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Biomedical subjects

K Nishio

Publications and source records attributed to K Nishio.

At least 55 records · Page 3Linked to original sources
PubMed2000

Transfer of iron-sulfur cluster from NifU to apoferredoxin.

Iron-sulfur proteins are present in a wide variety of organisms and are known to play important physiological roles, not only in electron transfer and metabolic reactions, but also in transcriptional regulation. However, little is known about how iron-sulfur clusters themselves are synthesized and assembled within polypeptides. Here we show that a [2Fe-2S] cluster-containing NifU of cyanobacterium Synechocystis PCC6803, SyNifU, possesses the ability to deliver its [2Fe-2S] cluster to an apoferredoxin without the aid of other proteinaceous or nonproteinaceous factor(s). Upon delivery the reconstituted holoferredoxin regained electron transfer ability. The [2Fe-2S] cluster contained within SyNifU was labile upon exposure to the iron-chelating reagent EDTA, suggesting that the iron-sulfur cluster is abnormally exposed to solvent. We propose that NifU serves as a scaffold for iron-sulfur cluster assembly and functions as a mediator for iron-sulfur cluster delivery.

Bacterial Proteins↗
PubMed2000

Developmental changes in the spatial expression of genes involved in myosin function in Dictyostelium.

We analyzed the spatial expression patterns of the genes involved in myosin function by in situ hybridization at the tipped aggregate and early culmination stages of Dictyostelium. Myosin heavy chain II mRNA was enriched in the anterior prestalk region of the tipped aggregates, whereas it disappeared from there and began to appear in both upper and lower cups of the early culminants. Similarly, mRNAs for essential light chain, regulatory light chain, myosin light chain kinase A, and myosin heavy chain kinase C were enriched in the prestalk region of the tipped aggregates. However, expression of these genes was distinctively regulated in the early culminants. These findings suggest the existence of mechanisms responsible for the expression of particular genes.

Animals↗
PubMed2000

Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells.

A tumor-suppressor gene, p16(INK4), which is deleted or mutated in tumors, regulates cell-cycle progression through a G(1)-S restriction point by inhibiting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found that ectopic p16(INK4) expression increased cellular sensitivity of human non-small-cell-lung-cancer (NSCLC) A549 cells to a selective growth-inhibitory effect induced by the topoisomerase-I inhibitor 11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT-11. This apoptosis was suppressed by the inhibitor of interleukin-1beta-converting enzyme (ICE/caspase-1) or ICE-like proteases, Z-Asp-CH2-DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3. In A549/p16-1 cells, cytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethylcoumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethylketone. These findings indicate that p16(INK) is positively involved in the activation pathway of the caspase-3 induced by CPT-11. The increased delay in S-phase progression and subsequent induction of apoptosis were observed in CPT-11-treated A549/p16-1 cells on the basis of DNA histograms. Specific down-regulation of the cyclin-A protein level in A549/p16-1 cells was observed after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16(INK4) expression inappropriately decreases cyclin A and thereby terminates CPT-11-induced G(2)/M accumulation, which is followed by increased apoptosis in p16(INK4)-expressing A549 cells.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Increased expression of the MRP5 gene is associated with exposure to platinum drugs in lung cancer.

To investigate the role of the multidrug resistance-associated protein (MRP1) homologue MRP5 in relation to platinum drug resistance, we examined the steady-state levels of the mRNAs for MRP5 in both lung cancer cell lines and peripheral mononuclear cells (PMN) after exposure to platinum drug and in normal lung and lung cancer tissue specimens. Firstly, we examined MRP5 gene expression levels in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) from 40 patients who had died from lung cancer. Next, we monitored MRP5 gene expression levels within 24 hr in both lung cancer cell lines incubated with cisplatin and in PMN from 10 previously untreated lung cancer patients after carboplatin administration alone. The MRP5 gene expression levels were assessed by quantitative reverse transcription polymerase chain reaction or RNase protection assay. The MRP5 expression levels in normal lung tissues and in tumors from patients exposed to platinum drugs during their lifetime were significantly higher than those in tissues from non-exposed patients. On the other hand, the MRP5 expression levels were not rapidly induced by platinum drugs either in lung cancer cell lines or in PMN within 24 hr. Our results suggest that increased expression levels of the MRP5 gene are associated with exposure to platinum drugs in lung cancer in vivo and/or the chronic stress response to xenobiotics.

