do not express tryptases, this is the first report of rodent mast cell tryptase expressed in the intestinal mucosa.

Amino Acid Sequence↗
PubMed1995

G protein beta gamma subunit activates Ras, Raf, and MAP kinase in HEK 293 cells.

Using transient transfection of HEK 293 cells, we have studied the activation of Ras, c-Raf, and MAP kinase by G protein-coupled receptors, activated G protein alpha subunit (G alpha), and beta gamma subunits (G beta gamma). The expression of constitutively activated Gs alpha, Gi alpha, and G11 alpha did not have any effect on MAP kinase phosphorylation. In contrast, overexpression of G beta gamma could stimulate the phosphorylation of MAP kinase and enhance the MEK kinase activity of c-Raf. Coexpression of dominant negative Ras inhibited G beta gamma-induced phosphorylation of MAP kinase. Furthermore, the GTP-bound form of Ras was increased by overexpression of G beta gamma. These results strongly suggest that the G beta gamma may play an important role in signaling from G protein-coupled receptors to the MAP kinase pathway, and the activation of Ras and c-Raf may be involved in this signaling cascade in HEK 293 cells.

Cell Line↗
PubMed1995

Mammalian 60-kDa stress protein (chaperonin homolog). Identification, biochemical properties, and localization.

Mammalian chaperonin homolog (HSP60) was purified from porcine livers cytosol using a tandem ATP-Sepharose column and Mono Q column chromatography. A partial amino acid sequence (96 amino acid residues) of this protein was determined and coincided with those of human HSP60 with 96.9% homology, which was deduced from the nucleotide sequence of the cDNA. The sequence of the NH2 termini of the purified protein (5 amino acid residues) coincided with the signal sequence of HSP60. These facts led to the identification of the 60-kDa liver protein with the chaperonin homolog. Dihydrofolate reductase was able to form a stable complex with the liver chaperonin homolog. The liver chaperonin homolog was detected by at least five spots around pI = 5.6 on two-dimensional gel electrophoresis. Immunoblotting studies using an antibody against chaperonin homolog showed that the chaperonin homolog was localized in the cytosol, mitochondrial, and nuclear fractions of porcine liver. The chaperonin homolog was localized both in the mitochondria and cytoplasm of rat kidneys at the electron microscopic level. The chaperonin homolog in the cytosol, but not in the other subcellular fractions, was cross-reacted with an antibody against the synthetic peptide corresponding to the signal peptide of HSP60 as well as the purified chaperonin homolog on immunoblotting. These results suggested that the functional chaperonin homolog in the cytosol may be transported into the mitochondria and the protein may be processed to mitochondrial HSP60 in the organella.

Amino Acid Sequence↗
PubMed1995

Structural organization and chromosomal assignment of the human prostacyclin receptor gene.

Prostacyclin receptor is a member of the prostanoid receptor family in the G protein-coupled receptor superfamily with seven transmembrane domains. We report here the isolation and structural organization of the human prostacyclin receptor gene. Southern blot analysis demonstrated a single copy of the human prostacyclin receptor gene in the human genome. The human prostacyclin receptor gene spanned approximately 7.0 kb and was composed of three exons separated by two introns. The first intron occurred in the 5'-untranslated region, 13 bp upstream to the ATG start codon. The second intron was located at the end of the sixth transmembrane domain, thereby separating it from the downstream coding region and the 3'-untranslated region. By primer extension analysis, the transcription initiation sites were mapped 870-872 bp upstream to the ATG start codon. The 1.2-kb human prostacyclin receptor 5'-flanking region lacked conventional TATA and CCAAT boxes, but it contained several cis-acting regulatory elements including an inverted CCAAT box (Y box) and two copies of SP-1 binding sites. Using human-rodent somatic hybrid cell DNA, the human prostacyclin receptor gene was assigned to human chromosome 19. The present study helps establish the genetic basis for prostacyclin receptor research and provides further insight into the molecular mechanisms underlying the prostanoid receptor family.

Amino Acid Sequence↗
PubMed1995

Activity in the human primary motor cortex related to arm and finger movements.

With the purpose of mapping representations of the finger and proximal arm in the human primary motor cortex (MI), we measured regional cerebral blood flow (rCBF) in eight right-handed normal volunteers during self-paced finger and proximal arm movements. Each movement activated two different fields in MI, one located deep in the anterior lip of the central sulcus and the other relatively close to the surface of the precentral gyrus. Arm fields were located higher than finger fields. A superficial arm field and a deep finger field partially, overlapped. The results indicate that there are two spatially separate motor representations of the finger and proximal arm in the human MI.

Adult↗
PubMed1995

Diol-bonded silica gel as a restricted access packing forming a binary-layered phase for direct injection of serum for the determination of drugs.

Direct serum injection for drug determinations can be achieved on a diol-bonded silica gel as a restricted access packing. The diol-bonded phase, 3-(2,3-dihydroxypropoxy)propylsilylsilica, contains two different functions, a hydrophilic function at the tip of the single chemical bond and a hydrophobic function on the inside part of the bond to form a "binary-layered phase" on the support surface. Proteins, as large molecules, contact only the hydrophilic surface of the diol phase, and they are eluted at the solvent front based on size-exclusion chromatography. On the other hand, small molecules such as synthetic drugs are retained on the internal hydrophobic function and separate based on reversed-phase chromatography. Accordingly, the diol-bonded silica gel performs as a restricted access packing for direct serum injection for the determination of relatively hydrophobic drugs.

Blood Chemical Analysis↗