Biomedical subjects

T Masuzawa

Publications and source records attributed to T Masuzawa.

At least 55 records · Page 3Linked to original sources

PubMed1998

Frequent coexpression of MRP/GS-X pump and gamma-glutamylcysteine synthetase mRNA in drug-resistant cells, untreated tumor cells, and normal mouse tissues.

Expression of the multidrug-resistance protein gene MRP, which confers non-P-glycoprotein-mediated multidrug resistance, has been found in many drug-resistant variants and tumor samples. Recent studies have demonstrated that MRP functions as an ATP-dependent transporter functionally related to the previously described glutathione-conjugate (GS-X) pump. We have shown recently that the MRP and gamma-glutamylcysteine synthetase (gamma-GCS) heavy subunit mRNA levels are coordinately overexpressed in cisplatin (CP)-resistant human leukemia cells (Ishikawa et al., J Biol Chem 271: 14981-14988, 1996) and frequently co-elevated in human colorectal tumors (Kuo et al., Cancer Res 56: 3642-3644, 1996). In the present study, we showed the coexpression patterns of thirteen additional human drug-resistant cell lines representing different tumor cell origins selected with different agents, except for one doxorubicin-selected line which demonstrated minor elevation in MRP mRNA with no detectable increase in gamma-GCS mRNA, suggesting that the increase of MRP mRNA preceded the increase in gamma-GCS mRNA. Furthermore, in seventeen randomly selected untreated tumor cell lines, the overall correlation coefficient between MRP and gamma-GCS mRNA levels was 0.861. In normal mice, the correlation coefficient of mrp and gamma-gcs mRNA was 0.662 in fourteen tissues (kidney and liver were not included) analyzed. Kidney and liver expressed low levels of mrp relative to gamma-gcs; however, these two tissues expressed high levels of a functionally related mrp homologue, mrp2 (cMoat or cMrp), which may have compensated for the underexpressed mrp in maintaining the total GS-X pump activities. Altogether, these results demonstrated the frequent coexpression of these two genes in various cell settings.

ATP-Binding Cassette Transporters↗

PubMed1998

Biphasic changes in F3/contactin expression in the gerbil hippocampus after transient ischemia.

We studied changes in expression of F3/contactin (F3), a neuron-specific adhesion molecule, in the gerbil hippocampus after transient forebrain ischemia for 5 min. By immunohistochemical techniques using F3 antibody, we found a biphasic change in immunoreactivity for F3 in the CA1 area after ischemia. Western blotting of F3 protein showed a similar biphasic change. F3 immunoblots decreased to 67% of the control at 1 week, but then they increased and attained 159% at 3 weeks and 152% at 5 weeks after ischemia. Immunoreactivity of a neurofilament (NF145) showed a similar biphasic change to F3 but to a lesser extent. In contrast, microtubule-associated protein 2 (MAP2) immunoreactivity uniformly decreased after ischemia. In situ hybridization revealed that F3 messenger RNA (mRNA) hybridization signals in CA1 area were greatly reduced 1 week after ischemia, while the signals in the CA3 area were unchanged and even increased 3 weeks after ischemia. Damage to CA3 neurons by hyperthermic ischemia blocked the F3 increase in area CA1. Our results suggest that the initial decrease in F3 following ischemia reflects loss of CA1 neurons and the late increase in F3, which shows that a similar time course with neurofilaments may be caused by neurite sprouting.

Animals↗

PubMed1998

Flow visualization as a complementary tool to hemolysis testing in the development of centrifugal blood pumps.

With a 250% scaled-up pump model, high speed video camera, and argon ion laser light sheet, flow patterns related to hemolysis were visualized and analyzed with 4 frame particle tracking software. Different flow patterns and shear distributions were clarified by flow visualization for pumps modified to have different hemolysis levels. A combination of in vitro hemolysis tests, flow visualization, and CFD analysis suggested a close relationship between hemolysis and high shear caused by small impeller/casing gaps. Because arbitrary cross sections can be illuminated by laser light sheet, flow visualization is a useful tool in finding locations related to hemolysis in the design process of rotary blood pumps.

