Biomedical subjects

H Takano

Publications and source records attributed to H Takano.

At least 73 records · Page 4Linked to original sources

PubMed2001

Incidence of mutation and deletion in topoisomerase II alpha mRNA of etoposide and mAMSA-resistant cell lines.

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. To further understand resistance to topoisomerase II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to VP-16 (etoposide) or mAMSA (m-amsacrine). Subsequently, a population of cells from each subline was exposed to three-fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration. Finally, 66 sublines were picked up. The frequency and nature of mutations in the topoisomerase II gene in the drug-selected cell lines were evaluated. In order to screen a large number of cell lines, an RNAse protection assay was developed and mismatches were observed in 13.6% of resistant cell lines (12% of resistant cell lines exposed to lower drug concentrations and 18.8% of resistant cell lines exposed to higher drug concentrations). Some of these mutations are located in vital regions of topoisomerase II (phosphorylation sites in the C-terminal or N-terminal, and nuclear localizing signal of topoisomerase II). Our findings suggest that mutations of topoisomerase II gene are an important and frequent mechanism of resistance to topoisomerase II inhibitors.

Amsacrine↗

PubMed2001

Expression of drug resistance genes in VP-16 and mAMSA-selected human carcinoma cells.

The cell lines described in the present study were isolated as part of an effort to understand resistance to topoisomerase (topo) II inhibitors. To that end, 50 sublines were isolated from four human breast cancer cell lines, i.e., MCF-7, T47D, MDA-MB-231, and ZR-75B. As an initial step, a concentration that would be lethal to the majority of cells (IC99) was selected for both VP-16 and mAMSA, for each cell line. The identification of an increasing number of putative drug resistance-related proteins provided the opportunity to examine expression of the corresponding genes in the selected cell lines. Northern blot analysis revealed different responses to the selecting agents in the different cell lines. Previous studies examining expression of multidrug resistance (MDR)-1 in resistant cell lines had found undetectable levels in all cells. In the ZR-75B sublines, increased expression of MDR-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) was observed, and when the relative levels of overexpression were compared, a high correlation was found. In contrast, increased expression of MRP was observed in some of the MDA-MB-231 sublines, without a concomitant increase in cMOAT expression. Finally, in both T47D and MCF-7 sublines, increased expression of cMOAT or MRP was observed infrequently, and where it occurred, was of a much smaller magnitude. In the analysis of expression of MRP, the highest levels were found in the ZR-75B and MDA-MB-231 sublines, with lower levels in the MCF-7 and T47D clones. Similarly, differences in the expression of topo IIalpha were observed among the sublines. Although the differences in expression appear to depend on the parental cell line from which the resistant sublines were derived, a strong correlation was observed between the expression of MRP and the levels of topo IIalpha. Cell lines with low levels of MRP had lower levels of topo IIalpha, while those with high levels of MRP maintained higher levels of topo IIalpha. While a reduced topo IIalpha level was common, there did not appear to be a compensating increase in the expression of topo IIbeta or topo I or casein kinase (CK) IIalpha in any of the cell lines. While the possibility that such compensation could occur has been discussed and even reported in some cell lines, such an adaptation was not observed in the present study, suggesting that it is not common.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Hypophosphorylation of topoisomerase IIalpha in etoposide (VP-16)-resistant human carcinoma cell lines associated with carboxy-terminal truncation.

