Biomedical subjects

S Itoh

Publications and source records attributed to S Itoh.

At least 181 records · Page 10Linked to original sources

PubMed1998

Mutagenic specificity of model estrogen-DNA adducts in mammalian cells.

Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic properties of the model estrogen-DNA adducts N2-[3-methoxyestra-1,3,5(10)-trien-6(alpha, beta)-yl]-2'-deoxyguanosine (dG-N2-3MeE) and N6-[3-methoxyestra-1,3, 5(10)-trien-6(alpha,beta)-yl]-2'-deoxyadenosine (dA-N6-3MeE) in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'TCCTCCTCXCCTCTC; X = dG, dA, dG-N2-3MeE, or dA-N6-3MeE) containing an unmodified or model estrogen lesion were inserted into single-stranded (ss) phagemid vectors. These ss vectors were transfected into COS-7 cells. The progeny plasmid obtained were used to transform Escherichia coli DH10B. The transformants were analyzed by oligodeoxynucleotide hybridization and sequencing to determine the mutation frequency and spectrum. Preferential incorporation of dCMP, the correct base, was observed opposite the dG-N2-3MeE lesion. Targeted mutations showing G --> T transversions were detected, along with a small number of G --> C transversions. When a dA-N6-3MeE-modified oligodeoxynucleotide was used, preferential incorporation of dTMP, the correct base, was also observed. Targeted mutations representing A --> T transversions were detected, accompanied by a small amount of A --> G transitions. The frequency of mutation observed opposite dA-N6-3MeE (17.5%) was 2.3 times higher than that observed opposite dG-N2-3MeE (7.5%). These results indicate that estrogen DNA adducts have mutagenic potential in mammalian cells.

Animals↗

PubMed1998

Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.

Smad proteins play a key role in the intracellular signalling of transforming growth factor beta (TGF beta), which elicits a large variety of cellular responses. Upon TGF beta receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGF beta. Here we report the identification of Smad3/Smad4 binding sequences, termed CAGA boxes, within the promoter of the human PAI-1 gene. The CAGA boxes confer TGF beta and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter was found to abolish TGF beta responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGF beta. In addition, TGFbeta induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 proteins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGF beta-responsive regions of several other genes suggests that this may be a widely used motif in TGF beta-regulated transcription.

Activins↗

PubMed1998

Quantitative detection method of triglycerides in serum lipoproteins and serum-free glycerol by high-performance liquid chromatography.

We have developed a simple and reliable method for quantitative detection of triglycerides (TG) in serum lipoproteins and serum-free glycerol (FG) by high-performance liquid chromatography (HPLC). After separation of serum constituents using a new gel-permeation column (TSK gel Lipopropak XL, Tosoh) and a new eluent (TSK eluent LP-2, Tosoh), TG and FG were detected by on-line reaction using a modified reagent which contained glycerol kinase, glycerol-3-phosphate oxidase and lipoprotein lipase. HPLC patterns showed five peaks corresponding to chylomicrons, very-low-density, low-density, high-density lipoproteins and FG. Absolute concentrations of TG in each lipoprotein fraction and serum FG were calculated from the corresponding peak areas using standard FG as a calibrator. Due to its very high sensitivity of peak detection, this method has become desirable for the analyses of lipoproteins of very low concentrations such as in cell culture systems. This technique will contribute to a better understanding of lipoprotein TG and serum FG distribution in human and nonhuman subjects.

Animals↗

PubMed1998

Model studies on calcium-containing quinoprotein alcohol dehydrogenases. Catalytic role of Ca2+ for the oxidation of alcohols by coenzyme PQQ (4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2, 7,9-tricarboxylic acid).

