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Biomedical subjects

K Ohkubo

Publications and source records attributed to K Ohkubo.

At least 73 records · Page 4Linked to original sources
PubMed1995

Localization of neutral endopeptidase (NEP) mRNA in human bronchi.

Neutral endopeptidase (NEP) may regulate peptide-induced inflammation in the respiratory tract. It is of interest to determine which respiratory resident cells express NEP. Trachea and bronchi from seven nonsmoking, nonasthmatic subjects were examined. NEP messenger ribonucleic acid (mRNA) was characterized by Northern blot hybridization of cultured human tracheobronchial epithelial and smooth muscle cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) in trachea and bronchi. In situ hybridization with biotin- and 35S-labelled antisense complementary ribonucleic acid (cRNA) probes was used to determine the distribution of NEP mRNA in human bronchial mucosa. NEP-immunoreactive material was detected using MEK10 murine monoclonal antibodies and the immunogold method with silver enhancement. NEP mRNA was 4.5 kb in size in the cultured human smooth muscle and epithelial cells by Northern blot analysis. No evidence was found by RT-PCR for truncated, alternatively spliced NEP mRNAs, such as del exon 16 or del exons 5-18 in human bronchus. NEP mRNA was detected by in situ hybridization in epithelial cells, submucosal glands, bronchial smooth muscle and endothelium. NEP-immunoreactive material was identified in the epithelium, submucosal glands, bronchial smooth muscle, and endothelium, demonstrating an excellent correlation between the distribution of NEP mRNA and the cell surface protein. NEP mRNA and immunoreactive material were excluded from epithelial goblet cell and submucosal gland mucous cell vacuoles. We conclude that the various sites of NEP protein and mRNA expression correlate with the locations of peptide receptors and NEP enzyme function, and are consistent with the hypothesis that NEP may regulate peptide-induced inflammation in human bronchi.

Base Sequence↗
PubMed1994

In vitro conversion of recombinant human proapolipoprotein A-I to apolipoprotein A-I.

We recently investigated the in vivo conversion of recombinant human proapolipoprotein A-I (rh-Met-proapo A-I) from E. coli to apolipoprotein (apo) A-I in rabbits. In vitro incubation of rh-Met-proapo A-I with rabbit serum produced mature apo A-I3 isoproteins, as determined by the immunoblotting method. However, at the time we were unable to chemically confirm a newly produced protein band which appeared at the position of human apo A-I3. Since then, we have confirmed the amino acid sequence of the protein using a membrane protein sequence technique, and have concluded that it corresponds to human apo A-I3.

Amino Acid Sequence↗
PubMed1994

Effect of recombinant human adult T cell leukemia-derived factor on rat lung reperfusion injury.

The formation of reactive oxygen species (ROS) is a major factor responsible for reperfusion injury in lungs. Adult T cell leukemia derived factor (ADF), a polypeptide made of 104 amino acids, is induced by a variety of stresses including X-ray, ultraviolet, H2O2, and mitogen. ADF has a reducing activity, which catalyzes the proton transfer between thiol-radical of cystein-containing proteins. Furthermore, ADF has a protective activity of ROS which are formed by xanthine oxidase and other alternative pathways in vitro. Using a rat in vivo model of lung ischemia, we examined the protective effect of recombinant human ADF (rhADF) against ischemia reperfusion injury of the lung. Ischemia, lasting for 75 min, was induced in the left lung of rats at 23 degrees C. The lung was then reperfused. These animals were divided into two groups: group 1 (n = 6, treatment with normal saline) and group 2 (n = 6, treatment with 28 micrograms/g of rhADF). One minute after the beginning of reperfusion, arterial oxygen tension (PaO2) decreased significantly in both groups (p < 0.01), without any significant intergroup difference (55.5 +/- 9.8, 49.8 +/- 8.6 mm Hg, respectively). Twenty minutes after reperfusion, PaO2 was significantly higher (p < 0.05) in group 2 (113.0 +/- 8.1 mm Hg) than in group 1 (72.3 +/- 13.6 mm Hg). The wet/dry weight ratio was significantly higher in group 1 (7.31 +/- 0.54) than in group 2 (5.82 +/- 0.36). Histologically, lung injury tended to be milder in group 2 than in group 1. These results suggest that rhADF has a protective effect against ischemia reperfusion injury of the rat lung.

