Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions

The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His–Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser²⁵⁰, which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser²⁵⁰ substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser²⁵⁰ phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys²⁶⁸ in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.     

Implications of corn prices on water footprints of bioethanol

Previously reported water footprints (WFPs) of corn ethanol have been estimated based on the assumption that corn ethanol feedstock could be supplied by the same states where the corn is grown. However, ethanol conversion facilities may choose out-of-state feedstock suppliers depending on the total price of feedstock they have to pay including both the corn price and transportation costs. The purpose of this study is to evaluate the WFPs and total water use (TWU) of corn ethanol considering an optimal allocation of corn with heterogeneous corn feedstock prices across states. The results show that the WFPs of corn ethanol are less than 100 l of water per liter of ethanol (Lw/Le) for all ethanol-producing states based on both the 2008 corn price and transportation costs for rail and truck. Results also reveal that WFPs are very sensitive to the market price of corn and that additional greenhouse gas emissions due to corn trade between states are not significant.     

Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift.HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.     

Aged garlic extract inhibits peroxynitrite-induced hemolysis

Nitric oxide (NO), which is synthesized by constitutive NO synthase (cNOS), plays important roles in physiological functions of the cardiovascular system. However, NO, which is synthesized by inducible NOS, is detrimental when it reacts with superoxide to form peroxynitrite. Peroxynitrite is recognized as a powerful oxidant, and results in vascular or tissue damage. We have previously reported that aged garlic extract (AGE) enhances NO production through cNOS stimulation. In the present study, we determined the effect of AGE, its fractions or constituents on peroxynitrite-induced hemolysis using rat erythrocytes. Incubation of rat erythrocytes with peroxynitrite (300 microM) for 30 min at 37 degrees C caused 4-fold hemolysis. AGE (0.14-0.57 %w/v) added to an erythrocyte suspension was found to reduce peroxynitrite-induced hemolysis in a concentration-dependent manner. Of the AGE fractions, a polar fraction and a low-molecular-weight fraction both suppressed the hemolysis to the same degree as that seen with AGE. S-allylcysteine, one of the major compounds in AGE, also reduced hemolysis at 1-10 mM dose-dependently. These data indicate that AGE and its compounds protect erythrocytes from membrane damage induced by peroxynitrite, suggesting that AGE could be useful for prevention of cardiovascular diseases associated with oxidative stress or dysfunction of NO production.     

Quantification of indole-3-acetic acid and amino acid conjugates in rice by liquid chromatography-electrospray ionization-tandem mass spectrometry

A method for quantifying indole-3-acetic acid (IAA) and its conjugates with the six amino acids, Ala, -Asp, -Ile, -Glu, -Phe and -Val, in rice (Oryza sativa) by using high-performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (HPLC-ESI-MS/MS) is described. Samples from the rice plant or callus were treated with 80% acetone in water containing 2.5 mM diethyl dithiocarbamate. Each extract was partially purified in C18 cartridge column for solid-phase extraction (SPE) and subjected to HPLC-ESI-MS/MS without converting the product. The detection limit was 3.8 fmol for IAA, and 0.4-2.9 fmol for the IAA amino acid conjugates. The method was applied to the analysis of IAA and its conjugates in rice seedlings, dehulled rice and calli, using 20-100 mg tissue samples.     

Migratory Responses of Benthic Diatoms to Light and Temperature Monitored by Chlorophyll Fluorescence

As an important adaptation for survival in the sediments of intertidal flats, benthic diatoms move up and down in response to a wide range of environmental stimuli. We investigated the vertical migration of two diatoms—Cylindrotheca closterium (Agradh) Kützing (B-25) and Nitzschia sp. (B-3)—under different combinations of light intensity and temperature conditions. An imaging pulse amplitude modulated (PAM) fluorometer was used to measure the minimum fluorescence (F ₀) in order to monitor variations in diatom biomass in surface sediments. Rapid light curves (RLCs) were applied to assess their photosynthetic activities. Both species had increased motility under higher temperatures, with the longer valved C. closterium being twice as fast as the shorter valved Nitzschia sp. The former was also influenced by exposure to light intensities of 100 or 250 μmol m⁻² s⁻¹, whereas the latter was not. Consequently, no light/temperature interaction effect was associated with the vertical migration of Nitzschia sp., perhaps because of its lower photosynthetic capacity and smaller cell size. Therefore, we conclude that motile benthic diatoms exhibit species-specific responses to light and temperature due to differences in their photosynthetic capability and morphological characteristics.     

Hwanggeumchal sorghum induces cell cycle arrest, and suppresses tumor growth and metastasis through Jak2/STAT pathways in breast cancer xenografts

Background

Cancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer.

Methodology/Principal Findings

Preliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both in vivo and in vitro to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model.

Conclusions/Significance

Usage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer.     

Discovery of genome-wide DNA polymorphisms in a landrace cultivar of Japonica rice by whole-genome sequencing

Molecular breeding approaches are of growing importance to crop improvement. However, closely related cultivars generally used for crossing material lack sufficient known DNA polymorphisms due to their genetic relatedness. Next-generation sequencing allows the identification of a massive number of DNA polymorphisms such as single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) between highly homologous genomes. Using this technology, we performed whole-genome sequencing of a landrace of japonica rice, Omachi, which is used for sake brewing and is an important source for modern cultivars. A total of 229 million reads, each comprising 75 nucleotides of the Omachi genome, was generated with 45-fold coverage and uniquely mapped to 89.7% of the Nipponbare genome, a closely related cultivar. We identified 132,462 SNPs, 16,448 insertions and 19,318 deletions between the Omachi and Nipponbare genomes. An SNP array was designed to validate 731 selected SNPs, resulting in validation rates of 95 and 88% for the Omachi and Nipponbare genomes, respectively. Among the 577 SNPs validated in both genomes, 532 are entirely new SNP markers not previously reported between related rice cultivars. We also validated InDels on a part of chromosome 2 as DNA markers and successfully genotyped five japonica rice cultivars. Our results present the methodology and extensive data on SNPs and InDels available for whole-genome genotyping and marker-assisted breeding. The polymorphism information between Omachi and Nipponbare is available at NGRC_Rice_Omachi (http://www.nodai-genome.org/oryza_sativa_en.html).     

4-Hydroxynonenal enhances MMP-2 production in vascular smooth muscle cells via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways

4-Hydroxynonenal (HNE) accumulates at atherosclerotic lesions, but its role in the progression of atherosclerosis is not clear. Considering the role of matrix metalloproteinases (MMP) in plaque destabilization, we investigated the mechanism by which HNE induces MMP production in vascular smooth muscle cells (VSMC). VSMC stimulated by HNE (1.0 microM) produced enzymatically active MMP-2 with an increased promoter activity, which was abolished by mutation of the NF-kappaB binding site in the promoter region. The increased NF-kappaB activity with subsequent MMP-2 production by HNE was significantly attenuated by transfection with Akt siRNA as well as by pretreatment with the PI3K/Akt inhibitors LY294002 (10 microM) and SH-5 (1.0 microM). The phosphorylation of Akt occurred as early as 5 min in VSMC exposed to HNE and was markedly attenuated by inhibition of mitochondrial reactive oxygen species (ROS). Furthermore, the impact of mitochondrial ROS on HNE-induced Akt phosphorylation with subsequent MMP-2 production was also demonstrated in mitochondrial function-deficient VSMC, as well as in cells transfected with manganese superoxide dismutase. Taken together, these results suggest that HNE enhances MMP-2 production in VSMC via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways.