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Biomedical subjects

M Ikawa

Publications and source records attributed to M Ikawa.

At least 19 recordsLinked to original sources
PubMed2001

Calmegin is required for fertilin alpha/beta heterodimerization and sperm fertility.

Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607-611). In the present study, we demonstrate that calmegin -/- sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin -/- sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in fertilin beta -/- sperm, we investigated the interaction of calmegin with fertilin beta. Using immunoprecipitation techniques, calmegin was found to bind to sperm membrane proteins, fertilin alpha and beta, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin alpha and beta. In the calmegin -/- mice, a loss of heterodimerization of fertilin alpha and beta was observed and fertilin beta was not detectable in mature sperm. The data not only explain why the calmegin and fertilin beta knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum.

ADAM Proteins↗
PubMed2001

Alkalinization of acrosome measured by GFP as a pH indicator and its relation to sperm capacitation.

We previously targeted EGFP (a mutant of green fluorescent protein) to the lumen of the mouse sperm acrosome and reported the time course of EGFP release during the acrosome reaction. In the study reported here, we estimated the pH within the mouse sperm acrosome utilizing the pH-dependent nature of EGFP fluorescence. The average intra-acrosomal pH was estimated to be 5.3 +/- 0.1 immediately after sperm preparation, gradually increasing to 6.2 +/- 0.3 during 120 min of incubation in TYH media suitable for capacitation. Spontaneous acrosome reactions were noted to increase concomitantly with acrosomal alkalinization during incubation. We also demonstrated that acrosomal antigens detected by monoclonal antibodies MN7 and MC41 did not dissolve following the acrosome reaction in pH 5.3 media, but dissolved at pH 6.2. These data suggest that acrosomal alkalinization during incubation conducive for sperm capacitation may function to alter acrosomal contents and prepare them for release during the acrosome reaction.

Acrosome↗
PubMed2001

Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice.

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.

Animals↗
PubMed2001

Cyclin G1 is involved in G2/M arrest in response to DNA damage and in growth control after damage recovery.

Cyclin G1 is one of the target genes of the transcription factor p53, and is induced in a p53-dependent manner in response to DNA damage. Although cyclin G1 has been implicated in a range of biological phenomena, its precise function remains unclear. Here we present an analysis of the physiological role of cyclin G1 using mice homozygous for a targeted disruption of the cyclin G1 gene. In order to clarify the role of cyclin G1 in the p53 pathway, downstream events such as apoptosis, cell growth and cell cycle checkpoint control were analysed in thymocytes and embryonic fibroblasts derived from cyclin G1-disrupted mice. No difference was detected in induction of apoptosis between mouse embryo fibroblasts (MEFs) derived from cyclin G1+/+ and cyclin G1-/- mice. Following irradiation, cyclin G1-/- MEFs proliferated more slowly and reached lower cell densities in culture dishes than cyclin G1+/+ MEFs. Analysis of cell survival showed that cyclin G1-/- MEFs were about twice as sensitive as cyclin G1+/+ MEFs to gamma radiation or UV radiation. Cyclin G1-/- mice were more sensitive to gamma radiation than wild-type mice. Flow cytometeric analysis revealed that the number of cyclin G1-/- MEFs in G2/M phase after irradiation was reduced by 50% relative to cyclin G1+/+ MEFs. Our results demonstrate that cyclin G1 plays roles in G2/M arrest, damage recovery and growth promotion after cellular stress.

Animals↗
PubMed2001

The effect of short-term tooth intrusion on human pulpal blood flow measured by laser Doppler flowmetry.

The purpose was to examine the effect of brief intrusive forces on human pulpal blood flow (PBF). Laser Doppler flowmetric measurements were made from 17 vital upper left central incisors of 17 participants who had clinically healthy tooth crowns and periodontal tissues. Brief intrusive forces (0.5,1,5 N; duration 20 s) were applied to the incisal edges of the examined teeth, and apical displacement of the teeth and the PBF were measured simultaneously. Recordings were made with and without an opaque rubber dam applied to the examined teeth. Intrusive force significantly reduced PBF flux both with and without the dam (P<0.05, Friedman analysis). The results indicate that transient apical displacement can reduce PBF.

