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Biomedical subjects

J Ando

Publications and source records attributed to J Ando.

At least 55 records · Page 3Linked to original sources
PubMed1998

Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges.

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca2+ concentration ([Ca2+]i). In a single bovine aortic EC, the [Ca2+]i rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca2+ wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca2+ wave was unaffected by the removal of extracellular Ca2+, demonstrating its dependence on intracellular Ca2+ release. Microinjection of heparin into the cell inhibited the ATP-induced Ca2+ responses, indicating that the Ca2+ wave is at least partly mediated by the inositol 1,4, 5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca2+ waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca2+]i increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca2+ waves in ECs.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Fluid shear stress increases the production of granulocyte-macrophage colony-stimulating factor by endothelial cells via mRNA stabilization.

To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm2 markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.5 times, respectively, the amount released by the static controls at 24 hours, but it had no significant influence on the release of granulocyte CSF or macrophage CSF. The results of reverse transcriptase-polymerase chain reaction demonstrated that GM-CSF mRNA began to increase as early as 2 hours after initiation of 15 dyne/cm2 shear stress and continued to increase with time, reaching a peak of about four times the control levels at 24 hours. This increase in GM-CSF mRNA levels in response to shear stress depended on protein synthesis, because it was blocked by cycloheximide. Neither nuclear run-on assay or luciferase assay using a reporter gene containing GM-CSF gene promoter showed any significant change in transcription of the GM-CSF gene even after 24-hour exposure to a shear stress of 15 dyne/cm2. Actinomycin D chase experiments using a competitive polymerase chain reaction showed that shear stress extended the half-life of GM-CSF mRNA from approximately 23 to 42 minutes in HUVECs. These findings suggest that fluid shear stress increases the production of GM-CSF in HUVECs via mRNA stabilization.

Base Sequence↗
PubMed1998

Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers.

To better understand the vascular activity of hemoglobin-based (Hb-based) oxygen carriers, the endothelial permeability characteristics of Hb derivatives having various molecular masses were defined by using monolayers of bovine endothelial cells cultured on microporous membranes. The endothelial permeability of unmodified bovine Hb was almost twice that of bovine serum albumin. Intramolecularly cross-linked human Hb showed slightly but significantly reduced permeability as compared with unmodified bovine Hb. Polyethyleneglycol modification or haptoglobin binding to Hb further reduced the permeability. These properties were intensified in conditions in which the endothelial barrier function was reduced by pretreatment with either interleukin-6 (100 ng/mL, 21 hours) or lipopolysaccharide (1 microg/mL, 10 hours). In contrast, there was little permeability of liposome-encapsulated Hb, and it was almost unaffected by the pretreatments. These data provide the first information that Hb derivatives with smaller molecular masses show larger transendothelial flux. Because Hb is a potent scavenger of endothelium-derived relaxing factor (EDRF), our observations support the idea that smaller Hb-based acellular oxygen carriers are potent vasoconstrictors as a result of abluminal EDRF scavenging.

Animals↗
PubMed1998

Genomic structure of the spermatid-specific hsp70 homolog gene located in the class III region of the major histocompatibility complex of mouse and man.

The Hsc70t gene is a Hsp70 homolog gene expressed constitutively in spermatids in mice. This gene is linked to two heat-inducible Hsp70 genes, Hsp70.1 and Hsp70.3, located in the MHC class III region. The syntenic region of human chromosome 6 contains the HSPA1B, HSPA1A, and HSPA1L genes. Here, we have isolated a HSPA1L cDNA clone from human testicular cells. The HSPA1L gene contained an intron 13 bp upstream of the initiating ATG. A similar genomic structure was found in the Hsc70t gene. The transcription initiation site of the Hsc70t gene was located at ca. 600 bp upstream of the heat-inducible Hsp70.3 gene, linked head-to-head. Sequence alignment of the mouse and human genes revealed that the human HSPA1L and HSPA1A genes were orthologous to the mouse Hsc70t and Hsp70.3 genes, respectively. Conserved sequence stretches observed in the 5' flanking region and the first exon of the spermatid-specific Hsp70 gene may be involved in regulation of the specific gene expression.

Animals↗
PubMed1998

An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells.

