Isao Uemura

Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism.

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.

Regulation of Mitochondrial Transport and Inter-Microtubule Spacing by Tau Phosphorylation at the Sites Hyperphosphorylated in Alzheimer's Disease

The microtubule-associated protein Tau is a major component of the neurofibrillary tangles that serve as a neuropathological hallmark of Alzheimers 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 Alzheimers 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 Alzheimers 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 Alzheimers disease.

MARKED DECREASE IN THE RIGIDITY OF STARFISH OOCYTES INDUCED BY 1‐METHYLADENINE1

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 (1‐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 μg/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.

Studies on Unequal Cleavage in Sea Urchins II. Surface Differentiation and the Direction of Nuclear Migration

Cortical features of the meso‐ and macromeres differ from those of the micromeres in sea urchins. At the end of the 8‐cell stage, the four animal cells have a continuous row of vesicles lining the free surface of the cell by transmission electron microscopy (TEM) and the nuclei and the resulting mitotic apparatuses (MA) remain at the cell centers and eventually divide equally into eight mesomeres. In the four vegetal cells, narrow gaps can be seen in the vesicular rows near the vegetal pole. The resting nuclei migrate to these gaps and on forming the spindles, they point directly to the gaps. The result is formation of vesicle‐free micromeres and vesicle‐covered macromeres by unequal divisions.

Peroxisome proliferator-activated receptor γ ligands stimulate myeloid differentiation and lipogenensis in human leukemia NB4 cells

Peroxisome proliferator‐activated receptor γ (PPARγ) plays a central role in adipocyte and macrophage differentiation. Pioglitazone (Actos, AD4833), an antidiabetic drug, and 15‐deoxy‐Δ12,14‐prostaglandin J2 (PGJ2) have recently been identified as synthetic and natural ligands for PPARγ, respectively. In this study, we examined the effects of PPARγ ligands on differentiation and lipogenesis in promyelocytic leukemia NB4 cells, in which PPARγ protein was expressed and ligand‐stimulated PPARγ‐specific transcription of adipocyte fatty‐acid binding protein was confirmed. Treatment with PPARγ ligand (AD4833 or PGJ2) alone markedly suppressed proliferation but did not induce differentiation. The combined treatment of the cells with PPARγ ligand and all‐trans retinoic acid (ATRA) synergistically induced myelocytic differentiation, as determined by nitroblue tetrazolium reducing ability and cell morphology. During these processes of differentiation, we observed marked accumulation of lipid droplets in the cytoplasm. The cellular triacylglycerol levels increased 2.7‐fold after treatment with the inducers. Simultaneously, BODIPY‐fatty acid was incorporated into the cytosol and concentrated in lipid droplets. The biosynthesis of triacylglycerol‐containing BODIPY‐fatty acids was increased twofold in differentiated cells. These findings clearly demonstrate that treatment with PPARγ ligands not only induced differentiation but also stimulated lipogenesis in NB4 cells, indicating a close association between differentiation and lipogenesis in PPARγ‐stimulated human myeloid cells.

Asymmetric formation and possible function of the primary pore canal in plutei of Temnopleurus hardwicki

The development and possible function of the primary pore canal (PPC) in plutei of the sea urchin Temnopleurus hardwicki was examined by immunochemistry, electron microscopy and microsurgery. Left and right PPC that extended from coelomic sacs in plutei contained a bundle of cilia with a 9 + 2 structure that was initially detected as a group of anti‐acetylated tubulin antibody‐binding granules in the epithelium of coelomic sacs in 28 h postfertilization (PF) prism larvae. The granules extended to be a bundle of fibers toward the larval dorsal surface, concurrent with formation of the PPC on both sides, over the next 4 h. The cilia in both PPC beat actively. However, the PPC on the right side disappeared by approximately 55 h PF, establishing left–right asymmetry by 60 h PF (the four‐arm pluteus stage). The numbers of cilia in the left and right PPC in 56 h PF plutei were five and eight, respectively. Microsurgical removal of the coelomic sac from both sides or the left side only from 26 h PF prism larvae decreased body width to 64 and 91% of normal width by 50 h PF pluteus stage, respectively, whereas that of the right PPC did not. These observations suggest that PPC contribute to the maintenance of normal body width, and that there is asymmetrical activity between the left and right PPC.