ATP-Binding Cassette Transporters↗
PubMed2000

Detailed structural analysis on both human MRP5 and mouse mrp5 transcripts.

The multidrug-resistant phenotype in tumor cells is attributed in part to anti-cancer drug efflux transporters such as the MRP family. The amino-terminal structure of MRP5 has not been refined. To determine the amino-terminal structure of a major transcript of the MRP5 gene, we performed primer extension analysis to determine a major transcriptional start site of this gene and compared the structure of human MRP5 and that of mouse mrp5. We successfully determined the structures of human MRP5 and mouse mrp5. Estimated amino acid sequences are 1437 and 1436 amino acids for human MRP5 and mouse mrp5 respectively, and were highly conserved (94.1%). We further showed that our previously identified SMRP mRNA was a splicing variant of the MRP5 gene, which was expressed in various human tissues, suggesting that a short form of MRP5 protein encoded by the SMRP mRNA may have a physiological role.

ATP-Binding Cassette Transporters↗
PubMed2000

Molecular determinants of UCN-01-induced growth inhibition in human lung cancer cells.

UCN-01 (7-hydroxystaurosporine) inhibits the growth of various malignant cell lines in vitro and in vivo. In this study, a human small cell lung carcinoma subline resistant to UCN-01, SBC-3/UCN, was established and characterized. SBC-3/UCN cells showed 8-fold greater resistance to the UCN-01-induced growth-inhibitory effect than the parent cells, SBC-3. No UCN-01-induced G1 accumulation in SBC-3 cells was observed in SBC-3/UCN cells and decreased expression of phosphorylated RB protein was found in SBC-3 cells. Neither basal expression nor induction of p21(Cip1) by UCN-01 treatment was detected in the SBC-3/UCN cell line. An inhibitory effect of UCN-01 on CDK2 activity, which is mediated by p21(Cip1)/CDK2 complex formation upon UCN-01 treatment, was observed in SBC-3 but not in SBC-3/UCN cells. SBC-3/UCN showed higher CDK6 activity than SBC-3 cells. UCN-01 did not inhibit the CDK4 and CDK6 activities in both cells. We screened the cell cycle regulatory molecules associated with G(1)/S progression and found a remarked decrease in interferon regulatory factor 1 (IRF-1), which is known to cooperate with p53 in p21(Cip1) induction. Our results suggest that p21(Cip1) regulation via the IRF-1-associated pathway may represent a major determinant of UCN-01-induced growth inhibition in human lung cancer cells.

Alkaloids↗
PubMed2000

Problems in the development of target-based drugs.

Numerous molecular targets of cancer chemotherapy have been identified based on progress made in molecular biology, and new categories of anticancer drugs have been developed. These include inhibitors for signal transduction, cyclin-dependent kinase, angiogenesis, and matrix metalloproteinase, gene therapy, etc. They are variously called target-based drugs, noncytotoxic drugs, or cytostatic drugs. Such drugs have interesting mechanisms of action and appear promising. However, preclinical and clinical evaluations are difficult. Some drugs have a direct antitumor effect, with demonstrated tumor shrinkage. Others show no direct cytotoxicity. The majority of recent phase I trials have evaluated the maximum tolerated dose, pharmacokinetics, adverse events, and antitumor effect. Unusual, unacceptable toxicities have been noted with some target-based drugs. Few phase I trials or preclinical studies have attempted to demonstrate target inhibition. So far very few studies has shown that there is a correlation between target inhibition and antitumor effect. In general, phase II studies are undertaken with compounds such as trastuzumab which have direct antitumor activity. Phase III trials of most target-based drugs are undertaken immediately after phase I studies since the design of appropriate phase II studies is difficult. The ultimate endpoint of phase III trials of target-based drugs is the same as that for cytotoxic drugs, such as improved cure and survival rates.

Antineoplastic Agents↗
PubMed2000

Corneal infection of herpes simplex virus type 2--induced neuronal apoptosis in the brain stem of mice with expression of tumor suppressor gene (p53) and transcription factors.