Cytosolic Ca2+ ([Ca2+]i) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca2+ can be triggered by depletion of intracellular Ca2+ stores, the mechanism (or mechanisms) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light-chain kinase (MLCK) in the regulation of Ca2+ signaling was investigated in agonist- and fluid flow-stimulated endothelial cells loaded with Ca2+-sensitive dyes. Bradykinin (BK) and thapsigargin caused an increase in [Ca2+]i followed by a sustained rise due to Ca2+ influx from extracellular space and shifted total myosin light-chain (MLC) from the unphosphorylated to the diphosphorylated form. ML-9 (100 microM), an inhibitor of MLCK, abolished Ca2+ influx and prevented MLC diphosphorylation in BK- and thapsigargin-treated cells, but did not affect Ca2+ mobilization from internal stores. Fluid flow stimulation (shear stress=5 dynes/cm2) increased [Ca2+]i and enhanced MLC phosphorylation. ML-9 also inhibited Ca2+ response and MLC phosphorylation in fluid flow-stimulated cells. The Ca2+ influx in response to BK was linearly correlated with the diphosphorylation of MLC in ML-9 treated cells. Effects of ML-5 and ML-7, analogs of ML-9, to inhibit Ca2+ influx paralleled their potencies to inhibit MLCK activity. These findings demonstrate that MLCK plays an essential role in regulating the plasmalemmal Ca2+ influx in agonist- and fluid flow-stimulated endothelial cells. This study is the first to report the close relationship between Ca2+ influx and MLC diphosphorylation.

Animals↗
PubMed1998

Maximizing care outcomes of a patient with impaired bladder function: a PI project in a rehabilitation unit.

At Jamaica Hospital Medical Center Rehabilitation Unit, approximately 50 percent of patients have impaired bladder function due to disease and trauma. In 1996, the rehabilitation staff identified an opportunity to improve care outcomes of patients with impaired bladder function. Using five steps for performance improvement from the Joint Commission on Accreditation of Healthcare Organizations, a team consisting of a physician, nurse, therapist, and psychologist was formed to explore strategies for improving care of these patients. The goal was to investigate the advantages of using noninvasive devices in maximizing care outcomes.

Adult↗
PubMed1998

Physiologic shear stress suppresses endothelin-converting enzyme-1 expression in vascular endothelial cells.

Shear stress dilates blood vessels and exerts an antiproliferative effect on vascular walls. These effects are ascribed to shear stress-induced, endothelium-derived vasoactive substances. Endothelin-converting enzymes (ECEs), the enzymes that convert big endothelin-1 (ET-1) to ET-1, have recently been isolated and the corresponding proteins have been termed ECE-1 and ECE-2. Furthermore, two isoforms of human ECE-1 have been demonstrated and termed ECE-1 alpha and ECE-1 beta. In this study, to elucidate the role of ECE-1 under shear stress we examined the effect of physiologic shear stress on the mRNA expression of ECE-1 and ET-1 in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical veins (HUVECs), and also ECE-1 alpha mRNA expression in HUVECs. ECE-1 mRNA expression was significantly downregulated by shear stress in 24 h, both in BAECs and HUVECs, in a shear stress intensity-dependent manner. The expression of ECE-1 alpha mRNA was also attenuated by shear stress in HUVECs. ET-1 mRNA expression showed a concordant decrease with ECE-1 mRNA expression. These results suggest that shear stress-induced gene regulation of ET-1 and ECE-1 mRNA expression can contribute to the decrease of ET-1 peptide level by shear stress.

Animals↗
PubMed1998

Intracellular Signal-transducing elements involved in transendothelial migration of lymphoma cells.