Topoisomerase IIalpha is a target for many chemotherapeutic agents in clinical use. To define mechanisms of resistance and regions crucial for the function of topoisomerase IIalpha, drug-resistant cell lines have been isolated following exposure to topoisomerase II poisons. Two resistant sublines, T47D-VP and MCF-7-VP, were isolated from human carcinoma cell lines following exposure to 300 or 500 ng / ml etoposide (VP-16). Cytotoxicity studies confirmed resistance to etoposide and other topoisomerase II poisons. KCl-sodium dodecyl sulfate (K-SDS) precipitation assays using intact cells showed reduced DNA-topoisomerase II complex formation following VP-16 or amsacrine (m-AMSA). RNAse protection analysis identified a deletion of 200 base pairs in the topoisomerase IIalpha cDNA of T47D-VP and rising dbl quote, left (low)AA insertion" in the topoisomerase IIalpha cDNA of MCF-7-VP. Reduced topoisomerase IIalpha mRNA and protein levels were observed in both cell lines. It was somewhat surprising to find that nuclear extracts from T47D-VP and MCF-7-VP cells had comparable topoisomerase II activity to that of parental cells. Analysis of the extent of phosphorylation demonstrated that topoisomerase IIalpha from the resistant cells was relatively hypophosphorylated compared to that of parental cells. In these cell lines, hypophosphorylation secondary to loss of a portion of the C-terminal domain of topoisomerase IIalpha mediated the restored activity, despite a fall in topoisomerase IIalpha mRNA and protein, and this resulted in cross resistance to topoisomerase II poisons.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Cyclooxygenase-2 does not mediate late preconditioning induced by activation of adenosine A1 or A3 receptors.

Recent studies have demonstrated that the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and the adenosine A3 receptor agonist N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) produce a delayed phase of protection against infarction similar to the late phase of ischemic preconditioning (PC). However, the mechanism for adenosine A1 or A3 receptor-induced late PC remains unknown. The goal of this study was to determine whether the delayed cardioprotective effects of adenosine A1 or A3 receptors are mediated by cyclooxygenase-2 (COX-2), which is an obligatory mediator of ischemic PC. We found that COX-2 protein expression (Western blotting) did not increase 24 h after the administration of either CCPA (100 microg/kg iv) or IB-MECA (300 microg/kg iv) compared with controls. To probe the role of constitutive COX-2 expression, conscious rabbits were subjected to 30-min coronary occlusion followed by 72-h reperfusion. Twenty-four hours before the occlusion, the rabbits were pretreated with CCPA (100 microg/kg iv) or IB-MECA (300 microg/kg iv). Both CCPA and IB-MECA resulted in a marked (approximately 47%) reduction in infarct size vs. controls [36.2 +/- 4.0% of the risk region (n = 9), 31.2 +/- 4.7% (n = 9), and 59.5 +/- 3.8% (n = 9), respectively; P < 0.05], similar to that induced by the late phase of ischemic PC [31.8 +/- 3.2% (n = 9)]. The selective COX-2 inhibitor N-(2-[cyclohexyloxy]4-nitrophenyl)methanesulfonamide (NS-398, 5 mg/kg), which abolished the protective effect of ischemic late PC, failed to block the protection of either CCPA or IB-MECA, indicating that COX-2 does not mediate the delayed protection of either CCPA or IB-MECA [CCPA + NS-398, 29.1 +/- 3.4% (n = 7); IB-MECA + NS-398, 34.9 +/- 2.9% (n = 8)]. NS-398 in itself did not affect infarct size [54.9 +/- 3.7% (n = 9)]. Taken together, these results demonstrate that, in contrast to ischemia-induced late PC, the mechanisms of adenosine A1 or A3 receptor-induced late PC is independent of COX-2.

Animals↗

PubMed2001

Effect of bulking agent on the reduction of NH3 emissions during thermophilic composting of night-soil sludge.

Night-soil sludge contains a high percentage of nitrogen and releases large amounts of ammonia when it is composted. In this research, three types of bulking agents, sawdust, perlite, and activated carbon, were used, and the effects of these bulking agents on the reduction of NH3 emissions during thermophilic composting were evaluated. It was found that not only is sawdust the most effective agent in reducing NH3 emissions, but that it also effectively promotes organic matter decomposition. Increases in the mixing ratio of sawdust in the range adopted in the present research, from 70% to 92% on a dry-weight basis, appeared to cause no adverse effects on the decomposition of sludge itself. And the higher the mixing ratio of sawdust, the more effective the reduction in NH3 emissions, however this effect was not enough to completely suppress these emissions, even at the highest mixing ratio.

