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Biomedical subjects

S Ihara

Publications and source records attributed to S Ihara.

At least 19 recordsLinked to original sources
PubMed2000

Evidence that 3'-phosphorylated polyphosphoinositides are generated at the nuclear surface: use of immunostaining technique with monoclonal antibodies specific for PI 3,4-P(2).

Phosphatidylinositol (PI) 3,4-P(2) is a phosphoinositide that has been shown to be important for signal transduction in growth factor stimulation. We have produced monoclonal antibodies specific for PI 3,4-P(2), which were able to detect PI 3,4-P(2) generated in 293T cells treated with H(2)O(2), or in MKN45/BD110 cells expressing activated PI 3-kinase in immunostaining. Prolonged treatment with 0.05% Tween 20 resulted in detection of staining not only at the plasma membrane, but also at the nuclear surface, indicating that 3'-phosphorylated phosphoinositides can be generated and function in the nucleus.

Animals↗
PubMed2000

High-frequency ultrasonic wire bonding systems

The vibration characteristics of longitudinal-complex transverse vibration systems with multiple resonance frequencies of 350-980 kHz for ultrasonic wire bonding of IC, LSI or electronic devices were studied. The complex vibration systems can be applied for direct welding of semiconductor tips (face-down bonding, flip-chip bonding) and packaging of electronic devices. A longitudinal-complex transverse vibration bonding system consists of a complex transverse vibration rod, two driving longitudinal transducers 7.0 mm in diameter and a transverse vibration welding tip. The vibration distributions along ceramic and stainless-steel welding tips were measured at up to 980 kHz. A high-frequency vibration system with a height of 20.7 mm and a weight of less than 15 g was obtained.

Journal Article↗
PubMed1999

Dedifferentiation of adenocarcinomas by activation of phosphatidylinositol 3-kinase.

Signet ring cell carcinoma is a malignant type of poorly differentiated adenocarcinomas in stomach, which is characterized by the occasional presence of signet ring-like cancer cells. We found that expression of constitutively active phosphatidylinositol 3-kinase (PI 3-kinase) in well differentiated adenocarcinoma cell lines induced the loss of cell-cell contact and some of the cells changed their shapes to signet ring cell-like, characterized by appearance of mucus droplets in the cytoplasm with well developed endplasmic reticulum and Golgi complexes. The active PI 3-kinase-expressing cells formed poorly differentiated tumors in nude mice, which were clearly different from those of the original cell lines. The PI 3-kinase activities detected in anti-phosphotyrosine immunoprecipitates were higher in several signet ring cell carcinoma-derived cell lines than in other adenocarcinoma cell lines. In addition, PI 3-kinase was found to be associated with a 200-kDa protein phosphorylated in tyrosine in 4 of 6 signet ring cells but not in other cell lines, suggesting that PI 3-kinase is possibly activated in these cells by binding to the 200-kDa protein. The 200-kDa protein-PI 3-kinase complex was exclusively fractionated in the membrane fractions. The specific activity of the PI 3-kinase immunoprecipitated with anti-phosphotyrosine antibody was approximately 3-fold higher than that with anti-PI 3-kinase antibody. These results suggest that PI 3-kinase in signet ring cell carcinoma is recruited to the membrane and activated by the binding to the 200-kDa protein.

Adenocarcinoma↗
PubMed1999

Evidence that a phosphatidylinositol 3,4,5-trisphosphate-binding protein can function in nucleus.

PIP3BP is a phosphatidylinositol 3,4,5-trisphosphate-binding protein (PIP3BP) abundant in brain, containing a zinc finger motif and two pleckstrin homology (PH) domains. Staining of rat brain cells with anti-PIP3BP antibody and determination of localization of PIP3BP fused to the green fluorescent protein (GFP-PIP3BP) revealed that PIP3BP was targeted to the nucleus. Targeting was dependent on a putative nuclear localization signal in PIP3BP. Generation of PIP3 in the nucleus was detected in H2O2-treated 293T cells, nerve growth factor (NGF)-treated PC12 cells, and platelet-derived growth factor (PDGF)-treated NIH 3T3 cells. Translocation of phosphatidylinositol 3-kinase (PI 3-kinase) to the nucleus and enhanced activity of PI 3-kinase in the nucleus fraction were observed after H2O2 treatment of 293T cells, suggesting that PI 3-kinase can be activated in the nucleus as well as in the membrane after appropriate stimulation of the cells. Co-expression of the constitutively active PI 3-kinase with PIP3BP resulted in exportation of the protein from the nucleus to the cytoplasm, suggesting that PIP3BP can function as a PIP3-binding protein in the intact cells. These results imply that there may be an unknown function of PI 3-kinase in the nucleus.

