Biomedical subjects
Publications and source records attributed to K Ohkubo.
We present a patient with superior vena cava (SVC) obstruction in whom two areas of increased hepatic enhancement within the left lobe were seen on abdominal computed tomography (CT). The significance of this case is that abnormal enhancements of the liver on abdominal CT in the regions described should be suggestive of an SVC obstruction on this basis alone.
The transcriptional activator CooA from Rhodospirillum rubrum contains a b-type heme that acts as a CO sensor in vivo. CooA is the first example of a transcriptional regulator containing a heme as a prosthetic group and of a hemeprotein in which CO plays a physiological role. In this study, we constructed an in vivo reporter system to measure the transcriptional activator activity of CooA and prepared some CooA mutants in which a mutation was introduced at Cys, His, Met, Lys, or Tyr. Only the mutations of Cys75 and His77 affected the electronic absorption spectra of the heme in CooA. The electronic absorption spectra, EPR spectra, and the transcriptional activator activity of the wild-type and mutant CooA proteins indicate that 1) the thiolate derived from Cys75 is the axial ligand in the ferric heme, but it is not coordinated to the CO-bound ferrous heme; 2) Cys75 is protonated or displaced in the ferrous heme; and 3) His77 is the proximal ligand in the CO-bound ferrous heme and probably also in the ferrous heme, but it is not coordinated to the ferric heme. NMR spectra reveal that the conformational change around the heme, which will trigger the activation of CooA by CO, takes place upon the binding of CO to the heme.
In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme-containing transcriptional activator, the heme environmental structure and the dynamics of the CO rebinding and dissociation have been examined in the absence and presence of its target DNA. In the absence of DNA, the Fe-CO and C=O stretching Raman lines of the CO-bound CooA were observed at 487 and 1969 cm-1, respectively, suggesting that a neutral histidine is an axial ligand trans to CO. The frequency of nu(Fe-CO) implies an open conformation of the distal heme pocket, indicating that the ligand replaced by CO is located away from the bound CO. When the target DNA was added to CO-bound CooA, an appearance of a new nu(Fe-CO) line at 519 cm-1 and narrowing of the main line at 486 cm-1 were observed. Although the rate of the CO dissociation was insensitive to the additions of DNA, the CO rebinding was decelerated in the presence of the target DNA, but not in the presence of nonsense DNA. These observations demonstrate the structural alterations in the heme distal site in response to binding of the target DNA and support the activation mechanism proposed for CooA, which is triggered by the movement of the heme distal ligand to modify the conformation of the DNA binding domain.
BACKGROUND: Aminopeptidases activate bradykinin and degrade many inflammatory peptides. OBJECTIVE: The objective of this study was to identify the types of aminopeptidase activities in human nasal mucosa. METHODS: Human nasal mucosa was homogenized (n = 12), and cytoplasmic (S2) and membrane-rich (P2) fractions were obtained. Several aminopeptidase (Ap) activities were defined by (1) substrate specificity with leucine-enkephalin (leu-Ap) and alanine-nitroanilide (ala-Ap), (2) inhibitor studies with puromycin and bestatin, (3) enzyme activity histochemistry (zymography), (4) immunohistochemistry, and (5) gel electrophoresis. Human volunteers had methacholine, histamine, and allergen nasal provocations to determine the mechanisms controlling nasal aminopeptidase secretion in vivo. RESULTS: P2 was the largest reservoir of puromycin-resistant aminopeptidase activity (630 pmol leu-enk/min/mg protein). S2 contained 32 pmol leu-enk/min/mg activity, with 80% representing puromycin-resistant activity and 20% puromycin-sensitive aminopeptidase (PS-Ap). Ala-Ap was detected in both P2 and S2 fractions and was localized by zymography to epithelial and gland cells. Anti-rat brain-soluble PS-Ap IgG detected immunoreactive material in epithelium, glands, and endothelium. In nasal provocation studies, leu-AP correlated with glandular exocytosis but not vascular leak. CONCLUSIONS: The predominant aminopeptidase in human nasal epithelial and submucosal gland cells was membrane-bound puromycin-resistant aminopeptidase. A novel soluble puromycin-resistant aminopeptidase and lower amounts of soluble PS-Ap were also detected.
