alpha-Synuclein stimulates differentiation of osteosarcoma cells: relevance to down-regulation of proteasome activity

Because a limited study previously showed that alpha-synuclein (alpha-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether alpha-syn might be involved in the regulation of tumor differentiation. For this purpose, alpha-syn and its non-amyloidogenic homologue beta-syn were stably transfected to human osteosarcoma MG63 cell line. Compared with beta-syn-overexpressing and vector-transfected cells, alpha-syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in alpha-syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In alpha-syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in alpha-syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of alpha-syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of alpha-syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of alpha-syn regulation of tumor differentiation and neuropathological effects of alpha-syn may considerably overlap with each other.     

A simple method to disperse eggs from lepidopteran scalelike egg masses and to observe embryogenesis

Various insect species lay tiny, thin- and soft-shelled eggs in a connected “egg mass”. Especially in several lepidopteran species, the structure of such clustered eggs is covered with complicated scale-like secretion, which has so far prevented analysis of individual embryos. However, few studies on methods to disperse egg clusters of such insects and to compare different methods have been carried out. To overcome these problems, we developed methods to separate egg masses into individual eggs, using two Tortricidae pests, Homona magnanima Diakonoff and Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae). The eggs were successfully separated from each other using potassium hydroxide and sodium hypochlorite. Although the separated eggs no longer continued their embryogenesis, fixation with heptane–paraformaldehyde, permeabilization with heptane–methanol, and staining with several dyes enabled easy observation of embryogenesis. This protocol is expected to be applicable to other insect taxa and will facilitate further morphological and genetic studies in insects that lay egg masses.     

Prevalence and species richness of trematode parasites only partially recovers after the 2011 Tohoku,Japan, earthquake tsunami

Trematode parasites have complex life cycles and use a variety of host species across different trophic levels. Thus, they can be used as indicators of disturbance and recovery of coastal ecosystems. Estuaries on the Pacific coast of northeastern Japan were heavily affected by the 2011 Tohoku earthquake tsunami. To evaluate the effect of the tsunami on the trematode community, we examined trematodes in the mud snail, Batillaria attramentaria, at five study sites (three sites severely exposed to the tsunami and two sites sheltered from the tsunami) in Sendai Bay for 2 years prior to and 8 years after the tsunami. While the trematode prevalence decreased at all study sites, the species richness decreased only at the sites exposed to the tsunami. Although parasitism increased over the study period post-tsunami, the community had not fully recovered 8 years after the event. Trematode community structure has changed every year since the tsunami and has not stabilised. This could be explained by the alteration of first and second intermediate host communities. Our study suggests that it will take more time for the recovery of the trematode community and the associated coastal ecosystem in the Tohoku region.     

Macrophage colony-stimulating factor is produced by human T lymphoblastoid cell line, CEM-ON: identification by amino-terminal amino acid sequence analysis

A subclone, designated CEM-ON, derived from an azaguanine-resistant human leukemic T cell line, CEM-AG(R), constitutively secretes a colony-stimulating factor (CSF) which stimulates the production of macrophages from murine bone marrow progenitor cells. This CSF has been purified from serum-free conditioned medium. Highly purified CEM-ON CSF with a specific activity of 4.7 X 10(7) units/mg protein was obtained. Amino-terminal sequence analysis showed that the first 27 amino acids were identical to the amino-terminal sequence of the M-CSF (CSF-1) based on the cDNAs for human M-CSF. On SDS-PAGE analysis, CEM-ON CSF had an apparent molecular weight of 33,000-43,000; following reduction with 2-mercaptoethanol, this migrated as a 20,000-24,000 subunit, suggesting a homodimer structure. These results show that a human T cell line, CEM-ON, secretes M-CSF into its medium.     

Mouse coq7/clk-1 orthologue rescued slowed rhythmic behavior and extended life span of clk-1 longevity mutant in Caenorhabditis elegans

The coq7/clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a regulatory role in biological rhythm and longevity. The mouse COQ7 is homologous to Saccharomyces cerevisiae COQ7/CAT5 that is required for the biosynthesis of coenzyme Q (ubiquinone), an essential messenger in mitochondrial respiration. In the present study, we characterized the expression and processing of mouse COQ7. We found that COQ7 is highly expressed in tissues with high energy demand such as heart, muscle, liver, and kidney in mice. Biochemical analysis revealed that COQ7 is targeted to mitochondria where it is processed to mature form. Transgenic expression of mouse coq7 completely rescued the slowed rhythmic behaviors of clk-1 such as defecation. In life-span analysis, transgenic expression reverted the extended life span of clk-1 to the comparable level with wild-type control. These data strongly suggested that coq7 plays a pivotal role in the regulation of biological rhythms and the determination of life span in mammalian species.     

Nuclear localization of yeast Nfs1p is required for cell survival

Saccharomyces cerevisiae Nfs1p is mainly found in the mitochondrial matrix and has been shown to participate in iron-sulfur cluster assembly. We show here that Nfs1p contains a potential nuclear localization signal, RRRPR, in its mature part. When this sequence was mutated to RRGSR, the mutant protein could not restore cell growth under chromosomal NFS1-depleted conditions. However, this mutation did not affect the function of Nfs1p in biogenesis of mitochondrial iron-sulfur proteins. The growth defect of the mutant was complemented by simultaneous expression of the mature Nfs1p, which contains the intact nuclear localization signal but lacks its mitochondrial-targeting presequence. These results suggest that a fraction of Nfs1p is localized in the nucleus and is essential for cell viability.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.