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Biomedical subjects

A Ishii

Publications and source records attributed to A Ishii.

At least 37 records · Page 2Linked to original sources
PubMed2001

Characterization and identification of exflagellation-inducing factor in the salivary gland of Anopheles stephensi (Diptera: Culicidae).

Gamete activation factor (GAF) induces exflagellation of Plasmodium microgametes. We found GAF in the salivary glands of female mosquitoes, Anopheles stephensi. The exflagellation was induced in a concentration-dependent manner in the supernatant of salivary gland's crude homogenate. The exflagellation-inducing activity in the salivary gland was higher than that in the midgut and the head. GAF in the salivary glands was found to be heat stable and low molecular weight (<3000 molecular weight). Analysis of the supernatant by capillary electrophoresis and UV absorbance profile showed that the salivary glands contained xanthurenic acid, which was previously identified as GAF in the head of A. stephensi. The exflagellation-inducing activity in the salivary gland declined immediately after a blood meal, implying that GAF was in the saliva, and was delivered into the midgut together with the blood and induced exflagellation in the midgut.

Animals↗
PubMed2001

Sensitive determination of four general anaesthetics in human whole blood by capillary gas chromatography with cryogenic oven trapping.

Four general anaesthetics, sevoflurane, isoflurane, enflurane and halothane, in human whole blood, have been found measurable with very high sensitivity by capillary gas chromatography-flame ionization detection (GC-FID) with cryogenic oven trapping upon injection of headspace (HS) vapor sample. To a 7-ml vial, containing 0.48 ml of distilled water and 20 microl of internal standard solution (5 microg), a 0.5-ml of whole blood sample spiked with or without anaesthetics, was added, and the mixture was heated at 55 degrees C for 15 min. A measure of 10 ml HS vapor was injected into the GC in the splitless mode at -40 degrees C oven temperature, which was programmed up to 250 degrees C. All four peaks were clearly separated; no impurity peaks were found among their peaks. Their extraction efficiencies were about 10%. The calibration curves showed good linearity in the range of 0.5-20 microg/ml; their detection limits were 10-100 ng/ml, which are almost comparable to those by previous reports. The coefficients of intra-day and day-to-day variations were 6.5-9.8 and 7.3-17.2%, respectively. Isoflurane or enflurane was also measured from whole blood samples in which three volunteers inhaled each compound.

Adult↗
PubMed2001

Validation and casework application of a Y chromosome specific STR multiplex.

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor's alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.

Alleles↗
PubMed2001

Roles of mast cells and sensory nerves in cutaneous vascular hyperpermeability and scratching behavior induced by poly-L-arginine in rats.

We investigated whether the polycation poly-L-arginine elicited cutaneous vascular hyperpermeability and scratching behavior and, if so, whether these responses involved mast cells and sensory nerves in rats. Intradermal injections of poly-L-arginine induced vascular hyperpermeability and scratching behavior. Combined treatment with chlorpheniramine and methysergide almost completely suppressed the poly-L-arginine (50 microg/site)-induced plasma leakage. Capsaicin desensitization and the tachykinin NK(1) receptor antagonist LY303870, (R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane, partially inhibited the leakage. In mast cell-deficient rats, poly-L-arginine only minimally induced plasma leakage. On the other hand, capsaicin desensitization and LY303870, but not chlorpheniramine or methysergide, suppressed the poly-L-arginine (200 microg/site)-induced scratching. Moreover, poly-L-arginine elicited the scratching even in mast cell-deficient rats. These results suggest that substance P is at least partly involved in both the cutaneous plasma leakage and the scratching behavior induced by poly-L-arginine. Moreover, mast cell-derived amines are suggested to be involved in the plasma extravasation but scarcely, if any, in the scratching behavior.

Animals↗
PubMed2001

Sensitive determination of pethidine in body fluids by surface ionization organic mass spectrometry.

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography (GC)/surface ionization organic mass spectrometry (SIOMS). Good linearity was obtained in the range of 0.625-25 ng/ml of whole blood and urine by mass chromatography, and in the range of 0.05-2 ng/ml of whole blood by selected ion monitoring (SIM). Pethidine and diphenylpyraline (internal standard) were extracted from body fluids with Bond Elut Certify cartridges; their recoveries were above 95%. The detection limits (signal-to-noise ratio=3) were estimated to be 0.2 ng/ml of whole blood or urine by mass chromatography, 0.02 ng/ml of whole blood by SIM.