Animals↗
PubMed1994

Angiotensin-converting enzyme in the human nasal mucosa.

Angiotensin-converting enzyme (ACE; EC 3.4.15.1) may participate in respiratory inflammatory diseases by regulating levels of inflammatory peptides such as bradykinin. The presence of ACE in the human nasal mucosa and in nasal secretions was determined by immunohistochemistry, measures of enzyme activity, and immunoblot. ACE activity was significantly more abundant in the membrane-rich fraction than in the soluble cytosolic fraction of nasal mucosal extracts (74.18 +/- 24.50 versus 3.99 +/- 1.83 pmol/min/mg protein, respectively, P < 0.01 by an enkephalin degradation assay; 89.16 +/- 16.17 versus 2.30 +/- 0.89 mU/mg protein, P < 0.01 by colorimetric assessment of Bz-Gly-Gly-Gly degradation). Topical application of histamine stimulated secretion of ACE activity into nasal lavage fluid (2.90 +/- 0.88 versus 1.53 +/- 0.45 U/liter after saline provocation, P < 0.05 by Bz-Gly-Gly-Gly assay). Allergen challenge also induced nasal secretion of ACE. In both histamine and allergen challenges, ACE release correlated closely with that of the vascular proteins IgG and albumin. Methacholine, a stimulant of glandular secretions, failed to augment ACE levels above baseline. ACE-immunoreactive material was localized by the immunogold technique with silver enhancement to the glycocalyx, between epithelial cells, and to interstitial, extracellular sites in the superficial lamina propria, with the highest intensity of staining immediately beneath the basement membrane. Some ACE was detectable in the mucus material of gland and duct lumens but not in gland cells themselves. Endothelial cells and some interstitial mononuclear cells also stained for ACE. ACE was identified by immunoblotting as a 150 kD band on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Immunological localization of neuropeptide-degrading enzymes in the nasal mucosa.

Neutral endopeptidase (NEP, EC 3.4.24.11), angiotensin-converting enzyme (ACE, EC 3.4.15.1) and carboxypeptidase N (CPN, EC 3.4.17.3) are potentially important enzymes which regulate the degradation of neuropeptides, such as bradykinin (BK) and substance P (SP), in the respiratory mucosa. Some neuropeptides are also degraded by these enzymes in vitro and in vivo. We investigated the localization of these enzymes in the human nasal mucosa by an indirect immunohistochemical technique (immunogold silver staining). NEP-immunoreactive areas were present in the epithelium, the serous cells of the submucosal glands, and the endothelial cells of small vessels. The epithelium and the serous cells were the predominant areas of NEP immunoreactivity in the nasal mucosa. ACE-immunoreactive areas were seen in the outer layer of the epithelium, the endothelial cells of vessels, and widely distributed in the superficial lamina propria. The endothelial cells of the vessels showed maximum positive intensity to ACE. CPN-immunoreactive areas were observed in the epithelium, the endothelium of vessels and the superficial lamina propria, except for the gland cells. The superficial lamina propria exhibited maximum immunoreactivity for CPN. We observed that the enzymes were widely distributed in the nasal mucosa. The epithelium, including the epithelial cells and glycocalyx, contains all three enzymes. These enzymes play an important role in the mucosal immunity of the respiratory mucosa by degrading active neuropeptides. These results show that NEP secretion is regulated by a glandular, cholinergic control. On the other hand, ACE and CPN secretion are regulated by vascular permeability.

