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Biomedical subjects

H Kaneko

Publications and source records attributed to H Kaneko.

At least 127 records · Page 7Linked to original sources
PubMed2000

Local thrombus formation in the site of intravenous injection of chlorpromazine: possible colloid-osmotic lysis of the local endothelial cells.

Since amphiphilic drugs are known to interact with biomembranes, we investigated local vessel damage and thrombosis which might be brought about by intravenous dosing using chlorpromazine (CPZ) as a representative compound. CPZ-induced hemolysis was suppressed by an increase in sucrose concentration in the medium, characterizing this hemolysis to be colloid-osmotic lysis, which includes the enhancement of membrane phospholipid fluidity and consequent small pore formation in the membranes. This was supported by the observation that hemolysis by filipin, not featuring the stage of small pore formation, was not affected by sucrose. [14C]Glucose-entrapping liposomes were degraded by CPZ, and this degradation was enhanced by an increase in the intravesicle glucose concentration. These results indicated that the compound could induce colloid-osmotic lysis in erythrocytes and artificial membrane vesicles. CPZ also injured cultured porcine aortic endothelial cells (PAEC), as evidenced by lactate dehydrogenase (LDH) leakage. This injury was also suppressed by increase in sucrose concentration in the medium, suggesting that colloid-osmotic lysis again occurred. When rats were intravenously injected with CPZ, local endothelial cell (EC) injury and associated thrombus formation were observed, suggesting that CPZ's action was also evident in vivo. To our knowledge, this is the first finding which suggests that an intravenously dosed amphiphilic drug can injure local ECs based on a colloid-osmotic lysis mechanism leading to thrombosis.

Animals↗
PubMed2000

Effects of hydrostatic pressure and temperature on growth and lipid composition of the inner membrane of barotolerant Pseudomonas sp. BT1 isolated from the deep-sea.

A barotolerant member of the genus Pseudomonas was isolated from deep-sea sediment obtained from the Japan Trench, at a depth of 4418 m. The growth temperature was found to affect the hydrostatic pressure range in which the bacterium could grow; the optimum hydrostatic pressure for growth shifted to a higher pressure with increasing temperature. We examined the lipid composition of the inner membrane of cells grown at various hydrostatic pressures and temperatures. The fatty acid components of the inner membrane lipids were C16:0, C16:1, C18:0, and C18:1. The phospholipid components of the inner membrane were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and phosphatidylserine. It is evident that the effects of elevated hydrostatic pressure are comparable to the effects of low temperature on both the fatty acid composition of the inner membrane lipids and the phospholipid composition of the inner membrane of this bacterium.

DNA, Ribosomal↗
PubMed2000

Signal through gp130 activated by soluble interleukin (IL)-6 receptor (R) and IL-6 or IL-6R/IL-6 fusion protein enhances ex vivo expansion of human peripheral blood-derived hematopoietic progenitors.

This study was designed to investigate the effects of a combination of soluble interleukin (sIL)-6 receptor (R) and IL-6 on the ex vivo expansion of human peripheral blood (PB)-derived hematopoietic progenitor cells in a short-term serum-free liquid suspension culture system, using PB-derived CD34(+)IL-6R(+/-) cells as a target. In combination with stem cell factor (SCF), IL-3, and sIL-6R/IL-6, the expansion efficiency (EE) for granulocyte/macrophage colony-forming unit (CFU-GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU-E) and mixed colony-forming unit (CFU-Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL-3 + sIL-6R/IL-6 + flt3 ligand (FL) most effectively expanded CFU-GM and CFU-Mix. The maximum EEs for CFU-GM and CFU-Mix were 208-fold and 42-fold, respectively. While the EE for BFU-E was 70-90-fold in the presence of SCF + IL-3 + sIL-6R/IL-6, FL significantly augmented the EE for CFU-GM and CFU-Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU-Mix. Interestingly, in combination with IL-3 and SCF, newly generated IL-6R/IL-6 fusion protein (FP) expanded PB-derived BFU-E and CFU-Mix twice more effectively than a combination of sIL-6R and IL-6. These results demonstrated that human PB-derived committed progenitors were effectively expanded in vitro using sIL-6R/IL-6 or FP, in combination with IL-3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL-6R and IL-6.

