Biomedical subjects

Y Okada

Publications and source records attributed to Y Okada.

At least 145 records · Page 8Linked to original sources

PubMed2001

Distribution and function of JCV agnoprotein.

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), encodes six major proteins including agnoprotein, the function of which is unknown. To explore its function, we initially studied the expression and localization of agnoprotein in both cultured cells and PML brain using immunohistochemical methods. Employing a specific polyclonal antibody, agnoprotein was found mostly in the cytoplasm of persistently infected JCI cells and in the finely elaborated cytoplasmic processes of oligodendroglial cells in PML brain. The immunohistochemistry indicated that the cytoplasm of oligodendroglial cells was relatively well-preserved in the demyelinated foci. Agnoprotein coprecipitated with tubulin in immunoprecipitation assays and the colocalization of agnoprotein with cytoplasmic tubulin was verified by double immunostaining with confocal microscopy. Transfection of an agnogene deleted JCV Mad1 strain [Mad1(Delta agno)] into the susceptible cell line failed to produce not only agnoprotein but also VP1 and large T mRNAs, whereas the wild-type JCV Mad1 resulted in the expression of both large T and VP1 mRNAs. The cytoplasmic agnoprotein was phosphorylated and when coexpressed with GST-EGFP, was also localized in the cytoplasm. Inhibition of protein kinase A by its inhibitor H-89, however, reversed the cytoplasmic localization of agnoprotein to the nuclear compartment. Our results suggest that JCV agnoprotein may "shuttle" between the nucleus and cytoplasm in a phosphorylation-dependent manner during viral replication.

Cell Line↗

PubMed2001

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

ATP-Binding Cassette Transporters↗

PubMed2001

Differential transduction mechanisms underlying NaCl- and KCl-induced responses in mouse taste cells.

The transduction mechanism of salt-induced responses of mouse taste cells was investigated using the patch clamp and the local stimulation techniques under quasi-natural conditions. Apically applied NaCl induced a voltage-independent current, which was partially suppressed by amiloride and Cd2+. In contrast, apically applied 0.5 M KCl induced an inwardly rectifying current (KCl-induced Iir). The KCl-induced Iir was unaffected by amiloride. The Iir was suppressed not only by external Ba2+ and Cs+, but also by a Cl- channel blocker, niflumic acid. The Er of the KCl-induced response was independent of the apical ionic concentration, but rather was close to the equilibrium potential of Cl- (E(Cl)) at the basolateral membrane. The KCl-induced Iir displayed a fast run-down under the conditions of the conventional whole cell clamp method, but not under the perforated patch conditions. Immunohistochemical localization of an inwardly rectifying Cl- channel protein, ClC-2, was observed in taste bud cells of the fungiform papillae. It is concluded that the transduction mechanism of NaCl-induced responses is completely different from that of KCl-induced responses in mouse taste cells.

Amiloride↗

PubMed2001

Non-synaptic transformation of gustatory receptor potential by stimulation of the parasympathetic fiber of the frog glossopharyngeal nerve.

When the glossopharyngeal nerve (GP) in the frog was strongly stimulated electrically, slow potentials were elicited from the tongue surface and taste cells in the fungiform papillae. Injection of atropine completely blocked these slow potentials. The present and previous data indicate that the slow potentials induced in the tongue surface and taste cells are due to a liquid junction potential between saliva secreted from the lingual glands due to parasympathetic fiber activity and an adapting solution on the tongue surface. Intracellularly recorded depolarizing receptor potentials in taste cells induced by 0.5 M NaCl and 3 mM acetic acid were enhanced by depolarizing slow potentials induced by GP nerve stimulation, but were depressed by the hyperpolarizing slow potentials. On average, the receptor potential of taste cells for 0.5 M NaCl was increased by 25% by the GP nerve-induced slow potential, but the receptor potential of taste cells for 3 mM acetic acid was decreased by 1% by the slow potential. These transformations of receptor potentials in frog taste cells were not due to a synaptic event initiated between taste cells and the efferent nerve fiber, but due to a non-synaptic event, a lingual junction potential generated in the dorsal lingual epithelium by GP nerve stimulation.

Animals↗

PubMed2001

Binding diversity of a noncovalent-type low-molecular-weight serine protease inhibitor and function of a catalytic water molecule: X-ray crystal structure of PKSI-527--inhibited trypsin.