Adult↗
PubMed2001

Revascularization of an immature permanent tooth with apical periodontitis and sinus tract.

A necrotic immature mandibular second premolar with periapical involvement in a 13-year-old patient was treated. Instead of the standard root canal treatment protocol and apexification, antimicrobial agents were used in the canal, after which the canal was left empty. Radiographic examination showed the start of apical closure 5 months after the completion of the antimicrobial protocol. Thickening of the canal wall and complete apical closure was confirmed 30 months after the treatment, indicating the revascularization potential of a young permanent tooth pulp into a bacteria-free root canal space.

Adolescent↗
PubMed2001

Effects of combination psychotropic drug treatment on heart rate variability in psychiatric patients.

The aim of this study was to evaluate the effects of combination psychotropic drug treatment on heart rate variability (HRV), which was mainly controlled by the parasympathetic nervous system. Mean R-R intervals (mRR) and coefficient of variation (CV), an index of HRV, were studied in 22 psychiatric patients and 21 age- and sex-matched healthy controls. Next, in the patient group focusing on both anticholinergic and antidopaminergic properties, combination psychotropic drug daily doses were converted into biperiden milligram equivalents (BPDeq) and chlorpromazine milligram equivalents (CPZeq), respectively. The relationship between mRR and CV and these equivalent dosages was examined. A significant reduction in both mRR (P < 0.05) and CV (P < 0.05) was found in the patient group. In addition, significant negative correlations were observed between the dose of BPDeq and mRR (P < 0.05), and between the dose of BPDeq and CV (P < 0.005). In contrast, no significant correlations were observed between the dose of CPZeq and either parameter. These findings suggest that the effects of combination psychotropic drug treatment on HRV are mainly due to their anticholinergic properties. Therefore, CV is a useful indicator to assess the parasympathetic activity of psychiatric patients under combination psychotropic drug treatment.

Adult↗
PubMed2001

A synthetic dDAF (CD55) gene based on optimal codon usage for transgenic animals.

The human DAF (CD55) gene was chosen as a representative molecule in a xenotransplantation study. The gene was synthesized in order to adapt its codons to those which are more frequent in mammals, especially pigs, and the expression levels were then examined in Chinese hamster ovarian (CHO) cells, swine endothelial cell (SEC) and transgenic mice. A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF (wtDAF) and delta-SCR1 wild-type DAF (Delta1wtDAF). Consistent with the in vitro data, the expression of DAF in mice that carry sDAF was higher than Delta1wtDAF in many organs, especially the pancreas. The sDAF showed a high level of expression in SEC and transgenic mice, suggesting that it will be useful in the development of transgenic pigs with high levels of expression.

Animals↗
PubMed2001

Expression of the endoplasmic reticulum molecular chaperone (ORP150) rescues hippocampal neurons from glutamate toxicity.

A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of mu-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.

Animals↗
PubMed2000

Homeobox gene Hex is essential for onset of mouse embryonic liver development and differentiation of the monocyte lineage.

Disruption of the mouse Hex gene resulted in embryonic lethality around embryonic age (E) 10.5, due to no substantial liver formation. Expression of albumin was detectable in heterozygous (Hex(+/-)) but not in homozygous (Hex(-/-)) [corrected] embryos at E8.5. Instead of liver bud formation at E9.5, a liver-like capsule structure was observed in Hex(-/-) [corrected] embryos. In Hex(-/-) [corrected] mutant liver, we found no hepatocytes but no signs of apoptotic cell death in the area. Expression of transcription factors involved in hepatocyte differentiation, hepatocyte nuclear factor (Hnf)3beta, Hnf6, Hnf4alpha and Hnf1alpha, were restricted to the capsule and internal matrix-like structure in the mutant liver and expression of a subset of these factors were reduced. Hematopoiesis of monocytes was impaired in mutant embryos while erythroid lineage was unaffected. These results indicate that Hex plays an essential role in progenitor cells which commit to the hepatic endoderm and in the hematopoietic differentiation of the monocyte lineage.

Albumins↗
PubMed2000

The class IV semaphorin CD100 plays nonredundant roles in the immune system: defective B and T cell activation in CD100-deficient mice.