To understand the mechanism of neuronal apoptosis induced by herpes simplex virus (HSV) infection in vivo, the distribution of viral antigen, the appearance of apoptotic bodies, and the expressions of the tumor suppressor gene p53 and several transcription factors such as c-fos, c-jun and NF-kappaB were examined immunohistochemically and histopathologically after corneal infection of mice with HSV type 2 strain 186. Five days after HSV infection, viral antigen was diffusely detected in the corneal epithelium, the trigeminal ganglion and the pars caudalis of the spinal trigeminal nucleus. Neuronal apoptosis was observed in the brain stem ipsilateral to the HSV-infected side with the immunoreactivities of c-fos, c-jun, NF-kappaB and p53. Dual-labeling immunohistochemical studies revealed that almost all of the viral antigen-positive neurons and glia in the brain stem also showed p53 immunoreactivity. On the other hand, no neuronal apoptosis but only with the expression of c-jun was found in the trigeminal ganglion. Our results suggest that the different expression of transcription factors between the brain stem and the trigeminal ganglion may influence the neuronal apoptosis induced by HSV infection.

Afferent Pathways↗
PubMed2000

Guillain-Barré syndrome in a child with systemic lupus erythematosus and anti-Ro/SSA and anti-La/SSB autoantibodies.

We report a 9-year follow-up of a girl with systemic lupus erythematosus (SLE) and probable Sjögren's syndrome. At the age of 7 years, the patient developed a chilblain-like eruption with features of SLE, including leucopenia, oral ulcers, positive rheumatoid and antinuclear antibodies and positive anti-dsDNA, anti-Ro/SSA and anti-La/SSB antibodies. At the age of 13 years she developed Guillain-Barré syndrome, which completely resolved with aggressive treatment, including high-dose corticosteroids and the use of plasma exchange followed by intravenous gammaglobulin.

Autoantibodies↗
PubMed2000

Quantitative intensity measurement of equal thickness fringes in Si and MgO crystal images with an energy-filtering transmission electron microscope using an imaging plate.

Quantitative measurement of intensity profiles of equal thickness fringes has been carried out in Si and MgO crystal images with an energy-filtering transmission electron microscope using an imaging plate. The crystals have a 90 degrees wedge-shape with [110] surfaces for Si and with [100] surfaces for MgO, and are observed under the exact axial incidence of a 200 keV electron beam along the [100] axis for Si and along the [110] axis for MgO. The intensities are measured in bright field and 022 and 040 dark field images for Si, and in bright field and 111, 002, 220, 113, 222, and 004 dark field images for MgO, with and without an energy slit having +/- 5 eV energy width for incident electrons. The intensity profiles obtained from the images are presented as standard experimental data for calculation of electron diffraction intensities. A few simulation programs for high-resolution transmission electron microscopy are checked by comparing the calculated diffraction intensities with the experimental data. The complex potential suitable for matching the data is discussed.

Journal Article↗
PubMed2000

A computational model for quantitative analysis of cell cycle arrest and its contribution to overall growth inhibition by anticancer agents.

Most anticancer agents induce cell cycle arrest (cytostatic effect) and cell death (cytotoxic effect), resulting in the inhibition of population growth of cancer cells. When asynchronous cells are to be examined, the currently used flow cytometric method can not provide checkpoint-specific and quantitative information on the drug-induced cell cycle arrest. Hence, despite its significance, no good method to analyze in detail the mechanism of cell cycle arrest and its contribution to overall growth inhibition induced by an anticancer agent has yet been established. We describe in this study the development of a discrete time (Markov model)-based computational model for cell cycle progression / arrest with transition probability (TP(i)) as a model parameter. TP(i) was calculated using model equations that include easily measurable parameters such as the fraction of cells in each cell cycle phase and population doubling time. The TP(i) was then used to analyze checkpoint-specific and quantitative changes in cell cycle progression. We also used TP(i) in a Monte-Carlo simulation to predict growth inhibition caused by cell cycle arrest only. Human SCLC cells (SBC-3) exposed to UCN-01 were used to validate the model. The model-predicted growth curves agreed with the observed data for SBC-3 cells not treated or treated at a cytostatic concentration (0.2 mM) of UCN-01, indicating validity of the present model. The changes in TP(i) indicated that UCN-01 reduced the G(1)-to-S transition rate and increased the S-to-G(2) / M and G(2) / M-to-G(1) transition rates of SBC-3 cells in a concentration- and time-dependent manner. When the model-predicted growth curves were compared with the observed data for cells treated at a cytotoxic concentration (2 mM), they suggested that 22% out of 65% and 32% out of 73% of the growth inhibition could be attributed to the cell cycle arrest effect after 48 h and 72 h exposure, respectively. In conclusion, we report here the establishment of a novel method of analysis that can provide checkpoint-specific and quantitative information about cell cycle arrest induced by an anticancer agent and that can be used to assess the contribution of cell cycle arrest effect to the overall growth inhibition.

