We previously reported that tau protein kinase I (TPKI) induced normal tau protein into a state of paired helical filaments (PHF); this is further confirmed here by tmmunoblot analysis using several antibodies. We also present the amino acid sequence of TPKI, which is identical to glycogen synthase kinase 38 (GSK3B). Moreover, we found that TPKI acttvity was inseparable from GSK3 acttvity throughout the purification procedure. These results indicate that TPKI is identical to GSK3/?.

Disruption of the presynaptically enriched polyphosphoinositide phosphatase synaptojanin 1 leads to an increase of clathrin-coated intermediates and of polymerized actin at endocytic zones of nerve terminals. These changes correlate with elevated levels of PI(4,5)P(2) in neurons. We report that phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), a major brain PI(4)P 5-kinase, is concentrated at synapses. Synaptojanin 1 and PIPKIgamma antagonize each other in the recruitment of clathrin coats to lipid membranes. Like synaptojanin 1 and other proteins involved in endocytosis, PIPKIgamma undergoes stimulation-dependent dephosphorylation. These results implicate PIPKIgamma in the synthesis of a PI(4,5)P(2) pool that acts as a positive regulator of clathrin coat recruitment and actin function at the synapse.

We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD-sC-). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD-sC-adeA-) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD-sC-DeltaargB adeA- or niaD-sC-DeltaargB adeB-) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD-sC-DeltaargB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.

Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.Autophagy is a protein degradation system that is conserved in eukaryotic cells and used to recycle macromolecules and aid cell survival under nutritional starvation conditions (12, 13). When autophagy is induced, bulk cytoplasm and/or organelles are sequestered within double-membrane vesicles termed autophagosomes. The outer membranes of the autophagosomes then fuse to the vacuolar/lysosomal membrane and deliver single-membrane vesicles, called autophagic bodies, into the lumina of the vacuoles/lysosomes. The subsequent breakdown of the vesicle membranes allows the degradation of the contents of the autophagic bodies by vacuolar hydrolases. ATG8 is an autophagy-related gene found in Saccharomyces cerevisiae that plays an important role in the formation of autophagosomes (9). Atg8 is localized in the membranes of preautophagosomal structures (PAS), autophagosomes, and autophagic bodies and has therefore been used as a marker of these organelles (33).In addition to helping cells to survive starvation, autophagy is involved in stress-induced differentiation and development. In S. cerevisiae diploid cells, atg mutations block starvationinduced sporulation (34). In Dictyostelium discoideum, starvation, overcrowding, and high temperature induce the formation of fruiting bodies and atg mutations block these multicellular developmental processes (25). Additionally, in Caenorhabditis elegans, atg mutations result in abnormal dauer development, which is also induced by starvation, overcrowding, high temperature and so on (17). Recent studies have suggested that autophagy might participate in diseases such as cancer, liver disease, muscula...

We previously reported that tau protein kinase II (TPKII) from bovine brain was composed of 30 kDa and 23 kDa subunits. The 30 kDa subunit of TPKII can be regarded as a catalytic subunit because of its ATP-binding activity. Antibodies directed against TPKII-phosphorylated tau also reacted with tau phosphorylated by cdc2 kinase obtained from starfish oocytes, indicating that TPKII and cdc2 kinase phosphorylate the same sites. We determined the amino acid sequence of the 30 kDa subunit and found it to be homologous with a cdc2-related lcmase, PSSALRE/cdkS. Moreover, an antibody against PSSALRE/cdkS reacted with the 30 kDa subunit. These results indicate that the 30 kDa subunit of TPKII is bovine homologue of PSSALRE/cdkS. Expression of the 30 kDa subunit mRNA was enhanced in juvenile rat brain. This result supports our previous hypothesis that the kinase works actively in juvenile brain.Tau protein; Paired helical filament; Alzheimer's disease; cdc2-relaed protein kinase; Cyclin-dependent kinase 5