Adult T-cell leukemia (ATL) is a peripheral T-cell neoplasm caused by human T-cell leukemia virus type I (HTLV-I). Despite the administration of combined intensive chemotherapy, the reported survival time of patients with acute and lymphoma types of ATL is less than 10 months. We therefore examine the effects of all-trans-retinoic acid (ATRA), 9-cis-RA and 13-cis-RA and tried to elucidate the mechanisms of inducing growth inhibition and apoptosis by these RAs using four ATL cell lines established in our laboratory. All the investigated RAs inhibited cell growth and the cells were arrested at the G1 phase. Apoptosis was induced in three out of four cell lines. Among the growth regulatory proteins examined, the level of p21Waf1/Cip1 protein was found to increase after RA treatment, thus resulting in pRb hypophosphorylation which also induced the arrest of the cells at the G1 phase. In addition, the p53 level decreased at the same time. Fas-FasL system and the downregulation of CD25 (IL-2R/alpha) expression did not seem to be involved. Based on these findings, the ability of RAs to induce a remission of ATL is thus strongly suggested.
The production of interleukin-8 (CINC: cytokine-induced neutrophil chemo-attractant) from different cell populations in the rat liver was studied and cells related to the initiation of CINC production in lipopolysaccharide (LPS)-injected endotoxaemic rats were characterized. Sinusoidal endothelial cells (16.4 +/- 10.6 ng/mL) produced significantly higher amounts of CINC in 24 h primary cultures compared with hepatocytes (0.9 +/- 0.9 ng/mL; P < 0.05) and Kupffer cells (6.5 +/- 5.1 ng/mL; P < 0.05). Lipopolysaccharide, tumour necrosis factor-alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha) stimulated different liver cell populations to produce CINC; LPS mainly stimulated Kupffer cells. TNF-alpha stimulated hepatocytes and IL-1 alpha stimulated all three types of cells. Intraperitoneal injection of LPS (4 mg/kg) caused CINC accumulation in non-parenchymal cells of the rat liver within 1 h of injection, as shown by immunohistochemical staining. In contrast, CINC-positive hepatocytes were not seen until 3 h after injection of LPS. Ethanol was not a direct inducer of CINC production by rat hepatocytes in vitro. These findings strongly suggest that non-parenchymal liver cells, including sinusoidal endothelial cells, are the main source of CINC. Our data also suggest that during endotoxaemia, CINC production is initiated by non-parenchymal cells and this is followed by production from hepatocytes.
N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.
To elucidate the presence and distribution of Endothelin-1 (ET-1) in tissues of human salivary glands, we performed an immunohistochemical analysis of ET-1 in 15 normal salivary glands, 8 adenolymphomas, 13 pleomorphic adenomas and 5 carcinomas, using the mouse monoclonal antibody against human ET-1. In normal glands, immunoreactivity for ET-1 was observed in the striated duct cells. In adenolymphomas, the columnar cells of the granular epithelium showed strong intracytoplasmic immunoreactivity. In carcinomas, moderate or strong immunoreactivity was observed in the tumor cells, whereas in pleomorphic adenomas, weak immunoreactivity was observed. A good relation was detected between the size of pleomorphic adenoma and ET-1 immunoreactivity, as well as between the duration of tumor in carcinoma and ET-1 immunoreactivity. The presence and distribution of ET-1 in salivary glands and salivary gland tumors suggests a possible role for ET-1 in the regulation of electrolytes and water transport in salivary glands, and as a growth-promoting factor for tumors.
Cytokines are potentially active biological peptides that are known to play an important role in several immune responses. Several studies have reported the existence of a variety of cytokines in the nasal mucosa of patients with allergic rhinitis. However, there are few reports on cytokines released into the nasal secretion. In the present study, we investigated the sources, and levels of three key proinflammatory cytokines namely, IL-6, IL-8, and GM-CSF in the nasal secretion, as well the mechanisms of their release, by ELISA and immunohistochemistry. Firstly, we examined the levels of IL-6, IL-8, and GM-CSF in the nasal secretion after in vivo nasal challenge with methacholine (MC), histamine (HI) and allergen (Ag) in patients with nasal allergy to house dust mite (HDMAR). Next, we examined the levels of IL-6, IL-8, and GM-CSF released, in vitro, after Ag challenge of nasal scrapings from patients with HDMAR. Finally, we examined the sources of these cytokines in the nasal mucosa, by immunohistochemistry. After MC challenge in patients with HDMAR, the concentration of IL-6, but not IL-8, and GM-CSF, was significantly greater on the challenged side than on the contralateral side. Ag and HI provocation induced significantly greater levels of IL-6 and IL-8 secretion in patients with HDMAR, on the challenged side than on the contralateral side. GM-CSF was only detected in the nasal secretion after Ag challenge. Immunoreactivity for IL-6 and IL-8 was very similar in that it was predominantly localised to the apical portion of epithelial cells, the superficial lamina propria, gland cells, and migrating cells. The immunoreactivity for GM-CSF varied slightly from that of IL-6 and IL-8: strong immunoreactivity was detected in the basal part of epithelial cells, basement membrane, glandular ducts, and migrating cells. These results suggest that the levels, sources, and mechanisms of release of IL-6, IL-8, and GM-CSF in the nasal secretion of patients with HDMAR do vary, but are important in the manifestation of the allergic reaction.