PKSI-527 is a noncovalent-type low-molecular-weight inhibitor. The X-ray crystal structure of the trypsin-PKSI-527 complex revealed a binding mode (Form II) different from the previously reported one (Form I) [Nakamura, M. et al. (1995) Biochem. Biophys. Res. Commun. 213, 583--587]. In contrast to the previous case, the electron density of the inhibitor revealed the whole structure clearly. Each structural part of the inhibitor in Forms I and II was differently located at the active site, although the modes of binding of the terminal amino group to the Asp189 carboxyl group were similar. This binding diversity, which is a characteristic of the noncovalent-type low-molecular-weight inhibitor, provides a suitable example for estimating the possible mechanism toward the enzymatic inhibition, together with the structural basis necessary for a specific inhibitor. The mode of binding in Form II reflects the inhibitor-specific situation and is in contrast with the substrate-mimetic binding mode for Form I. Based on the generally accepted catalytic mechanism for serine protease, we propose that a water molecule located at the catalytic site plays an important role in blocking the catalytic function of the reactive Ser193 OH group.

Animals↗

PubMed2001

Catecholamines play a role in the production of interleukin-6 and interleukin-1alpha in unburned skin after burn injury in mice.

OBJECTIVE: To investigate the effects of catecholamines on the production of interleukin (IL)-6 and IL-1alpha in unburned skin after a burn injury. DESIGN: Randomized, controlled study. SETTING: Animal laboratory. SUBJECTS: Anesthetized female C57BL/6 mice. INTERVENTIONS: A full-thickness burn injury on 20% of total body surface area was produced on the clipped dorsum, which was followed by resuscitation with lactated Ringer's solution (0.1 mL/g). Phentolamine mesylate (10 mg/kg) or propranolol hydrochloride (10 mg/kg) was administered intraperitoneally before the burn injury. MEASUREMENTS AND MAIN RESULTS: Epinephrine and norepinephrine increased the production of IL-6 but not of IL-1alpha in normal abdominal skin, and these increases were reversed by a beta-blocker (propranolol hydrochloride) but not an alpha-blocker (phentolamine mesylate). A 20% full-thickness burn injury of the skin increased the plasma concentration of epinephrine 30 mins after the injury in female C57BL/6 mice, but not norepinephrine or dopamine. The burn also increased IL-6 production in unburned abdominal skin. The administration of a beta-blocker before a 20% burn injury suppressed the increased IL-6 production in unburned abdominal skin as determined by both protein level and mRNA expression. Pretreatment with the alpha-blocker was less effective. CONCLUSIONS: We propose that the neuroendocrine system activated by a 20% burn injury participates in the increased IL-6 production in the unburned skin.

Adrenergic alpha-Antagonists↗

PubMed2001

Alteration of integrins by interleukin-1alpha in human pancreatic cancer cells.

INTRODUCTION: Adhesion of tumor cells to extracellular matrix (ECM) proteins plays an important role in tumor invasion and metastasis. AIMS: To investigate the expression of integrins in human pancreatic cancer cell lines and its alteration by interleukin (IL)-1alpha to examine the mechanism of adhesion of metastatic human pancreatic cancer cells to ECM proteins. METHODOLOGY: The expression of integrin subunits and their alteration by IL-1alpha were examined by flow-cytometric analysis and cellular enzyme-linked immunosorbent assay in three metastatic human pancreatic cancer cell lines (AsPC-1, BxPC-3, and SW1990) and two nonmetastatic cancer cell lines (PaCa-2 and PANC-1). In addition, assays of cancer cell adhesion to ECM proteins were performed to investigate if increased integrin expression actually affected the adhesive interaction between cancer cells and the putative integrin ECM ligands. RESULTS: The alpha(6) subunit expressed in metastatic cancer cells was enhanced by IL-1alpha. Metastatic cancer cells also showed preferential adherence to laminin compared with nonmetastatic cancer cells, and this was enhanced by IL-1alpha. CONCLUSION: In pancreatic cancer, the enhancement of alpha(6)beta(1) integrin by IL-1alpha through IL-1 receptor type I, as well as the expression of alpha(6)beta(1) integrin, plays an important role in metastasis formation.

Antigens, CD↗

PubMed2001

The citrus flavonoid, nobiletin, inhibits peritoneal dissemination of human gastric carcinoma in SCID mice.