3T3 Cells↗
PubMed1999

Breast cancer with choriocarcinomatous features: A case report with cytopathologic details.

A case of breast cancer with choriocarcinomatous features (BCCF) is reported. The patient was a 38-year-old Japanese female with a long history of schizophrenia. Her nursing staff noticed a palpable mass in her right breast, which showed rapid growth. Following cytopathologic confirmation of a malignant breast tumor, she underwent mastectomy with ipsilateral axillary lymph node dissection. Histologic examination revealed BCCF, which was positive for placental alkaline phosphatase and human chorionic gonadotropin (HCG) by immunohistochemistry. The serum HCG level was high. She died 7 months postoperatively, with multiple metastases of BCCF to the chest wall, lung and liver.

Adult↗
PubMed1999

Dose-dependency and rate of decay of efficacy of Resovist on MR images in a rat cirrhotic liver model.

RATIONALE AND OBJECTIVES: To investigate the dose-dependency and half-life of decay of the signal reduction by super-paramagnetic iron oxides on three different spin-echo images in a rat model of cirrhosis. METHODS: For normal and cirrhotic liver, signal intensities were measured 15 minutes after injection of Resovist (range, 1-40 mumol Fe/kg) on three different spin-echo images. Subsequently, recovery of signal intensity was monitored up to 2 weeks after injection. RESULTS: The dose-dependency of efficacy was somewhat less at all doses and imaging parameters in cirrhotic liver. However, the submaximal effect was obtained at a dose of 20 mumol Fe/kg, the same as in normal liver. The rate of decay of the efficacy in cirrhotic liver was similar to or faster than that in normal liver. CONCLUSIONS: The dose-dependency and rate of decay of efficacy of Resovist in cirrhosis were similar to those in normal liver, although the efficacy was less in cirrhotic liver and both the dose-dependency and rate of decay of efficacy were dependent on imaging parameters.

Animals↗
PubMed1999

Preparation of recombinant human monoclonal antibody Fab fragments specific for Entamoeba histolytica.

Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.

Amino Acid Sequence↗
PubMed1999

Bacterial expression of a neutralizing mouse monoclonal antibody Fab fragment to a 150-kilodalton surface antigen of Entamoeba histolytica.

A mouse monoclonal antibody (MAb) (EH3015, IgG1 with a K light chain) prepared by hybridoma technology recognizes a 150-kD surface antigen of Entamoeba histolytica and inhibits adherence and cytotoxicity of the ameba to mammalian cells. The genes encoding the light chain and the Fd region of the heavy chain of the MAb were cloned and expressed in Escherichia coli. The plasmid used was designed for the expression of Fab with a hexa-histidine tag in the periplasmic space. Recombinant Fab fragments were purified and analyzed by an indirect immunofluorescence antibody test and Western immunoblot. The specificity of the recombinant Fab fragment was comparable with the parent whole IgG. In addition, the Fab fragments significantly inhibited the adherence of E. histolytica to erythrocytes. These results suggest that the production of a neutralizing MAb in Escherichia coli is practical and efficient with this expression system.

Amino Acid Sequence↗
PubMed1999

Bacterial expression of a human recombinant monoclonal antibody fab fragment against hepatitis B surface antigen.

