Yutaka Toyoda

Mice deficient for the IL-3/GM-CSF/IL-5 βc receptor exhibit lung pathology and impaired immune response, while βIL3 receptor-deficient mice are normal

The receptors for IL-3, GM-CSF, and IL-5 share a common beta subunit (beta c), and mice have an additional IL-3 beta subunit (beta IL3). We have independently generated mice carrying null mutations of each molecule. beta c mutant bone marrow showed no response to GM-CSF or IL-5, whereas IL-3 stimulation of beta c or beta IL3 mutant bone marrow was normal. beta c mutant mice showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis, and also exhibited low basal numbers of eosinophils. Infection of beta c mutant mice by Nippostrongylus brasiliensis resulted in the absence of blood and lung eosinophilia. Animals repopulated with beta c mutant bone marrow cells showed slower leukocyte recovery and reduced eosinophil numbers. These data define the role of beta c in vivo, and show a phenotype that is likely to be the cumulative effect of loss of GM-CSF and IL-5 stimulation.

Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes

To investigate the involvement of mitogen-activated protein kinase (MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using myelin basic protein (MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0-20 h of culture. An abrupt increase was observed at metaphase I (30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.

Production of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific T cell clones

Abstract The protective effect of a mouse hepatitis virus type-4 (MHV-4)-specific CD8+ cytotoxic T cell clone and a CD4+ helper T cell clone was examined by the adoptive transfer into brains of mice lethally infected with MHV-4. Mice survived acute encephalitis if more than 5 × 105 cells of either type of the virus-specific T cell clones had been transfered into H-2-matched recipients by 1 day post-infection. Although the adoptive transfer of both types of the T cell clones suppressed viral growth and viral antigen-positive cells in the brains, a significant inhibition of virus replication by the cytotoxic T cell clone was detected prior to that induced by the helper T cell clone. Histologically, cell destruction was prominent in the brains of mice which received the cytotoxic T cell clone. These results demonstrate that both the CD8+ cytotoxic T cell and the CD4+ helper T cell can protect mice from a lethal MHV-4 infection in the central nervous system.

Histone H1 kenase activity during in vitro fertilization of pig follicular oocytes matured in vitro

Abstract The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouths MB752/1 medium and were then inseminated in vitro with frozenthawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 μg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P

Analysis of the role of egg integrins in sperm-egg binding and fusion.

Sperm‐egg fusion is believed to be mediated via specific molecular interactions. Integrin α6β1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin α6β1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin α6β1 in sperm‐egg interactions was examined by biochemical and immunocytochemical analyses. To identify egg molecules that specifically interact with sperm, we first incubated sperm with biotin‐labeled egg surface proteins. Under this condition, solubilized proteins from eggs inhibited sperm‐egg fusion. Western blot analysis under reducing conditions indicated that a major‐labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti‐integrin α6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin α6 subunit. To investigate the potential involvement of integrin α6β1 in sperm‐egg fusion, we next examined the localization of integrin α6 and β1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm‐egg fusion, the integrin α6 and β1 subunits were accumulated at the sperm binding site. The frequency of cluster formation was closely related to that of sperm‐egg fusion, indicating that integrin receptors are accumulated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin α6β1 is involved in sperm‐egg binding leading to fusion via direct association of the integrin α6 with sperm. Mol. Reprod. Dev. 56:412–423, 2000.

No dosage effect of recombinational hotspots in the mouse major histocompatibility complex.

