Histone H4 lysine 20 of Saccharomyces cerevisiae is monomethylated and functions in subtelomeric silencing

Histones undergo post-translational modifications that are linked to important biological processes. Previous studies have indicated that lysine methylation correlating with closed or repressive chromatin is absent in the budding yeast Saccharomyces cerevisiae, including at H4 lysine 20 (K20). Here we provide functional evidence for H4 K20 monomethylation (K20me1) in budding yeast. H4 K20me1 is detectable on endogenous H4 by western analysis using methyl-specific antibodies, and the signal is abrogated by H4 K20 substitutions and by competition with H4 K20me1 peptides. Using chromatin immunoprecipitation, we show that H4 K20me1 levels are highest at heterochromatic locations, including subtelomeres, the silent mating type locus, and rDNA repeats, and lowest at centromeres within euchromatin. Further, an H4 K20A substitution strongly reduced heterochromatic reporter silencing at telomeres and the silent mating type locus and led to an increase in subtelomeric endogenous gene expression. The correlation between the location of H4 K20me1 and the effect of the H4 K20A substitution suggests that this modification plays a repressive function. Our findings reveal the first negative regulatory histone methylation in budding yeast and indicate that H4 K20me1 is evolutionarily conserved from simple to complex eukaryotes.     

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone and its interaction with human serum albumin

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone (α1) with high stereoselectivity and good yield was developed. The feature of its binding to human serum albumin (HSA) was also investigated under simulative physiological conditions via fluorescence and UV–vis absorption spectroscopy and molecular modeling methods. The results revealed that α1 caused the fluorescence quenching of HSA by the formation of α1–HSA complexes. Hydrophobic interactions played a major role in stabilizing the complex, which was in good agreement with the results of the molecular modeling study. In addition, the effect of common ions on the binding constants of α1–HSA complexes at room temperature was also discussed. All the experimental results and theoretical data indicated that α1 bound to HSA and was effectively transported and eliminated in the body. Such findings may provide useful guidelines for further drug design.     

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone and its interaction with human serum albumin

An improved synthetic approach to 7-[3-amino-4-O-(α-l-mycarosyl)-2,3,6-trideoxy-α-l-lyxo-hexopyranosyl]daunorubicinone (α1) with high stereoselectivity and good yield was developed. The feature of its binding to human serum albumin (HSA) was also investigated under simulative physiological conditions via fluorescence and UV-vis absorption spectroscopy and molecular modeling methods. The results revealed that α1 caused the fluorescence quenching of HSA by the formation of α1-HSA complexes. Hydrophobic interactions played a major role in stabilizing the complex, which was in good agreement with the results of the molecular modeling study. In addition, the effect of common ions on the binding constants of α1-HSA complexes at room temperature was also discussed. All the experimental results and theoretical data indicated that α1 bound to HSA and was effectively transported and eliminated in the body. Such findings may provide useful guidelines for further drug design.     

Genetic variation and covariation in floral allocation of two species of Schiedea with contrasting levels of sexual dimorphism

The evolution of sexual dimorphism depends in part on the additive genetic variance-covariance matrices within females, within males, and across the sexes. We investigated quantitative genetics of floral biomass allocation in females and hermaphrodites of gynodioecious Schiedea adamantis (Caryophyllaceae). The G-matrices within females (G(f)), within hermaphrodites (G(m)), and between sexes (B) were compared to those for the closely related S. salicaria, which exhibits a lower frequency of females and less-pronounced sexual dimorphism. Additive genetic variation was detected in all measured traits in S. adamantis, with narrow-sense heritability from 0.34-1.0. Female allocation and floral size traits covaried more tightly than did those traits with allocation to stamens. Between-sex genetic correlations were all <1, indicating sex-specific expression of genes. Common principal-components analysis detected differences between G(f) and G(m) , suggesting potential for further independent evolution of the sexes. The two species of Schiedea differed in G(m) and especially so in G(f) , with S. adamantis showing greater genetic variation in capsule mass and tighter genetic covariation between female allocation traits and flower size in females. Despite greater sexual dimorphism in S. adamantis, genetic correlations between the two sexes (standardized elements of B) were similar to correlations between sexes in S. salicaria.     

Effects of external beam radiation on in vitro formation of Abeta1-42 fibrils and preformed fibrils

Plaques containing fibrillar amyloid-beta (Abeta) are a characteristic finding in Alzheimer's disease. Although plaque counts correlate poorly with the extent of cognitive deficits in this disorder, fibrillar Abeta can promote neuronal damage through a variety of mechanisms. External beam radiotherapy has been reported to be an effective treatment for tracheobronchial amyloidosis, in which amyloid is deposited as submucosal plaques and tumor-like masses in the trachea and/or bronchi. Radiotherapy's effectiveness in this disorder is thought to be due to its toxicity to plasma cells, but direct effects of radiotherapy on amyloid may also be involved. On this basis, whole-brain radiotherapy has been suggested as a treatment for Alzheimer's disease. The objective of this study was to determine the effects of external beam radiation on preformed Abeta1-42 fibrils and on the formation of these fibrils. Using the Thioflavin-T assay, no effects of radiation were found on either of these parameters. Our results in this in vitro study suggest that whole-brain irradiation is unlikely to directly reduce plaque counts in the Alzheimer's disease brain. This treatment might still lower plaque counts indirectly, but any potential benefits would need to be weighed against its possible neurotoxic effects, which could induce further cognitive deficits.     

Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus

The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.     

Identification of a single amino acid required for APOBEC3 antiretroviral cytidine deaminase activity

During studies of APOBEC3 (A3) anti-human immunodeficiency virus type 1 (anti-HIV-1) mechanisms, we identified a single cysteine at position 320 (C320) that disrupts A3DE activity. This residue is located in the recently identified DNA binding domain in A3G. Replacing C320 with a corresponding tyrosine from A3F (Y307) increased A3DE antiviral activity more than 20-fold. Conversely, replacing A3F Y307 with a cysteine or inserting a similar cysteine into A3B or A3G disrupted the anti-HIV activity of A3. Further investigation uncovered that C320 significantly reduces A3DE catalytic activity.