The microtubule-associated protein Tau is a major component of the neurofibrillary tangles that serve as a neuropathological hallmark of Alzheimer's disease. Tau is a substrate for protein phosphorylation at multiple sites and occurs in tangles in a hyperphosphorylated state. However, the physiological functions of Tau phosphorylation or how it may contribute mechanistically to Alzheimer's pathophysiology are not completely understood. Here, we examined the function of human Tau phosphorylation at three sites, Ser199, Ser202, and Thr205, which together comprise the AT8 sites that mark abnormal phosphorylation in Alzheimer's disease. Overexpression of wild-type Tau or mutated forms in which these sites had been changed to either unphosphorylatable alanines or phosphomimetic aspartates inhibited mitochondrial movement in the neurite processes of PC12 cells as well as the axons of mouse brain cortical neurons. However, the greatest effects on mitochondrial translocation were induced by phosphomimetic mutations. These mutations also caused expansion of the space between microtubules in cultured cells when membrane tension was reduced by disrupting actin filaments. Thus, Tau phosphorylation at the AT8 sites may have meaningful effects on mitochondrial movement, likely by controlling microtubule spacing. Hyperphosphorylation of the AT8 sites may contribute to axonal degeneration by disrupting mitochondrial transport in Alzheimer's disease.

In vitro fertilization (IVF) systems using isolated male and female gametes have been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, as the female gametophytes of plants are deeply embedded within ovaries. In this study, a rice IVF system was established to take advantage of the abundant resources stemming from rice research for investigations into the mechanisms of fertilization and early embryogenesis. Fusion of gametes was performed using a modified electrofusion method, and the fusion product, a zygote, formed cell wall and an additional nucleolus. The zygote divided into a two-celled embryo 15-24 h after fusion, and developed into a globular-like embryo consisting of an average of 15-16 cells by 48 h after fusion. Comparison of the developmental processes of zygotes produced by IVF with those of zygotes generated in planta suggested that zygotes produced by IVF develop and grow into early globular stage embryos in a highly similar manner to those in planta. Although the IVF-produced globular embryos did not develop into late globular-stage or differentiated embryos, but into irregularly shaped cell masses, fertile plants were regenerated from the cell masses and the seeds harvested from these plants germinated normally. The rice IVF system reported here will be a powerful tool for studying the molecular mechanisms involved in the early embryogenesis of angiosperms and for making new cultivars.

Ultrastructural changes induced by heavy metals (cadmium, zinc, and copper) and polyphosphate metabolism were studied in Chlamydomonas acidophila. Transmission electron microscopy indicated that cadmium led to the most drastic morphometric changes. An increase in number and volume of starch grains and vacuoles as well as the presence of electron dense deposits in vacuole and membrane whorls were observed. Energy-dispersive X-ray analysis revealed that vacuolar deposits inside cells treated with cadmium contained phosphate and cadmium. These ultrastructural changes were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo (31)P-nuclear magnetic resonance. It was also observed that cadmium treatment caused polyphosphate degradation and increased vacuolar short-chains and orthophosphates.

Extreme rigidity of immature starfish oocytes as measured by compression method was found to decline during the early phase of their maturation when induced by 1-methyladenine (I-MeAde). The onset of this decrease in stiffness occurred within 5 to 9 min of 1-MeAde treatment, well before the breakdown of the germinal vesicle, progressively declining to reach a minimum stiffness after 20 min. Dithiothreitol, known as an artificial maturation-inducing agent, caused a similar change. The stiffness is thus expected to serve as a quantitative indicator of the early process of cytoplasmic events, which would induce the breakdown of the germinal vesicle. Cytochalasin B (3 pg/ml) also reduced the stiffness, but unlike the former two agents, the effect was reversible, and did not interfere with the process of maturation. Due to the effect of cytochalasin B, it became possible to enucleate immature oocytes by centrifugal force. Non-nucleate fragments thus obtained still maintained their marked stiffness, which was decreased by the action of 1-MeAde, with a time-course similar to that of intact oocytes.It is known that I-methyladenine, the hormone which initiates maturation of starfish oocytes (9, 1 I ) by acting on the cell surface (10, 18), induces the production of a 'maturationpromoting factor' in the cytoplasm. This in turn triggers the breakdown of the germinal vesicle (15, 16). It is most likely that there are some other accompanying cellular processes initiated also by 1-methyladenine, which interract and result in cytoplasmic maturation of the oocytes.HIRAMOTO and his colleagues (1 9, 22) noticed marked changes in the mechanical properties of the cytoplasm, which occur prior to the breakdown of the germinal vesicle (GVBD). In the present study, detailed investigation of oocyte rigidity, which we postulate as serving as a quantitative indicator of the process of maturation, was carried out. MATERIALS AND METHODS Materialswith cold sea water (15-20°C) until use. Astropecten scoparius and Asterias amurensis were also employed in some experiments. Solutions(DTT) in 0.25 M NaCI, and 2 mg cytochalasin B in ml dimethylsulfoxide, were prepared.Starfishes were collected during their respective breeding seasons, and stored in aquaria supplied Materials mainly used were the oocytes of Asterina pectinifera.As stock solutions, I mM 1-methyladenine (I-MeAde) in de-ionized water, 0.5 M dithiothreitol These were diluted * This paper is dedicated to the memory of the late Professor Jean Clark Dan, 315

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