Biomechanical Phenomena↗

PubMed1998

Development of a flow estimation and control system of an implantable centrifugal blood pump for circulatory assist.

A bypass flow rate estimation and control system (BECS) for an implantable centrifugal blood pump (ICBP) has been developed in our institute. The estimated flow rate (EF) of the ICBP was derived from the electric power consumption, the rotating speed of a motor, and the blood viscosity presumed by the hematocrit and body temperature. The error in the EF was 0.5 +/- 0.4 L/min in in vivo experiments for 40 days. The rotating speed of the motor was controlled automatically every 200 ms to bring the EF in accord with the desired flow rate (DF). The reactivity and accuracy of the BECS were investigated in in vitro and in vivo experiments. The ICBP was operated by the BECS in a mock circuit in parallel with a pulsatile ventricular assist device (PVAD) to simulate left heart bypass. The reactivity was evaluated by changing the DF from 7 L/min to 5 L/min at an afterload of 160/97 mm Hg. To evaluate the accuracy of the BECS, the ICBP was driven under the aortic pressure of 110/85 mm Hg in the abdominal wall of an adult goat (70 kg). The DF was set at 5 L/min for 4 min for the goat in an awake condition. It took 13 s to change the flow rate in the in vitro experiment. The measured flow rate (MF) was maintained at 5.0 +/- 0.2 L/min by the BECS in vivo. In conclusion, the BECS has moderate reactivity and accuracy.

Abdominal Muscles↗

PubMed1998

Prolonged nonpulsatile left heart bypass with reduced systemic pulse pressure causes morphological changes in the aortic wall.

We investigated the morphological changes in the aorta due to reduced systemic pulse pressure in prolonged nonpulsatile left heart bypass (LHB). Nineteen adult goats were divided into 3 groups, the nonpulsatile group in which nonpulsatile LHB was conducted, the pulsatile group in which pulsatile LHB was conducted, and the control group used as the normal control. The average aortic pulse pressures were 12, 47, and 37 mm Hg, respectively. The descending aorta was subjected to morphological examination. In the nonpulsatile group, the wall was significantly thinner, and the volume ratio of smooth muscle cells (SMCs) was much lower. In terms of the SMC type classification, the proportion of SMCs with low activity and low contractility was higher, and the cell density of the SMCs was increased compared to those in the other groups. These results indicate that prolonged nonpulsatile LHB causes morphological atrophic changes in the aorta.

Animals↗

PubMed1998

Development of design methods of a centrifugal blood pump with in vitro tests, flow visualization, and computational fluid dynamics: results in hemolysis tests.

There are few established engineering guidelines aimed at reducing hemolysis for the design of centrifugal blood pumps. In this study, a fluid dynamic approach was applied to investigate hemolysis in centrifugal pumps. Three different strategies were integrated to examine the relationship between hemolysis and flow patterns. Hemolytic performances were evaluated in in vitro tests and compared with the flow patterns analyzed by flow visualization and computational fluid dynamic (CFD). Then our group tried to establish engineering guidelines to reduce hemolysis in the development of centrifugal blood pumps. The commercially available Nikkiso centrifugal blood pump (HPM-15) was used as a standard, and the dimensions of 2 types of gaps between the impeller and the casing, the axial and the radial gap, were varied. Four impellers with different vane outlet angles were also prepared and tested. Representative results of the hemolysis tests were as follows: The axial gaps of 0.5, 1.0, and 1.5 mm resulted in normalized index of hemolysis (NIH) values of 0.0028, 0.0013 and 0.0008 g/100 L, respectively. The radial gaps of 0.5 and 1.5 mm resulted in NIH values of 0.0012 and 0.0008 g/100 L, respectively. The backward type vane and the standard one resulted in NIH values of 0.0013 and 0.0002 g/100 L, respectively. These results revealed that small gaps led to more hemolysis and that the backward type vane caused more hemolysis. Therefore, the design parameters of centrifugal blood pumps could affect their hemolytic performances. In flow visualization tests, vortices around the impeller outer tip and tongue region were observed, and their patterns varied with the dimensions of the gaps. CFD analysis also predicted high shear stress consistent with the results of the hemolysis tests. Further investigation of the regional flow patterns is needed to discuss the cause of the hemolysis in centrifugal blood pumps.