We constructed an in vivo reporter system to measure the activity of CooA as the transcriptional activator and showed that the recombinant CooA was active as the transcriptional activator in the presence of CO even in E. coli cells. A dominant positive mutant of CooA, in which Met131 was replaced by Leu, was isolated by a random mutagenesis with this reporter system. The electronic absorption spectra of M131L mutant were identical to those of wild type CooA in oxidized (Fe3+), reduced (Fe2+), and CO-bound (CO-Fe2+) state, indicating that the coordination structure and environment of the heme were not changed by this mutation. Methionine at position 131 was the carboxyl-terminal end of the heme-binding domain of CooA, which would be adjacent to the hinge region connecting the heme-binding domain and the DNA-binding domain.
We identified a novel gene, kf-1, highly expressed in the normal cerebellum but not in the cerebral cortex, the expression of which could have been augmented in the cerebral cortex of a sporadic Alzheimer's disease patient. We cloned human and mouse entire kf-1 cDNAs encoding conserved 79 kDa proteins containing a zinc-binding RING-H2 finger motif at the carboxy-terminus as found in acetylcholine receptor-associated protein (RAPsyn). The 3'-untranslated regions are highly conserved between human and mouse as to constitute a common mRNA secondary structure. In situ hybridization analysis of mouse brain sections revealed strong kf-1 expression in the cerebellum and hippocampus. We propose that KF-1 is involved in membranous protein-sorting apparatus similarly to RAPsyn. We mapped the human kf-1 gene to 2p11.2.
The aim of this study was to compare the hemodynamic effects of three nitric oxide (NO) donors [i.e., (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409), (+/-)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-6-methyl-5-nitro-3-he ptenyl]-3-pyridinecarboxamide (FR 146801) and isosorbide dinitrate (ISDN)] in rats. In in vitro experiments, FK409 had a higher spontaneous NO-releasing rate in solution and more potent vasorelaxant activity in isolated rat aorta than other drugs. FR146801 and ISDN showed almost the same vasorelaxant activity. In in vivo experiments, FK409 significantly decreased hematocrit at 1.0 mg/kg p.o., whereas FR146801 and ISDN significantly decreased it at 10 mg/kg p.o., suggesting that these NO-donating agents cause significant plasma volume expansion. However, only FK409 showed significant hypotensive effects immediately after oral administration even at 0.32 mg/kg; FR146801 and ISDN did not cause any significant hypotension at 10 mg/kg, suggesting that FK409 induces much more potent arterial vasodilation than other drugs. These findings suggest that NO donors induce significant plasma volume expansion and that the differences in the selectivities between these effects and their hypotensive effects is probably produced by their different NO-releasing activities.
Peripheral schwannomas are nerve sheath neoplasms that consist of focal proliferation of Schwann cells. We reviewed the MRI findings in 17 patients with pathologically proved peripheral schwannomas. When compared with the signal intensity of muscle, that of the mass was isointense or hyperintense on T1-weighted images and hyperintense in all 17 tumors on T2-weighted images. All of the masses showed heterogeneous enhancement following the intravenous injection of Gd-DTPA on T1-weighted images. On T1-weighted images, hyperintensity was observed in the tumors that contained predominantly hypercellular Antoni type A tissue, while isointensity was observed in the tumors that contained predominantly hypocellular Antoni type B tissue. Relatively high signal intensity seen on T2-weighted images and Gd-DTPA enhanced T1-weighted images was observed in the tumors that contained predominantly Antoni type B tissue when compared with the signal intensity of tumors that contained predominantly Antoni type A tissue. A capsule was pathologically identified in 15 of 17 tumors. MRI correctly identified the presence of a capsule in 11 of 15 tumors and the absence of a capsule in one of 2 tumors. Thus the diagnostic accuracy was 71% (12/17). The cause of 4 false negative results appeared to be a hemorrhage or cystic change around the peripheral portion of the tumor, and it appeared to be a chemical artifact in one false positive result. Thus the appearance of MRI may suggest the cellular type of schwannoma, Antoni type A or B. However, prediction of the presence or absence of tumor capsule may be relatively difficult with MRI.
