Genetic studies in the fission yeast Schizosaccharomyces pombe have established that a critical element required for the G2----M-phase transition in the cell cycle is encoded by the cdc2+ gene. The product of this gene is a serine/threonine protein kinase, designated p34cdc, that is highly conserved functionally from yeast to man2 and has a relative molecular mass of 34,000 (34 K). Purified maturation-promoting factor (MPF) is a complex of p34cdc2 and a 45K substrate that appears in late G2 phase and is sufficient to drive cells into mitosis. This factor has been identified in all eukaryotic cells, and in vitro histone H1 is the preferred substrate for phosphorylation. The increase in the activity of H1 kinase in M-phase is associated with a large increase in total cell protein phosphorylation which is believed to be a consequence of MPF activation. We show here that the H1 kinase activity of p34cdc2 oscillates during the cell cycle in Xenopus, and maximal activity correlates with the dephosphorylated state of p34cdc2. Direct inactivation of MPF in vitro is accompanied by phosphorylation of p34cdc2 and reduction of its protein kinase activity.

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Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into 2 components: 1) RGC survival, and 2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased 4-5 folds at the protein level by 3-5 days after nerve injury.Subsequently, Bcl-2 levels increased 1.7 folds, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody 3 inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.

In the adult mammalian central nervous system (CNS), axonal injury leads to retrograde neuronal degeneration and death in cell soma.1,2) For instance, transection of the rat optic nerve (ON) induces cell death of retinal ganglion cells (RGCs) within 2 weeks. 3,4) In contrast, injury to the fish ON never leads to widely spreading death of RGCs. Goldfish regrow the axons of their RGCs towards the tectum and finally restore their vision, even after ON transection. 5,6) It has been shown that some rat RGCs can regenerate their axons through peripheral nerve grafts.7) This result implies that the inability of CNS neurons to regenerate in mammals is mainly due to a surrounding inhibitory environment, but not to the nature of the neurons themselves. Therefore, two approaches to the reconstruction of injured ONs in mammals have been studied intensively: 1) the use of nerve grafts or transplantation of stem cells [8][9][10] and 2) studying the regeneration mechanism of the mammalian peripheral nervous system (PNS) and fish CNS.11-13) Our goal here is aimed to introduce the works of goldfish ON regeneration after injury and show up the difference between fish and mammals.Since the first description of fish ON regeneration by Sperry's group in the 1950s, there has been a significant increase in the understanding of morphological changes in RGCs and factors which promote neurite outgrowth in optic axons after ON transection. In contrast, there is little knowledge regarding the molecular (genetic) mechanisms and cellular signaling pathways involved in fish ON regeneration after injury. Very recently, we apply a molecular cloning technique to find out ON regenerating molecules in goldfish after nerve injury. We obtain cDNA clones that are upregulated in the fish retina and tectum during ON regeneration. In this article, we review the neurite outgrowth-promoting factors reported by other investigators and our strategies for searching ON regenerating molecules in the goldfish visual system after injury. MORPHOLOGICAL ASPECTS OF FISH ON REGENER-ATIONIn goldfish, unmyelinated sproutings occur at the cut end of the ON within 3 d, and then bundles of 20-30 axonal sproutings penetrate into the cutting edge by 6 d after ON lesion.14) Outgrowth of the leading axons proceeds at 0.3 mm/d after axotomy.15) The regenerating fibers first reach the optic lobe at 10-12 d after ON lesion, and the plexiform layer formed by the regenerating fibers is visible in all areas of the optic tectum at 14-18 d after ON lesion.16) Although the retinotectal connection is formed 20-40 d after ON section, the regenerated retinotectal projection lacks topographic order for up to 40 d. Thereafter, the retinotectal topography slowly improves over several months.17) Thus, the retinotectal connection is initially very rough and then becomes very refined after a long time. This refinement process of the regenerating fibers can be traced using local injections of wheat germ agglutinin conjugated to horse radish peroxidase (WGA-HRP) in the retina. After goldfish ON...