Alkaloids↗
PubMed2000

Effect of steroid on hyperoxia-induced ICAM-1 expression in pulmonary endothelial cells.

Intercellular adhesion molecule-1 (ICAM-1) of the vascular endothelium plays a key role in the development of pulmonary oxygen toxicity. We studied the effect of steroid on hyperoxia-induced ICAM-1 expression using cultured endothelial cells in vitro. Human pulmonary artery endothelial cells (HPAECs) were cultured to confluence, and then the monolayers were exposed to either control (21% O(2)-5% CO(2)) or hyperoxic (90% O(2)-5% CO(2)) conditions with and without a synthetic glucocorticoid, methylprednisolone (MP). MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression in a dose-dependent manner. Neutrophil adhesion to hyperoxia-exposed endothelial cells was also inhibited by MP treatment. In addition, MP attenuated hyperoxia-induced H(2)O(2) production in HPAECs as assessed by flow cytometry. An electrophoretic mobility shift assay demonstrated that hyperoxia activated nuclear factor-kappaB (NF-kappaB) but not activator protein-1 (AP-1) and that MP attenuated hyperoxia-induced NF-kappaB activation dose dependently. With Western immunoblot analysis, IkappaB-alpha expression was decreased by hyperoxia and increased by MP treatment. These results suggest that MP downregulates hyperoxia-induced ICAM-1 expression by inhibiting NF-kappaB activation via increased IkappaB-alpha expression.

Cell Adhesion↗
PubMed2000

Roles of ICAM-1 for abnormal leukocyte recruitment in the microcirculation of bleomycin-induced fibrotic lung injury.

To assess the importance of endothelial intercellular adhesion molecule-1 (ICAM-1) in microvascular leukocyte kinetics in diseased lungs, we investigated the transitional changes in ICAM-1 expression, vascular diameter, and leukocyte behavior in rat pulmonary microcirculation during the development of acute lung injury (ALI) and chronic fibrosis (FIB) evoked by bleomycin (BLM). Observations were made in the isolated perfused lung with a real-time confocal laser luminescence microscope. Microvascular cell kinetics were evaluated by measuring the behavior of fluorescence- labeled leukocytes and erythrocytes in the presence or absence of anti-ICAM-1 monoclonal antibody (1A29). Arteriolar ICAM-1 showed little change at any time after BLM treatment. Venular ICAM-1 was first enhanced at the initial phase of ALI followed by the second upregulation at the early phase of FIB. Capillary ICAM-1 showed a sustained increase at both ALI and FIB. Arteriolar and venular diameters were not altered but capillary diameter decreased during ALI and early FIB stages. Although firm adherence of leukocytes to arteriolar and venular walls was not observed, rolling leukocytes were increased in venules both at the initial phase of ALI and at the early phase of FIB. The leukocyte rolling in venules correlated well with transitional changes in ICAM-1 and was inhibited by 1A29. Sustained entrapment of leukocytes in capillaries was attributed to changes in vascular diameter as well as augmented ICAM-1. In conclusion, ICAM-1 plays an important role in microvascular leukocyte recruitment in both ALI and FIB in the BLM-injured lung.

Animals↗
PubMed2000

[Conquering drug resistance in lung cancer].

The emergence of drug resistance of the tumors is the critical problem in lung cancer. The multifactorial mechanisms of cisplatin resistance are major problems. The reversal agents against MDR have been evaluated clinically. Increased adverse effects and modulation of pharmacokinetics in combined with cytotoxic drugs are the problems in clinical setting. The new ABC transporters related with MDR were cloned and characterized. Modulators for these transporters could be evaluated their clinical efficacy. High-frequent mutations of beta-tubulin in clinical samples are reported as a predictive marker for paclitaxel. Target-based drugs might conquer the resistance to conventional drugs. Prediction of the drug sensitivity and resistance in each patient will be expected using high-throghput gene screening system.

ATP-Binding Cassette Transporters↗
PubMed1999

Chloroplast chaperonins: evidence for heterogeneous assembly of alpha and beta Cpn60 polypeptides into a chaperonin oligomer.

Higher plant chloroplasts contain two chaperonin 60 family proteins, Cpn60alpha and Cpn60beta, which are known to have divergent amino acid sequences. Although Cpn60alpha and Cpn60beta are present in roughly equal amounts and copurify in their native states, a heterogeneous ensemble of the chaperonin oligomer has not yet been demonstrated. We separately purified Cpn60alpha and Cpn60beta proteins from spinach leaves as the monomeric form. Either antibody raised against one chaperonin 60 protein could coimmunoprecipitate the other chaperonin 60 protein in their oligomeric state but not in its monomeric state, suggesting that the chloroplast Cpn60alpha and Cpn60beta polypeptides actually reside in the same chaperonin oligomer in the stroma. Moreover, the chaperonin oligomers migrated as at least two distinct bands on the native gel electrophoresis, each of which contained both chaperonin 60 proteins. These results suggest that chloroplast chaperonin oligomers might be composed of at least two distinct sets of two chaperonin proteins.

Amino Acid Sequence↗
PubMed1999

Repair of segmental bone defects using bioactive bone cement: comparison with PMMA bone cement.

We developed a bioactive bone cement (BABC) that consists of apatite and wollastonite containing glass ceramic (AW-GC) powder and bisphenol-A-glycidyl dimethacrylate (Bis-GMA) based resin. In the present study, the effectiveness of the BABC for repair of segmental bone defects under load-bearing conditions was examined using a rabbit tibia model. Polymethylmethacrylate (PMMA) bone cement was used as a control. A 15-mm length of bone was resected from the middle of the shaft of the tibia, and the tibia was fixed by two Kirschner wires. The defects were replaced by cement. Each cement was used in 12 rabbits; six rabbits were sacrificed at 12 and 25 weeks after surgery, and the tibia containing the bone cement was excised and tension tested. At both the intervals studied, the failure loads of the BABC were significantly higher than those of the PMMA cement. The BABC was in direct contact with bone, whereas soft tissue was observed between the cement and bone in all PMMA cement specimens. Results indicated that the BABC was useful as a bone substitute under load-bearing conditions.

Animals↗
PubMed1999

Effect of bioactive filler content on mechanical properties and osteoconductivity of bioactive bone cement.

We took three types of bioactive bone cement (designated AWC, HAC, and TCPC), each with a different bioactive filler, and evaluated the influence of each filler on the mechanical properties and osteoconductivity of the cement. The cements consisted of bisphenol-a-glycidyl methacrylate-based (Bis-GMA based) monomers as an organic matrix, with a bioactive filler of apatite/wollastonite containing glass-ceramic (AW-GC) or sintered hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP) powder. Each filler was mixed with the monomers in proportions of 50, 70, and 80% (w/w), giving a total of nine cement subgroups. The nine subgroups were designated AWC50, AWC70, AWC80, HAC50, HAC70, HAC80, TCPC50, TCPC70, and TCPC80. The compressive and bending strengths of AWC were found to be higher than those of HAC and TCPC for all bioactive filler contents. We also evaluated the cements in vivo by packing them into the intramedullary canals of rat tibiae. To compare the osteoconductivity of the cements, an affinity index was calculated for each cement; it equaled the length of bone in direct apposition to the cement, expressed as a percentage of the total length of the cement surface. Microradiographic examination up to 26 weeks after implantation revealed that AWC showed a higher affinity index than HAC and TCPC for each filler content although the affinity indices of all nine subgroups (especially the AWC and HAC subgroups) increased with time. New bone had formed along the AWC surface within 4 weeks, even in the cement containing AW-GC filler at only 50% (w/w); observation of the cement-bone interfaces using a scanning electron microscope showed that all the cements had directly contacted the bone. At 4 weeks the AWC had bonded to the bone via a 10 micron-thick reactive layer; the width of the layer, in which partly degraded AW-GC particles were seen, became slightly thicker with time. On the other hand, in the HAC- and TCPC-implanted tibiae, some particles on the cement surface were surrounded by new bone and partly absorbed or degraded. The results suggest that the stronger bonding between the inorganic filler and the organic matrix in the AWC cements gave them better mechanical properties. The results also indicate that the higher osteoconductivity of AWC was caused by the higher reactivity of the AW-GC powder on the cement surface.

Animals↗