Analgesics, Opioid↗
PubMed2001

A role of tensin in skeletal-muscle regeneration.

Regeneration of skeletal muscle requires the activation, proliferation, differentiation and fusion of satellite cells to generate new muscle fibres. This study was designed to determine the role of tensin in this process. Cardiotoxin was used to induce regeneration in the anterior tibial muscles of tensin-knockout and wild-type mice. From histological analysis, we found that the regeneration process lasted longer in knockout than in wild-type mice. To investigate the mechanism involved in this delay, we examined each regeneration step in animals and cultured primary cells. We found fewer proliferating myogenic cells identified by bromodeoxyuridine and desmin double labelling in knockout mice on the first 2 days after injury. Expression of myosin, paxillin, dystrophin and dystrophin-associated proteins were delayed in knockout mice. Withdrawal from the cell cycle was less efficient in isolated knockout myoblasts, and the fusion capacity was reduced in these cells as well. These defects in regeneration most likely contributed to the 9-fold increase of centrally nucleated fibres occurring in the non-injected knockout mice. Our results demonstrated clearly that tensin plays a role in skeletal-muscle regeneration.

Animals↗
PubMed2001

A novel serum protein that is selectively produced by cytotoxic lymphocytes.

Cytotoxic lymphocytes such as CTL and NK cells play principal roles in the host defense mechanisms. Monitoring these effector cells in vivo is helpful to understand the immune responses in disorders such as cancer and infectious diseases. In this study, we identified a novel secretory protein, killer-specific secretory protein of 37 kDa (Ksp37), as a Th1-specific protein by a subtractive cloning method between human Th1 and Th2 cells. In peripheral blood leukocytes, Ksp37 expression was limited to Th1-type CD4(+) T cells, effector CD8(+) T cells, gammadelta T cells, and CD16(+) NK cells. Most of these Ksp37-expressing cells coexpressed perforin, indicating that Ksp37 is selectively and commonly expressed in the lymphocytes that have cytotoxic potential. Ksp37 was released at constant rate from both unstimulated and stimulated PBMCs in vitro and also detected in normal human sera. In healthy individuals, serum Ksp37 levels were significantly higher in children (mean +/- SD; 984 +/- 365 ng/ml for age 0-9) than in adults (441 +/- 135 ng/ml for age 20-99), consistent with reported differences in the absolute counts of blood T and NK cells between children and adults. In patients with infectious mononucleosis, transient elevation of serum Ksp37 levels was observed during the early acute phase of primary EBV infection. These results suggest that Ksp37 may be involved in an essential process of cytotoxic lymphocyte-mediated immunity and that Ksp37 may also have clinical value as a new type of serum indicator for monitoring cytotoxic lymphocytes in vivo.

Adolescent↗
PubMed2001

Asymmetric activation of chiral alkoxyzinc catalysts by chiral nitrogen activators for dialkylzinc addition to aldehydes: super high-throughput screening of combinatorial libraries of chiral ligands and activators by HPLC-CD/UV and HPLC-OR/RIU systems.

Asymmetric catalysts, prepared by chiral ligand exchange or chiral modification, can evolve further into highly activated catalysts through engineering with chiral activators. Two new methodologies for "super high-throughput screening" (SHTS) of chiral ligands and activators have been developed as a combination of HPLC-CD/UV (CD/ UV = circular dichroism/ultraviolet spectroscopy) or -OR/RIU (OR/RIU = optical rotation/refractive index unit) with a combinatorial chemistry (CC) factory. With these techniques, the % ee of the product is determined within minutes without separation of the enantiomeric products by using a nonchiral stationary phase. Therefore, those SHTS techniques combined with our 'asymmetric activation' concept can provide a powerful strategy for finding the best activated chiral catalyst.

Journal Article↗
PubMed2001

The role of saliva of Anopheles stephensi in inflammatory response: identification of a high molecular weight neutrophil chemotactic factor.