Air Pollution↗

PubMed2001

Detection of nasopharyngeal carcinoma: fast short time inversion recovery images compared with fat suppression, contrast enhanced T(1) weighted spin echo images.

The aim was to compare fast short time inversion recovery (FSTIR) images and fat suppression, contrast enhanced T(1) weighted (FSCE T1W) spin echo images in the diagnosis of nasopharyngeal carcinoma (NPC). 102 MR studies were obtained with a 1.0 T or a 1.5 T system in 28 patients with NPC. The MR studies comprised both FSTIR and FSCE T1W images. FSTIR and FSCE T1W images were compared for detection of NPC by means of a receiver operating characteristic (ROC) analysis. The areas under the ROC curves of FSTIR and FSCE T1W images showed no statistical difference (0.87 vs 0.87). There was also no statistical difference in the sensitivity, specificity and accuracy of each sequence (0.74 vs 0.77, 0.81 vs 0.77 and 0.79 vs 0.77, respectively). Both sequences had the same performance for detection of NPC. FSTIR is as useful as FSCE T1W images, especially in the detection of recurrent tumours, but without the cost of contrast medium.

Adult↗

PubMed2001

Effects of losartan on blood pressure and humoral factors in a patient who suffered from anaphylactoid reactions when treated with ACE inhibitors during LDL apheresis.

In a patient who was taking an angiotensin-converting-enzyme inhibitor, low-density lipoprotein (LDL) apheresis with dextran-sulfate cellulose provoked hypotension accompanied by lacrimation and blurred vision. Hypotension was eliminated by changing the anticoagulant from heparin to a protease inhibitor, nafamostat mesilate. A study was undertaken to clarify whether an antagonist of angiotensin type 1-receptor, losartan, could be safely used in the same patient during LDL apheresis treatment. Blood pressure and humoral factors were compared between the apheresis sessions with losartan and those without. Although angiotensin II and bradykinin plasma levels during LDL apheresis were significantly greater with losartan than without, blood pressure reduction by losartan was mild and unpleasant symptoms were not induced. Losartan was thus safely used for this patient during treatment by LDL apheresis. The greater rise in bradykinin levels during apheresis with losartan might be ascribable to angiotensin type 2-receptor stimulation.

Aldosterone↗

PubMed2001

Evaluation of renal biopsy samples of patients with diabetic nephropathy.

OBJECTIVE: To evaluate the usefulness of renal biopsy in the overall management of patients with diabetes mellitus (DM), we examined the relationship between the clinical parameters and histopathological findings of renal biopsy samples. METHODS: Renal biopsy specimens were obtained from 109 type 2 diabetic patients with proteinuria. Samples were divided into the following two groups: Diabetic Nephropathy (DN) group (n=80) had typical diabetic lesions without other renal diseases, complication group (n=29) had diabetic lesions with other renal diseases. Furthermore, DN group was subdivided into two subgroups: slow progressive group (SP group, n=32), the level of serum creatinine (s-Cr) was normal at the time of renal biopsy and three years after renal biopsy, and fast progressive group (FP group, n=14), the level of s-Cr was normal at the time of renal biopsy but more than doubled three years after renal biopsy. RESULTS: The level of total protein was significantly lower and HbA1c significantly higher in the DN group than in the Complication group. However, other clinical parameters were not significantly different between the two groups. Urinary protein, systolic and diastolic blood pressure in FP group were significantly higher than in SP group. The percentage of sclerotic glomeruli, the severity of mesangial expansion, tubular injury and cell infiltration were significantly greater in FP than in SP group. CONCLUSIONS: Our results indicated that a complete evaluation of renal pathology in DM could not be made by clinical parameters only, and that the progression of DN could be accurately predicted by histopathological evaluation. Therefore, this study emphasizes the importance of renal biopsy in the overall management of patients with DM and/or DN.

Adult↗

PubMed2001

Unique action determinants of double acting topoisomerase inhibitor, TAS-103.

