In Vivo Turnover of Tau and APP Metabolites in the Brains of Wild-Type and Tg2576 Mice: Greater Stability of sAPP in the β-Amyloid Depositing Mice

The metabolism of the amyloid precursor protein (APP) and tau are central to the pathobiology of Alzheimer''s disease (AD). We have examined the in vivo turnover of APP, secreted APP (sAPP), Aβ and tau in the wild-type and Tg2576 mouse brain using cycloheximide to block protein synthesis. In spite of overexpression of APP in the Tg2576 mouse, APP is rapidly degraded, similar to the rapid turnover of the endogenous protein in the wild-type mouse. sAPP is cleared from the brain more slowly, particularly in the Tg2576 model where the half-life of both the endogenous murine and transgene-derived human sAPP is nearly doubled compared to wild-type mice. The important Aβ degrading enzymes neprilysin and IDE were found to be highly stable in the brain, and soluble Aβ40 and Aβ42 levels in both wild-type and Tg2576 mice rapidly declined following the depletion of APP. The cytoskeletal-associated protein tau was found to be highly stable in both wild-type and Tg2576 mice. Our findings unexpectedly show that of these various AD-relevant protein metabolites, sAPP turnover in the brain is the most different when comparing a wild-type mouse and a β-amyloid depositing, APP overexpressing transgenic model. Given the neurotrophic roles attributed to sAPP, the enhanced stability of sAPP in the β-amyloid depositing Tg2576 mice may represent a neuroprotective response.     

Synthesis of sialic acid derivatives having a CC double bond substituted at the C-5 position and their glycopolymers

Glycomonomers of sialic acid in which the acetamide group at C-5 was converted into two kinds of CC double bond substituents were prepared and the fully protected glycomonomers were directly polymerized before deprotection steps. Radical polymerization with acrylamide in DMF in the presence of ammonium persulfate and N,N,N’,N’-tetramethylethylenediamine proceeded smoothly and gave corresponding sialopolymers. Interestingly glycomonomers had hemagglutination inhibitory activities not only for H1N1 but also for H3N2 of human influenza virus strains.     

Bioorganic synthesis of end-capped anti-HIV peptides by simultaneous cyanocysteine-mediated cleavages of recombinant proteins

Bioorganic synthesis of N- and C-terminal end-capped peptides by two simultaneous S-cyanocysteine-mediated cleavages of recombinant proteins is described. This approach is demonstrated in the preparation of anti-HIV fusion inhibitory peptides.     