To investigate the molecular mechanisms underlying transendothelial migration of tumor cells, an essential process for their hematogenous dissemination, we developed an in vitro model system that allows the separate monitoring of cell adhesion and transmigration processes. This system uses a human pre-B lymphoma cell line, Nalm-6, and a cultured mouse endothelial cell line, KOP2.16. Nalm-6 cells rapidly adhered to KOP2.16 and subsequently transmigrated underneath them. Using this model, we examined the effects on transendothelial migration, of various reagents which specifically interfere with the function of intracellular signal transduction molecules. Treatment of Nalm-6 cells with wortmannin (WMN), herbimycin A, pertussis toxin, or C3 exoenzyme of Clostridium botulinum, which specifically inhibit P13 kinase and/or myosin light chain kinase, herbimycin-sensitive tyrosine kinases, heterotrimeric G proteins, and the small G proteins, and the small G proteins rho/rac, respectively, reduced transmigration in a dose-dependent manner, Pretreatment of KOP2.16 endothelial cells with WMN also reduced transmigration in a dose-dependent manner. Binding of Nalm-6 binding to KOp2.16 was not affected, even when Nalm-6 or KOP2.16 cells were pretreated with these inhibitors, indicating that the reduction of transmigration was not due to a reduction of Nalm-6 to KOP2.16. These results also indicate that the signal transduction pathway(s) involved in transmigration can be dissociated from that of adhesion. Our results support the notion that endothelial cells are not a passive barrier in lymphoma extravasation, but that they assist lymphoma cell extravasation.

Androstadienes↗
PubMed1998

Schedule-dependent synergism and antagonism between paclitaxel and methotrexate in human carcinoma cell lines.

Paclitaxel and methotrexate are active against a variety of solid tumors. Because of differences in their mechanisms of action and toxicity profiles, the combination of these two agents has clinical potential. Clinical studies of this combination are in progress. We studied the optimal schedule of paclitaxel and methotrexate in combination at various schedules in vitro using human lung cancer A549, breast cancer MCF7, ovarian cancer PA1, and colon cancer WiDr cells. Cells were simultaneously exposed to paclitaxel and methotrexate for 24 h and sequentially exposed to paclitaxel for 24 h followed by methotrexate for 24 h or vice versa. Cell growth inhibition after 5 days was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of drug combinations at the concentration of drug that produced 80% cell growth inhibition (the IC80 level) were analyzed by the isobologram method. The simultaneous exposure to paclitaxel and methotrexate produced additive to antagonistic effects in the A549 and PA1 cells, and antagonistic effects in the MCF7 and WiDr cells. The sequential exposure to paclitaxel followed by methotrexate produced additive effects in all four cell lines. The reverse sequence produced synergistic effects in the A549, MCF7, and WiDr cells, and additive effects in the PA1 cells. These findings suggest that a sequential administration of methotrexate followed by paclitaxel may be the appropriate schedule for this combination. On the basis of the observed in vitro synergism, further in vivo and clinical studies are necessary to clarify the toxicity and proposed antitumor effects of this schedule.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Cloning of cDNAs encoding G protein-coupled receptor expressed in human endothelial cells exposed to fluid shear stress.

A cDNA library of human umbilical vein endothelial cells exposed to fluid shear stress was constructed to search for functional endothelial genes expressed under flow conditions, and cDNAs encoding members of the G protein-coupled receptor (GPCR) family were cloned by a polymerase chain reaction (PCR) method using degenerate oligonucleotide primers. One of the two GPCR clones obtained was edg-1, and the other clone is a novel gene named FEG-1 that encodes a 375-amino acid protein similar to the receptors for both angiotensin II and chemokines. Reverse transcriptase-PCR showed that the FEG-1 and edg-1 mRNA levels in endothelial cells increased markedly in response to fluid flow. This suggests that FEG-1 and edg-1 may be receptor genes that play important roles in the regulation of endothelial function under physiological blood flow conditions.

Amino Acid Sequence↗
PubMed1997

Thirteen-week subchronic toxicity study of thiamphenicol in F344 rats.

A 13-week subchronic toxicity study of thiamphenicol (TAP) was performed in F344 rats. The minimum lethal dose was estimated to be greater than 10 g/kg body weight, when a single dose of 4-10 g/kg of TAP was given orally. In the subchronic toxicity study, groups of 12 F344 rats of each sex were given solutions containing 0 (control), 125, 250 and 500 ppm of TAP as their drinking water ad libitum for 13 weeks. Body weight gain was significantly suppressed in both sexes of the 250 and 500 ppm groups. Slight suppression of erythropoiesis was observed in the highest-dose group along with slightly reduced spermatogenesis in the testes of the males. In addition, spermatogranulomas were found in the epididymis of both middle- and highest-dose groups. The no observed adverse effect level (NOAEL) was concluded to be 125 ppm (daily doses of 9.0 mg/kg in males and 11.6 mg/kg in females). From the above described results, doses of 250 and 125 ppm were selected as appropriate for a 2-year carcinogenicity study.