Mechanistic studies on the action of calcium-containing quinoprotein alcohol dehydrogenases have been performed by using a series of PQQ model compounds in anhydrous organic media. The PQQ model compounds are shown to form 1:1 complexes with a series of alkaline earth metal ions by spectroscopic methods and theoretical calculations. The site of coordination of the PQQ molecule to the metal ions in solution is indicated to be the same as in the case of enzymatic systems. It has also been found that Ca2+ binds to the quinone most strongly among the alkaline earth metal ions. Formation of the C-5 hemiacetal derivatives with methanol, ethanol, and 2-propanol is also investigated spectrophotometrically to show that the alcohol addition to the quinone is enhanced in the presence of the metal ions. In this case as well, Ca2+ shows the highest efficiency for the enhancement of the C-5 hemiacetal formation. Addition of a strong base such as DBU into an MeCN solution containing the Ca2+ complex of the PQQ model compounds and the alcohols leads to the redox reactions to afford reduced PQQ derivatives and the corresponding aldehydes. On the basis of detailed kinetic studies on the redox reactions, including structural effects of PQQ analogues and metal ion effects, we propose the addition-oxidative elimination mechanism through the C-5 hemiacetal intermediate.

2-Propanol↗

PubMed1998

Bone marrow and liver mutagenesis in lacZ transgenic mice treated with hexavalent chromium.

The mutagenic effects of the hexavalent chromium compound K2CrO4 in lacZ transgenic mice (Muta Mouse) were investigated at two sampling times. K2CrO4 was administered intraperitoneally to five male mice per treatment group at a single dose of 40 mg/kg. The animals were sacrificed on days 1 and 7 after the treatment. Mutant frequencies in the bone marrow and liver were analyzed by the positive selection method using Escherichia coli C (galE-) strain and phenyl beta-D-galactoside. K2CrO4 induced a significant increase in mutant frequency in the bone marrow on day 1, but not on day 7 after the treatment. In the liver, on the other hand, a significant induction in the mutant frequency was seen on day 7, whereas no induction was observed on day 1. The reason for the different responses to the mutagenic activity of K2CrO4 between these organs may be related to their cell turnover rates. The mutations induced by K2CrO4 in the bone marrow may have occurred in more differentiated cells than stem cells, and the rapid proliferative activity may have caused a rapid decrease in mutated cells by day 7. These results suggest that experiments on mutagenesis should be done with more than one sampling point, a short expression time in addition to a longer one, so as to detect mutations induced in organ with high cell proliferation.

Animals↗

PubMed1998

The effect on immunocytes of anodic oxide titanium after hydrothermal treatment.

All dental root implants come in contact with the oral epithelium, and many complex factors are found to arise in this region. In order to perform a successful dental root implantation, it is necessary to clarify the interaction of the dental root implant material with the host defense mechanisms involved in the specific and nonspecific immune responses to many antigens in oral bacteria and their components. Recently, focusing on developing the dental root implant, the Nikon Corporation improved the surface characteristics of pure titanium even further by developing a hydroxyapatite (HA) layer formed on an anodic titanium oxide film containing Ca and P via hydrothermal treatment (SA treatment). However, since little is known about the effect of SA-treated pure titanium (HA/Ti) on the defense mechanisms of the oral membrane epithelium, we investigated (1) the in vitro proliferation of murine splenic B lymphocytes on the surface of HA/Ti in the presence of three lipopolysaccharide (LPS) concentrations and (2) interleukin-1alpha (IL-1alpha) production by the reaction of human peripheral blood mononuclear cells (PBM cells) on the surface of HA/Ti under the same concentrations. After culture, murine splenic lymphocytes were measured by uptake of 3H-thymidine, and cytokine release (IL-1alpha) from PBM cells was measured by ELISA. Results showed that HA/Ti had hardly any effect on the LPS-induced proliferation of B lymphocytes and IL-1alpha production. In vitro investigations of the effects of HA/Ti on the LPS-induced proliferation of murine splenic B lymphocytes and IL-1alpha from PBM cells might be a useful way of elucidating the defense mechanism between implants and the oral epithelium.