Exo- and endocytotic trafficking of SCAMP2

Exo- and endocytotic membrane trafficking is an essential process for transport of secretory proteins, extracellular glycans, transporters and lipids in plant cells. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco BY-2 cells as a model system, we recently demonstrated that SCAMP2 positive structures containing secretory materials are transported from the Golgi apparatus to the plasma membrane (PM) and/or cell plate. This structure is consisted with clustered vesicles and was thus named the secretory vesicle cluster (SVC). Here, we have utilized the reversible photoswitching fluorescent protein Dronpa1 to trace the movement of SCAMP2 on the PM and cell plate. Activated SCAMP2-Dronpa fluorescence on the PM and cell plate moved into the BY-2 cells within several minutes, but did not spread around PM. This is consistent with recycling of SCAMP2 among endomembrane compartments such as the TGN, PM and cell plate. The relationship between SVC-mediated trafficking and exo- and endocytosis of plant cells is discussed taking into account this new data and knowledge provided by recent reports.Key words: SVC, secretory vesicle cluster, secretory carrier membrane protein 2, SCAMP2, exocytosis, endocytosis, dronpa, trans-Golgi network, Golgi apparatus, pectin, secretory protein, plasma menbrane, endosome, endomembrane systemExo- and endocytosis are essential events for cellular division and expansion. During exocytosis, lipids, proteins and polysaccharides are synthesized and/or modified in the Golgi apparatus and sorted into secretory vesicles at the trans-Golgi network (TGN) for transport to the PM² or extracellular space. Secretory carrier membrane proteins (SCAMPs) are a group of transmembrane proteins that plays vesicle trafficking between Golgi apparatus and PM in higher eukaryotic cells.³ Recently it was reported that in BY-2 cells, the rice SCAMP1 is localized to the PM and clathrin-coated tubularvesicular structures that were likely the early endosomal compartment.⁴ The same protein is also targeted to the cell plate in dividing cells.⁵ We have recently reported that another member of the SCAMP family, SCAMP2 from tobacco, is localized to the TGN, PM, cell plate and previously uncharacterized SVC organelles, which are an intermediate organelle between the TGN and PM.⁶Both SCAMP1 and SCAMP2 appear to be recycled between the PM and intracellular compartments. This was suggested by data using stelyl dye FM4-64 as an endocytotic marker, fluorescent-tagged SCAMP proteins and protein trafficking inhibitors such as brefeldin A and 2,3-butanedione monoxime. We reported that SCAMP2 is exported to the PM from dotted structures in the cells, and back from the PM via the acto-myosin pathway but do not transport FM4-64 positive early endosome.⁶ As SCAMP2 did not localize on multivesicular bodies, endocytic vesicles may be directly transported to TGN or Golgi.⁶ However, this data was obtained using inhibitors that disrupt the trafficking system, and thus we have now investigated the endocytotic transport in the absence of inhibitors.Dronpa is a reversible photo-switching fluorescent protein. Using 488 and 405 nm laser light this protein can be converted between fluorescent and non-fluorescent forms within milliseconds.¹ In order to test whether SCAMP2 returned to internal compartments from the PM, and to characterize the initial compartment of endocytosis, we expressed Dronpatagged SCAMP2 (SCAMP2-Dronpa) in tobacco BY-2 cells. The fluorescence of SCAMP2-Dronpa was similar to that for SCAMP2-YFP and -mRFP fusions⁶ (Fig. 1A, upper part). To visualize the endocytic transport of SCAMP2-Dronpa, we first erased the majority of Dronpa fluorescence by illumination with 488 nm laser and then activated the protein at a part of the PM by 405 nm illumination using confocal laser scanning microscope (LSM) (Fig. 1A, upper right part). The fluorescence was then traced by 30 minutes interval up to 90 minutes (Fig. 1A, lower pictures). SCAMP2 signals at the PM did not spread laterally in the PM and decreased over the time. In parallel, signals were detected in the cytosol and some of them appeared as puncta (Fig. 1A, arrowheads). This observation is consistent with our proposal that SCAMP2 is recycled back into the intracellular compartment from the PM, possibly through the TGN without passing through the early endosome.⁶Open in a separate windowFigure 1Time-lapse images of BY-2 cells expressing ScamP2-Dronpa. Fluorescence of Dronpa (mBL) tagged ScamP2 in the cells was erased by 488 nm laser and then a spot of Pm (a) or cell plate (B) was activated by 405 nm diode laser. these data were obtained by LSm510 meta, 63x oil lens, Argon laser with 488-nm excitation and a 505 nm LP filter (Zeiss). Arrowheads indicate dotted structures. Bar = 20 μm.During cytokinesis, cell wall materials and membrane proteins accumulate in the cell plate.^7–9 It has been shown that clathrin-coated vesicles (CCVs) and their constituents such as adapter proteins and dynamins are associated with cell plate membrane.¹⁰ However, it is not clear whether these molecules on the cell plate are re-used in daughter cells or are degraded at the cell plate. We thus investigated the movement of SCAMP2-Dronpa fluorescence on the cell plate during cytokinesis. Fluorescence of SCAMP2-Dronpa within late metaphase cells was first erased, followed by activation of SCAMP2-Dronpa specifically on the cell plate (Fig. 1B). Following a 15 min of incubation, SCAMP2-Dronpa associated fluorescence on the cell plate moved into intracellular structures within daughter cells. This confirmed our previous observation that SCAMP2 was transported to the trans-Golgi/TGN or intracellular structures from the cell plate during the cytokinesis.⁶Transmission electron microscope and LSM studies have revealed that CCVs are present in cell plates.¹⁰ Recent tomographic observation suggested that early- and late TGNs having CCVs exist not only in the cell plate region but also other places of the plant cell.¹¹ We found that immature SVCs, which might be identical to late TGN, are converted to mature SVCs by budding CCVs.⁶ Therefore, transport from the Golgi apparatus located inside of the cells to the PM or cell plate is mediated by SVCs, which are generated as immature SVCs from the TGN and converted to mature SVCs by budding CCVs during transport. Eventually, the mature SVC fuses with the PM and/or expanding cell plate (Fig. 2, left), after which CCVs are generated from the expanded cell plate to recycle SCAMPs and other molecules back to the daughter cells.Open in a separate windowFigure 2A model of the exocytotic pathway and SCAMP2 trafficking in plant cells. From the Golgi apparatus or tGn, at least two distinct compartments, such as maSc and SVc are generated for secretion. ScamP2 locates in the SVc and is transported to the Pm or cell plate. thereafter, SCAMP2 is recycled back to the TGN via clathrin-mediated endocytosis.     

Auto-/heterotrophic endosymbiosis evolves in a mature stage of ecosystem development in a microcosm composed of an alga,a bacterium and a ciliate

We investigate an ecological mechanism by which endosymbiotic associations evolve, with a particular focus on the relationship between the evolution of endosymbiosis between auto- and heterotrophic organisms, and the stages of ecosystem development. For this purpose we conducted a long-term microcosm culture composed of three species, a green alga (Chlorella vulgaris), a bacterium (Escherichia coli), and a ciliated protozoan (Tetrahymena thermophila) for 3 years. During this culture T. thermophila cells harboring Chlorella cells emerged by phagocytotic uptake, and increased in frequency, reaching ca. 80–90%. This level was maintained in the late stage of ecosystem dynamics. Analysis of the ecosystem dynamics in the microcosm revealed that a complex causal process through direct/indirect interactions among ecosystem components led to reduction in dissolved O₂ and food (E. coli) available to the T. thermophila, which gave a selective advantage to the organisms in the endosymbiotic association. This result suggests that the endosymbiosis evolves in a mature stage of ecosystem development, where reproduction and survival of prospective partner organisms is highly resource-limited and density-dependent, favoring efficient matter/energy transfers among participating organisms due to physical proximity. Consequently, a complex web of interactions and pathways of matter/energy flow in ecosystem evolves from an initially simple one.     