Animals↗
PubMed1997

Negative transcriptional regulation of the VCAM-1 gene by fluid shear stress in murine endothelial cells.

To explore the mechanism of shear stress-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1) expression in murine endothelial cells (ECs), we examined the effect of shear stress on VCAM-1 gene transcription and assessed the cis-acting elements involved in this phenomenon. VCAM-1 mRNA expression was downregulated at the transcriptional level as defined by nuclear run-on assay and transient transfection of VCAM-1 promoter-luciferase gene constructs. The luciferase assay on the VCAM-1 deletion mutants revealed that the cis-acting element is contained between -694 and -329 bp upstream from the transcription initiation site. Gel shift assay using overlapping oligonucleotide probes of this region showed that oligonucleotides containing a double AP-1 consensus sequence (TGACTCA) formed distinct complexes with nuclear proteins extracted from shear-stressed cells. Mutation of either one or both of two AP-1 consensus sequences completely abolished the ability of the promoter to respond to shear stress. These results suggest that fluid shear stress downregulates the transcription of the VCAM-1 gene via an upstream cis-element, a double AP-1 consensus sequence, in murine lymph node venule ECs.

Animals↗
PubMed1997

Shear stress augments expression of C-type natriuretic peptide and adrenomedullin.

Shear stress is known to dilate blood vessels and exert antiproliferative effects on vascular walls: these effects have been ascribed to shear stress-induced upregulation of endothelium-derived vasoactive substances, mainly nitric oxide and prostacyclin. We have demonstrated the significance of C-type natriuretic peptide (CNP) as a novel endothelium-derived relaxing peptide (EDRP) that shares a cGMP pathway with nitric oxide. Adrenomedullin is a recently isolated EDRP that elevates intracellular cAMP as prostacyclin does. To elucidate the possible role of these EDRPs under shear stress, we examined the effect of physiological shear stress on CNP mRNA expression in endothelial cells derived from the human umbilical vein (HUVECs), bovine aorta (BAECs), and murine lymph nodes (MLECs) as well as adrenomedullin mRNA expression in HUVECs. CNP mRNA was stimulated prominently in HUVECs under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, sixfold increase compared with that in the static condition; 24 hours, 30-fold increase). Similar results were obtained in BAECs (4 hours, twofold increase; 24 hours, threefold increase) and MLECs (4 hours, threefold increase; 24 hours, 10-fold increase). Augmentation of CNP mRNA expression that was dependent on shear stress intensity was also observed (5 dyne/cm2, 2.5-fold increase of static; 15 dyne/cm2, 4.5-fold increase). Increased CNP secretion was also confirmed by the specific radioimmunoassay for CNP. Adrenomedullin mRNA expression in HUVECs increased under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, 1.2-fold increase of static: 24 hours, threefold increase) and shear stress intensity-dependent manner (15 dyne/cm2, threefold increase compared with that at 5 dyne/cm2). These results suggest that the coordinated augmentation of mRNA expression of these novel EDRPs may constitute shear stress-dependent vasodilator and antiproliferative effects.

Adrenomedullin↗
PubMed1996

Differential display and cloning of shear stress-responsive messenger RNAs in human endothelial cells.

To investigate the effect of shear stress on endothelial gene expression, we performed differential display of mRNAs from cultured human umbilical vein endothelial cells either incubated under static conditions or exposed to shear stress (15 dynes/cm2) for 6 h in a flow-chamber. Around 4% of the total number of mRNAs detected were either up- or down-regulated by shear stress. DNA sequencing of some of these shear stress-responsive mRNAs revealed homology of several clones to known gene sequences and many other clones for unknown genes. Known genes, including those for human laminin B1 chain, H(+)-ATP synthase coupling factor 6, lysyl oxidase, myosin light chain kinase, and interleukin-8 receptor, were upregulated by shear stress, while the gene encoding NADH dehydrogenase was down-regulated. The present results suggest that shear stress can change the expression of numerous genes in endothelial cells, far more than reported to date, and that mRNA differential display is quite useful for cloning known and unknown shear stress-responsive genes.

Antigens, CD↗
PubMed1996

Contribution of sustained Ca2+ elevation for nitric oxide production in endothelial cells and subsequent modulation of Ca2+ transient in vascular smooth muscle cells in coculture.