Mosquito bites can elicit dermal hypersensitivity reactions, but little is known about the chemotactic factors for host leukocytes in mosquito saliva. In this study, we determined that saliva from a malarial vector mosquito, Anopheles stephensi, possesses intense neutrophil chemotactic activity. In contrast, the midgut extract had only marginal neutrophil chemotactic activity. Eosinophil chemotactic activity was detected in the midgut but not in the saliva. According to the results of size-exclusion HPLC on a G3000SW column and Western blot analysis, the apparent molecular weight (MW) of the main neutrophil chemotactic factor (NCF) was estimated to be 200 kDa. NCF could bind with IgG from the pooled serum of Solomon islanders, whereas not with that of healthy Japanese. NCF activity was increased upon heating to 56 degrees C for 30 min or protease digestion, whereas it was affected by periodate treatment. Protease-digested NCF and naive NCF bound to lentil lectin-Sepharose, and both were eluted with a competitive sugar, methyl-alpha-D-glucoside. These results indicate that A. stephensi saliva-derived NCF is a high MW glycoprotein, and its protein moiety is important for neutrophil chemotactic activity. This NCF is thought to contribute to the inflammatory reactions through the accumulation of neutrophils at the site of the mosquito bite.

Allergens↗
PubMed2001

Superoxide-dependent and -independent pathways are involved in the transmission blocking of malaria.

Superoxide plays a crucial role in innate immunity to various pathogens. We examined the role of superoxides in the transmission of malaria using gp91phox knockout (X-CGD) mice that lack the ability to produce superoxide. Mosquitoes that fed on X-CGD mice infected intraperitoneally with Plasmodium berghei NK65 ANKA formed more oocysts than did those that fed on control mice at any day after infection. The number of oocysts peaked on day 5 post-infection in X-CGD and control mice and then decreased significantly after day 5 post-infection. However, on day 7 post-infection, the infectivity of gametocytes in X-CGD mice was significantly higher than that in control mice. These results show that two pathways, superoxide-dependent and -independent, are involved in the host systems regulating the transmission of malaria and inhibiting gametocyte development.

Animals↗
PubMed2001

Distribution of glucose-6-phosphate dehydrogenase mutations in Southeast Asia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand conformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G-->A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G-->A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T-->C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G-->A (G6PD Viangchan) was observed mainly in Lao, and 592 C-->T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A-->G (G6PD Gaohe), 1003 G-->A (G6PD Chatham), 1360 C-->T (G6PD Union), 1376 G-->T (G6PD Canton), and 1388 G-->A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.

Amino Acid Substitution↗
PubMed2001

Bacteria expressing single-chain immunotoxin inhibit malaria parasite development in mosquitoes.

Single-chain immunotoxins are ideal tools to selectively kill infectious agents. In applying this technology to block transmission of malaria parasites in the mosquito vector, we have constructed a single-chain immunotoxin composed of a single-chain antibody fragment (scFv) directed to Pbs2l on the surface of Plasmodium berghei ookinetes linked to a lytic peptide, Shiva-1. The single-chain immunotoxin was expressed in Escherichia coli, and the protein was purified by a Ni-NTA column. The single-chain immunotoxin was initially shown to exhibit greater killing properties for P. berghei ookinetes in vitro compared with the scFv or synthetic Shiva-1 peptide alone. In an attempt to block malaria transmission by genetically engineered bacteria, recombinant E. coli harboring the single-chain immunotoxin gene were introduced into the mosquito midgut by membrane feeding. The number of infected mosquitoes and their oocyst densities were significantly reduced when the mosquitoes were subsequently allowed to feed on P. berghei-infected mice. These results indicate not only that a single-chain immunotoxin with enhanced parasiticidal activity could form a basis for the development of more effective malaria therapeutic agents, but also that introduction of genetically engineered bacteria into anopheline mosquitoes may offer a practical approach to the regulation of malaria transmission.

Amino Acid Sequence↗
PubMed2001

Sensitive determination of midazolam and identification of its two metabolites in human body fluids by column-switching capillary high-performance liquid chromatography/fast atom bombardment-mass spectrometry.

Midazolam is a benzodiazepine and is widely prescribed for preanesthesia or general anesthesia. Overdose or intoxication cases of midazolam have been reported. In Japan, smuggled midazolam tablets could be involved in some criminal cases. Midazolam and its two metabolites were extracted by the solid-phase extraction method using Bond Elut SCX cartridges. The compounds were analyzed by on-line capillary high-performance liquid chromatography/fast atom bombardment-mass spectrometry. Midazolam and its two metabolites were well separated on the chromatogram, and each mass spectra gave [M+H](+) ion as a base peak. Deuterium-labeled midazolam was synthesized as an internal standard; it has enabled precise and reproducible quantitation of midazolam in blood samples. The calibration curve showed excellent linearity in the range of 2-200 ng/ml in spiked serum. The detection limit was 300 pg/ml (signal-to-noise ratio=3). The whole blood and urine samples from the victim of a homicide case were analyzed, and the midazolam concentration in the whole blood was estimated to be 163 ng/ml. The present method should be useful in clinical and forensic toxicology, because of its high sensitivity and specificity.

