Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta

Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneural gene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. These observations support a model for Notch activation where the Delta-Notch interaction is followed by endocytosis of Delta and transendocytosis of the Notch extracellular domain by the signaling cell. This facilitates intramembranous cleavage of the remaining Notch receptor, release of the Notch intracellular fragment, and activation of target genes in neighboring cells.     

Statistical inference from single channel records: two-state Markov model with limited time resolution

Though stochastic models are widely used to describe single ion channel behaviour, statistical inference based on them has received little consideration. This paper describes techniques of statistical inference, in particular likelihood methods, suitable for Markov models incorporating limited time resolution by means of a discrete detection limit. To simplify the analysis, attention is restricted to two-state models, although the methods have more general applicability. Non-uniqueness of the mean open-time and mean closed-time estimators obtained by moment methods based on single exponential approximations to the apparent open-time and apparent closed-time distributions has been reported. The present study clarifies and extends this previous work by proving that, for such approximations, the likelihood equations as well as the moment equations (usually) have multiple solutions. Such non-uniqueness corresponds to non-identifiability of the statistical model for the apparent quantities. By contrast, higher-order approximations yield theoretically identifiable models. Likelihood-based estimation procedures are developed for both single exponential and bi-exponential approximations. The methods and results are illustrated by numerical examples based on literature and simulated data, with consideration given to empirical distributions and model control, likelihood plots, and point estimation and confidence regions.     

In Vivo Epigenomic Profiling of Germ Cells Reveals Germ Cell Molecular Signatures

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Highlights? Modified small-scale ChIP-seq method applicable to small number of cells ? Genome-wide maps of H3K4me3, H3K27me3, H3K27ac, and H2BK20ac of germ cells in vivo ? Identification of active and inactive regulatory elements in germ cells in vivo ? Germ cell H3K27me3 regions are enriched for retrotransposon repeats     

Methylation changes of lysine 9 of histone H3 during preimplantation mouse development

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-m2K9) during pronucleus development, while the maternal genome stained intensively. Such H3-m2K9 asymmetry between the parental pronuclei was independent of HP1b localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-m2K9. A conspicuous increase in H3-m2K9 level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-m2K9 was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.     

Genetically engineered biosynthesis of macrolide derivatives including 4-amino-4,6-dideoxy-L-glucose from Streptomyces venezuelae YJ003-OTBP3

Two sugar biosynthetic cassette plasmids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003- OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ Na+] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12- membered ring aglycon (10-deoxymethynolide, 1) and 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.     

In Vitro and in Vivo Effects of Nitrofurantoin on Experimental Toxoplasmosis

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.     

Effects of Treadmill Exercise on Cell Proliferation and Differentiation in the Subgranular Zone of the Dentate Gyrus in a Rat Model of Type II Diabetes

In the present study, we investigated the effects of a treadmill exercise on serum glucose levels and Ki67 and doublecortin (DCX) immunoreactivity, which is a marker of cell proliferation expressed during cell cycles except G0 and early G1 and a marker of progenitors differentiating into neurons, respectively, in the subgranular zone of the dentate gyrus (SZDG) using a type II diabetic model. At 6 weeks of age, Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats were put on a treadmill with or without running for 1 h/day/5 consecutive days at 22 m/min for 5 weeks. Body weight was significantly increased in the control (without running)-ZDF rats compared to that in the other groups. In the control groups blood glucose levels were increased by 392.7 mg/dl in the control-ZDF rats and by 143.3 mg/dl in the control-ZLC rats. However, in the exercise groups, blood glucose levels were similar between the exercise-ZLC and ZDF rats: The blood glucose levels were 110.0 and 118.2 mg/dl, respectively. Ki67 positive nuclei were detected in the SZDG in control and exercise groups. The number of Ki67 positive nuclei was significantly high in exercise groups compared to that in the control groups. In addition, Ki67 positive cells were abundant in ZLC groups compared to those in ZDF groups. DCX-immunoreactive structures in the control-ZDF rats were lower than that in the control-ZLC rats. In the exercise groups, DCX-immunoreactive structures (somata and processes with tertiary dendrites) and DCX protein levels were markedly increased in both the exercise-ZLC and ZDF rats compared to that in the control groups. These results suggest that a treadmill exercise reduces blood glucose levels in ZDF rats and increases cell proliferation and differentiation in the SZDG in ZLC and ZDF rats compared to those in control groups.     

Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.