O6-methylguanine-DNA methyltransferase (MGMT), gamma-glutamylcysteine synthetase (gamma-GCS), and glutathione (GSH) are found to participate in resistance to TAS-103, a topoisomerase I/II inhibitor. In 13 human cancer cell lines, MGMT expression correlated with IC50 for TAS-103, whereas gamma-GCS expression inversely correlated with the IC50 value, suggesting MGMT may work to decrease TAS-103 activity but gamma-GCS may increase it. A reduced gamma-GCS and GSH, and an increased MGMT were associated with the development of resistance in A549 and DLD cells, and gamma-GCS inhibition by buthionine sulphoximine increased the TAS-103 resistance, whereas MGMT inhibition by both O6-benzyl-guanine and MGMT-antisense transfection sensitized cells to TAS-103.

Aminoquinolines↗

PubMed2001

Oxygen metabolism under various bypass flow conditions during cardiopulmonary support in awake goats.

Despite its wide clinical application, patient recovery from cardiopulmonary support (CPS) is not necessarily satisfactory. To clarify what influence CPS has on organ perfusion, we investigated the oxygen metabolism under various bypass flow (BF) conditions in a series of chronic animal CPS experiments. The CPS system, which consists of a pulsatile ventricular assist device and a compact artificial lung was installed without anesthesia in 6 adult goats weighing 49-51 kg. BF was adjusted stepwise from 0% to 50%, 75%, 90%, and 100% of total systemic blood flow (TSF) by balancing the pulmonary arterial flow. The animals' TSF and oxygen delivery (DO2) were sufficiently maintained throughout the experiments. The oxygen consumption (VO2) and the oxygen extraction rate (ExO2) increased from 178+/-14 to 342+/-19 ml/min, and from 28+/-2% to 64+/-1%, respectively, in proportion to the increase of CPBF dependency from 0% to 100%. The blood lactate level did not change appreciably even at 90% BF from 5.7+/-0.3 to 11.2+/-1.2 mg/dl, but drastically elevated to 23.5+/-4.6 mg/dl at the total bypass. This indicates that CPS leads to a relative lack of oxygen and can induce organ dysfunction due to increasing VO2 and ExO2 in proportion to the increase of BF dependence even if TSF and DO2 are sufficiently maintained.

Analysis of Variance↗

PubMed2001

[Cardiovascular abnormalities as a cause of hypertension].

This review is an attempt to highlight evidence that may implicate the cardiovascular abnormalities in the pathogenesis of hypertension. Many physiological, pharmacological, and biochemical studies have been conducted in in vitro and in vivo systems. Since blood pressure can rise in response to an increase in cardiac output and/or a rise in peripheral resistance, abnormalities may be present in one or more of the multiple factors that affect these two parameters in hypertension. These multiple factors include various neurohumoral factors. Increased levels of various vasoconstrictor neurohumoral factors have been found in patients with hypertension. Vasoconstrictor neurohumoral factors such as catecholamines, angiotensin II, and endothelin-1 induce vascular smooth muscle cells(VSMCs) proliferation and contraction. On the other hand, vasodilator neurohumoral factors such as natriuretic peptides and adrenomedulin inhibit VSMCs proliferation. Both neurohumoral factors mutually interact and develop hypertension.

Adrenomedullin↗

PubMed2001

Characterization of the spontaneous electrical and contractile activity of smooth muscle cells in the rat upper urinary tract.