Animals↗

PubMed1998

Computational fluid dynamic analyses to establish design process of centrifugal blood pumps.

To establish quantitative, efficient design theories for centrifugal blood pumps, computational fluid dynamics (CFD) analyses were compared to the results of flow visualization tests and hemolysis tests, mainly on the Nikkiso centrifugal blood pump. The results turned out to coincide in the velocity vector plots. CFD analysis revealed that the smaller the gap is, the greater the shear stress becomes. This tendency becomes even greater with a radial gap change. Hemolysis study also indicated that the smaller the gap is, the greater the hemolysis. CFD analysis in comparison with hemolysis tests could be a useful index for developing blood pumps in the future.

Biomechanical Phenomena↗

PubMed1998

Infectivity and arthritis induction of Borrelia japonica on SCID mice and immune competent mice: possible role of galactosylceramide binding activity on initiation of infection.

We investigated the relationship between the binding activity to galactosylceramide (GalCer) and the arthritis induction activity of Borrelia japonica. The B. japonica strains maintained the ability to induce arthritis in inbred C3H/HeN and immunodeficient SCID mice, but the ability was lower than that of Borrelia burgdorferi sensu stricto virulent strain 297. Histopathological changes were restricted to the joints, and a marked effusion of polymorphonuclear neutrophils into the joint space was found. The binding activity of B. japonica strains to GalCer was lower than that of the virulent strain 297 but higher than that of the high-passage strain 297. The lower infectivity and virulence of B. japonica may explain its lower binding ability to GalCer.

Animals↗

PubMed1998

Lyme disease Borrelia species in northeastern China resemble those isolated from far eastern Russia and Japan.

Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.

Base Sequence↗

PubMed1998

Change in Ca2+ sensitivity of cerebrovascular smooth muscle in experimental chronic cerebral vasospasm.

The mechanism of chronic cerebral vasospasm was investigated by examining the relation between cytosolic Ca2+ level ([Ca2+]i) and muscle contraction in vascular smooth muscle after experimental vasospasm produced by the two-hemorrhage method in the canine basilar artery. [Ca2+]i and tension were recorded simultaneously with a fluorimeter using the arterial strips with a Ca2+ indicator, fura-2. High K+ concentration (72.4 mM) and U-46619 (thromboxane A2 analogue, 10(-8) M) were used as stimulants. The increase in contractile tension per [Ca2+]i per unit cross-sectional area was calculated, and compared for the vasospasm and the control groups. The increase in [Ca2+]i in the vasospasm group was smaller than that in the control group, so the increase in tension per [Ca2+]i per unit sectional area was larger in the vasospasm group. This value was much larger for the contraction induced by U-46619 than that induced by high K+ concentration in both groups. These results suggest that there is an increase in the basal Ca2+ sensitivity of the contractile elements in vasospastic vessels. In addition, the U-46619-induced increase in Ca2+ sensitivity seemed to be augmented in the vasospasm group.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

Lyme disease spirochetes in ticks from northeastern China.

During May 1996, field surveys on Lyme disease spirochetes were conducted in Beijing, Shenyang, Fushun, and Inner Mongolia in northeastern China. The ticks collected consisted of 3 genera and 12 species. Of these, Ixodes persulcatus was dominant in sun-exposed vegetation in forests in Inner Mongolia; 57 Borrelia strains (55/123 unfed adults and 2/5 immature stages fed on a rodent) were obtained from this tick by BSK culture. Additionally, 2/2 Apodemus peninsulae were positive. Ixodes nipponensis, Ixodes pavlovskyi, Haemaphysalis douglasi, and Haemaphysalis megaspinosa, newly recorded in China, and other Haemaphysalis spp. were all negative for Borrelia. Based on a polymerase chain reaction restriction fragment length polymorphism analysis of the 45 strains successfully subcultured, these were classified as 29 Borrelia garinii and 16 Borrelia afelii. These strains seemed to be more closely related to Japanese strains in genetic features than to those from Europe. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested more diversity in both genospecies, but Borrelia burgdorferi sensu stricto was not found.

