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Biomedical subjects

S Ihara

Publications and source records attributed to S Ihara.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1997

Dual control of neurite outgrowth by STAT3 and MAP kinase in PC12 cells stimulated with interleukin-6.

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). We explored the signals required for neurite outgrowth of PC12 cells by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. The mutants incapable of activating the MAP kinase cascade failed to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibited neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolished the ability to activate STAT3 did not inhibit, but rather stimulated neurite outgrowth. This mutant did not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimicked NGF pretreatment, and NGF suppressed the IL-6-induced activation of STAT3, supporting the idea that STAT3 might regulate the differentiation of PC12 cells negatively. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF.

Animals↗
PubMed1997

Expression of a constitutively active phosphatidylinositol 3-kinase induces process formation in rat PC12 cells. Use of Cre/loxP recombination system.

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.

Animals↗
PubMed1997

Phosphatidylinositol-3 kinase is involved in ruffled border formation in osteoclasts.

Phosphatidylinositol (PI)-3 kinase has been implicated in several aspects of intracellular membrane trafficking, although the detailed mechanism is yet to be established. We previously reported that wortmannin (WT), a selective inhibitor of PI-3 kinase, inhibited the bone-resorbing activity of osteoclasts (Nakamura et al., 1995, FEBS Lett., 361:79-84). In this study, we examined how PI-3 kinase was involved in membrane trafficking in osteoclasts which are primary bone-resorbing cells. Osteoclasts exhibit a highly polarized cytoplasmic organization, the ruffled border. Ruffled borders are formed by numerous deep membrane invaginations, on which vacuolar H(+)-ATPase (V-ATPase) is localized in a high density. Immunoelectron microscopic analyses revealed that PI-3 kinase was specifically present along ruffled border membranes and the limiting membranes of associated intracellular vacuoles in rat authentic osteoclasts. WT and LY294002, another inhibitor of PI-3 kinase, caused the accumulation of numerous acidic vacuoles which were stained with acridine orange in murine osteoclast-like multinucleated cells formed in vitro. An electron microscopic examination showed that these vacuoles contained V-ATPase along their limiting membranes and appeared to be derived from the Golgi apparatus as ruffled border precursors. A time course study revealed that WT-induced vacuoles began to accumulate in the region close to the apical membrane and were finally distributed throughout the cytoplasm. Removal of WT from the culture medium resulted in the disappearance of vacuoles in the cytoplasm, leading to the formation of ruffled borders. During the culture period, some vacuoles were observed to fuse with the ruffled border membrane. A pit formation assay on dentine slices also showed that the pit-forming activity of osteoclast-like cells was recovered by the removal of WT from the assay. These results suggest that PI-3 kinase plays an important role in ruffled border formation in osteoclasts, probably in the fusion of membrane vacuoles with the plasma membrane.

Androstadienes↗
PubMed1997

Genetic analysis of a clinical isolate of human cytomegalovirus exhibiting resistance against both ganciclovir and cidofovir.

Both ganciclovir-sensitive and -resistant human cytomegaloviruses (HCMV) were isolated from a patient with aplastic anemia complicated with CMV retinitis and encephalitis. Ganciclovir-resistant clinical isolate, 93-1R, also showed cross-resistance against (s)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine (cidofovir). Molecular analysis of plaque-cloned strains revealed that a single nucleotide substitution at 2160 (C to T) resulted in amino acid substitution at codon 501 from leucine to phenylalanine in the DNA polymerase gene. This mutation at codon 501 was easily identified by means of AluI digestion of the selected PCR product. The same mutation existed in the DNA fragment amplified from the patient's brain, suggesting that cross-resistant mutant 93-1R caused encephalitis. Furthermore, ganciclovir-resistant 93-1R-3 replicated much faster and was released more efficiently into the culture medium than ganciclovir-sensitive 91-7S-1.

Antiviral Agents↗
PubMed1996

Identification of interleukin-6 as a factor that induces neurite outgrowth by PC12 cells primed with NGF.