PURPOSE: We morphologically and electrophysiologically identified the cells that generate the electrical activity underlying the peristaltic contractions of the rat upper urinary tract. MATERIALS AND METHODS: Electron microscopy and tension recording techniques were used to characterize the smooth muscle cells underlying spontaneous contractions in the wall of the rat ureter, and proximal and distal renal pelvis. Intracellular microelectrodes, containing 4% neurobiotin were used to record data from the cells of the renal pelvis, which were later viewed on a confocal microscope. RESULTS: Spontaneous myogenic contractions (average 22.3 +/- 2.2 minutes(-1)) originated in the proximal renal pelvis and propagated into the distal renal pelvis and ureter in 6 preparations. Smooth muscle cells in the renal pelvis and ureter were typical in appearance with greater than 85% of their sectional area containing clumped contractile filaments. In contrast, contractile fibrils occupied only 65% of the sectional area of the smooth muscle cells within the most proximal region of the renal pelvis (pelvicaliceal junction). In strips of the renal pelvis spindle shaped cells 83 to 200 microm. long fired spontaneous action potentials (6 minutes(-1)) consisting of an initial spike, a quiescent plateau phase and abrupt hyperpolarization to a peak diastolic potential of -60 mV. Other spindle shaped cells 94 to 112 microm. long displayed small membrane transients (15 minutes(-1)) 9 to 19 mV. in amplitude, firing from a diastolic potential of -40 mV. CONCLUSIONS: It is likely that the spontaneous contractile activity of the rat upper urinary tract arises from the discharge of action potentials in typical smooth muscle cells of the proximal renal pelvis that are directly driven by the spontaneous membrane oscillations of atypical smooth muscle cells.

Action Potentials↗

PubMed2001

[Radiological assessment of the structural relationship between the trachea and the innominate artery in physically disabled and scoliotic children].

Studies were made on the physically disabled and scoliotic children who underwent tracheostomy. The purpose was to clarify factors that might lead to the tracheo-innominate artery fistula, by use of three-dimensional helical CT (3 DCT). In a case of right scoliosis, the tracheo-innominate artery fistula may be caused by the left-ward displacement of the trachea from the vertebra, having the innominate artery ride over the trachea and compressing it. In a case of the physically disabled and scoliotic children, there may be more compression on the trachea by the innominate artery along with the worsening scoliosis. Therefore, we consider it necessary to examine the structural relationship between the trachea, the vertebra, and the innominate artery periodically by 3 DCT.

Adolescent↗

PubMed2001

Altered topoisomerase IIalpha and multidrug resistance-associated protein levels during drug selection: adaptations to increasing drug pressure.

To understand resistance to topoisomerase II inhibitors, we used four cancer cell lines (ZR-75B, MDA-MB-231, T47D, and MCF-7) and performed a single-step selection process to isolate 50 clones resistant to topoisomerase II inhibitors. Of these, 26 were isolated with VP-16 and 24 with mAMSA. Sixteen of these isolates (four from each cell line; two selected with VP-16 and two with mAMSA) were further exposed to higher drug concentrations. Characterization of the resistant sublines revealed the adaptation that occurs with increasing drug concentration during in-vitro selections. Reduced topoisomerase IIalpha mRNA level was observed in the majority of the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity. Other isolates showed increased levels of multidrug resistance-associated protein (MRP). With advancing resistance, MRP expression was increased further, concomitantly with some recovery in topoisomerase IIalpha expression and topoisomerase II activity. In these sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These results extend previous studies demonstrating how cellular adaptation to increasing drug pressure utilizes more than one mechanism. Reduced expression of topoisomerase IIalpha occurs early in the selection process. MRP overexpression can occur early or can help to confer high levels of resistance. In the latter case, MRP overexpression allows some recovery of topoisomerase II activity without loss of high drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

[Bone marrow plays an important role in joint destruction in patients with rheumatoid arthritis].

In iliac bone marrow the absolute number of mononuclear cells (MNCs) was increased in RA patients compared with the non-RA controls. In CD8 positive cell and myeloid cell fractions, significant differences were recognized between RA patients and non-RA controls. The presence of abnormal myeloid lineage cells in epiphyseal bone marrow adjacent to joints affected with severe RA was shown. Stroma cell lines from RA bone marrow with nursing activity were established and shown to play a pivotal role in the pathogenesis in RA bone marrow. Histologic study also shows that subchondral region expressing tissue-damaging proteinases plays an important role in joint destruction in RA.

English Abstract↗

PubMed2000

Direct interaction of p53 with the Y-box binding protein, YB-1: a mechanism for regulation of human gene expression.