Journal Article↗
PubMed2001

A case of death due to rescue action by a power shovel after being buried alive.

We report a rare case of death due to rescue using a power shovel. A 41-year-old female was accidentally buried alive by a landslide of the earth and sand upon working at a construction site. One of her colleagues started to save her using a power shovel. However, she was dug out dead at the spot about 10 min after the accident with marked head and face injuries. The autopsy disclosed that there was extensive laceration across the face and head with marked skull bone fractures. Around these injuries, extensive hemorrhage could be observed as a vital reaction. Asphyxial death had to be taken into consideration, because she was buried under the earth and sand for about 10 min; but we finally judged that the cause of her death was head injury by the power shovel inflicted during the attempted rescue.

Journal Article↗
PubMed2001

The expression of dystrophin and alpha1-syntrophin during skeletal muscle regeneration.

The expression of dystrophin and alpha1-syntrophin in rat tibialis anterior muscles were evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. Immunohistochemical studies were performed in cryosections of muscles on days 1, 3, 5, 7, 10, 14, 21 and 28 after injection of cardiotoxin. Western blot analysis was also examined in muscle on days 1, 3, 5, 7, 10, 14, 21 and 28. In immunohistochemical studies, dystrophin was stained weakly at the sarcolemma of some regenerating muscle fibers on day 3, and by day 10 it was stained strongly on almost all regenerating muscle fibers. Alpha1-syntrophin was stained weakly at the sarcolemma of some regenerating fibers on day 5, and by day 14 it was detected on all regenerating muscle fibers. In Western blot analysis, dystrophin (DYS1) and alpha1-syntrophin (alpha1S) were completely absent on day 1. Re-expression of DYS1 and alpha1S was visible by day 5 and accelerated thereafter. The Western blots of DYS1 and alpha1S were densitometrically analyzed on each day. The protein levels on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. From the sequential line based on these data, the following results were obtained on the chronological course of DYS1 and alpha1S. DYS1: 25% of the protein level on day 28 was reached by 3.5 days, 50% was reached by 5.3 days, and 90% was reached by 6.9 days. Alpha1S: 25% of the protein level on day 28 was reached by 4.6 days, 50% was reached by 6.0 days, and 90% was reached by 12.5 days. In this study, DYS1 regenerated earlier than alpha1S at the sarcolemma of regenerating muscle fibers.

Animals↗
PubMed2001

Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse.

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

Actin Depolymerizing Factors↗
PubMed2001

Modulation of graft-versus-tumor effects in a murine allogeneic bone marrow transplantation model by tumor-derived transforming growth factor-betaI.

Although graft-versus-leukemia effects in allogeneic bone marrow transplantation (alloBMT) are well documented, graft-versus-tumor (GVT) effects are poorly defined. To investigate the latter, we established a murine model of breast cancer using TS/A, a transforming growth factor (TGF)-beta1-secreting breast cancer cell line of BALB/c origin. In the setting of disparate (parent into F1) alloBMT, no appreciable GVT was identified. To assess whether TGF-beta1 secreted by the tumor might inhibit the antitumor response, TGF-beta1 antisense vector was transfected into the TS/A breast cancer cell line. Mice were inoculated with either TGF-beta1 antisense transfected or the mock transfected cell line and underwent syngeneic or alloBMT. No evidence of GVT was appreciated for the mock-transfected breast cancer cell line as assessed by an absence of a statistically significant difference in survival between syngeneic and alloBMT groups. However, there was a highly statistically significant survival difference between allogeneic versus syngeneic bone marrow transplantation groups inoculated with the TGF-beta1 antisense-transfected cell line (P = .00001) as well as when comparing the survival of mice that received alloBMT for TGF-beta1 antisense-transfected tumor versus mock-transfected tumor (P = .0008). These data suggest that (1) GVT exists against the antisense-transfected breast cancer cells in this experimental model and (2) TGF-beta1 may be involved in suppressing antitumor responses in the setting of alloBMT for breast cancer.

Animals↗