A Mobile Secretory Vesicle Cluster Involved in Mass Transport from the Golgi to the Plant Cell Exterior

Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post-Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells.     

Light-induced and apoptosis-like cell death in the unicellular eukaryote, Blepharisma japonicum

The unicellular eukaryote, Blepharisma japonicum, is a light-sensitive ciliated protozoa. It possesses a photoreceptor pigment called blepharismin that plays critical roles in defensive behavior against predators and step-up photophobic response. In addition, the pigment generates reactive oxygen species such as singlet oxygen and hydroxyl radicals which contribute to photodynamic action. Previous studies reported that intense light (>300 W m⁻²) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3-30 W m⁻²) only induced pigment extrusion and photooxidation. We have found that moderate light (5 W m⁻²) induced apoptosis-like cell death. Microscopically it was found that >3 h of moderate light irradiation induced macronuclear condensation and plasma efflux without cell swelling. Single cell gel electrophoresis assay showed that DNA fragmentation occurred between 1 and 3 h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light-irradiated cells contained DNA fragments of 180-200 and 360-400 bp, which were seen as apoptosis ladders.     

Identification and Characterization of the Na+/H+ Antiporter Nhas3 from the Thylakoid Membrane of Synechocystis sp. PCC 6803

Na⁺/H⁺ antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na⁺/H⁺ antiporters, nhaS1–5 and sll0556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na⁺/H⁺ antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in E. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na⁺ concentrations. NhaS3 had no K⁺/H⁺ exchange activity itself but enhanced K⁺ uptake from the medium when expressed in an E. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO₂ to high CO₂ conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO₂ consumption caused by the ceasing of O₂-evolving photosynthesis. This is the first report of a Na⁺/H⁺ antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H⁺, Na⁺, and K⁺ in supporting the conversion of photosynthetic products and in the supply of energy in the dark.Na⁺/H⁺ antiporters are integral membrane proteins that transport Na⁺ and H⁺ in opposite directions across the membrane and that occur in virtually all cell types. These transporters play an important role in the regulation of cytosolic pH and Na⁺ concentrations and influence proton or sodium motive force across the membrane (1, 2). In Escherichia coli, three Na⁺/H⁺ antiporters (NhaA, NhaB, and ChaA) have been described in detail. Of these, NhaA is the functionally best characterized transporter. The crystal structure of NhaA has been resolved (3). In addition, mutants of nhaA, nhaB, and chaA as well as the triple mutant have been generated (4). The triple mutant was shown to be hypersensitive to extracellular Na⁺. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains six genes encoding Na⁺/H⁺ antiporters (NhaS1–5 and sll0556). NhaS1 (slr1727) has also been designated SynNhaP (5, 6). Null mutants of nhaS1, nhaS2, nhaS4, and nhaS5 have been generated; however, a null mutant of nhaS3 could not be obtained, indicating that it is an essential gene (6–8). By heterologous expression in E. coli, Na⁺/H⁺ exchange activities could be shown for NhaS1–5 (5, 6). Inactivation of nhaS1 and nhaS2 results in retardation of growth of Synechocystis (5, 6). It has been reported that in these mutants the concentration of Na⁺ in cytosol and intrathylakoid space (lumen) increases and impairs the photosynthetic and/or respiratory activity of the cell (9, 10). Therefore the Na⁺ extrusion by Synechocystis Na⁺/H⁺ antiporters similar to E. coli NhaA, NhaB, and ChaA is essential for the adaptation to salinity stress.In contrast to the case in E. coli, Na⁺ is an essential element for the growth of some cyanobacteria (11, 12). Interestingly, the Na⁺/H⁺ antiporter homolog NhaS4 was identified as an uptake system for Na⁺ from the medium during a screen for mutations in Synechocystis that result in lack of growth at low Na⁺ concentrations (7). The requirement of a Na⁺ uptake antiporter for cell growth is consistent with the physiology of Synechocystis. Specifically, photoautotrophic bacteria like cyanobacteria share some components (plastoquinone, cytochrome b₆f, and c₆) of the thylakoid membrane for electron transport for both photophosphorylation and respiratory oxidative phosphorylation. Na⁺/H⁺ antiporters therefore may coordinate both H⁺ and Na⁺ gradients across the plasma and thylakoid membranes to adapt to daily environmental changes (11). It remains to be determined whether the six Na⁺/H⁺ antiporters are localized to the plasma membrane or to the thylakoid membrane in Synechocystis. Information on the membrane localization will also provide information on the physiological role in Synechocystis. In this study, we explored the membrane localization of NhaS3, the role of specific amino acid residues for its function, and the effect of CO₂ concentration and circadian rhythms on the expression pattern of nhaS3 to gain insight into the physiological role of NhaS3 in Synechocystis.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.