Animals↗
PubMed2000

Characterization of the nuclear localization signal in the DNA helicase responsible for Bloom syndrome.

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, photosensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS. The BLM protein is homologous to DNA helicase and has two basic amino acid clusters in its C-terminal region. Previously, we reported that the distal arm of these basic amino acids clusters in the BLM protein functioned as the nuclear localization signal (NLS) of the protein. In this study, we generated plasmid constructs for expression of enhanced green fluorescent protein (EGFP) fused with various BLM protein variants having a mutation with deletions or substitutions in the basic amino acid and analyzed the subcellular localization of the expressed proteins. The EGFP-fused protein containing the basic amino acid cluster region proximal to the C-terminus of BLM helicase was localized exclusively in the nucleus. However, the EGFP-BLM proteins that lacked either Arg1344 or Lys1346 distributed in both the cytoplasm and the nucleus equally. Deletion of Arg1347 also resulted in localization in both the nucleus and cytoplasm, and substitution of Arg1344, Lys1346, Arg1347 or Arg1348 with non-basic amino acids reduced the nuclear localization of BLM protein. Mouse BLM protein which also migrate to the nucleus has two basic amino acid clusters in the C-terminus and the basic amino acids (Lys1346-Pro1347-Lys1348-Arg1349-Arg1350) proximal to the C-terminus are conserved between mouse and human. These findings suggest that the Arg1344-Ser1345-Lys1346-Arg1347 sequence at the C-terminus of the human BLM protein is essential for nuclear localization of this protein.

Adenosine Triphosphatases↗
PubMed2000

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes.

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.

Analysis of Variance↗
PubMed2000

[Evaluation of antiviral agents for adenovirus using the MTT method in vitro].

PURPOSE: Currently, there is no effective treatment for adenoviral conjunctivitis. We evaluated the antiviral inhibitory effect of four antiviral agents against adenovirus (ADV) in vitro. METHODS: Viruses used for the experiment were ADV type 4 (ADV 4), type 8 (ADV 8) and type 37 (ADV 37). We examined four antiviral agents, i.e., cidofovir (HPMPC), zalcitabine (ddC), foscarnet (PFA), and acyclovir (ACV). 50% effective concentration (EC50), 50% cytotoxic concentration (CC50) and selectivity index (SI) of compounds were determined for ADV infection in HEp-2 cells using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. RESULTS: HPMPC and ddC showed an inhibitory effect against all three ADVs. In particular, ddC showed more potent and selective inhibition against ADV than HPMPC. PFA and ACV were ineffective against ADV. CONCLUSIONS: HPMPC and ddC were inhibitory against ADV 4, ADV 8 and ADV 37 replication in vitro. The MTT method is rapid and simple for the screening of antiviral agents. We think this method is also very useful for the screening of anti-ADV agents.

Acyclovir↗
PubMed2000

A comparison of the multigroup and collocation methods for solving the low-energy neutron Boltzmann equation.

A low-energy neutron transport algorithm for use in space-radiation protection is developed. The algorithm is based upon a multiple energy group analysis of the straight ahead Boltzmann equation utilizing a mean value theorem for integrals. The algorithm developed is then verified by using a collocation method solution on the same straight ahead Boltzmann equation. This algorithm was then coupled to the existing NASA Langley HZETRN (high charge and energy transport) code through the evaporation source term. Evaluation of the neutron fluence generated by the February 23, 1956 solar particle event for an aluminum-water shield-target configuration is then compared with the LAHET Monte Carlo calculation for the same shield-target configuration. The algorithm developed showed a great improvement in results over the unmodified HZETRN solution. A bidirectional modification of the evaporation source produced further improvement of the fluence.

Algorithms↗
PubMed1999

Mutations of the IL-12 receptor beta2 chain gene in atopic subjects.

