Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.     

Gain of function by phosphorylation in Presenilin 1-mediated regulation of insulin signaling

During pregnancy, activation of the maternal immune system results in inflammation in the foetal nervous system. The causative agents are pro-inflammatory cytokines like interleukin-1β (IL-1β), produced by the foetus. In this study, we examine the effect of IL-1β on the proliferation and differentiation of neural progenitor cells (NPCs) to better understand its potential effects on the developing brain. We find that the IL-1β receptor (IL-1R1) is expressed in the ventral mesencephalon of the developing brain. Furthermore, IL-1R1 is expressed on Nestin-positive, Sox-2-positive NPCs. IL-1β treatment reduced the numbers of proliferating NPCs, an effect prevented by the IL-1R1 receptor antagonist. LDH and MTT assays, and western blot analysis for cleaved caspase 3 and poly(ADP-ribose) polymerase, confirmed that this was not due to an increase in cell death but rather an induction of differentiation. To further study the effects of IL-1β on cell fate determination, we differentiated NPCs in the presence and absence of IL-1β. Il-1β promoted gliogenesis and inhibited neurogenesis, an effect that required p38-MAPK kinase signalling. In summary, these data show that exposure of NPCs to IL-1β affects their development. This necessitates an examination of the consequences that maternal immune system activation during pregnancy has on the cellular architecture of the developing brain.     

Effect of thiazole orange doubly labeled thymidine on DNA duplex formation

Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ～ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/ . It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.     

The ensemble of hetero-proteins in inorganic nanochannels

The assembly and proper alignment of two heterofluorescent proteins (sGFP and DsRed) in the mesoporous channels of ethanol-treated FSM6.2 (a folded-sheet mesoporous material with a pore diameter of 6.2 nm) was confirmed using a fluorescence resonance energy transfer (FRET) technique. The sGFP-DsRed-FSM6.2 conjugate showed a large decrease in the emission of donor (sGFP) fluorescence, indicating that the conjugate functions as an energy transfer system through the combination of the two heteroproteins, due to the successful encapsulation of the sGFP-DsRed pairs in the mesopores. Fluorescence spectral analysis demonstrated that the proteins were highly dispersed and homogeneously encapsulated in the mesopores of FSM6.2, even at high concentration, although they spontaneously aggregated and showed a red shift in solution at the concentration corresponding to that in the conjugate. Furthermore, an increase in the amount of sGFP and DsRed adsorbed to the pores of FSM6.2 led to a decrease in the distance between these proteins, resulting in enhancement of FRET efficiency.     

Identification of a second DNA binding site in the human Rad52 protein

Rad52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.     

Biosynthesis and secretion of salusin-alpha from human cells

Salusins originally identified using bioinformatics analyses have been shown to act on the cardiovascular and endocrine systems. Although the hypotensive activity of salusin-alpha is limited, it exerts a significant anti-atherosclerotic effect via suppression of foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1. Furthermore, serum salusin-alpha levels show a close negative correlation with the severity of atherosclerotic diseases. However, biosynthesis and secretion of salusin-alpha peptide from cultured mammalian cells have not been demonstrated to date. We examined the expression, synthesis and release of salusin-alpha in human-derived cell lines. Preprosalusin mRNA and protein were detected ubiquitously in all cells tested, whereas the processing of preprosalusin into salusin-alpha peptide is dependent upon each cell type. Immunohistochemical study revealed the most abundant salusin-alpha-like immunoreactivity to be present in HeLa cells which released salusin-alpha-like immunoreactivity into the culture supernatant. Analysis of extracted conditioned media from HeLa cells by reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a single immunoreactive component that co-eluted with authentic salusin-alpha. These results present the first evidence that salusin-alpha is biosynthesized and released from human-derived cells.     

Lim15/Dmc1 enhances DNA topoisomerase II catenation activity independent of sequence homology

We reported previously that Coprinus cinereus Lim15/Dmc1 (CcLim15), a meiosis-specific recA-like protein, could specifically activate C. cinereus DNA topoisomerase II (CcTopII). In particular, it enhanced the catenation activity of CcTopII in vitro at the meiotic prophase stage (Iwabata K, Koshiyama A, Yamaguchi T, Sugawara H, Hamada NF, Namekawa HS, Ishii S, Ishizaki T, Chiku H, Nara T, Sakaguchi K, Nucleic Acids Res, 33:5809–5818, 2005). In this study, the interaction between CcTopII and CcLim15, especially during catenation, was investigated in detail using atomic force microscopy. We demonstrated earlier that CcLim15 enhanced the catenation activity of CcTopII in a dose-dependent manner. When using two different-sized plasmid rings (5.4 and 3 kbp), which did not have any homologous sequence regions, equal proportions of homologous and heterologous catenanes were produced, suggesting that CcLim15 causes an increase in catenation activity irrespective of the presence of homologous sequences between the rings. We also showed that CcLim15 works as a DNA-condensing agent. Therefore, we speculate that CcLim15 may work as a DNA-condensing factor specific to the zygotene event and that CcTopII is likely to resolve tangles when the chromosomes initiate pairing at multiple sites by CcLim15.     

Coenzyme Q10 as a potent compound that inhibits Cdt1–geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q₁₀ (CoQ₁₀) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ₁₀, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ₁₀ bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ₁₀ had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ₁₀ inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ₁₀ was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ₁₀ could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed.     

Plant DNA polymerase lambda, a DNA repair enzyme that functions in plant meristematic and meiotic tissues.

Little is known about the functions of DNA polymerase lambda (Pol lambda) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol lambda may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol lambda in a higher plant, rice (Oryza sativa L. cv. Nipponbare) (OsPol lambda). OsPol lambda had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol lambda. Northern blot analysis showed that the level of OsPol lambda expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol lambda is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol beta and terminal deoxyribonucleotidyl transferase.