A 45-year-old woman was incidentally suspected to have megacolon. Chest X-rays showed elevated left diaphragm due to colonic gas, and the heart was deviated to the midline. Barium enema revealed marked dilation of the sigmoid colon, confirming the diagnosis of megacolon. Maximal diameter of the sigmoid colon was 23 cm, but she had no gastrointestinal symptoms. During the work up for megacolon, the presence of myotonic dystrophy was suspected. She had hatchet face, but was not bald. Muscles of the neck and extremities were slightly atrophic. There was percussion myotonia of the tongue and both hands, and grip myotonia of the hands. Laboratory examinations showed impaired glucose tolerance and low level of serum IgG. EMG showed myotonic discharges and myopathic units in the limbs. Brain CT imaging revealed a thick skull. Cases of myotonic dystrophy associated with marked megacolon are rare in Japan. Megacolon presents a high risk for ileus, volvulus, and rupture, and myotonic dystrophy is associated with a high operative and anesthesic risk. Megacolon, therefore, is an important complication to look for in the management of myotonic dystrophy.
Percutaneous vertebroplasty is a radiologically guided therapeutic procedure that consists of percutaneous injection of surgical cement into a vertebral osteolytic metastatic lesion. This procedure has the advantage of providing rapid pain relief and bone strengthening. We report a 46-year-old male patient with osteolytic L3 vertebral metastasis from lung adenocarcinoma treated by percutaneous vertebroplasty under CT and fluoroscopic guidance. After this treatment, definite pain relief and stabilization of the vertebra were achieved. Vertebroplasty for osteolytic metastasis may be a minimally invasive procedure that provides immediate, long-term pain relief and contributes to spinal stabilization.
We examined whether p.o. biological activities of (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409), a spontaneous nitric oxide (NO) releaser, and the derivatives, i.e., (+/-)-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-5-nitro-3-hexenyl]-3 - pyridinecarboxamide (FR144420) and (+/-)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-6-methyl-5- nitro-3-heptenyl]-3-pyridinecarboxamide (FR146801), could be accounted for by their NO-releasing rates and p.o. absorption manners. These compounds spontaneously released NO with the rates in the rank order of FK409 > FR144420 > FR146801. Total contribution of these drugs as NO donors in vivo was almost the same from the determination of urinary nitrite/nitrate (NOx) levels after p.o. administration of the compounds at 10 mg/kg to rats. Plasma NOx level after p.o. administration of FK409 at 10 mg/kg to rats reached maximal level at 120 min, and decreased gradually. On the other hand, plasma NOx levels time-dependently increased during 360 min after p.o. administration of FR144420 and FR146801 at the same dose. FK409 and FR144420 showed hypotensive effects immediately after p.o. administration at 10 mg/kg to rats, and the maximum response of FR144420 was less and the duration of the effect was longer than those of FK409, respectively. On the other hand, FR146801, which is most stable in solution, did not show any significant hypotensive effect during 240 min after p.o. administration at the same dose. In conclusion, the response and the duration of biological activity after p.o. administration of three spontaneous NO releasers can be closely accounted for by their NO-releasing rates and p.o. absorption manners.
Explore the source record for details and available documents.
We report that (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409) decomposes and releases nitric oxide (NO) spontaneously in solution. (+/-)-N-[(E)-4-Ethyl-3-[(Z)-hydroxyimino]-5-nitro-3-hexen-1- yl]-3- pyridinecarboxamide (FR144420) was synthesized with the aim of discovering a compound with longer duration of effects in vivo, compared with FK409. FR144420, like FK409, released NO spontaneously in solution, but the amount of NO released from FR144420 during a 5-min incubation was half the amount from FK409. In addition, FR144420 spontaneously decomposed and generated nitrite, which is an oxidative metabolite of NO, at half the rate of FK409. In a vasorelaxant study with isolated rat aorta, FR144420 had a weaker potency than FK409 (EC50 = 54 and 8.1 nM, respectively). In in vivo studies, FR144420 decreased mean blood pressure immediately after intravenous and oral administration to conscious rats. The maximum hypotensive effects of FR144420 were less than those of FK409. However, the durations of FR144420-induced (i.v. and p.o.) hypotensive effects were longer than those of FK409-induced effects. In conclusion, FR144420 is more stable and releases NO more slowly in solution than does FK409. In in vivo experiments, FR144420 showed a longer duration of effects than FK409. FR144420 may be very useful for investigating the in vivo actions of NO.