Animals↗

PubMed1997

Extensive RNA editing and possible double-stranded structures determining editing sites in the atpB transcripts of hornwort chloroplasts.

Three nonsense codons and an unusual initiation codon were located within the putative coding region of the atpB gene of chloroplast DNA of the hornwort Anthoceros formosae. Nucleotide sequencing of cDNA prepared from transcripts revealed extensive RNA editing. The unusual initiation codon ACG was changed to AUG and three nonsense codons were converted into sense codons. In total 15 C residues of the genomic DNA were replaced by U residues in the mRNA sequences, while 14 U residues were replaced by C residues. This is the highest number of editing events for a chloroplast mRNA reported so far. Partial editing was also shown in a cDNA clone where 23 sites were edited but six sites remained unedited, representing the existence of premature mRNA. The expected two-dimensional structure of the mRNA shows the existence of a sequence complementary to every editing site, which can produce continuous base pairing longer than 5 bp, suggesting that mispairing in the double strand is the site determinant for RNA editing in Anthoceros chloroplasts. Comparison of the cDNA sequence with other chloroplast genes suggests that the mechanism arose in the first land plants and has been reduced during evolution.

Amino Acid Sequence↗

PubMed1997

Transient induction of the MRP/GS-X pump and gamma-glutamylcysteine synthetase by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea in human glioma cells.

Treatment of human glioma A172 cells with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), an alkylating antitumor agent the primary target of which has been thought to be DNA, resulted in elevated expression of mRNA for multidrug resistance-associated protein (MRP) within the first 2 h and then a decrease in expression 24 h after the treatment. Western blot analyses revealed that levels of MRP in these ACNU-treated cells paralleled mRNA levels. Membrane vesicles prepared from ACNU-treated cells also displayed elevated transport activities for leukotriene C4, a known substrate for MRP. Gamma-glutamylcysteine synthetase (gamma-GCS) mRNA expression was coinduced with MRP by ACNU. Because gamma-GCS is the rate-limiting enzyme involved in the de novo biosynthesis of glutathione, increases in glutathione were also transiently induced by ACNU. These results demonstrate for the first time that the expression of functional MRP and gamma-GCS can be transiently coinduced by ACNU. Multiple short exposures (1 h) of ACNU following a long duration (1 week) of drug-free conditions resulted in the development of an ACNU-resistant population (designated A172R) that overexpressed MRP/gamma-GCS mRNA and had elevated transport activities for leukotriene C4. A172R exhibited cross-resistance to the antitumor drug doxorubicin and heavy metal sodium arsenate but not to cisplatin. Our results also demonstrate that intermittent treatments of human glioma cells with ACNU can lead to the development of MRP-related multidrug resistance. These results, taken together, reveal a possible new mechanism of the development of drug resistance for the antitumor nitrosoureas.

ATP-Binding Cassette Transporters↗

PubMed1997

Posttranscriptional regulation of MRP/GS-X pump and gamma-glutamylcysteine synthetase expression by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea and by cycloheximide in human glioma cells.

Treatment of human glioma A172 cells with 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethy-3-nitrosourea (ACNU) for 2 to 4 hr resulted in a 2- to 3-fold increase in steady-state levels of multidrug resistance-associated protein (MRP) and gamma-glutamylcysteine synthetase (gamma-GCS) mRNA. Nuclear run-on assays revealed a less than 0.5-fold increase in transcription rates of these genes under the same treatment conditions, suggesting that posttranscriptional regulation plays an important role for the increased mRNA levels. In the absence of ACNU, rates of MRP and gamma-GCS mRNA degradation were similar in A172 cells as determined by incubating cells with the RNase inhibitor, Actinomycin D. ACNU treatments resulted in increased MRP mRNA stability. Induction of MRP and gamma-GCS mRNA by ACNU apparently did not require de novo protein synthesis as determined by the use of protein synthesis inhibitor cycloheximide (CHX). However, CHX alone could induce accumulation of gamma-GCS mRNA, also by posttranscriptional mechanism. Taken together, these results demonstrate that (i) posttranscriptional regulation is primarily involved in the induction of MRP and gamma-GCS expression by ACNU and CHX in human glioma cells; and (ii) despite the fact that these two genes have been reported to be frequently co-expressed, their responses to the treatments of RNA and protein synthesis inhibitors are not the same.