The Y-box binding protein, YB-1, belongs to a family of multifunctional proteins which regulate gene expression on both transcriptional and translational levels. The tumor suppressor gene p53 displays growth suppressive properties by regulating gene expression through transcriptional regulation. We now demonstrate that YB-1 directly interacts with p53 using an in vitro pull-down assay. Using immunochemical co-precipitation methods, we also found that the two proteins are bound in vivo. Deletion analysis showed that three independent domains of YB-1, one at the N-terminal and two at the C-terminal, interact with p53. Conversely, a 14 amino acid sequence at the C-terminal of p53 was required for its interaction with YB-1. Gel mobility shift assays showed that the interaction of YB-1 with p53 stimulated the sequence-specific DNA binding of p53 to its consensus sequence. By contrast, this interaction inhibited the binding of YB-1. Using a p53-responsive p21 promoter linked to a reporter gene, it can be shown that antisense expression of YB-1 inhibits the induction of this promoter by p53 in transient transfection assays. These findings delineate a straightforward mechanism for gene expression through p53-YB-1 interaction.

CCAAT-Enhancer-Binding Proteins↗

PubMed2000

In vitro self-renewal division of hematopoietic stem cells.

Little is known about how hematopoietic stem cells (HSCs) self-renew. We studied the regeneration of HSCs in culture. Effects of various cytokines on cell division of CD34(-/low) c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) bone marrow cells of the mouse were first evaluated in serum-free single cell culture. We then performed a competitive repopulation assay on divided cells to ask if such cell division involved self-renewal of HSCs. In the presence of stem cell factor (SCF), thrombopoietin (TPO) induced a first cell division of CD34(-)KSL cells more efficiently than did interleukin (IL)-3 or IL-6. Multilineage repopulating cells were detected in a significant proportion of cells derived from single cells in culture with TPO and SCF, although this culture condition led to a substantial decrease in HSC number. These regenerated repopulating cells could be further transplanted into secondary recipients. When paired daughter cells were separately studied, one of a pair gave rise to repopulating cells with self-renewal potential, suggesting asymmetric self-renewal division. This study provides evidence that one HSC regenerates at least one HSC in culture.

Animals↗

PubMed2000

Peroxisome proliferator-activated receptor activators inhibit lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal rat cardiac myocytes.

Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Recently, PPAR activators have been shown to inhibit the production of proinflammatory cytokines in macrophages or vascular smooth muscle cells. It has been reported that tumor necrosis factor-alpha (TNF-alpha) expression is elevated in the failing heart and that TNF-alpha has a negative inotropic effect on cardiac myocytes. Therefore, we examined the effects of PPARalpha and PPARgamma activators on expression of TNF-alpha in neonatal rat cardiac myocytes. Northern blot analysis revealed expression of PPARalpha and PPARgamma mRNA in cardiac myocytes. Immunofluorescent staining demonstrated that both PPARalpha and PPARgamma were expressed in the nuclei of cells. When cardiac myocytes were transfected with PPAR responsive element (PPRE)-luciferase reporter plasmid, both PPARalpha and PPARgamma activators increased the promoter activity. Cardiomyocytes were stimulated with lipopolysaccharide (LPS), and the levels of TNF-alpha in the medium were measured by ELISA. After exposure to LPS, the levels of TNF-alpha significantly increased. However, pretreatment of myocytes with PPARalpha or PPARgamma activators decreased LPS-induced expression of TNF-alpha in the medium. Both PPARalpha and PPARgamma activators also inhibited LPS-induced increase in TNF-alpha mRNA in myocytes. In addition, electrophoretic mobility shift assays demonstrated that PPAR activators reduced LPS-induced nuclear factor-kappaB activation. These results suggest that both PPARalpha and PPARgamma activators inhibit cardiac expression of TNF-alpha in part by antagonizing nuclear factor-kappaB activity and that treatment with PPAR activators may lead to improvement in congestive heart failure.

Animals↗