We have screened for a factor that induces neurite outgrowth by PC12 cells only after NGF pretreatment in the supernatants of 38 transformed cell lines, finding three positive clones. The factor showing the strongest activity was purified and identified as IL6. In this NGF-IL6 system, the process of neurite outgrowth by PC12 cells can be separated into NGF-dependent and IL6-dependent steps. The IL6-dependent step requires RNA synthesis, suggesting that IL6 induces new gene expression depending on NGF-priming. These findings suggest that the gene expression during the differentiation process is regulated by at least two signals.

Animals↗
PubMed1996

A novel differentiation factor for PC12 cells from culture supernatant of mouse hepatocyte cell line MLE-15A2.

We have found a factor that induces neurite outgrowth of rat PC12 cells in the culture supernatant of the cell line MLE-15A2. This factor was designated as MDDF. The factor was sensitive to protease, dithiothreitol, and high-temperature treatments. The apparent molecular mass was 80 kDa on Superdex 200 gel filtration. No significant tyrosine phosphorylation was detected after MDDF stimulation in Western blotting analysis with anti-phosphotyrosine antibody, suggesting that the signal transduction may not be mediated by a tyrosine kinase cascade that is involved in signaling of most of the known factors. Activation of MAP kinase was very weak and was seen only 5 min after stimulation, suggesting that prolonged activation of MAP kinase was not required for neurite outgrowth induced by MDDF. Because the biochemical characteristics of MDDF are different from those of any known peptide factors that induce neurite outgrowth of PC12 cells, MDDF may be a novel differentiation factor for PC12 cells.

Animals↗
PubMed1996

[A case of abdominal tuberculous lymphadenitis diagnosed by percutaneous needle biopsy under ultrasound control and followed up by ultrasound imaging].

A case of abdominal tuberculous lymphadenitis diagnosed by percutaneous needle biopsy under ultrasound control and followed up by ultrasound imaging was reported. A 63 years-old male was admitted to a certain hospital complaining of dizziness and diplopia. Chest roentgenogram showed bilateral infiltrative shadows in the upper lung fields and Mycobacterium tuberculosis was detected in cultures from specimens of gastric aspiration. Abdominal ultrasound examination and computed tomography showed abdominal lymph nodes swelling. Needle biopsy under ultrasound control was performed, and the specimens showed necrosis and Mycobacterium tuberculosis was positive by culture. He was diagnosed as lung tuberculosis with abdominal tuberculous lymphadenitis and admitted to our hospital for anti-tuberculous chemotherapy. Ultrasound examination done every two weeks showed diminution in size of the lymph nodes after a month of anti-tuberculous therapy. For the diagnosis of abdominal lymph node swelling, a needle biopsy under ultrasound control is safe and useful technique, and a ultrasound examination is also valuable to follow up the course of abdominal tuberculous lymphadenitis.

Abdomen↗
PubMed1995

Mixed cultures comprising syngeneic and allogeneic mouse keratinocytes as a graftable skin substitute.

Using inbred mouse strains C3H/He and BALB/c and an anti-class I MHC monoclonal antibody, we examined the graftability of mixed cultures composed of syngeneic and allogeneic mouse keratinocytes. Keratinocytes from neonatal C3H/He (syngeneic donor) and BALB/c (allogeneic donor) mice were cultured for 7 days, both individually and at the inoculum ratio of 1:1, 1:3, or 1:15. The resulting sheets of keratinocytes were implanted subcutaneously into adult C3H/He mice and morphological evaluation was made on the 4th, 7th, and 14th days after grafting. The control cultures of 100% allogeneic keratinocytes were rejected, whereas epidermal reconstruction without typical signs of rejection took place in all grafts derived from the mixed cultures, including the 1:15 cultures. However, immunofluorescence staining showed that, within the grafts, the allogeneic keratinocytes were gradually replaced by syngeneic cells. Thus, these results suggested that graftable skin substitutes may be produced by co-culturing a small amount of autologous keratinocytes with allogeneic keratinocytes, which are readily available.

Animals↗