The flavonoid nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), found in Citrus depressa Rutaceae, a popular citrus fruit in Okinawa, Japan, reportedly inhibits the production of pro-matrix metalloproteinase (proMMP)-1, 3, and 9 in rabbit synovial fibroblasts in vitro. In the present study, we demonstrated the inhibitory effects of nobiletin on the proliferation of the cancer cell line, TMK- 1, and its production of MMPs. In the SCID mouse model, we found that nobiletin inhibited the formation of peritoneal dissemination nodules from TMK-1. The enzymatic activity of MMP-9 expressed in culture medium obtained from a co-culture of TMK-1 and mouse fibroblastic cells was inhibited by nobiletin in a concentration-dependent manner. In the SCID mouse model, total weight of dissemination nodules was significantly lower in the treated group compared with the vehicle control group (0.07 g vs. 0.78 g, P = 0.0059). The total number of dissemination nodules was also significantly lower than in the vehicle control group (7.5 vs. 69.3 / body, P = 0.0001). These results suggest that nobiletin may be a candidate anti-metastatic drug for prevention of peritoneal dissemination of gastric cancer.

Animals↗

PubMed2001

Acute respiratory failure caused by leukaemic infiltration of the lung of a dog.

A seven-year-old crossbred male dog with a suspected leukaemic condition was referred for investigation and treatment. A bone marrow aspirate revealed an acute myeloid leukaemia. Combination chemotherapy was administered and the dog initially improved, but 18 days after the initiation of therapy its body condition deteriorated and the animal developed acute respiratory distress. On postmortem examination, extensive leukaemic pulmonary infiltrates were evident.

Animals↗

PubMed2001

Interaction of JC virus agno protein with T antigen modulates transcription and replication of the viral genome in glial cells.

In addition to encoding the structural and regulatory proteins, many viruses encode auxiliary proteins, some of which have been shown to play important roles in lytic and latent states of the viruses. The human neurotropic JC virus (JCV) genome encodes an auxiliary protein called Agno whose function remains unknown. Here, we investigated the functional role of JCV Agno protein on transcription and replication of the viral genome in glial cells. Results from transfection of human glial cells showed that Agno protein suppresses both T-antigen-mediated transcription of the viral late gene promoter and T-antigen-induced replication of viral DNA. Affinity chromatography and coimmunoprecipitation assays demonstrated that the Agno protein and T antigen physically interact with each other. Through the use of a series of deletion mutants, we demonstrated that the T-antigen-interacting region of Agno protein is localized to its amino-terminal half and the Agno-interacting domain of T antigen maps to its central portion. Furthermore, utilizing various Agno deletion mutants in functional studies, we confirmed the importance of the Agno-T antigen interaction in the observed down-modulation of T antigen function upon viral gene transcription and DNA replication by Agno protein. Taken together these data suggest that the Agno protein of JCV, which is produced late during the late phase of the lytic cycle, can physically and functionally interact with the viral early protein, T antigen, and downregulate viral gene expression and DNA replication. The importance of these observations in the lytic cycle of JCV is discussed.

Amino Acid Sequence↗

PubMed2001

Sera from patients with Kawasaki disease induce intercellular adhesion molecule-1 but not Fas in human endothelial cells.

BACKGROUND: Kawasaki disease (KD) is an acute vasculitis of unknown etiology occurring in childhood, characterized by abnormalities of the immune system including elevations of proinflammatory cytokines in the serum. We investigated the effect of serum from patients with KD on the expression of intercellular adhesion molecule-1 (ICAM-1) and Fas by human umbilical vein endothelial cells (HUVEC). METHODS: Confluent monolayers of HUVEC were incubated with sera from patients in the acute or convalescent phase of KD. Expression of ICAM-1 and Fas by HUVEC was assessed by flow cytometry. Concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in sera from patients with KD were measured by an immunoradiometric assay and an enzyme-linked immunosorbent assay, respectively. RESULTS: Sera from patients in the acute phase of KD produced significantly greater ICAM-1 expression by HUVEC than sera from patients in the convalescent phase. In contrast, KD sera did not induce Fas expression. While the mean serum concentration of TNF-alpha in patients in the acute phase of KD was significantly higher than in those in the convalescent phase, IL-1beta concentrations did not differ between the acute and convalescent phases. Exposure of HUVEC to recombinant human TNF-alpha increased the expression of both ICAM-1 and Fas, but a much lower concentration was required for an effect upon ICAM-1. Exogenous TNF-alpha did not induce apoptosis in HUVEC. CONCLUSIONS: These results suggest that increased expression of ICAM-1 by endothelial cells might be involved in the pathogenesis of acute KD, and that TNF-alpha might induce ICAM-1 expression.

