Targeted substrate degradation by an engineered double RING ubiquitin ligase

Recognition of the substrates by ubiquitin ligases is crucial for substrate specificity in the ubiquitin-proteasome proteolytic pathway. In the present study, we designed a double RING finger ubiquitin ligase to direct the ubiquitin machinery to a specific substrate. The engineered ligase contains the RING finger domains of both BRCA1 and BARD1 linked to a substrate recognition site PCNA, which is known to interact with cyclin-dependent kinase inhibitor p57. The double RING finger ubiquitin ligase formed a homo-oligomer complex and exhibited significant ligase activity. Co-transfection of the ligase reduced the expression of transfected p57 to the background level in a proteasome-dependent manner and restored the colony formation ability of U2OS cells that is otherwise inhibited by overexpressed p57. The results indicate the ability of the engineered double RING ubiquitin ligase to target the intended substrate. By redesigning the substrate recognition site, expression of engineered double RING ubiquitin ligases may provide a useful tool for removing many different gene products at the protein level.     

Identification of the site of inhibition of mitogenic signaling by oncogenic ras-p21 by a ras effector peptide

We have previously found that a ras switch 1 domain peptide (PNC-7, residues 35–47) selectively blocks oocyte maturation induced by oncogenic (Val 12–containing) ras-p21 protein and also blocks c-raf–induced oocyte maturation. We now find that oncogenic ras-p21 does not inhibit oocyte maturation of a constitutively activated raf protein (raf BXB) that is lacking most of the first 301 amino terminal amino acids, including the major ras binding domain and accessory ras-binding regions. We also find that a dominant negative raf that completely blocks c-raf–induced maturation likewise does not block raf-BXB–induced maturation. We conclude that PNC-7 blocks ras by binding to the amino-terminal domain of raf and that raf BXB must initiate signal transduction in the cytosol.     

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.     

How fruit abundance affects the chimpanzee party size: a comparison between four study sites

We examined the relationship between fruit abundance and chimpanzee (Pan troglodytes) party size by comparing data from four study sites: the Kalinzu Forest Reserve, Uganda, the Djinji Camp and Guga Camp in the Ndoki Forest, Congo, and Kahuzi-Biega National Park, Democratic Republic of Congo. Although the difference in the fruit abundance between the sites was responsible for the difference in the party size between the sites, the seasonal changes in fruit abundance did not explain the changes in the party size in each study site. Across the four study sites, there were significant correlations of the mean and minimum of monthly party size with the mean of monthly fruiting-tree density, and a significant correlation of the maximum of monthly party sizes with the minimum of monthly fruiting-tree density. We proposed a hypothesis that (1) the monthly fruit abundance affects the monthly party size in the sites where the fruit availability is as low as to limit the party size during a major part of a year, while (2) the party size does not increase with the increase in the monthly fruit abundance, but is affected by other social factors, in the sites where the minimum of monthly fruit abundance is high enough for chimpanzees to form parties of an adequate size. Electronic Publication     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.     

Bone morphogenetic protein 2 may be a good predictor of success in oocyte fertilization during assisted reproductive technology

The aim of this study was to determine whether the bone-morphogenetic proreins-2 (BMP-2) or -7 (BMP-7) levels in follicular fluid (FF) are associated with success in oocyte fertilization during assisted reproductive technology (ART) treatment. Twenty-four cycles in 24 patients who underwent oocyte retrieval in Sugiyama clinic were included in this retrospective study. The patients were divided into two groups according to the success or failure of fertilization. FF samples were obtained from a single follicle in each patient, and the levels of BMP-2, BMP-7, Anti-Müllerian hormone (AMH), estradiol and progesterone from FF were measured, and evaluated in relation to the ART outcomes. The BMP-2 levels correlated positively with the AMH levels in FF (r(2) = 0.4928), but there was no statistically significant difference between BMP-7 and AMH levels. The BMP-2 and BMP-7 levels had no relation with either progesterone or estradiol levels, but BMP-2 levels in the fertilized group were significantly higher than those in the unfertilized group (P < 0.05). The BMP-2 levels in FF positively correlated with the AMH levels in FF, and those in the fertilized group were significantly higher than in the unfertilized group. Therefore, the BMP-2 levels in FF could be a predictive marker for fertilization.     

Fluorescent analysis of excess electron transfer through DNA

The DNA base stack provides unique features for the efficient long-range charge transfer. For the purpose of investigating excess electron transfer process through DNA, we developed a new method for fluorescence analysis of excess electron transfer based on reductive cleavage of a disulfide bond and a thiol-specific fluorescent probe. Excess electron transfer was detected by monitoring the fluorescence of emissive pyrene monomer generated by the reaction of pyrene maleimides with the cleaved disulfide bond (thiols). Mechanism of reductive cleavage of disulfides through excess electron transfer and subsequent reaction with the fluorescent probes were discussed. This facile and sensitive detection by fluorescence method can be applied for mechanistic study of excess electron transfer.     

Starch phosphorus content in potato (Solanum tuberosum L.) cultivars and its effect on other starch properties

The research presented herein provides valuable data with respect to the phosphorus content of starches from many potato (Solanum tuberosum L.) cultivars using an energy-dispersive X-ray fluorescence technique. In all starches examined, the phosphorus content ranged from 308 to 1244 ppm. Furthermore, the estimation of the starch characteristics of representative samples differing manifestly in their phosphorus content indicated that enhancing the starch phosphate resulted in significant increases in the swelling power, peak viscosity, and breakdown and significant but small increases in the onset and peak temperatures of gelatinization. Other starch quality parameters, such as the amylose content, median granule size, and the gelatinization enthalpy, did not change significantly due to the degree of phosphate substitution of starch.