The class IV semaphorin CD100/Sema4D differentially utilizes two distinct receptors: plexin-B1 in nonlymphoid tissues, such as brain and kidney, and CD72 in lymphoid tissues. We have generated CD100-deficient mice and demonstrated that they have functional defects in their immune system, without apparent abnormalities in other tissues. The number of CD5(+) B-1 cells was considerably decreased in the mutant mice, whereas conventional B cells and T cells appeared to develop normally. In vitro proliferative responses and immunoglobulin production were reduced in CD100-deficient B cells. The humoral immune response against a T cell-dependent antigen and in vivo priming of T cells were also defective in the mutant mice. These results demonstrate nonredundant and essential roles of CD100-CD72 interactions in the immune system.

Animals↗
PubMed2000

Relationship between EEG dimensional complexity and neuropsychological findings in Alzheimer's disease.

The aim of the present study was to examine the region-specific correlations between electroencephalography (EEG) dimensional complexity (DC), a measure of non-linear dynamics, and neuropsychological performance in 25 right-handed patients with Alzheimer's disease (AD). Electroencephalography recording sites were according to the international 10-20 system. Neuropsychological tests included Wechelor Adult Intelligence Scale-Revised (WAIS-R) (Full-scale Intelligence Quotient (FIQ), Verbal Intelligence Quotient (VIQ), Performance Intelligent Quotient (PIQ)); Mini-Mental State Examination; Raven's Coloured Progressive Matrices (RCPM); Rey Auditory-Verbal Learning Test (RAVLT); seven word pairs-revised Miyake Paired-Associate Word Learning Test; Benton Visual Retention Test; and Rey Complex Figure Test. A partial correlational analysis was carried out, controlling for age and sex (P< 0.005). The FIQ, VIQ and RCPM scores were found to be significantly correlated with DC at the F3, C3 and T3 electrodes. Significant correlations were also found between RAVLT scores and DC at the C3, P3 and T5 electrodes. The study on AD indicated region-specific correlations between DC and neuropsychological performance: one between the DC value in the left frontal, central and mid-temporal areas and intellectual function; and another between the DC value in the left central, parietal and post-temporal areas and verbal memory. Dimensional complexity would therefore seem to be a useful indicator for the assessment of neuropsychological deficits in AD.

Aged↗
PubMed2000

Mice with markedly reduced PACAP (PAC(1)) receptor expression by targeted deletion of the signal peptide.

In an attempt to study the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC(1)) receptor (PAC(1)R) function in vivo and to produce a mouse model with altered expression of PAC(1)R, we have used gene targeting in embryonic stem cells to disrupt exon 2 of the PAC(1)R gene, which contains the ATG translation start site and the signal peptide. Un-expectedly, active transcription of PAC(1)R mRNA was detected in the mutant mice; however, exon 1 was spliced to exon 3 (skipping exon 2), and (125)I-PACAP27 binding in brain was greatly reduced. PAC(1)R exon 2(-/-) mice were viable, fertile, and morphologically and histologically indistinguishable from their wild-type counterparts. We next examined the ligand binding and cell surface expression of the mutant receptor lacking the signal peptide in transfected COS-7 cells. (125)I-PACAP27 binding of the mutant receptor was approximately one-tenth of that in the wild-type receptor. Although the wild-type receptor was expressed abundantly in both the plasma membrane and the cytoplasm around the nucleus, the mutant receptor was expressed in the plasma membrane with a markedly reduced level. Digestion of the membranes with endoglycosidase F greatly reduced the size of the wild-type receptor but only slightly reduced that of the mutant receptor. These results demonstrate that the signal peptide is required for efficient cell surface expression and N-linked glycosylation of the PAC(1)R. However, the mutant receptors still functionally coupled to adenylate cyclase in COS-7 cells, suggesting the presence of sufficient spare receptors such that the mutant receptors are capable of activating the second messenger system. We suggest that the mutant mice with markedly reduced PAC(1)R expression can serve as a useful animal model or cell culture system for further studies in PAC(1)R function.

Animals↗
PubMed2000

Synthesis of a new Cre recombinase gene based on optimal codon usage for mammalian systems.