Animals↗

PubMed1998

Intracellular signaling of osteogenic protein-1 through Smad5 activation.

Smad proteins play pivotal roles in the intracellular signaling of the multifunctional transforming growth factor-beta (TGF-beta) family members downstream of serine/threonine kinase type I and type II receptors. Smad2 and Smad3 are specific mediators of TGF-beta and activin, while Smadl and Smad5 are involved in bone morphogenetic protein-2 (BMP-2) and BMP-4 signaling. Here we report that osteogenic protein-1 (OP-1), also termed BMP-7, binds predominantly to BMPR-IB in the rat osteoprogenitor-like cell line, ROB-C26. Smad1, Smad5, and Smad8, but not Smad2 and Smad3, were found to stably interact with the kinase-deficient BMPR-IB after it was phosphorylated by the BMPR-II kinase. In ROB-C26 cells, which express Smad2, Smad3, Smad4, and Smad5, OP-1 was found to stimulate the phosphorylation of Smad5. Whereas transfection of wild-type Smad5 enhanced the OP-1-induced response, transfection of wild-type Smad2 had no effect on OP-1 signaling. A Smad5-2SA mutant, in which the two most carboxy-terminal serine residues were mutated to alanine residues, was found to act as a dominant negative inhibitor of OP-1-induced responses upon its transfection into various cell types, including ROB-C26 cells, in contrast to ectopic expression of a Smad2-2SA mutant which was without effect. Smad5, therefore, is a key component in the intracellular signaling of OP-1.

Amino Acid Sequence↗

PubMed1998

[Repair of intrathoracic visceral damage using video-assisted thoracoscopic surgery for blunt chest trauma and rib fixation at the site of mini-thoracotomy].

We treated three patients with intrathoracic visceral damage caused by severely dislocated fractured ribs resulting from blunt trauma by using video-assisted thoracoscopic surgery (VATS) and rib fixation through a mini-thoracotomy. Under general anesthesia and unilateral respiration, the thoracic cavity was inspected with a thoracic video scope through the port inserted through the thoracic drainage opening which was made upon arrival at hospital. As the visceral damage seemed restorable under VATS, a mini-thoracotomy was positioned just above the rib fracture. Two thoracic ports were inserted through the site of rib fracture or through the intercostal space and then VATS was performed using three ports. After the restoration of intrathoracic visceral damage, the fractured rib was fixated using a bioabsorbable poly-L-lactide rib fixation pin or a marlex mesh. Lung injuries were sutured and ligated under VATS in two of our cases and a spur of the fractured rib was shaved in one case. Only severely dislocated ribs were fixated through the mini-thoracotomy in all cases. Air leakage stopped just after this procedure and there were no complications. The rib fixation and bone regeneration were excellent after this procedure. The advantages of this method are the visceral restoration under VATS through a mini-thoracotomy and the ability to perform rib fixation without injuries to the intercostal muscle, artery, vein or nerve. This operative procedure is recommended for intrathoracic visceral damage caused by severely dislocated rib fracture.

Adult↗

PubMed1998

Allergic conjunctivitis as a risk factor for regression and haze after photorefractive keratectomy.