Interleukin-12(IL-12) promotes cell-mediated Th1 responses and production of IFN-gamma that downregulates IgE production. The signal of IL-12 is transduced through the IL-12 receptor (IL-12R) and Stat4. Twenty-four of 75 atopic individuals with high levels of IgE showed insufficient IFN-gamma production by peripheral blood mononuclear cells (PBMCs) following stimulation with IL-12 but not that with phytohemagglutinin (PHA). Interestingly, 10 of the above 24 subjects were found to be heterozygous for truncated (2496 del 91) or missense (1577 A to G and 2799 A to G) mutations of IL-12R beta2 chain gene (IL-12R beta2). Insufficient phosphorylation of Stat4 was also demonstrated in these 10 individuals. This is the first report showing that reduced IFN-gamma production following IL-12 stimulation is associated with the heterozygous IL-12R beta2 mutations in atopic subjects.

Adolescent↗
PubMed1999

ATM is upregulated during the mitogenic response in peripheral blood mononuclear cells.

Patients with the human genetic disorder ataxia-telangiectasia (A-T) are characterized by immunodeficiency and a predisposition to develop lymphoid malignancies. The gene mutated in A-T patients, ATM, codes for a high molecular weight protein that is implicated in DNA damage recognition and cell cycle control. The ATM protein does not change in amount or cellular distribution throughout the cell cycle or in response to DNA damaging agents. Because peripheral blood mononuclear cells (PBMCs) are largely in a state of quiescence and can be readily stimulated to enter a proliferative phase and because A-T cells exhibit growth abnormalities and senescence, indicative of a general intracellular defect in signalling, we chose PBMCs to examine the relationship of ATM to the proliferative status of the cell. We show here that ATM protein is present at low levels in freshly isolated PBMCs and increases approximately 6-fold to 10-fold in response to a mitogenic stimulus, reaching a maximum after 3 to 4 days. A similar, but delayed response, was evident in the presence of serum only. This increase in ATM protein was accompanied by an increase in ATM kinase activity. While expression of ATM protein increased during proliferation, ATM mRNA expression was unchanged in stimulated and unstimulated cells and there was no evidence for increased ATM protein stability in the phytohemagglutinin (PHA)-treated cells. In keeping with the reduced levels of ATM in quiescent cells, the extent of radiation-induction of the p53 pathway was significantly lower than in mitogen-stimulated cells. Basal levels of p21 were elevated in quiescent cells, and the response to radiation was negligible or reduced compared with proliferating cells over a 2-hour period. Overall, the data suggest that the increase in ATM protein in proliferating cells is due to posttranscriptional regulation and points to a role for ATM in more general signalling.

Ataxia Telangiectasia↗
PubMed1999

Ethylnitrosourea-induced mutation and molecular analysis of transgenic mice containing the gpt shuttle vector.

