Quantitative proteomic analysis of rat liver for carcinogenicity prediction in a 28-day repeated dose study

The potential of quantitative proteomic analysis to predict carcinogenicity of chemical compounds was investigated. Using 2D-DIGE, we analyzed the effects of 63 chemical compounds on protein expression in the rat liver after 28 daily doses. Types of carcinogens were categorized depending on the species and organ specificity. The carcinogen characteristic proteins for each classification were identified by Welch's t value. For evaluation of the predictive concordance we used support vector machines. The rat hepatic carcinogen-specific classification gave higher concordance than the other classification. The generalization performance was measured by leave-one-out cross-validation. For genotoxic and non-genotoxic compounds, a concordance of 79.3 and 76.5%, respectively, was obtained by the top 30 ranked proteins with Welch's t value. Furthermore, we found that the increase of the expression level of the stress response proteins as the common feature of poorly predicted chemical compounds in the leave-20%-out cross-validation. Quantitative proteomics could be promising technique for developing biomarker panels that can be used for carcinogenicity prediction. The list of proteins identified in this study and the zoomed gel images of the top ranked proteins in statistic analysis are provided in Supplementary Data.     

A novel method for analyzing phosphoproteins using SELDI-TOF MS in combination with a series of recombinant proteins

A novel SELDI-TOF MS-based method for analyzing phosphoproteins was developed using a series of recombinant wild-type and mutant ribosomal P2 proteins. We demonstrated that the phosphorylation status of the overexpressed proteins in cells was easily and rapidly confirmed using this method. The ribosomal P2 protein contained two phosphorylation sites, which were sequentially phosphorylated in vivo. We also quantitatively detected the phosphoprotein by using SELDI-TOF MS.     

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.     

Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...     

RhoGDIβ affects HeLa cell spindle orientation following UVC irradiation

The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)-irradiated surviving cells. N-terminal truncated Rho GDP-dissociation inhibitor β (RhoGDIβ), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA-mediated knockdown of RhoGDIβ significantly attenuated UVC-induced misorientation. Subsequent expression of wild-type RhoGDIβ, but not a noncleavable mutant, RhoGDIβ (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIβ impacts spindle orientation in response to DNA damage.     

Site-directed mutagenesis of two amino acid residues in cytochrome b559 α subunit that interact with a phosphatidylglycerol molecule (PG772) induces quinone-dependent inhibition of photosystem II activity

Photosynthesis Research - X-ray crystallographic analysis (1.9-Å resolution) of the cyanobacterial photosystem II (PSII) dimer showed the presence of five phosphatidylglycerol (PG) molecules...     

Production of the plant polyketide curcumin in Aspergillus oryzae: strengthening malonyl-CoA supply for yield improvement

The filamentous fungus Aspergillus oryzae was recently used as a heterologous host for fungal secondary metabolite production. Here, we aimed to produce the plant polyketide curcumin in A. oryzae. Curcumin is synthesized from feruloyl-coenzyme A (CoA) and malonyl-CoA by curcuminoid synthase (CUS). A. oryzae expressing CUS produced curcumin (64 μg/plate) on an agar medium containing feruloyl-N-acetylcysteamine (a feruloyl-CoA analog). To increase curcumin yield, we attempted to strengthen the supply of malonyl-CoA using two approaches: enhancement of the reaction catalyzed by acetyl-CoA carboxylase (ACC), which produces malonyl-CoA from acetyl-CoA, and inactivation of the acetyl-CoA-consuming sterol biosynthesis pathway. Finally, we succeeded in increasing curcumin yield sixfold by the double disruption of snfA and SCAP; SnfA is a homolog of SNF1, which inhibits ACC activity by phosphorylation in Saccharomyces cerevisiae and SCAP is positively related to sterol biosynthesis in Aspergillus terreus. This study provided useful information for heterologous polyketide production in A. oryzae.     

Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Production of soluble granulocyte colony-stimulating factor receptors from myelomonocytic cells

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b-CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b-CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.