Humans↗
PubMed1994

[Report of a case of small cell lung cancer associated with Lambert-Eaton myasthenic syndrome and subacute cerebellar degeneration--with a review of the Japanese literature].

We report a patient with small cell lung cancer associated with Lambert-Eaton myasthenic syndrome (LEMS) and subacute cerebellar degeneration (SCD). The patient was a 71-year-old man suffering from weakness of the limbs and a gait disturbance who developed limb ataxia and dysarthria one month after admission. Electrophysiologic studies confirmed the diagnosis of Lambert-Eaton myasthenic syndrome. Chest X-rays 2 months after admission revealed an abnormal shadow, and small cell lung cancer was diagnosed on the basis of biopsy specimens. Anti-voltage-gated calcium channel antibody was positive. Anti-Yo and -Hu antibodies were negative. The patient was treated by plasmapheresis and chemotherapy, which resulted in a transient improvement in the LEMS symptoms but not in the SCD. Fifteen cases of LEMS associated with SCD have been reported in the Japanese literature, and all were accompanied by small cell lung cancer. We discuss the frequency of association with LEMS and SCD and the effects of plasmapheresis and chemotherapy in both diseases.

Aged↗
PubMed1994

[Fluticasone propionate reduced the production of GM-CSF, IL-6 and IL-8 generated from cultured nasal epithelial cells].

Accumulation of mast cells and eosinophils in the nasal epithelial layer occurs in nasal allergic reactions and nasal polyps. We have already demonstrated that fluticasone propionate (FP) inhibits the accumulation of mast cells and eosinophils locally, and also improves the nasal symptoms of patients with allergic rhinitis. We hypothesized that cytokines generated from nasal epithelial cells possibly contribute to the accumulation of cells and eosinophils in the nasal epithelial layer. In this experiment we examined the inhibitory effect of FP on the production of GM-CSF, IL-6 and IL-8 by culturing of nasal epithelial cells in vitro. Our results show that FP significantly reduces the level of GM-CSF, IL-6 and IL-8 in the supernatant of culture media of nasal epithelial cells for a period of 6 days. In addition, preincubation of nasal epithelial cells with FP for 6 days causes a significant reduction of GM-CSF levels in the supernatant of culture-media of cultured nasal epithelial cells during the subsequent period of 6 days without FP. These results provide evidence that FP inhibits the accumulation of mast cells and eosinophils in the mucoepithelial layer of the nasal membrane.

Administration, Topical↗
PubMed1994

[Study of endothelin: distribution in the airway and release from nasal epithelial cells].

In order to clarify the existence and the role of endothelin in the respiratory tract, we investigated the distribution of endothelin in the respiratory mucosa by immunohistochemistry. The endothelin release from cultured nasal epithelial cells of allergic mucosa and polyp mucosa was also studied by RIA. Endothelin was distributed in the epithelium, endothelium of vessels, and submucosal glands in both nasal and bronchial mucosa. Moreover, in bronchial mucosa smooth muscles of bronchus had also positive staining of endothelin. On the other hand, 1.8 x 10(-11) pg of endothelin was released to culture medium from single cultured epithelial cell of allergic nasal mucosa, and 2.8 x 10(-11) pg of endothelin was released to culture medium from that of nasal polyp. These increase of endothelin was correlated with an increase of cultured epithelial cells from both nasal allergy mucosa and nasal polyp, of which the cell number was increased depend on the concentration of FCS. These results indicate that endothelin in the respiratory mucosa acts on not only control of blood flow and air flow, but also has some reaction which was related on the mucosal epithelium such as epithelial cilially movement. Endothelin has some role in inflammation because endothelin was increased more in nasal polyp culture medium than in allergic mucosa culture medium.

Cell Count↗
PubMed1993

In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) to apolipoprotein AI in rabbits.