Acute Disease↗

PubMed2001

Increased expression of transforming growth factor-beta1 in small airway epithelium from tobacco smokers and patients with chronic obstructive pulmonary disease (COPD).

Tobacco smoke is believed to cause small airway disease and then chronic obstructive pulmonary disease (COPD), but the molecular mechanisms by which small airway obstruction occurs remain unknown. To study the gene expression levels of transforming growth factor (TGF)-beta1, a potent fibrogenic factor, in small airway epithelium from smokers and patients with COPD, we harvested highly pure samples of epithelial cells from small airways under direct vision by using an ultrathin bronchofiberscope BF-2.7T (outer diameter 2.7 mm with a biopsy channel of 0.8 mm in diameter). The expression levels of TGF-beta1 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of TGF-beta1 corrected by beta-actin transcripts were significantly higher in the smoking group and patients with COPD than those in nonsmokers (p < 0.01). Furthermore, among smokers and patients with COPD, TGF-beta1 mRNA levels correlated positively with the extent of smoking history (pack-years) and the degree of small airway obstruction as assessed by measurements of flow-volume curves. Immunocytochemistry of the cells demonstrated more intense stainings for TGF-beta1 in samples from smokers and patients with COPD than from nonsmokers. Spontaneously released immunoreactive TGF-beta1 levels from cultured epithelial cells were more elevated in subjects with a history of smoking and patients with COPD than in nonsmokers. Our study showed a close link between smoking and expression of TGF-beta1 in small airways. Our results also suggested that small airway epithelial cells might be involved in obstructive changes found in smokers and patients with COPD.

Analysis of Variance↗

PubMed2001

ApoE knockout mice expressing human matrix metalloproteinase-1 in macrophages have less advanced atherosclerosis.

Matrix metalloproteinase-1 (MMP-1), or interstitial collagenase, has been hypothesized to contribute to the progression of the human atherosclerotic lesions by digesting the fibrillar collagens of the neointimal ECM. The apolipoprotein E knockout (apoE0) mouse model develops complex atherosclerotic lesions, but mice do not possess a homologue for MMP-1. To provide an in vivo evaluation of the role of MMP-1 in atherogenesis, we created a transgenic mouse model that expresses this enzyme specifically in the macrophage, under the control of the scavenger receptor A (SCAV) enhancer/promoter. The MMP-1 transgenic mice were crossed into the apoE0 background and fed an atherogenic diet for 16-25 weeks. Surprisingly, the transgenic mice demonstrated decreased lesion size compared with control littermates. The lesions of the transgenic animals were less extensive and immature, with fewer cellular layers and a diminished content of fibrillar collagen. There was no evidence of plaque rupture. Our data suggest that remodeling of the neointimal extracellular matrix by MMP-1 is beneficial in the progression of lesions.

Animals↗

PubMed2001

Clinical application of an ultrasonically activated scalpel in neurosurgery.

Ultrasonically activated scalpel (UAS) is a surgical device utilizing ultrasonic energy to cut and coagulate tissues simultaneously. The actions and features of UAS are considerably different from those of conventional ultrasonic cavitational aspirators (UCA) that have been used frequently in the neurosurgical field. The UAS has been used routinely in a number of general surgeries, including laparoscopic surgeries and open surgeries of the lung and liver. However, neurosurgical application of UAS has not been reported. We used UAS in surgical resection of intracranial tumors consisting of five meningiomas, two gliomas, and one metastatic brain tumor, and monitored thermal changes in tissues using thermoencephaloscopy to investigate the possibility of heat injury by UAS. It was used to achieve both dissection of the attachment of meningiomas and internal decompression of the tumors and allowed efficient resection of various tumors with minimal bleeding by adjustment of the combination of coagulation and cutting powers at five different positions. The attachment of meningiomas was coagulated and cut easily and safely by UAS. Moreover, infrared thermographic study showed that the degree of temperature increase and the area of elevated temperature were extremely limited in UAS. The current study suggests that it is a promising device in neurosurgery.

Body Temperature↗

PubMed2001

Antioxidant effect of capsaicin on lipid peroxidation in homogeneous solution, micelle dispersions and liposomal membranes.

