Shunzo Kondo

Roles of jumonji and jumonji family genes in chromatin regulation and development

The jumonji (jmj) gene was identified by a mouse gene trap approach and has essential roles in the development of multiple tissues. The Jmj protein has a DNA binding domain, ARID, and two conserved jmj domains (jmjN and jmjC). In many diverse species including bacteria, fungi, plants, and animals, there are many jumonji family proteins that have only the jmjC domain or both jmj domains. Recently, Jmj protein was found to be a transcriptional repressor. Several proteins in the jumonji family are involved in transcriptional repression and/or chromatin regulation. Most recently, one of the human members has been shown to be a histone demethylase, and the jmjC domain is essential for the demethylase activity. Meanwhile, more and more evidence indicating that the jumonji family proteins play important roles during development is accumulating. Many proteins in the jumonji family may regulate chromatin and gene expression, and control development through various signaling pathways. Here, we highlight the roles of jmj and jumonji family proteins in chromatin regulation and development. Developmental Dynamics 235:2449–2459, 2006.

Neurotransmitter Release from Synaptotagmin-Deficient Clonal Variants of PC 12 Cells

Synaptotagmin (p65) is an abundant synaptic vesicle protein of neurons and contains regions similar to the regulatory domain of protein kinase C. These domains are thought to be involved in calcium-dependent interaction with membrane phospholipids during exocytosis. To assess the functional role of synaptotagmin, synaptotagmin-deficient clonal variants of PC12 cells were isolated. All of the variant cells released catecholamine and adenosine triphosphate in response to elevated intracellular concentrations of calcium, which suggests that synaptotagmin is not essential for secretion of catecholamine and adenosine triphosphate from PC12 cells.

jumonji gene is essential for the neurulation and cardiac development of mouse embryos with a C3H/He background.

The recessive mutant mouse jumonji (jmj), obtained by a gene trap strategy, shows neural tube defects in approximately half of homozygous embryos with a BALB/cA and 129/Ola mixed background, but no neural tube defects with BALB/cA, C57BL/6J, and DBA/2J backgrounds. Here, we show that neural tube and cardiac defects are observed in all embryos with a C3H/HeJ background. In addition, abnormal groove formation and prominent flexure are observed on the neural plate with full penetrance, suggesting that abnormal groove formation leads to neural tube defects. We found morphogenetic abnormalities in the bulbus cordis (future outflow tract and the right ventricle) of homozygous embryo hearts. Moreover, myocytes in the ventricular trabeculae show hyperplasia with cells filling the ventricles. Together with the observation that the jmj gene is expressed in the neural epithelium of the head neural plate and in myocytes in the bulbus cordis and trabeculae, the results show that the jmj gene plays essential roles in the normal development of the neural plate, morphogenesis of bulbus cordis, and proliferation of trabecular myocytes on a C3H/He background.

2, 3, 7, 8-tetrachlorodibenzo-p-dioxin induces apoptosis in the dorsal midbrain of zebrafish embryos by activation of arylhydrocarbon receptor

Neurotoxic effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) has not been fully elucidated, despite the known potent agonist of arylhydrocarbon receptor (AhR), which activation induces cytochrome P450 1A and several representative toxicities of halogenated aromatic hydrocarbons. In the present study, the effects of TCDD on cell death in zebrafish embryos (Danio rerio) during the early stage of development were investigated. As shown by terminal transferase-mediated nick-end-labeling staining, TCDD exposure significantly increased the occurrence of pycnotic cell death (PCD), especially in the dorsal midbrain (optic tectum). The ultrastructures of these pycnotic cells showed apoptotic features such as condensation and cleavage of chromatin. TCDD-induced PCD was mimicked by beta-naphthoflavone (AhR agonist), and inhibited by alpha-naphthoflavone (AhR antagonist). These results suggest that AhR activation can induce apoptosis in the central nervous system during development.

Protein kinase C and Ca2+/calmodulin-dependent protein kinase II phosphorylate a novel 58-kDa protein in synaptic vesicles

A monoclonal antibody was made using the spleen cells of a mouse immunized with chick synaptic membranes and designated as mAb 1D12. It immunoprecipitated 25% of the omega-conotoxin binding protein but no dihydropyridine binding protein solubilized from chick brain membranes. By immunoblotting, a polypeptide of 58-kDa was identified as the antigen of this antibody in chick, rat, rabbit and guinea pig brain. Immunohistochemical observation indicated the immunoreactivity of mAb 1D12 to be localized in the synaptic regions of central and peripheral neurons. In peripheral organs, there was additional staining in the distal portions of nerve fibers. Immunoelectron microscopy showed immunoreactivity to be located in synaptic vesicle and presynaptic plasma membranes. In the subcellular fractionation of rat brain, 58-kDa protein was recovered in the fractions of synaptic vesicles and plasma membranes but not soluble proteins. This protein could be extracted from membranes by Triton X-100 but treatment with EDTA, acid, base or high salt failed to have such effect. Solubilized 58-kDa protein of rat brain was purified by immunoaffinity chromatography using mAb 1D12. Both protein kinase C and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated purified 58-kDa protein, and maxima of 0.47 and 0.94 mol of phosphates, respectively, were incorporated per mol of 58-kDa protein. 58-kDa protein was not phosphorylated by either cAMP-dependent or cGMP-dependent protein kinase. When present in membranes, it was also phosphorylated by protein kinase C and CaM kinase II. Possible involvement of 58-kDa protein in the protein kinase C and CaM kinase II-mediated regulation of synaptic transmission in central and peripheral neurons is discussed.