PURPOSE: To analyze the relation between allergic conjunctivitis and the results of photorefractive keratectomy performed with an excimer laser in myopic eyes. METHODS: Fifty-seven myopic eyes in 57 Japanese patients were classified into three groups: a normal group (30 eyes of 30 patients), a treatment group composed of eyes with allergic conjunctivitis that were treated with fluorometholone and cromolyn sodium eyedrops from month 3 until the end of the 12- to 18-month follow-up period (16 eyes of 16 patients), and a no-treatment group composed of eyes with allergic conjunctivitis that received no allergic treatment until the end of the follow-up period (11 eyes of 11 patients). Preoperative and postoperative examinations included evaluation of corrected and uncorrected visual acuity and grading of corneal haze. RESULTS: In the no-treatment group, the mean corneal haze score +/- SD of 0.8 +/- 0.98 was significantly greater than the normal group score of 0.38 +/- 0.49 (P = .02). There was no significant difference in the haze score between the treated and normal groups. A refractive outcome of +/- 1 diopter was obtained in 30 (100%) of the 30 patients in the normal group, 15 (93.8%) of 16 patients in the treatment group, and four (36.4%) of the 11 patients in the no-treatment group. Visual acuity was 20/40 or better after photorefractive keratectomy in 30 patients (100%) in the normal group, 15 patients (93.8%) in the treatment group, and six patients (54.5%) in the no-treatment group. CONCLUSION: These findings suggest that untreated allergic conjunctivitis is a significant risk factor for haze and myopic regression after photorefractive keratectomy.

Adult↗

PubMed1998

Cytogenetic clonality analysis in monosomy 7 associated with juvenile myelomonocytic leukemia: clonality in B and NK cells, but not in T cells.

It remains unclear which lymphoid lineages are involved in juvenile myelomonocytic leukemia (JMML). We report a JMML patient who acquired monosomy 7 after intensive chemotherapy. In this case, the expression of monosomy 7 was analyzed in T, B and natural killer (NK) cells highly purified from peripheral blood mononuclear cells of the patient. The fluorescence in situ hybridization method revealed the expression of monosomy 7 in B cells, but not T cells. Half of the NK cells expressed monosomy 7; when NK cells were divided into CD2- and CD2+ populations, this abnormality was positive in 91.1% of CD2- NK cells but in only 14.7% of CD2+ NK cells. These results suggest that, in this JMML patient who acquired monosomy 7 after intensive chemotherapy, B cells and half of NK cells, but not T cells, have monosomy 7.

B-Lymphocytes↗

PubMed1998

Beta-decay half-lives and level ordering of 102m,gRh.

Beta-decay half-lives of the ground state and an isomer of 102Rh have been determined 207.3(17) d and 3,742(10) y, respectively, by gamma-ray decay curves following each beta-decay. It has been found that a state (2-) which has a shorter half-life (207.3 d) is the ground state from the result that the half-life of the 41.9 keV isomeric gamma-transition was equal to 3.742 y. It has also been confirmed that the 41.9 keV transition is certainly an isomeric transition with X-gamma coincidence measurement.

Beta Particles↗

PubMed1998

A case of pathophysiologic study in Kimura's disease: measurement of cytokines and surface analysis of eosinophils.

BACKGROUND: Kimura's disease is a rare but distinctive eosinophilic inflammatory disorder of unknown etiology; few reported case studies have focused on the immunopathologic background of this unique disease. OBJECTIVE: To define better the immunopathogenetic features of Kimura's disease, we attempted to quantitatively analyze values of cytokines and soluble interleukin-2 receptor (sIL-2R) in peripheral blood (PB), as well as perform surface immunophenotypic analysis of eosinophils from a Japanese patient with chronic relapsing Kimura's disease. RESULTS: Granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and sIL-2R were elevated, and newly expressed antigens on eosinophils CD4, CD25, and HLA-DR were found to be involved in the pathophysiology of this disorder. CONCLUSIONS: Kimura's disease may be a disease in which activated lymphocytes release cytokines, and these released cytokines, such as GM-CSF and TNF-alpha cause eosinophil activation. These processes may be related to the pathogenesis of this disorder.

Aged↗

PubMed1998

Identification of estrogen-modified nucleosides from calf thymus DNA reacted with 6-hydroxyestrogen 6-sulfates.