The antioxidant activity of capsaicin (CAP) was measured in the oxidation of methyl linoleate (ML) in homogeneous solution, of ML micelles in aqueous dispersions and also of soybean phosphatidylcholine liposomal membrane, and was compared to that of alpha-tocopherol (alpha-TOH) which is one of the most important antioxidants in vivo. The reactivity of CAP toward galvinoxyl (a model phenoxyl radical) in acetonitrile solution was found to be much smaller than that of alpha-TOH, suggesting that the radical scavenging activity of CAP is much weaker than that of alpha-TOH. In fact, in homogeneous acetonitrile solution where the antioxidant activity is determined primarily by the chemical activity of the antioxidant toward peroxyl radicals, CAP inhibited the oxidation of ML much less efficiently than alpha-TOH and a clear induction period was not observed. The antioxidant activity of CAP was found to be about 60 times smaller than that of alpha-TOH in homogeneous solution. However, in micelle oxidation, the difference in antioxidant activity of the two antioxidants was much smaller than in homogeneous solution. Furthermore, in the membrane, CAP inhibited the oxidation almost as effectively as alpha-TOH. These results suggest that CAP can act as an antioxidant in the biomembrane.

Acetonitriles↗

PubMed2001

Functional organisation of anterior thoracic stretch receptors in the deep-sea isopod Bathynomus doederleini: behavioural, morphological and physiological studies.

The relationship between segmental mobility and the organisation of thoracic stretch receptors was examined in the deep-sea isopod Bathynomus doederleini, which shows a developed adaptive behaviour during digging. The movements of segments during digging were analysed from video recordings, which showed that a large excursion occurred in the anterior thoracic segments. Dye-fills of axons revealed four types of thoracic stretch receptor (TSR): an N-cell type (TSR-1), a differentiated N-cell type (TSR-2), a muscle receptor organ (MRO)-type with a long, single receptor muscle (TSR-3) and an MRO-type with a short, single receptor muscle (TSR-4 to TSR-7). Physiologically, TSR-1 and TSR-2 are tonic-type stretch receptors. TSR-3 to TSR-7 show two kinds of stretch-activated responses, a tonic response and a phasico-tonic response in which responses are maintained as long as the stretch stimulus is delivered. Both TSR-2, with a long muscle strand, and TSR-3, with a single, long receptor muscle, have a wide dynamic range in their stretch-activated response. In addition, TSR-2 is controlled by an intersegmental inhibitory reflex from TSR-3. These results suggest that, although TSR-1 has no receptor muscle and TSR-2 has a less-differentiated receptor-like muscle, they are fully functional position detectors of segmental movements, as are the MRO-type receptors TSR-3 to TSR-7.

Animals↗

PubMed2001

Hematological studies on black cumin oil from the seeds of Nigella sativa L.

The methanol soluble portion of black cumin oil, which is prepared by compression of seeds of Nigella sativa L., showed inhibitory effects on arachidonic acid (AA)-induced platelet aggregation and blood coagulation. By bioactive assay of AA-induced platelet aggregation, the methanol soluble part was purified to isolate a new compound 2-(2-methoxypropyl)-5-methyl-1,4-benzenediol (1) and two known compounds, thymol (2), carvacrol (3), having very strong inhibitory activity. Further, we then examined the isolated compounds (1-3) and eight related compounds by the screening test for AA-induced platelet aggregation. Compounds possessing aromatic hydroxyl and acetoxyl group had more potent activity than aspirin, which is well known as a remedy for thrombosis.

Animals↗

PubMed2001

Isolation of plasma kallikrein by high efficiency affinity chromatography and its characterization.

We established a simple method for the purification of human plasma kallikrein (PK) by affinity chromatography and characterized it by analytical reverse phase-HPLC and Time of Flight Mass Spectroscopy (TOF-MS). The affinity resin (PKSI-Toyopearl) was synthesized using a selective synthetic inhibitor of plasma kallikrein (PKSI-527) as a ligand. The resin was found to have the highest efficiency in PK purification when the coupling ratio of PKSI-527 per resin was 9-14 micromol/g. PK was purified 466-fold with a yield of 83% from acetone-activated human plasma by affinity chromatography. The purity of PK thus obtained was confirmed by reverse phase-HPLC with a linear gradient of acetonitrile. The molecular weight of the purified PK was determined to be 86,151 by TOF-MS.

Chromatography, Affinity↗