Organogenesis of the liver, thymus and spleen is affected in jumonji mutant mice

The recessive mutant mouse jumonji (jmj), obtained by a gene trap strategy, shows neural tube defects in approximately half of homozygotes with a Balb/cA and 129/Ola mixed background. Here, we show that no neural tube defects are observed with a Balb/cA background. We also found hypoplasia of the liver, thymus and spleen with full penetrance with a Balb/cA background. In the livers of homozygous embryos we found excessive cell death in the peripheral region. In both the thymus and spleen, the accumulation of hematopoietic cells is affected in mutant embryos. These phenotypes were also observed with C57BL/6J and DBA/2J backgrounds, suggesting that the jmj gene plays an essential role in the organogenesis of these tissues.

Induction of Thymocyte Apoptosis by Ca2+-independent Protein Kinase C (nPKC) Activation and Its Regulation by Calcineurin Activation

Glucocorticoids appear to participate in apoptosis of unselected CD4+CD8+thymocytes. Activation of Ca2+-independent novel protein kinase C (nPKC) precedes glucocorticoid-induced thymocyte apoptosis, while proper levels of Ca2+-dependent protein kinase C (cPKC) and calcineurin activities contribute to rescue thymocytes. To clarify the role of nPKC in thymocyte apoptosis, murine thymocytes were stimulated with the diterpene diester, ingenol 3,20-dibenzoate (IDB). IDB induced selective translocation of nPKC-δ, -ε, and -θ and PKC-μ from the cytosolic fraction to the particulate fraction and induced morphologically typical apoptosis through de novo synthesis of macromolecules. The apoptosis was also induced by thymeleatoxin, a diterpene ester, at relatively high concentrations that induced translocation of cPKC, nPKC-θ, and PKC-μ. The IDB- or thymeleatoxin-induced death was inhibited by non-isoform-selective PKC inhibitors, but not by their structural analogs with weak PKC-inhibitory activity or the selective inhibitor of cPKC and PKC-μ, Gö 6976. The death was also inhibited by calcium ionophore ionomycin at concentrations within a narrow range. The range corresponded to the concentration range that contributes to the inhibition of glucocorticoid-induced apoptosis. The antiapoptotic effect was canceled by the immunosuppressant FK506 but not by rapamycin. These results indicate that activation of nPKC, especially nPKC-θ, induces apoptosis in thymocytes and that calcineurin activation regulates the apoptosis.

Prodigiosin 25-C uncouples vacuolar type H+-ATPase, inhibits vacuolar acidification and affects glycoprotein processing

Prodigiosin 25‐C inhibited the accumulation of 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methyldipropylamine and acridine orange in the acidic compartments of baby hamster kidney cells with little perturbation of cellular ATP levels. In rat liver lysosomes, prodigiosin 25‐C inhibited the proton pump activity with an IC50 of approximately 30 nM, but did not affect ATPase activity up to 1 μM. It also delayed the transport of vesicular stomatitis virus G protein and induced a drastic swelling of Golgi apparatus and mitochondria. These results indicate that prodigiosin 25‐C raises the pH of acidic compartments through inhibition of the proton pump activity of vacuolar type H+‐ATPase, thereby causing the functional and morphological changes to the Golgi apparatus.

A src family tyrosine kinase inhibits neurotransmitter release from neuronal cells.

Tyrosine kinases are expressed in many tissues, particularly in the central nervous system, and regulate various cellular functions. We report here that a src family tyrosine kinase-specific inhibitor, PP2, enhances neurotransmitter release from PC12 cells and primary cultured neurons. PP2 enhances only Ca2+-dependent release; it does not affect basal release. These effects result from an enhancement of vesicular exocytosis and not from the reuptake or refilling of neurotransmitters because Ca2+-dependent secretion of an exogenously expressed reporter protein, the human growth hormone (hGH), is also enhanced by PP2. Overexpression of constitutive active v-src, but not of a kinase-inactive mutant, suppressed Ca2+-dependent release. In PP2-treated cells, Pyk2, paxillin, and some other proteins showed a decrease in tyrosine phosphorylation, and the enhancement of tyrosine phosphorylation of these proteins in response to Ca2+ influx was also reduced. Electron and fluorescence microscopy showed that PP2 treatment induced morphological change and decreased phalloidin reactivity at the filopodium-like structures on the processes of PC12 cells. Interestingly, inhibition of actin polymerization with cytochalasin D and latrunculin A enhanced Ca2+-dependent, but not basal, release. It is possible that a src family tyrosine kinase, through the regulation of actin dynamics, has an inhibitory function to regulate neurotransmitter release.

The Drosophila Bruton’s Tyrosine Kinase (Btk) Homolog Is Required for Adult Survival and Male Genital Formation

ABSTRACT We isolated a Drosophila fickleP(ficP) mutant with a shortened copulatory duration and reduced adult-stage life span. The reduced copulatory duration is ascribable to incomplete fusion of the left and right halves of the apodeme that holds the penis during copulation.ficP is an intronic mutation occurring in theBtk gene, a gene which encodes two forms (type 1 and type 2) of a Bruton’s tyrosine kinase (Btk) family cytoplasmic tyrosine kinase as a result of alternative exon usage. TheficP mutation prevents the formation of the type 2 isoform but leaves expression of the type 1 transcript intact. Ubiquitous overexpression of the wild-type cDNA by using a heat shock 70 promoter during the late larval or pupal stages rescued the life span and genital defects in the mutant, respectively, establishing the causal relationship between the ficP phenotypes and the Btk gene mutation. The stage specificity of the rescuing ability suggests that the Btk gene is required for the development of male genitalia and substrates required for adult survival.