To elucidate the intracellular Ca2+ (Ca2+i ) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca2+i reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca2+i-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca2+i of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO2-. The order of potency of the agonists in terms of the peak Ca2+i in ECs was bradykinin (100 nM) > ATP (10 microM) > ionomycin (50 nM) > thapsigargin (1 microM). In contrast, the order in reference to both the extent of Ca2+i reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by NG-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca2+i response in cocultured VSMCs to the isoculture level. In ECs, when the Ca2+ influx was blocked by Ni2+ or by chelating extracellular Ca2+, all four agonists markedly decreased NO production, the half decay time of the Ca2+i degenerating phase, and the area under the Ca2+i curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca2+i curve but not to the Ca2+i peak level. Thus, the sustained elevation of Ca2+i in ECs, mainly a result of Ca2+ influx, determines the active NO production and subsequent Ca2+i reduction in adjacent VSMCs. Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca2+i in ECs, and markedly decreased the Ca2+i in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T. (1992) J. Biol. Chem. 267, 20377-20382).

Animals↗
PubMed1996

A matrix attachment region (MAR)-binding activity due to a p114 kilodalton protein is found only in human breast carcinomas and not in normal and benign breast disease tissues.

A M(r) 114,000 protein (p114) that specifically binds to nuclear matrix attachment DNA (matrix attachment region, MAR) from a breast carcinoma cell line SK-BR-3 was purified to near homogeneity. p114 strongly binds to a wild-type A+T-rich MAR probe with high unwinding propensity with a dissociation constant (Kd) of 10(-9), while it exhibits substantially reduced binding to a mutated A+T-rich non-MAR probe, which lacks unwinding propensity. This binding specificity and affinity is similar to the previously cloned thymocyte-associated MAR-binding protein SATB1. By Southwestern blot analysis, the MAR-binding activity of p114 is detectable in human breast carcinomas but is undetectable in normal breast tissues, benign breast diseases, and immortalized epithelial MCF-10A cells. Thus, the MAR-binding activity of p114 is not merely reflecting cell proliferation, but it strongly associates with breast carcinomas. The p114 MAR-binding activity was found in all 43 human breast carcinoma specimens tested, without exception. Much stronger p114 MAR-binding activity was detected in poorly differentiated than well-differentiated carcinomas. p114 may be a reliable diagnostic and possibly prognostic marker for breast cancer.

Base Sequence↗
PubMed1996

Schedule-dependent interaction between paclitaxel and 5-fluorouracil in human carcinoma cell lines in vitro.

We assessed the cytotoxic interaction between paclitaxel and 5-fluorouracil administered at various schedules against four human carcinoma cell lines, A549, MCF7, PA1 and WiDr. The cells were exposed simultaneously to paclitaxel and to 5-fluorouracil for 24 h or sequentially to one drug for 24 h followed by the other for 24 h, after which they were incubated in drug-free medium for 4 and 3 days respectively. In another experiment, the cells were exposed simultaneously to both agents for 5 days. Cell growth inhibition was determined by MTT reduction assay. The effects of drug combinations at IC80 were analysed by the isobologram. The cytotoxic interaction of paclitaxel and 5-fluorouracil was definitely schedule dependent. Simultaneous exposure to paclitaxel and 5-fluorouracil for 24 h showed mainly subadditive effects in A549, MCF7 and WiDr cell lines, whereas it showed additive effects in PA1 cells. Sequential exposure to paclitaxel followed by 5-fluorouracil showed additive effects in all cell lines. Sequential exposure to 5-fluorouracil followed by paclitaxel showed subadditive effects in A549, MCF7 and PA1 cells. Whereas it showed additive effects in WiDr cells. These findings suggest that maximum cytotoxic effects can be obtained when paclitaxel precedes 5-fluorouracil. Interestingly, the continuous (5-day) exposure to paclitaxel and 5-fluorouracil had additive effects in A549, PA1 and WiDr cells, indicating that the prolonged simultaneous administration of these agents may circumvent the antagonistic interaction produced by short-term simultaneous administration. These findings may be useful in clinical trials of combination chemotherapy with paclitaxel and 5-fluorouracil.

Antimetabolites, Antineoplastic↗