In vivo conversion of recombinant human proapolipoprotein AI (rh-Met-proapo AI) from E. coli to apolipoprotein (apo) AI was investigated. rh-Met-proapo AI was labeled with 125I, and then administered intravenously to rabbits. Blood was sampled periodically for 6 days. The plasma decay curves of radioiodinated rt-Met-proapo AI were similar to those of human mature apo AI (fractional catabolic rate (FCR); 1.018 +/- 0.090/day vs. 0.976 1 0.031/day, respectively). In vivo conversion of rh-Met-proapo AI to mature apo AI was examined by autoradiography of the isoelectric focusing (IEF) slab gel, i.e., the HDL fraction from each sampling point was semiquantitatively applied to IEF. It was found that the radioactivity of rh-Met-proapo AI migrated to more acidic isoproteins, the conversion was complete within 24 h, and the FCR of rh-Met-proapo AI was 9.20 +/- 1.34/day. Although the plasma decay curves of both human pro (rh-Met-proapo AI) and mature apo AI were significantly steeper than those of rabbit mature apo AI4 and apo AI5 (FCR; 0.703 +/- 0.027/day and 0.795 +/- 0.031/day, respectively), the conversion rate of human rt-Met-proapo AI to mature apo AI in rabbit was assumed to be 1:1. In vitro incubation of rh-Met-proapo AI with rabbit serum produced mature apo AI isoproteins, as determined by the apo AI immunoblotting method. Prediction of the amino acid sequence at the NH2 terminus of rabbit proapo AI showed that the prosegment consisted of an alpha helix with a high probability of a beta turn at Pro9, which is close to that in humans. Thus, (1) the proteolytic cleavage of proapo AI is an extracellular event, (2) the converting enzyme in rabbits can also process human proapo AI, (3) this converting enzyme does not specifically and directly attack the Gln6-Asp7 bond which links the carboxyl-terminal residue of the hexapeptide to the amino-terminal residue of human mature apo AI. The conformation of proapo AI at the NH2 terminus (alpha helix of the prosegment and a beta turn at Pro9) may have a key role in this cleavage, and (4) the examination of rh-Met-proapo AI in rabbits helps to explain the early events of HDL biogenesis.

Amino Acid Sequence↗
PubMed1993

Mast cell quantitation in nasal polyps, sinus mucosa and nasal turbinate mucosa.

The distribution and abundance of mast cells in nasal polyps, the maxillary sinus mucosa of patients with sinusitis and the turbinate mucosa of allergic rhinitis was microscopically examined using different methods of fixation. In the epithelium of the surface and the ducts of nasal polyps (n = 8), the mean number of mast cells was over 20,000 per mm3 using Mota's fixation and the increase was correlated with the epithelial thickness (P < 0.05). On the other hand those of the maxillary sinus mucosa (n = 6) and the nasal turbinate mucosa (n = 7) were less than 6,000 per mm3. In the subepithelial layer or areas deeper than the area with the glands, however, mast cell counts were less than 3,200 per mm3 in all diseases. More than 70-90 per cent of all mast cells in the epithelium of the mucosal surface and the ducts of the polyp, the maxillary sinus mucosa and nasal turbinates were formalin sensitive. Most of the mast cells in the subepithelial and deeper areas were formalin resistant in all diseases. These results suggest that conditions for mast cell growth differ between polyps and the other diseases, and that the conditions which affect mast cells may contribute to polyp development.

Adolescent↗
PubMed1993

Development and evaluation of a needle for percutaneous ethanol injection therapy.

To avoid the disadvantages associated with use of the traditional percutaneous transhepatic cholangiography (PTC) needle in ultrasonically guided percutaneous ethanol injection therapy (PEIT) for hepatocellular carcinoma, the authors developed a needle with a three-sided tip and three holes within 3 mm of the tip. In comparison with a PTC needle, the PEIT needle passed into the liver along a straighter line with less resistance and reflected a higher echo from the tip.

Ethanol↗
PubMed1993

Identification of neutral endopeptidase mRNA in human nasal mucosa.