Novel transgenic mice were developed in order to study the in vivo mutagenesis. The transgenic mice carried pCGK shuttle vector, which contained the Escherichia coli gpt gene as a mutational target, the kanamycin-resistant gene (Kanr) and cos region derived from bacteriophage lambda. The shuttle vector can be recovered from the transgenic mouse genome into the gpt-deficient E. coli by an in vitro packaging method and is selectable as a Kanr phenotype. Mutations induced at the gpt gene can be easily detected with a selective agent, 6-thioguanine (6-TG). In the previous study, the pCGK shuttle vector was incorporated into Chinese hamster CHL/IU cells and the resultant transgenic cell line was shown to be a useful system to study in vitro mutagenesis at the gpt gene. Therefore, an advantage of the shuttle vector is that in vivo mutational data obtained from the transgenic mouse can be compared with those of transgenic cell line in vitro. A transgenic CD-1 mouse line, designated as #128, that carried approximately 50 copies of pCGK shuttle vectors, was selected among 4 transgenic mouse lines. To investigate the sensitivity of the #128 line, the transgenic mice were treated with a single intraperitoneal injection of 250 mg/kg of N-ethyl-N-nitrosourea (ENU) or with 50 mg kg-1 day-1 of ENU for 5 consecutive days, and bone marrow, spleen and liver were dissected to investigate their mutational responses. The background mutant frequency was between 18x10(-6) and 75x10(-6) among all tissues tested. ENU induced significant increases in the mutant frequency above the background level in all three tissues at 14 days after single or 5-day treatment with the chemical. The increases in the mutant frequencies in bone marrow, spleen and liver were 6.4- to 6.8-fold, 3.0- to 5.6-fold and 3.0- to 3.3-fold, respectively. The shuttle vector DNA was recovered from the bone marrow of both spontaneous and ENU-treated mice and the gpt gene was amplified by polymerase chain reaction. The amplified DNA was subject to DNA sequence analysis. Out of 79 spontaneous and 52 ENU-induced mutants, the gpt gene could be amplified from 28 spontaneous and 46 ENU-induced mutants. DNA sequence analysis showed that predominant mutations were identified as A:T to T:A transversions (22 out of 46 sequenced mutants) and G:C to A:T transitions (9/46) in ENU-induced mutants, whereas G:C to T:A transversions (7 out of 28 sequenced mutants) were predominant in spontaneous mutants. These results demonstrate that this transgenic mouse, in combination with the transgenic CHL/IU cell line, is a useful system to study in vivo and in vitro mutational events at the same target gene.

Amino Acid Substitution↗
PubMed1999

Pathophysiological role of endothelins in pulmonary microcirculatory disorders due to intestinal ischemia and reperfusion.

BACKGROUND: This study was conducted to investigate pulmonary microcirculatory disorders caused by intestinal ischemia reperfusion (IIR), and the pathophysiological roles of endothelin (ET) in acute lung injury (ALI). METHODS: Male rats were pretreated with normal saline or a nonselective ET receptor antagonist (TAK-044) and subjected to IIR (60 min of intestinal ischemia and 180 min of reperfusion). The right upper lobe of the lung was examined by intravital confocal microscopy. RESULTS: The size of arterioles and venules was not significantly reduced during IIR, but the functional capillary density (FCD) decreased significantly. TAK-044 improved the pulmonary microhemodynamics, inhibiting the accumulation of leukocytes, the pulmonary edema, and the decrease of FCD. CONCLUSIONS: In the early stage of IIR, pulmonary microhemodynamics seemed more likely to be disturbed by the decrease of FCD, than by arteriolar or venular vasoconstriction. ETs decrease the FCD, promoting the interaction between leukocytes and pulmonary vessels.

Animals↗
PubMed1999

The time-course of preparatory spinal and cortico-spinal inhibition: an H-reflex and transcranial magnetic stimulation study in man.

In a previous study where reaction-time methods were combined with transcranial magnetic stimulation (TMS) of the motor cortex, cortico-spinal excitability was shown to reflect time preparation. Provided that subjects can accurately estimate time, the amplitude of motor-evoked potentials (MEPs) diminish progressively during the interval separating the warning signal from the response signal (i.e., the foreperiod). On the other hand, several experiments have demonstrated that the amplitude of the Hoffman (H) reflex elicited in prime movers diminishes during the foreperiod of reaction-time tasks. The aim of the present study was to compare the time course of the respective decrements of H-reflex and MEP amplitude during a constant 500-ms foreperiod. The subjects (n=8) participated in two experimental sessions. In one session, H-reflexes were induced in a tonically activated, responding hand muscle, the flexor pollicis brevis, at different times during the foreperiod of a visual-choice reaction-time task. In the other session, motor potentials were evoked in the same muscle by TMS of the motor cortex delivered in the same behavioral conditions and at the same times as in the first session. The results show that both H-reflexes and MEPs diminish in amplitude during the foreperiod, which replicates and extends previous findings. Interestingly, the time constants of the two decrements differed. There was a facilitatory effect of both electrical and magnetic stimulations on the subject's performance: reaction time was shorter for the trials during which a stimulation was delivered than for the no-stimulation trials. This facilitation was maximal when the stimulations were delivered simultaneously with the warning signal and vanished progressively with stimulation time.

Adult↗