Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.     

Polyamines stabilize left-handed Z-DNA: using X-ray crystallographic analysis, we have found a new type of polyamine (PA) that stabilizes left-handed Z-DNA

There are many great reports of polyamine stabilization of the Z-DNA by bridge conformation between neighboring, symmetry-related Z-DNA in the packing of crystals. However, polyamine binding to the minor groove of Z-DNA and stabilizing the Z-DNA structure has been rarely reported. We proved that the synthesized polyamines bind to the minor groove of Z-DNA and stabilize the conformation under various conditions, by X-ray crystallographic study. These polyamines consist of a polyamine nano wire structure. The modes of the polyamine interaction were changed under different conditions. It is the first example that the crystals consisted of metal free structure. This finding provides a basis for clarifying B-Z transition mechanics.     

Abnormal phosphorylation of Ser409/410 of TDP-43 in FTLD-U and ALS

A monoclonal antibody specific for phosphoserines 409 and 410 of TDP-43 (mAb pS409/410) has been produced. It strongly stained TDP-43-positive inclusions in brain of patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but did not stain nuclei, in which normal TDP-43 is localized. It did not recognize TDP-43 rapidly extracted from brains of rats at various developmental stages, strongly suggesting that phosphorylation of Ser409/410 is an abnormal event. Analysis of postmortem changes of TDP-43 revealed that the amounts of Sarkosyl-insoluble, urea-soluble full-length TDP-43 and a 35 kDa N-terminal fragment increased time-dependently.     

Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells

Interferon (IFN) is one of the potent antiproliferative cytokines and is used to treat some selected cancers. IFN arrests the growth of Burkitt Iymphoma derived cell line Daudi cells in the G1 phase. G1-to-S progression is controlled by positive and negative regulatory genes. Therefore, we investigated the effects of IFN on G1-controlling genes. Expression of cyclin-dependent kinases (Cdks 2, 3, 4, 5, 6), MO 15/Cdk7, and cyclins E and H was studied to assess positive regulators, while p15^Ink4B, p16^Ink4, p18, p21^CipI, and p27^Kip1 were assessed as negative regulators. Cdks 2, 4, 6 and cyclin E were markedly down-regulated. MO15/Cdk7 expression showed little change, but its regulatory subunit (cyclin H) was down-regulated like cyclin E. Expression of p15^Ink4B and p16^Ink4 was not observed. p18 was induced until 48 h and its expression returned to the initial level at 72 h. In contrast, p21^Cip1 mRNA expression remained at the baseline level throughout IFN treatment, while the expression of p27^Kip1 increased at 48 and 72 h. Taken together, these data indicate that IFN changes the messenger RNA of G1-controlling genes towards the suppression of G1-to-S transition.     

Proteolysis of non-phosphorylated and phosphorylated tau by thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.     

BEHAVIOR OF CHLOROPLAST NUCLEOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) IN SYNCHRONIZED CULTURE1

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12:12 h light: dark regimen. They increased in size during the light period, while nuclear division, chloroplast division and cytokinesis occurred during the dark period. Zoospores were liberated toward the end of the dark period. Changes in profile and distribution of chloroplast nucleoids were followed with a fluorescence Microscope after fixation with 0.1%(w/v) glutaraldehyde followed by staining with 4′.6-diamidino-2-phenylidole (DAPI), a DNA fluorochrome. About ten granular nucleoids were dispersed in the chloroplast at the beginning of the light period (0 h). Within 4 h the nucleoids aggregated around the pyrenoid giving a compact profile. The formation of the compact aggregate of cp-nucleoids around the pyrenoid occurred with maximal frequency twice during the light period. Toward the end of the light period the nucleoids were transformed into the form of threads interconnected with fine fibrils spreading throughout the chloroplast. Initially the thread-like nucleoids fluoresced only faintly. The fluorescence of some parts of the threadlike form became brighter over a period of 6 h; these nucleoids were divided into daughter chloroplasts during chloroplast division. Soon after chloroplast division, these thread-like nucleoids were transformed into about 20 granular forms, which were gradually combined to form about ten larger granular bodies in zoospores immediately prior to liberation from mother cells. Fixation of cells with glutaraldehyde at high concentrations or treatment of cells with protease significantly modified the profiles of DAPI-stained nucleoids. The different morphologies of chloroplast nucleoids are discussed in relation to changes in configuration of their protein components.     

Ammonia- and Light-Induced Degradation of Paramylum in Euglena gracilis

Ammonium ions (ammonia) induced, independent of light, the degradationof paramylum (beta-l,3-glucan) accumulated in Euglena gracilisvar. bacillaris under photoautotrophic and dark-organotrophicconditions. Part of the paramylum in darkorganotrophically growncells, however, was preserved without breakdown in the darkin the presence of ammonium ions and under carbon-starvation,being degraded on exposure to light. (Received March 20, 1987; Accepted September 7, 1987)     

All genomic mutations in the antimicrobial surfactant-resistant mutant, Escherichia coli OW66, are involved in cell resistance to surfactant

The spontaneous antimicrobial surfactant-resistant mutant, Escherichia coli OW66, has been isolated, and its physiological properties have been characterized in our previous paper (Ishikawa et al., J Appl Microbiol 92:261–268, 2002b). This report revealed that strain OW66 had seven mutations in their chromosomal DNA by comparative genomic hybridization microarray, and that their alternative functions were involved in cell resistance to antimicrobial surfactants. These mutations were located in oppB, ydcR, IVR(vacJ-yfdC), rpoN, rpoB, rpoC, and soxR. Furthermore, seven of the single-mutated isogenic strains and seven of the six-mutated isogenic strains were constructed from strains OW6 (NBRC106482) and OW66, respectively, through homologous recombination, and their resistances to an antimicrobial surfactant were measured using the minimum inhibitory concentration method. These results revealed that all six-mutated strains were more sensitive than strain OW66, and that the soxR66 mutation was independently involved in antimicrobial surfactant resistance of E. coli cells. Expression of soxR66 and soxS was increased in both strains OW66 and OW6-soxR66 without the surfactant treatment by the quantitative real time-polymerase chain reaction analysis, compared with strain OW6. Two-dimensional polyacrylamide gel electrophoresis analysis also revealed that some proteins in the soxRS regulon, including Mn-SOD, were overexpressed in both strains OW66 and OW6-soxR66. These results indicate that the soxR66 mutation leads to the constitutive expression of the soxRS regulon, resulting in the acquired resistance of E. coli cells to an antimicrobial surfactant.     

Methylene blue and dimebon inhibit aggregation of TDP-43 in cellular models

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are major neurodegenerative diseases with TDP-43 pathology. Here we investigated the effects of methylene blue (MB) and dimebon, two compounds that have been reported to be beneficial in phase II clinical trials of Alzheimer’s disease (AD), on the formation of TDP-43 aggregates in SH-SY5Y cells. Following treatment with 0.05 μM MB or 5 μM dimebon, the number of TDP-43 aggregates was reduced by 50% and 45%, respectively. The combined use of MB and dimebon resulted in a 80% reduction in the number. These findings were confirmed by immunoblot analysis. The results indicate that MB and dimebon may be useful for the treatment of ALS, FTLD-U and other TDP-43 proteinopathies.