Neutral endopeptidase (NEP) may regulate peptide-induced inflammation in the upper respiratory tract. NEP mRNA was detected by Northern blotting in poly(A)+ mRNA prepared from human turbinates. NEP mRNA bands were detected at 3.9 and 1.8 kb. In situ hybridization identified NEP mRNA in epithelial cells, serous cells of submucosal glands, and vessel walls. The vacuoles of goblet and mucous cells were not stained, but it is likely that the cytoplasm of these cells contained some NEP mRNA. Radioactive in situ hybridization with 35S-UTP appeared to be more sensitive than nonradioactive in situ hybridization with biotin-UTP and immunogold detection. The NEP mRNA-containing cells identified by in situ hybridization were the same as those identified by immunohistochemistry to contain immunoreactive NEP. These NEP-containing cells have been previously shown to possess peptide receptors. This is consistent with the hypothesis that NEP on cells bearing peptide receptors may regulate neuropeptide-induced inflammation in human nasal mucosa.

Biotin↗
PubMed1993

Nasal secretion of the ozone scavenger uric acid.

Uric acid, an important scavenger of ozone, has been identified as the major low molecular weight antioxidant in baseline and cholinergically induced nasal secretions. The purpose of this study was to determine the specific tissue source of uric acid in airway secretions. The secretion of uric acid is increased by cholinergic stimulation and correlates closely with the secretion of lactoferrin (a nasal glandular protein), suggesting that submucosal glands are involved. Indeed, nasal turbinate tissue was found to contain uric acid. However, careful analysis of nasal turbinate tissue failed to reveal the presence of xanthine oxidase, the enzyme responsible for uric acid synthesis. These data suggest that uric acid might be taken up secondarily by glands from plasma. This possibility was strengthened by the observation that lowering the plasma urate level with probenecid concomitantly lowered urate secretion. These findings are consistent with the hypotheses that the principal source of uric acid in nasal secretions is plasma and that uric acid is taken up, concentrated, and secreted by nasal glands.

Albumins↗
PubMed1993

Endothelin in human nasal mucosa.

Endothelin (ET), a potent vasoconstrictor and bronchoconstrictor peptide synthesized by endothelial and epithelial cells, was examined for its potential functions in human inferior turbinate nasal mucosal tissue by four techniques: (1) immunoreactive ET was localized in the mucosa by immunohistochemistry; (2) receptors for ET were identified by autoradiography employing [125I]ET; (3) ET-1 mRNA was localized by in situ hybridization; and (4) the secretory functions of ET were examined by the release of mucous and serous cell products after the addition of ET to human nasal turbinates in short-term cultures. Specific ET-1-immunoreactive material was found most extensively in small muscular arteries and in serous cells in submucosal glands. ET-1 was also found to a lower extent in the walls of venous sinusoids. [125I]ET-1 binding sites were localized by autoradiography to submucosal glands and to venous sinusoids and small muscular arterioles. mRNA for ET-1 was found most extensively in the venous sinusoids and to a lesser extent in small muscular arteries. In mucosal explant cultures, ET-1 and ET-2 stimulated lactoferrin and mucous glycoprotein release from serous and mucous cells, but ET-3 was inactive. The observations indicate that in the human nasal mucosa, ET is present in the vascular endothelium and the serous cells in submucosal glands and acts on glandular ET receptors to induce both serous and mucous cell secretion. It is also likely that ET plays a role in the regulation of vasomotor tone.

Binding Sites↗
PubMed1993

Human nasal mucosal neutral endopeptidase (NEP): location, quantitation, and secretion.

