Cucumis sativus secretes 4′-ketoriboflavin under iron-deficient conditions

A new compound in cucumber, Cucumis sativus, nutrient solution that appears under iron-deficient conditions, but not under ordinary culture conditions, has been revealed by HPLC analysis. The chemical structure of this compound was identified using LC-MS and NMR techniques as that of 4′-ketoriboflavin. This is the first report to show that 4′-ketoriboflavin can be found in metabolites from organisms.     

Fine root turnover of Japanese white birch (<Emphasis Type="Italic">Betula platyphylla</Emphasis> var. <Emphasis Type="Italic">japonica</Emphasis>) grown under elevated CO<Subscript>2</Subscript> in northern Japan

Key message

Elevated CO ₂ reduced fine root dynamics (production and turnover) of white birch seedlings, especially grown in volcanic ash soil compared with brown forest soil.

Abstract

Increased atmospheric CO₂ usually enhances photosynthetic ability and growth of trees. To understand how increased CO₂ affects below-ground part of trees under varied soil condition, we investigated the responses of the fine root (diameter <2 mm) dynamics of Japanese white birch (Betula platyphylla var. japonica) which was planted in 2010. The three-year-old birch seedlings were grown in four experimental treatments comprising two levels of CO₂, i.e., ambient: 380–390 and elevated: 500 μmol mol^?1, in combination with two kinds of soil: brown forest (BF) soil and volcanic ash (VA) soil which has few nutrients. The growth and turnover of fine roots were measured for 3 years (2011–2013) using the Mini-rhizotron. In the first observation year, live fine root length (standing crop) in BF soil was not affected by CO₂ treatment, but it was reduced by the elevated CO₂ from the second observation year. In VA soil, live fine root length was reduced by elevated CO₂ for all 3 years. Fine root turnover tended to decrease under elevated CO₂ compared with ambient in both soil types during the first and second observation years. Turnover of fine root production and mortality was also affected by the two factors, elevated CO₂ and different soil types. Median longevity of fine root increased under elevated CO₂, especially in VA soil at the beginning, and a shorter fine root lifespan appeared after 2 years of observation (2011–2012). These results suggest that elevated CO₂ does not consistently stimulate fine root turnover, particularly during the plant seedlings stage, as it may depend on the costs and benefits of constructing and retaining roots. Therefore, despite the other uncontrollable environment factors, carbon sequestration to the root system may be varied by CO₂ treatment period, soil type and plant age.

     

Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.     

c-kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina

Retinal progenitor cells are believed to display altered proliferation and differentiation during retinal development, suggesting that retinal progenitor cell populations are not homogeneous. However, the composition of progenitor cell populations is not known, due in part to the lack of known surface markers identifying distinct stages of retinal progenitor cells. We found a dramatic change in the expression profile of the cell surface antigens c-kit and stage-specific embryonic antigen-1 (SSEA-1) in retinal progenitor cells during development. While SSEA-1 was expressed early in development, c-kit expression peaked in late stage progenitor cells. The identification of these developmental markers enabled us to characterize distinct sub-populations of retinal progenitor cells. Progenitor cell subpopulations expressing either SSEA-1, c-kit, or both showed different proliferation and differentiation abilities. Although SSEA-1-positive cells were augmented by beta-catenin signaling, c-kit-positive cells were positively regulated by Notch signaling. Taken together, our data suggest that c-kit and SSEA-1 can be used to spatiotemporally differentiate retinal progenitor populations that have intrinsically distinct characteristics. Prolonged expression of c-kit by a retrovirus resulted in the promotion of proliferation and the appearance of nestin-positive cells in the presence of the c-kit ligand, stem cell factor (SCF). This suggests a role for c-kit, Notch, and the beta-catenin signaling network in retinal development.     

Re-expression of proteins involved in cytokinesis during cardiac hypertrophy

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved by increase in cell volume. In some species, cardiomyocytes undergo an additional incomplete mitosis in the first postnatal week, where karyokinesis takes place in the absence of cytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosin ring are known to be important for cytokinesis. Here we demonstrate for the first time that small GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II and Citron Kinase show a developmental stage specific expression in heart, with high levels being expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of many regulatory and cytoskeletal components involved in the formation of the actomyosin ring may be responsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytes after birth. Interestingly, when the myocardium tries to adapt to the increased workload during pathological hypertrophy a re-expression of proteins involved in DNA synthesis and cytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to divide despite this upregulation of the cytokinetic machinery. The inability to undergo complete division could be due to the presence of stable, highly ordered and functional sarcomeres in the adult myocardium or could be because of the inefficiency of degradation pathways, which facilitate the division of differentiated embryonic cardiomyocytes by disintegrating myofibrils.     

Screening procedure for the analysis of distigmine bromide in serum by high-performance liquid chromatography-electrospray ionization mass spectrometry

A screening procedure was developed for the identification and quantification of distigmine bromide in serum samples by using liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS). In this method, distigmine bromide was analyzed in 0.5 mL serum by using pancuronium bromide as the internal standard, and gradient elution was performed using a reversed-phase column and a mixture of 10 mM-ammonium formate and methanol as the mobile phase. A highly sensitive assay could be performed with simple solid phase extraction using a cation exchange cartridge column by carrying out selected ion monitoring analysis in the positive ion detection mode. The procedure was validated in terms of linearity (0.9973 at 2.5 ng/mL). The inter- and intra-day precisions (coefficient of variation; CV%) were <8.5% and < 9.7%, respectively. The analytes were evaluated for stability and were found to be stable in serum for 1 week at 4 degrees C and 4 weeks at -30 degrees C, and successfully applied to in the analysis of two overdose cases. This method is sensitive and useful for the detection, quantification, and confirmation of distigmine bromide in serum.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit

We investigated whether the assembly/disassembly of the 26S proteasome is regulated by phosphorylation/dephosphorylation. The regulatory complex disassembled from the 26S proteasome was capable of phosphorylating the p45/Sug1/Rpt6 subunit, suggesting that the protein kinase is activated upon dissociation of the 26S proteasome or that the phosphorylation site of p45 becomes susceptible to the protein kinase. In addition, the p45-phosphorylated regulatory complex was found to be incorporated into the 26S proteasome. When the 26S proteasome was treated with alkaline phosphatase, it was dissociated into the 20S proteasome and the regulatory complex. Furthermore, the p45 subunit and the C3/alpha2 subunit were cross-linked with DTBP, whereas these subunits were not cross-linked by dephosphorylating the 26S proteasome. These results indicate that the 26S proteasome is disassembled into the constituent subcomplexes by dephosphorylation and that it is assembled by phosphorylation of p45 by a protein kinase, which is tightly associated with the regulatory complex. It was also revealed that the p45 subunit is directly associated with the 20S proteasome alpha-subunit C3 in a phosphorylation-dependent manner.