Two estrogen sulfates, pyridinium 3-methoxyestra-1,3, 5(10)-trien-6alpha-yl sulfate (3MeE-6alpha-S) and its 6beta-isomer (3MeE-6beta-S), synthesized as model compounds to demonstrate the carcinogenesis of estrogen, were found to react with calf thymus DNA to produce steroid-modified DNA adducts. Digestion of the DNA by nuclease P1 and phosphodiesterase I followed by alkaline phosphatase gave a deoxyribonucleoside fraction, of which N2-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyguanosine, N2-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyguanosine, N6-[3-methoxyestra-1,3, 5(10)-trien-6beta-yl]deoxyadenosine, and N6-[3-methoxyestra-1,3, 5(10)-trien-6alpha-yl]deoxyadenosine (identified as a base adduct) were identified using HPLC by comparing them with authentic specimens prepared by reacting dG and dA with both sulfates. No steroid-dC adduct was detected in the digestion products of the DNA adduct, although dC reacted with the sulfates to form N4-[3-methoxyestra-1,3,5(10)-trien-6beta-yl]deoxycytidine. These results mean that estrogen 6-sulfate has an ability to modify DNA via the amino group of a guanine or adenine residue in DNA. The present studies imply that a sequential metabolism (hydroxylation and sulfation) at the C6-position of the estrogen molecule causes damage to DNA.

Animals↗

PubMed1998

Effect of preinduction of heat-shock proteins on acetic acid-induced small intestinal lesions in rats.

Bowel dysfunction such as irritable bowel syndrome caused by stress is well described. Previous reports suggest that stress is known to cause the release of endogenous substances such as catecholamine, beta-endorphine, 5-hydroxytryptamine, corticotropin-releasing factor, and thyrotropin-releasing hormone (TRH). However, the role played by these neurohormonal mediators in bowel dysfunction under stress conditions is not well known. We investigated the influence of water-immersion stress or TRH administration on the expression of 60-kDa, 72-kDa, and 90-kDa heat-shock proteins (HSP60, HSP72, and HSP90, respectively) in rat small intestinal mucosa by Western blot and immunohistochemical analyses. The cytoprotective function of preinduced HSPs on experimentally induced mucosal damage also was studied. In order to investigate the influence of preinduction of HSP60 on small intestinal damage, the small intestinal lumen was perfused with 1.5% acetic acid 1 ml/min for 15 min with or without pretreatment with water-immersion stress or TRH administration. Expression of HSP60 was significantly increased by water-immersion stress or TRH administration in the small intestinal mucosa, whereas HSP72 and HSP90 did not increase. Interestingly, expression of this protein showed the biphasic peak pattern after water-immersion stress or TRH administration. Each peak was observed 3-6 hr and 21-24 hr after the initiation of water-immersion stress or TRH administration. Immunohistochemical study also showed a significant increment of HSP60 in both the cytoplasm and nuclei of the small intestinal mucosal cells. No histopathologic alteration was observed in rat small intestinal mucosa after each treatment. Small intestinal damage caused by 1.5% acetic acid perfusion was not influenced by preinduction of HSP60. We demonstrated that water-immersion stress or TRH administration specifically induced HSP60, although preinduction of this protein did not show a cytoprotective function in the small intestinal mucosa.

Acetic Acid↗

PubMed1998

Molecular cloning and regulation of a novel guinea pig cytochrome P450 (CYP3A20) which differs from guinea pig CYP3A14 in only two amino acid residues.

A full length cDNA clone of cytochrome P450, encoding 503 amino acid residues, was isolated from a male guinea pig liver cDNA library. The sequence was highly homologous to other members in the CYP3A subfamily and was designated CYP3A20. CYP3A20 and CYP3A14, another guinea pig CYP3A, shared 99.4% nucleotide and 99.6% deduced amino acid sequence homology. There were only two amino acid differences between CYP3A20 and CYP3A14. No significant induction of CYP3A20 mRNA in the livers from male guinea pigs treated with dexamethasone was observed by S1 mapping analysis although CYP3A14 mRNA was induced. The expression of CYP3A20 mRNA in the livers did not change between 5 and 10 weeks after birth while that of CYP3A14 mRNA in livers significantly increased at 10 weeks. This is the first report that the two highly resembling forms of cytochrome P450 display differently regulated expression from each other.