The origin of the Cre recombinase gene is bacteriophage P1, and thus the codon usages are different from in mammals. In order to adapt this codon usage for mammals, we synthesized a "mammalian Cre recombinase gene" and examined its expression in Chinese hamster ovarian tumor (CHO) cells. Significant increases in protein production as well as mRNA levels were observed. When the recombination efficiency was compared using CHO cell transfectants having a cDNA containing loxP sites, the "mammalian Cre recombinase gene" recombined the loxP sites much more efficiently than the wild-type Cre recombinase gene.

Amino Acid Sequence↗
PubMed1999

Green fluorescent protein-transgenic mice: immune functions and their application to studies of lymphocyte development.

Green fluorescent protein (GFP) transgenic (GFP+) mice express GFP in most tissues except erythrocytes and hair. Immune responses of GFP+ mouse and their application to studies of lymphocyte development were investigated. Flow cytometric analyses revealed that differentiation patterns of lymphocytes from GFP+ mice are equivalent to those from parental C57BL/6 mice. There was no difference in mature T-cell proliferative ability in response to allogeneic stimulator cells or anti-CD3epsilon stimulation between GFP+ and C57BL/6 mice. Furthermore, the anti-OVA antibody response of GFP+ mice was also the same as that of C57BL/6 mice. Taken together, these results show no immunological differences between GFP+ and C57BL/6 mice. Bone marrow transplantation and in vitro thymus reconstitution experiments were performed in an attempt to apply the GFP+ mice to the analysis of lymphocyte development. When bone marrow cells from GFP+ mice were transplanted. T and B lymphocytes containing GFP developed normally in scid recipients. Next we examined intrathymic T-cell development by hanging drop culture methods. GFP+ and CD4+8+ immature T-cells developed normally from bone marrow cells in the reconstituted thymus. The experimental system using hematopoietic cells from GFP+ mice is a powerful tool for visualizing lymphocyte development.

Animals↗
PubMed1999

Roles of integrins and CD44 on the adhesion and migration of fetal liver cells to the fetal thymus.

Adhesion and migration of mouse fetal liver (FL) cells to the thymus were investigated using cells from green fluorescent protein transgenic (GFP+) mice. FL cells from GFP+ embryos at 12 gestational days (E12) of mice were incubated with 2'-deoxyguanosine-treated fetal thymus lobe (from E14) by thymic repopulation (hanging drop) culture methods. GFP+ cells were observed in the thymus lobe at the end of the repopulation culture period. A large part of the infiltrated cells expressed CD44 until day 2 of culture on a permeable membrane, then lost the expression. CD25 expression was observed from day 1 to day 4. Around day 8, GFP+ cells became both CD4+ and CD8+. The results support the early observation of the sequential expression of CD44, CD25, and CD4/8 during the early stages of thymocyte development. When anti-CD44 mAb was added at the beginning of the repopulation culture period, GFP+ FL cells adhered to the surface of the thymus lobe but did not migrate into the thymus. Pretreatment of the thymus with hyaluronidase or hyaluronate produced results similar to the results of anti-CD44 treatment. On the other hand, the addition of anti-integrin alpha4 mAb inhibited adhesion to the thymus, and almost no GFP+ cells were seen on the surface of the thymus lobe. The data suggest that integrin alpha4 and CD44 play different roles, i.e., integrin alpha4 is required for the adhesion of FL cells to the thymus lobe and CD44 is required for the migration of the cells into the thymus.

Animals↗
PubMed1999

Migration of exogenous immature hematopoietic cells into adult mouse brain parenchyma under GFP-expressing bone marrow chimera.

Bone marrow transplantation with GFP-expressing cells from GFP-transgenic mice resulted in migration of GFP-positive cells into peripheral tissues and brain parenchyma. Most of these cells were observed as colony-like clusters. GFP-positive clusters in the brain were stained by antibody for ER-MP12, but those in the peripheral tissues were not. Since ER-MP12 antigen has been reported as a marker for murine early-stage myeloid precursor, this might suggest that some parts of phagocytic cells in the brain parenchyma such as microglia are derived from undifferentiated pluripotent hematopoietic cells.

Animals↗