The Fab fragment was cloned from the monoclonal cell line TAPC301-CL4, which was produced using the Epstein-Barr virus (EBV) transformation method. This cell line produces a human monoclonal antibody (CL4MAb) against the hepatitis B surface antigen (HBsAg). This MAb was shown to have hepatitis B virus (HBV) neutralizing activity in chimpanzees. The Fab fragment was produced by subjecting the heavy and light chain antibody genes of the TAPC301-CL4 cell line to reverse transcription-polymerase chain reaction, cloning the products in the plasmid vector pFab1-His2 and introducing the plasmid into bacteria. Sequence analyses of the CL4Fab fragment revealed that the light and heavy chains belong to the Vk3a and VH3 groups of the immunoglobulin (Ig) family, respectively. An enzyme-linked immunosorbent assay confirmed that specificity of the recombinant CL4Fab antibody against HBsAg was the same as that of the parental MAb. Flow cytometric analysis using PLC/PRF/5 (Alexander) cells, which express HBsAg, showed the reactivities of the CL4MAb and CL4Fab antibody were the same. These results suggest that the recombinant CL4Fab antibody produced by Escherichia coli using the new vector-primer system developed for human IgG Fab fragments has a very high affinity for the HBsAg and may be useful clinically. A source for generation of human MAb for human therapy with very stable and specific expression was thus produced by isolating antibodies from EBV-transformed cell lines.

Amino Acid Sequence↗
PubMed1998

A possible role of RGS9 in phototransduction. A bridge between the cGMP-phosphodiesterase system and the guanylyl cyclase system.

In the current concept of phototransduction, the concentration of cGMP in retinal rod outer segments is controlled by the balance of two enzyme activities: cGMP phosphodiesterase (PDE) and guanylyl cyclase (GC). However, no protein directly mediates these two enzyme systems. Here we show that RGS9, which is suggested to control PDE activity through regulation of transducin GTPase activity (He, W., Cowan, C. W., and Wensel, T. G. (1998) Neuron 20, 95-102), directly interacts with GC. When proteins in the Triton X-100-insoluble fraction of bovine rod outer segments were isolated by two-dimensional gel electrophoresis and binding of GC to these proteins was examined using a GC-specific antibody, proteins (55 and 32 kDa) were found to interact with GC. However, the activity of GC bound to the 55-kDa protein was not detected. This observation was elucidated by the finding that the 55-kDa protein inhibited GC activity in a dose-dependent manner. Amino acid sequence showed that five peptides derived from the 55-kDa protein were identical to corresponding peptides of RGS9. Together with other biochemical characterization of the 55-kDa protein, these observations indicate that the 55-kDa protein is RGS9 and that RGS9 inhibits GC. RGS9 may serve as a mediator between the PDE and GC systems.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Human monoclonal anti-HCMV neutralizing antibody from phage display libraries.

Human cytomegalovirus (HCMV) infection in immunocompromised patients causes considerable morbidity and mortality. Although ganciclovir prophylaxis reduces the incidence of HCMV disease, severe side effects raise serious problems. Thus, the development of new strategies for prophylaxis are clearly needed, and human monoclonal antibodies offer a potential alternative. We describe the cloning, using the phage display system, of a recombinant human Fab fragment against HCMV. A phage display library with 4 x 10(6) clones was panned three times against lysates of HCMV-infected cells, and screened by ELISA. Of six antigen-binding clones, one monoclonal antibody reacted strongly to HCMV. In immunostaining analysis, this Fab was able to stain HCMV-infected cells from 24 h post-infection (pi) through to 96 h pi, but not at 6 h pi. In the presence of cytosine arabinoside, HCMV-infected cells were not stained, even at 24 h pi. These results indicate that an HCMV protein that was recognized by the Fab was synthesized in the late phase of infection. In addition, this Fab exhibited neutralizing activity: at 1 microg/ml it reduced HCMV plaque formation by 50%. The Fab was able to neutralize three HCMV strains, but it did not neutralize HSV-1 or -2 infection.

Amino Acid Sequence↗
PubMed1998

Histodifferentiation of hair follicles in grafting of cell aggregates obtained by rotation culture of embryonic rat skin.

We have previously reported reconstruction of hair follicles from a single cell suspension of rat fetal upper lip by a two-step culture method consisting of rotation and flotation cultures. Rotation sorted out the cells and flotation facilitated histodifferentiation. In the present study, we added grafting procedures to the previous method to see whether cell aggregates obtained this way were graftable, and whether the grafting promoted histodifferentiation. The aggregates before and after flotation were grafted, and differentiation of hair follicles comparable to those in vivo was confirmed 10 days after grafting. There was no difference in the degree of differentiation between the two kinds of grafts. The grafting procedure therefore resulted in an appreciable increase in histodifferentiation even when aggregates obtained after flotation were grafted.

Animals↗
PubMed1998

Downstream of phosphatidylinositol-3 kinase, a multifunctional signaling molecule, and its regulation in cell responses.

