Masaaki Ohnuma

Cloning and Expression of the Platelet-Specific Collagen Receptor Glycoprotein VI

Platelet glycoprotein VI (GP VI) was purified from platelet membranes and its internal amino acid sequences were determined. The cloned cDNA of GP VI indicates an open reading frame coding for 20 amino acid signal sequences and a mature protein of 319 amino acids. Its extracellular region has two Ig-like domains and a mucin-like, Ser/Thr-rich region, suggesting that GP VI is a member of the paired Ig-like receptor family. GP VI-transfected cells contained convulxin-(reactive) and antibody against recombinant GP VI-reactive protein bands that migrated at the same position as platelet GP VI in SDS/PAGE-electroblotting. These data indicate that the protein deduced from the cloned cDNA corresponds to platelet GP VI.

Identification and Expression Analysis of Upregulated Genes in the Resting Egg-Producing Water Flea (Daphnia pulex)

Water fleas (Daphnia pulex) normally produce subitaneous eggs that initiate development immediately after oviposition. However, in response to habitat degradation, resting eggs are produced, which are enclosed in a sturdy outer envelope (ephippium) and can survive in harsh environments for an extended time. To understand the molecular mechanism underlying resting egg production in D. pulex, we investigated the genes whose expression patterns played a role in the production and identified the following six candidate genes: Dpfa-1, Dpfa-2, Dpep-1, Dpep-2, Dpep-3, and Dpep-4. These six genes displayed > 40-fold higher expression levels in resting egg-producing animals compared with those in subitaneous egg-producing animals at the period when the ovaries were mature. Dpfa-1 and Dpfa-2 were expressed in the fat cells, and their expression patterns were synchronized with the development of resting egg oocytes in the ovary. In contrast, Dpep-1–4 were expressed in the morphologically altered epidermal cells of the brood chamber with the formation of the ephippium, and their expression patterns were also related to ephippium formation. Our results suggest that the former two genes encode the resting egg-specific components produced by fat cells and that the latter four genes encode the components related to the ephippium formation synthesized by epidermal cells.

Phorbol ester treatment of two megakaryoblastic cell lines, CMK and UT-7, induces specific protein composition changes with non-coincident time-courses.

The expressions of platelet-specific glycoprotein(GP)s Ib, IIb and IIIa were analyzed in 2 megakaryoblastic cell lines: CMK and UT-7. Phorbol-12 myristate 13-acetate (PMA) treatment of CMK induced expressions of GPs IIb and IIIa that peaked on the 4th day post-treatment, while treated UT-7 cells showed maximal levels of these GPs during the 6-8th days. Antibody staining to detect the formation of GPIb alpha after PMA induction showed the presence of the intact GP in UT-7 and the degraded form in CMK. In UT-7, synthesis of GPIIb mRNA increased on days 4-6 after PMA treatment, in parallel with the increase of GPIIb. PMA increased the cytoskeletal protein (actin and myosin) contents in both lines, but in contrast to the two GPs, the increase in these proteins started immediately after PMA addition to the cells. Cell surface proteins of CMK and UT-7 cells were rapidly modified after PMA induction. Especially notable were the degradations of 93-kDa and 140-kDa proteins that occurred on days 1-2 after PMA treatment. These studies demonstrate that the expression of platelet-specific proteins shows a different time dependency than the increment of cytoskeletal proteins, indicating that the syntheses of these two classes of proteins are most likely induced through different mechanisms.