The sites of meiotic recombination in the proximal region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. Some MHC haplotypes derived from Asian wild mice increase the frequency of recombination at such hotspots when heterozygous with standard laboratory haplotypes. The wm7 and cas3 haplotypes, have a hotspot close to the Lmp-2 gene (Lmp-2 hotspot), and the cas4 haplotype has a hotspot about 100 kilobase (kb) proximal, close to the Pb gene (Pb hotspot). To examine the effect of a double dose of hotspots, we estimated the rate of recombination and determined the location of the breakpoints in crosses of wm7/cas3 and wm7/cas4. In 3570 backcross progeny we identified 29 new recombinants in the H-2K to Ab interval, at a frequency of 0.81%. This frequency is 40-fold higher than in crosses between laboratory haplotypes and very similar to those previously obtained in crosses between these wild and standard laboratory haplotypes. Thus, a double dose of hotspots has no additive effect on the frequency of meiotic recombination. The site-specificity of recombination was also conserved. Twenty-three breakpoints were confined within 5.4 kb in the Lmp-2 hotspot, and six breakpoints from the cas4 cross were located in the Pb hotspot, which we have now confined to a 15 kb segment.

Recombination in the class III region of the mouse major histocompatibility complex.

The sites of meiotic recombination in the class II region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. To search for hotspots in the class III region, we mapped combiantional break-points of 79 Ab: H2-D recombinants with 11 DNA markers; these included Tnx, the gene for an extracellular matrix protein, tenascin X, the Notch-related Int3 gene, and a microsatellite marker, D17Mit13, none of which had previously been mapped precisely. The results gave the gene order Eb-61.11-Int3-Tnx-Cyp21/C4-Bf-Hsp68c-D17Mit13-Tnfa/Tnfb-D. The crossover sites in 40 of the 79 recombinants were cofiend within the Eb/Int3:Tnx/Cyp21 interval. The result demonstrated that an unequal distribution of recombination is a general feature of the mouse MHC, suggesting the presence of a recombinational hotsopt within the Int3:Tnx interval.

Structural changes in the oviductal wall during the passage of unfertilized cumulus‐oocyte complexes in mice

Background: Little information is available on the structural relationship of cumulus‐oocyte complexes and the oviductal wall during the transport of cumulus‐oocyte complexes. Then, morphological changes of the oviductal wall during the passage of unfertilized cumulus‐oocyte complexes was examined chronologically in ICR mice 25–27 days of age injected with PMSG and hCG.

Acute and late disease induced by murine coronavirus, strain JHM, in a series of recombinant inbred strains between BALB/cHeA and STS/A mice

Abstract To examine the genetic control of acute and late disease induced by a murine coronavirus, strain JHM (JHMV), BALB/cHeA, STS/A, F1 hybrids and 13 recombinant inbred (RI) strains between BALB/cHeA and STS/A mouse strains were inoculated intracerebrally with 100 pfu of JHMV. All the BALB/cHeA mice died within 2 weeks from acute encephalitis. In contrast, STS/A mice were shown to be partially resistant, with a mortality rate of 30%, longer survival times and lower rates of viral production. The mortality rates, survival times and viral titers of F1 hybrids and the RI strains varied, suggesting involvement of multiple genes. STS/A, F1 hybrid and RI mice surviving the acute infection occasionally developed severe paraparesis about 1 month post-infection. In these mice, vacuolar degeneration, astrocytosis, the absence of perivascular cuffing and minimal demyelination were found in the central nervous system. No infectious virus could be recovered from these mice. Although the paralysis of delayed onset was limited to STS/A, F1 hybrid and eight of the 13 RI strains, the incidence varied significantly among the RI strains. These results may suggest that JHMV-induced late disease is also under multifactorial control. The pathogenesis of JHMV infection is discussed.

Mouse Hepatitis Virus-Specific CD8+ Cytotoxic T Lymphocytes Induce Apoptosis in Their Target Cells

CD8+ cytotoxic T lymphocytes (CTL) have been suggested to play an important role in virus clearance and development of demyelination during mouse hepatitis virus (MHV) infection in mice1. Production of antiviral cytokines such as γ-IFN and direct cytolysis of virus-infected cells have been considered to be the major role of CTL, but their precise mechanisms in MHV infection remain unclarified. We have previously established CTL line, P1 1D which specifically lyses cells infected with MHV, strain JHM (JHMV)2 and saves mice from lethal JHMV infection3. To clarify the detailed mechanism of CTL during MHV infection, we examined morphological changes of JHMV-infected target cells in vitro cocultured with anti-MHV CTL line, P1 1D.