Phosphatidylinositol (PI)-3 kinase is an enzyme that phosphorylates the D-3 position of PI and its derivatives. It is activated immediately after growth factor or differentiation factor stimulation, suggesting that PI-3 kinase is involved in signal transduction of the stimulation. PI-3 kinase appears to play various important roles including signaling to the nucleus, vesicle transport, and rearrangement of the cytoskeleton since many cell responses which require these events are affected by inhibition or activation of PI-3 kinase. To understand how PI-3 kinase can act in such multiple ways, it is important to identify the factors downstream of PI-3 kinase. In this review, we discuss the factors downstream of PI-3 kinase and the methods used to identify them. Recent studies revealed that some proteins involved in vesicle transport or in rearrangement of the cytoskeleton are regulated by the phospholipids generated by PI-3 kinase, implying the mechanism by which PI-3 kinase regulates these cell responses.

Animals↗
PubMed1998

Matrix-producing carcinoma of the breast: case report with radiographical and cytopathological features.

A case of matrix-producing carcinoma (MPC), which is a unique variant of breast cancer, is described with cytopathological and radiographical findings. A 75-year-old female presented with a palpable mass in her right breast. After confirmation of the cytopathological diagnosis, the patient received a right mastectomy with ipsilateral axillary lymph node dissection. The histopathological examination revealed MPC. The Union Internationalis Contra Cancrum postoperative clinical stage was IIB. The patient is well and without disease 18 months after the surgery.

Aged↗
PubMed1998

Microinjection of activated phosphatidylinositol-3 kinase induces process outgrowth in rat PC12 cells through the Rac-JNK signal transduction pathway.

We have previously shown that sustained phosphatidylinositol (PI)-3 kinase activity is necessary for neurite outgrowth of PC12 cells induced by nerve growth factor (NGF). Microinjection of a constitutively active mutant of PI-3 kinase induced process formation suggesting that PI-3 kinase is indeed involved in the neurite outgrowth. However, the processes appeared to be incomplete neurites as they had very poor organization of F-actin and GAP43 antigen. The microtubule network was enhanced in the process-bearing cells and process formation was inhibited by colchicine suggesting that microtubules play an important role in process formation downstream of PI-3 kinase. These cell responses were inhibited by dominant-negative mutants of Rac and Sek1/SAPK but not by a dominant-negative mutant Ras and PD98059, a MAP kinase kinase (MEK) inhibitor, suggesting that not the Ras-MAP kinase pathway but the Rac-Jun N-terminal kinase (JNK) pathway is involved in process formation.

Animals↗
PubMed1998

[Neutralizing human antibody specific for human cytomegalovirus in E. coli expression system].

We isolated neutralizing human Fab fragment specific for human cytomegalovirus (HCMV) by phage display system. Fab libraries were constructed from peripheral lymphocyte of healthy individual. In several clones reacted for HCMV-infected HEL cells, one clone, designated 13-3, stained HCMV infected cells at 96 hrs post infection. It didn't react to cells at 6 hrs post infection or infected cells in the presence of AraC, meaning that 13-3 recognized protein synthesized at late times of infection. It also neutralized HCMV, Towne strain, efficiently. This neutralizing activity was specific for HCMV and no effect for HSV-1 and 2.

Animals↗
PubMed1998

Use of a glycoprotein gB promoter for expression of genes inserted into the human cytomegalovirus genome.

We attempted to utilize the human cytomegalovirus (HCMV) as an expression vector by replacing the dispensable genes of the viral genome with foreign genes. The selection of a promoter to be fused to the foreign gene is important to achieve a high expression rate in the recombinant virus. We selected the glycoprotein B (gB) promoter of HCMV as a target of analysis because gB is one of the most abundantly synthesized components in cell culture. The gB promoter, fused to the E. coli lacZ gene, was introduced into the HCMV HindIII-O fragment region by homologous recombination. It was confirmed that the gB promoter-lacZ construct was inserted in the targeted site of HCMV. The expression of the lacZ gene in the recombinant virus infection was initiated 24 h after infection and increased until 120 h post infection. The lacZ gene expression was inhibited by the presence of cytosine arabinoside. These observations indicate that the expression of the lacZ gene is under the control of the late promoter of gB.

Cell Line↗