Rika Suzuki

Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans.

Male embryonic mice with mutations in the X-linked aristaless-related homeobox gene (Arx) developed with small brains due to suppressed proliferation and regional deficiencies in the forebrain. These mice also showed aberrant migration and differentiation of interneurons containing γ-aminobutyric acid (GABAergic interneurons) in the ganglionic eminence and neocortex as well as abnormal testicular differentiation. These characteristics recapitulate some of the clinical features of X-linked lissencephaly with abnormal genitalia (XLAG) in humans. We found multiple loss-of-function mutations in ARX in individuals affected with XLAG and in some female relatives, and conclude that mutation of ARX causes XLAG. The present report is, to our knowledge, the first to use phenotypic analysis of a knockout mouse to identify a gene associated with an X-linked human brain malformation.

Peg1/Mest imprinted gene on chromosome 6 identified by cDNA subtraction hybridization

Parthenogenesis in the mouse is embryonic lethal partly because of imprinted genes that are expressed only from the paternal genome. In a systematic screen using subtraction hybridization between cDNAs from normal and parthenogenetic embryos, we initially identified two apparently novel imprinted genes, Peg1 and Peg3. Peg1 (paternally expressed gene 1) or Mest, the first imprinted gene found on the mouse chromosome 6, may contribute to the lethality of parthenogenones and of embryos with a maternal duplication for the proximal chromosome 6. Peg1/Mest is widely expressed in mesodermal tissues and belongs to the alpha/beta hydrolase fold family. A similar approach with androgenones can be used to identify imprinted genes that are expressed from the maternal genome only.

Characterization of two tobacco cell lines selected to grow in the presence of either ionic Al or insoluble Al-phosphate

A tobacco cell line tolerant to ionic A1 (AIT) was selected at a frequency of 10-6 to 10-7 by plating the wild-type cells on agar medium containing 2.5 mm AICI3 and 0.1 mM phosphate at pH 4.0. Another cell line which is able to grow with insoluble Al-phosphate as a sole source of phosphate (IPU) was also selected by subculturing the wild-type cells for more than 10 passages in suspension culture in the presence of 2 mM phosphate and 4 mM A1C13 at pH 5.0. The IPU cell line also tolerated ionic A1 but the AIT line showed poor growth with insoluble Al-phosphate as a sole source of phosphate. Both cell lines excreted citrate into the media. The rate of citrate excretion by the IPU cell line, on a fresh weight basis, was higher than that by the AIT and wild-type cell lines.

The calcium‐independent protein kinase C participates in an early process of CD3/CD28‐mediated induction of thymocyte apoptosis

Thymocyte negative selection eliminates self‐reactive clones and involves both a T‐cell receptor (TCR)/CD3‐mediated signal and a costimulatory signal, which can be delivered via CD28. Anti‐CD3/anti‐CD28‐triggered apoptosis in isolated CD4+CD8+ thymocytes in vitro provides a basic model for negative selection. Effects of isoform‐selective and non‐isoform‐selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca2+‐independent PKC isoforms during the first 2–3 hr of culture is essential for inducing apoptosis, and that Ca2+‐dependent PKC isoforms may be influential, but not essential, for apoptosis. To assess the CD3/CD28‐mediated activation of PKC in the apoptotic process, we prepared CD4+CD8+ thymocytes (without contamination with cells that had received negative or positive selection signals in vivo) by establishing TCR‐transgenic mice with RAG‐2‐deficient and non‐selecting major histocompatibility complex (MHC) backgrounds, in addition to a CD4+CD8+ thymocyte‐enriched population from normal mice. Translocation of Ca2+‐independent PKC from the cytosolic fraction to the particulate fraction of CD4+CD8+ thymocytes was induced by CD3/CD28‐mediated stimulation, but not by CD3‐ or CD28‐mediated stimulation alone, and peaked 2 hr after the start of culture. The kinase activity of the translocated Ca2+‐independent PKC was dependent on cofactors in vitro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolytic PKC fragment, was responsible for the activity. Immunoblotting analysis indicated that the nPKC‐θ isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC‐α isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the θ isoform) in CD4+CD8+ thymocytes is involved in a pathway for negative selection.

Trypsin-like Hatching Enzyme of Mouse Blastocysts: Evidence for Its Participation in Hatching Process before Zona Shedding of Embryos'

Effects of twelve protease inhibitors on hatching of mouse embryos were investigated. Mouse hatching was strongly or moderately inhibited by trypsin inhibitors including p‐toluenesulfonyl‐Lys‐CH2Cl (TLCK) and chicken ovomucoid, while inhibitors for chymotrypsin and elastase showed weak or no inhibition. These results indicate the participation of a trypsin‐like protease in the hatching of mouse embryos as a hatching enzyme., Since TLCK is the strongest and an irreversible inhibitor for the enzyme, timing of the participation of the hatching enzyme in the hatching process was examined by pulse treatment of embryos with TLCK before and during the zona shedding. The results indicated that a trypsin‐like hatching enzyme functions before, but not during, the zona shedding of embryos, especially during a 15 h period immediately before the beginning of the shedding.

Inhibition of Mouse Blastocyst Hatching by Subsite-Specific Trypsin Inhibitors, Peptidyl Argininals1

To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P4)‐Leu(P3)‐Leu(P2)‐argininal(P1)] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P4 position inhibited the hatching more strongly than those containing acetyl group or unprotected N‐terminal amino acid. Among five Z‐Leu‐P2‐argininals, a derivative containing a P2 Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P2 position followed in this order. Then, we synthesized four Z‐P3‐Ser‐argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P3 position was the strongest inhibitor, and the Leu‐, Pro‐, and Ala‐containing derivatives were ranked in this order. Thus, among Z‐dipeptidyl‐argininals tested, Z‐Phe‐Ser‐argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P3)‐Ser(P2)‐Arg(P1) sequence as a substrate.

Enhancement of Noradrenergic Phenotype Expression in Transgenic Mice Overexpressing V-1, A Cytoplasmic Ankyrin Repeat Protein

Catecholaminergic cells exhibit three types of transmitter phenotypes: dopaminergic, noradrenergic and adrenergic. The catecholaminergic phenotypes are thought to be determined by a phenotype-specific coordinate regulation of gene expression of the four catecholamine synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). However, molecular mechanisms that control the coordinate transcriptional regulation of these genes remain to be fully elucidated, although the second messenger systems and transcription factors involved in catecholaminergic cell-specific gene expression for catecholamine synthesizing enzymes have been so far identified and extensively investigated (Zetterstrom et al., 1997; Morin et al., 1997; Lo et al., 1999). Recently, an ankyrin repeat protein designated V-1 has been suggested to act as a possible coordinate regulator for the expression of genes encoding catecholamine synthesizing enzymes, since it has been demonstrated that stable overexpression of V-1 protein in the catecholaminergic cell line, PC12D cell, augments the expression of transcripts encoding TH, AADC and DBH, and as a resuit, catecholamine production is upregulated (Yamakuni et al., 1998). V-1 is a 12 kDa protein containing 2.5 copies of the ankyrin repeat, which has been originally isolated as a developmentally regulated protein from murine cerebellum (Taoka et al., 1992).