Neutral endopeptidase (E.C.3.4.24.11, enkephalinase, NEP) is a potentially important enzyme capable of regulating the activity of neuropeptides released in the respiratory mucosa. In order to confirm the existence of NEP in the human respiratory mucosa, inferior nasal turbinate mucosae obtained at surgery and nasal secretions induced by topical provocations with methacholine, histamine, and allergen were analyzed for: (1) NEP activity (pmol product/min/ml) by enzymatic degradation of [3H]leu-enkephalin, (2) the presence of NEP-immunoreactive material by Western blot analysis, and (3) cellular localization of NEP distribution by immunohistochemistry. NEP activity in human nasal secretions obtained after normal saline challenge was 0.15 +/- 0.06 pmol/min/ml. Secretion increased to 0.86 +/- 0.26 pmol/min/ml after methacholine provocation and 1.69 +/- 0.74 pmol/min/ml after histamine provocation. The increase in NEP activity in methacholine-induced secretions was prevented by atropine (0.13 +/- 0.06 pmol/min/ml). After methacholine, histamine, and antigen nasal provocation, the kinetics of NEP appearance correlated more closely to the glandular marker, lactoferrin, than with the vascular markers albumin and IgG. In homogenates of nasal mucosa, the membrane fraction contained significantly more NEP on a per mg protein basis than did the soluble fraction (227.6 +/- 50.52 versus 9.61 +/- 3.18 pmol/min/mg protein, respectively, P < 0.01, n = 6). NEP in the membrane fraction was detected as a single band migrating at 97 kD on Western blots using antibodies specific for NEP and the common acute lymphoblastic leukemia antigen (CALLA). Immunoreactive NEP was localized to serous cells of the submucosal glands, epithelial cells, and endothelial and myoepithelial cells of small vessels. Staining for NEP in the serous cells was of the same intensity as that in epithelial cells. These results indicate that 97 kD NEP-immunoreactive material exists in discrete locations in the nasal mucosa, including the epithelium, serous cells of the submucosal glands, and vessel walls, and that NEP activity is detected as a minor component in nasal secretions enriched by glandular products. In addition to the modulating functions of NEP on neuropeptide-mediated activities on vessels and glands, it is possible that NEP in secretions plays a role in regulating mucosal responses to luminal neuropeptides or other as yet uncharacterized NEP substrates.

Blotting, Western↗
PubMed1992

Portal hypertensive gastropathy in patients with cirrhosis.

Portal hypertensive gastropathy is a recently recognized important complication of cirrhosis. In the present study, the clinical features, portohepatic hemodynamics, and hepatic function were investigated in a series of 47 patients with cirrhosis. Mild gastropathy was found in 15 patients (32%) and severe gastropathy in 17 patients (36%). The presence of gastropathy seemed to be independent of age, sex, cause of cirrhosis, or grade of gastroesophageal varices. However, severe gastropathy was associated with an increase in portal venous pressure gradient (vs. control, P less than 0.01; vs. mild gastropathy, P less than 0.01), an increase in hepatic sinusoidal resistance (vs. control, P less than 0.01; vs. mild gastropathy, NS), and a decrease in hepatic blood flow (vs. control, P less than 0.01; vs. mild gastropathy, NS). In addition, patients with severe gastropathy had impaired metabolic activity of the liver, which was assessed by intrinsic clearance of indocyanine green (vs. control, P less than 0.01; vs. mild gastropathy, NS). These observations may have important therapeutic implications in patients with cirrhosis and portal hypertensive gastropathy.

Aged↗
PubMed1992

Collagenous deposits on explanted intraocular lenses.

An immunohistochemical study of type I collagen in deposits on the surface of two intraocular lenses (IOLs) explanted from human eyes was conducted. Type I collagen-immunoreactive proteinaceous deposits with cells were found around the haptics of an iris-supported IOL. A few such deposits and what appeared to be macrophages were observed on the optic. A few cells (presumably macrophages and giant cells) were observed on a posterior chamber IOL, whereas proteinaceous deposits that reacted positively to the antibody were not identified. Type I collagen-immunoreactive deposits on the iris-supported IOL were thought to be the products of fibroblastic cells, originating from iris tissue, that attached directly to the haptics and helped stabilize the implant.

Aged↗