Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells

Immunodeficient CD8 knockout mice were infected with Sarcocystis neurona merozoites, in order to determine the role of CD8 cells in protective immunity. Using a direct agglutination test, all infected mice seroconverted by selected time points. Infected mice developed splenomegaly and bilateral lymphadenopathy. Histological changes included marked follicular development in the spleen, endothelitis and moderate perivascular inflammation in the liver, and meningoencephalitis in the brain. Infected brains were positive for S. neurona by polymerase chain reaction. Corresponding to histopathological changes, there were decreased numbers of B-cells in the spleen. The mice did not have significant memory (CD44hi/CD4) or effector (CD45RBhi/CD4) populations present at the time of euthanasia. Flow cytometry confirmed the lack of CD8 cells. Taken together, these data support previous studies suggesting a critical role for CD8 cells in the prevention of menigoencephalitis in S. neurona-infected mice.     

Partial biopterin deficiency disturbs postnatal development of the dopaminergic system in the brain

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.     

Heart-specific Deletion of CnB1 Reveals Multiple Mechanisms Whereby Calcineurin Regulates Cardiac Growth and Function

Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca²⁺ following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca²⁺ handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca²⁺-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the α-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca²⁺-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Immunofluorescent Identification of Melanocytes in Murine Hair Follicles

Summary Immunocytochemical identification of skin cells are difficult due to numerous endogenous autofluorescent components within the cell and the environment. This is particularly evident in hair follicles. This paper reports on a serendipitous modification to an existing method which results in a drastically reduced background fluorescence. Immediately after antigen retrieval, sections exposed to 0.3% hydrogen peroxide in methanol for 30 min at room temperature exhibited low background fluorescence, increased antigenicity and revealed quantifiable numbers of melanocytes. This method is applicable to both human and mouse melanocytes particularly in the hair follicle.     

Disruption of the mitotic kinesin Eg5 gene (Knsl1) results in early embryonic lethality

Eg5, a member of the widely conserved kinesin-5 family, is a plus-end-directed motor involved in separation of centrosomes, and in bipolar spindle formation and maintenance during mitosis in vertebrates. To investigate the requirement for Eg5 in mammalian development, we have generated Eg5 deficient mice by gene targeting. Heterozygous mice are healthy, fertile, and show no detectable phenotype, whereas Eg5^−/− embryos die during early embryogenesis, prior to the implantation stage. This result shows that Eg5 is essential during early mouse development and cannot be compensated by another molecular motor.