Amino Acid Sequence↗

PubMed1998

Lack of mutagenicity of levofloxacin in lacZ transgenic mice.

The mutagenic effects of levofloxacin in lacZ transgenic mice (Muta Mouse) have been investigated. Male Muta-Mouse mice were administered levofloxacin i.p. at a dose of 300 or 600 mg/kg. The higher dose corresponded to half the LD50 of the compound in ddY strain mice. The mutant frequencies in the bone marrow, liver (day 10 only), testis and sperm were examined by the positive selection method for lacZ mutations on days 1 and 10 after treatment. Levofloxacin did not induce any statistically significant increase in mutant frequency in any of the examined tissues at either dose level or at either sampling time. The mutant frequency increases over the spontaneous values in the bone marrow, liver, testis and sperm were 1.0- to 1.2-fold, 0.9-fold, 0.5- to 1.0-fold and 0.9- to 1.3-fold respectively. N-Ethyl-N-nitrosourea (100 mg/kg as a positive control), on the other hand, induced significant increases in mutant frequencies in both somatic cells (bone marrow and liver) and germ cells (testis and sperm) on day 10 after treatment. The mutagenic potency for ENU was bone marrow >> liver approximately testis approximately sperm (50.1-fold, 3.4-fold, 2.9-fold and 2.3-fold respectively over the spontaneous values). Levofloxacin was not mutagenic in the lacZ transgenic mice under the present experimental conditions.

Animals↗

PubMed1998

Expression and characterization of dog CYP2D15 using baculovirus expression system.

Dog CYP2D15 was expressed in Sf9 cells with a recombinant baculovirus. Infection of Sf9 insect cells with a recombinant dog CYP2D15-virus resulted in the expression of a protein which cross-reacted with a polyclonal antibody against a dog CYP2D15-specific peptide. The difference spectrum of CO-complex of reduced P450 of the infected cell microsomes had a maximal absorbance at 449 nm. The specific content of P450 was calculated to be 0.56 nmol/mg of Sf9 cell microsomal protein. Although the expressed dog CYP2D15 showed high catalytic activity for the hydroxylations of bunitrolol and imipramine at low substrate concentration (10 microM), the catalytic activity for that of debrisoquine (50 microM) was extremely low as compared with that of CYP2D from other species. Dog liver microsomes also showed bunitrolol and imipramine hydroxylase activities, but not debrisoquine hydroxylase activity at the same substrate concentrations. In addition, the expressed CYP2D showed high catalytic activity for imipramine N-demethylation. Thus, our study reveals that the expressed dog CYP2D15 engages in high catalytic activity and has a unique substrate specificity from other CYP2D subfamilies. Western blot analysis suggested that the dog CYP2D15 contents were less than 4% of the total liver P450 content, assuming that 100% of expressed CYP2D15 incorporated heme.

Animals↗

PubMed1998

Low-voltage backscattered electron imaging of non-coated biological samples in a low-vacuum environment using a variable-pressure scanning electron microscope with a YAG-detector.

A variable-pressure scanning electron microscope (VP-SEM) equipped with a high-sensitive backscattered electron (BSE) detector of the YAG type was applied to studies of biological tissue samples. The rat kidney and trachea were fixed, dehydrated in ethanol, critical point-dried and examined in the VP-SEM under a specimen chamber pressure of 1 to 150 Pa. The high-resolution surface images of the non-coated specimens were obtained in a low-vacuum (1-20 Pa) environment at an accelerating voltage of 5 kV, while the images at 10-20 kV contained information beneath the surface of the specimens. The observation in the VP-SEM equipped with YAG detector in a low-vacuum (1-20 Pa) environment at low (3-5 kV) accelerating voltages is useful for the three-dimensional analysis of the surface morphology of biological non-coated samples.

Animals↗