Association of the sea urchin EGF-related peptide, EGIP-D, with fasciclin I-related ECM proteins from the sea urchin Anthocidaris crassispina

Exogastrula‐inducing peptides (EGIP) of the sea urchin Anthocidaris crassispina are endogenous peptides related to epidermal growth factor (EGF), which induce exogastrulation in the embryo. Recently, a protein(s) from sea urchin embryos that binds to one of the EGIP, EGIP‐D (EGIP‐D‐binding protein, EBP) was purified. The isolation and characterization of the cDNA clones for two EBP proteins (EBP‐α and EBP‐β) is reported. The two EBP proteins were highly similar in structure to each other; both possessed putative cell‐binding sites and two repeated sequences characteristically seen in the insect neuronal cell adhesion protein, fasciclin I. The EBP showed similarity with other sea urchin proteins HLC‐32, Bep1, and Bep4. It has been confirmed that bacterially expressed EBP proteins associate with EGIP‐D as does native EBP, suggesting the interaction between EGF‐related proteins and fasciclin I‐related proteins. An EBP transcript of 1.4 kb was strongly expressed in immature ovaries but not in immature testes. A somewhat lower level of the transcript existed in unfertilized eggs and the amount gradually declined to an almost undetectable level by the pluteus stage. The EBP proteins were present throughout embryonic development at nearly constant levels. Although most of the proteins were distributed rather evenly in the cytoplasm, a small portion was detected on the apical surface of blastomeres and ectodermal cells, showing that EBP are components of the hyaline layer.

Disruption of mitochondria is an early event during dolichyl monophosphate-induced apoptosis in U937 cells.

Abstract Dolichyl monophosphate (Dol-P) is involved in the attachment of carbohydrate chains to proteins in the formation of N-linked glycoprotein. We found that this compound induces apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5–20 min), reduction in mitochondrial transmembrane potential (Δψm) and translocation of apoptosis-inducing factor (1–3 hr), caspase-3-like protease activation (2–4 hr), chromatin condensation and DNA ladder formation (3–4 hr) were observed successively. In this study, we examined mitochondrial morphological changes by electron microscopy and Δψm by JC-1 from immediately after treatment of Dol-P. After 5 min of treatment, we observed clearly that mitochondrial cristae began to be disrupted ultrastructurally and almost all the cristae were disintegrated after 1 hr of treatment. The Δψm of Dol-P treated cells was reduced to 34% as compared with that of control cells immediately after treatment and was quartered within 1 hr. The reduction in Δψm was not inhibited by cyclosporin A, N-acetyl-L-cysteine and vitamin E. These results indicate that mitochondrial disruption is one of the first triggering events of Dol-P-induced apoptosis.

Localization of an Exogastrula‐Inducing Peptide (EGIP) in Embryos of the Sea Urchin Anthocidaris crassispina

Exogastrula‐inducing peptides (EGIPs) are present in the unfertilized eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. The localization of EGIP‐D during embryogenesis has been explored using polyclonal antibodies against EGIP‐D. Immunofluorescent staining revealed that EGIP‐D is stored in the cytoplasm of immature oocytes and is concentrated into vesicles in unfertilized eggs. At fertilization, the vesicles containing EGIP‐D (EGIP‐vesicles) migrate to the cortical surface of the zygotes and are distributed in a ring‐like pattern at the apical surface of blastomeres, disappearing from basal surfaces and those adjacent to neighboring cells, during development from cleavage stages to larval stages. Mesenchyme cells also contain the vesicles but no such polarized distribution of vesicles is apparent. Acidic vesicles with a similar polarized distribution were examined by staining with acridine orange, which revealed that acidic vesicles were in close proximity to the surface of eggs at fertilization and were then distributed in a ring‐like pattern at the apical surface of blastomeres as are the EGIP‐vesicles. Furthermore, immunoelectron microscopy revealed that EGIP‐D is present in vesicles that are located at the apical surface of blastomeres. The significance of the localized distribution of EGIP‐D is discussed in relation to its function.

ELECTRON MICROSCOPIC OBSERVATIONS ON THE EXTRA-GRANULAR ZONE OF THE EMBRYO OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS*

“Extra‐granular zone” proposed by Dan and Dan (3) was studied with the electron microscope. Two stages are noticed during the process of its formation; at first, a relatively yolk granule‐free zone constitutes the egg periphery which should correspond to that observed by Dan and Dan, and later, this zone is occupied by a layer of the vacuoles (about 1.3 μm in mean diameter) with highly electron‐transparent inclusions which migrated to the egg surface from the inner cytoplasm after fertilization. These vacuoles are scarcely found in the micromeres of 16‐cell stage embryo.