ATP-Binding Cassette Transporters↗

PubMed1997

Relationship between pathogenicity of Coxiella burnetii isolates and gene sequences of the macrophage infectivity potentiator (Cbmip) and sensor-like protein (qrsA).

Coxiella burnetii, the Q fever agent, is an obligate intracellular bacterium and survival in phagolysosomes is an important virulence factor. The present study was performed to determine the relationship between its pathogenicity and genes related to its survival in macrophages, i.e. macrophage infectivity potentiator and Q fever agent regulatory sensor-like protein. The sequence similarity was more than 99% among Japanese, European and American strains, and no relationship was found between pathogenicity in guinea pigs and these genes.

Bacterial Proteins↗

PubMed1997

Characterization of Borrelia garinii isolated from Lyme disease patients in Hokkaido, Japan, by sequence analysis of OspA and OspB genes.

The outer surface proteins OspA and OspB genes of clinical Borrelia garinii isolates (JEM1-8) from Hokkaido, Japan were sequenced. One strain, JEM4, has a single ospA gene which is similar to European B. garinii strains PBr (sequence homology value: 94.1%) and T25 (91.2%). Five of the other seven strains exhibit a homologous C-terminus (300 bp) on both ospA and ospB genes (88.7-97.3%). The other two strains seem to be derived from the five strains by ospA or ospB alterations. In a phylogenetic analysis based on ospA, these strains could be classified into B. garinii, but formed independent branches and separated from the typical B. garinii isolates from Europe and Russia.

Amino Acid Sequence↗

PubMed1997

Homogeneity of Borrelia japonica and heterogeneity of Borrelia afzelii and 'Borrelia tanukii' isolated in Japan, determined from ospC gene sequences.

Borrelia afzelii, B. japonica, and 'B. tanukii' isolated from various sources and geographical origins in Japan were characterized by restriction fragment length polymorphism (RFLP) analysis and sequencing analysis of the outer surface protein C (OspC) amplicon. B. afzelii and 'B. tanukii' generated variable RFLP patterns and differences in ospC gene sequence were confirmed. In contrast, 26 isolates of B. japonica generated one OspC RFLP type, and sequence similarity between B. japonica ranged from 96.4 to 99.7%. These finding suggests that B. japonica is unique in comparison with other members of B. burgdorferi sensu lato species with respect to homogeneity of the ospC gene.

Animals↗

PubMed1997

Cyclic changes in NMDA receptor activation in hippocampal CA1 neurons after ischemia.

We studied N-methyl-D-aspartate (NMDA) receptor-mediated synaptic potentials in CA1 pyramidal neurons using hippocampal slices of gerbils after transient forebrain ischemia. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and bicuculline, stimulation of Schaffer collateral/commissural fibers induced field excitatory postsynaptic potentials (fEPSP) activated by NMDA receptors. We found that in many slices after ischemia, prolonged low-frequency stimulation (0.1-10 Hz) caused repeated depression and potentiation of the NMDA-mediated fEPSP. Changes in fEPSP amplitude were dependent on stimulus frequency and the cycle frequency ranged from 0.08 to 2.5 cycles/min. These cyclic changes were blocked by application of BAPTA-AM, a membrane-permeable Ca2+ chelator, but were little affected by application of verapamil or by lowering the Ca2+ in bathing solution. Intracellular recordings from CA1 neurons revealed that low-frequency stimulation caused periodic depolarizations of membrane potential accompanied by depression of the excitatory postsynaptic potentials. The cyclic changes of fEPSPs were blocked by inhibitors of protein kinase C (PKC) but were unaffected by inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMKII) or myosin light-chain kinase (MLCK). These results suggest that stimulus-dependent NMDA-receptor activation, mediated by PKC, takes place in the postischemic CA1 neurons and that the cyclic change may reflect abnormal intracellular Ca2+ signaling processes leading to neuronal degeneration.

Animals↗