Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.     

Sclerochronological study of the gigantic inoceramids Sphenoceramus schmidti and S. sachalinensis from Hokkaido,northern Japan

Here, we present the first sclerochronological investigation of shells of the gigantic inoceramids Sphenoceramus schmidti and S. sachalinensis from the middle Campanian cold seep carbonate‐bearing strata of the Yezo Basin in Hokkaido (northern Japan). Stable carbon (δ¹³C) and oxygen (δ¹⁸O) isotope values were measured in the aragonitic and calcitic shell layers of both species and compared to those of other co‐occurring benthic (mainly bivalves and gastropods) and demersal molluscs (ammonites). Sedimentological and stable isotope data suggest that these bivalves lived near cold seeps and were exposed to high H₂S level in the seawater. The inoceramid shells exhibited higher δ¹³C and lower δ¹⁸O values than the coeval non‐cold seep molluscs. We ascribed the anomalous isotopic pattern to a combination of vital and environmental effects determined by the hosting of chemosymbionts and the exposure to warm interstitial waters. Inoceramid δ¹³C minima coincided with growth lines and likely reflect changes in nutrient supply by the chemosymbionts. Absolute temperatures estimated from δ¹⁸O values of Sphenoceramus schmidti and S. sachalinensis were, on average, ca. 4–5°C warmer than those reconstructed for the non‐seepage environment (19.3 ± 0.7°C). Short‐term δ¹⁸O fluctuations of the inoceramid material indicate local temperature ranges of up to 5.2°C, that is four times larger than those reconstructed from the benthic and demersal fauna (1.3°C). In general, our data suggest that the stable carbon and oxygen isotope values of the studied Sphenoceramus spp. were strongly affected by short‐term fluctuations in seepage activity and do not reflect seasonal fluctuations.     

Stochastic thermodynamic limit on E. coli adaptation by information geometric approach

Biological systems process information under noisy environment. Sensory adaptation model of E. coli is suitable for investigation because of its simplicity. To understand the adaptation processing quantitatively, stochastic thermodynamic approach has been attempted. Information processing can be assumed as state transition of a system that consists of signal transduction molecules using thermodynamic approach, and efficiency can be measured as thermodynamic cost. Recently, using information geometry and stochastic thermodynamics, a relationship between speed of the transition and the thermodynamic cost has been investigated for a chemical reaction model. Here, we introduce this approach to sensory adaptation model of E. coli, and examined a relationship between adaptation speed and the thermodynamic cost, and efficiency of the adaptation speed. For increasing external noise level in stimulation, the efficiency decreased, but the efficiency was highly robust to external stimulation strength. Moreover, we demonstrated that there is the best noise to achieve the adaptation in the aspect of thermodynamic efficiency. Our quantification method provides a framework to understand the adaptation speed and the thermodynamic cost for various biological systems.     

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.     

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.     

Cloning of a new aquaporin (AQP10) abundantly expressed in duodenum and jejunum

A new aquaporin (AQP10) was identified in human small intestine. This gene encoded a 264-amino-acid protein with high sequence identity with AQP3 (53%), 9 (52%), and 7 (43%). These AQPs constitute one subfamily of AQP family that is differentiated from the other subfamily of AQP (AQP0, 1, 2, 4, 5, 6, and 8) by sequence homology. Ribonuclease protection assay and Northern blotting demonstrated almost exclusive expression of AQP10 mRNA in the duodenum and jejunum. In situ hybridization localized it in absorptive jejunal epithelial cells. Xenopus oocytes expressing AQP10 exhibited an increased osmotic water permeability in a mercury-sensitive manner. Although AQP10 belongs to the AQP subfamily, which has been characterized by permeability to water and neutral solutes such as urea and glycerol, it was not permeable to urea nor glycerol. The specific expression of AQP10 suggests its contribution to the water transport in the upper portion of small intestine.     

Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers involved in the intracellular signal transduction of a wide variety of extracellular stimuli. These signals regulate many biological processes including cell proliferation, differentiation, migration, and apoptosis. Recently, significant progress has been achieved in the molecular basis underlying cyclic nucleotide regulation of cell proliferation. This review summarizes our knowledge of the signaling pathways regulated by cyclic nucleotides in arterial smooth muscle cells.     

Brief exposure to low-pH stress causes irreversible damage to the growing root in Arabidopsis thaliana: pectin-Ca interaction may play an important role in proton rhizotoxicity

The viability of Arabidopsis thaliana (strain Landsberg) roots exposed to a low pH (4.5 or 4.7) solution that contained 100 microM CaCl(2) was examined by staining with fluorescein diacetate-propidium iodide. The elongation zone of growing roots lost viability within 1-2 h following exposure to low pH, but non-growing roots showed no damage under the same treatment. Low-pH damage in growing roots was irreversible after 1 h incubation at pH 4.5 as judged by regrowth in growing medium at pH 5.6. Growing lateral roots also lost viability in the same treatment, whereas non-growing lateral roots remained viable during and after the treatment. The low-pH damage was ameliorated by the simultaneous application of calcium, indicating the involvement of a calcium-requiring process in overcoming proton toxicity. At pH 5.0, growing roots required 25 microM of calcium to maintain elongation, and at pH 4.8 and pH 4.5 more than 250 microM and 750 microM, respectively. The low-pH damage was ameliorated by divalent cations in the order of Ba2+, approximately Sr2+>/=Ca2+>Mg2+. The monovalent cation K+ showed no ameliorative effect, but borate showed a strong ameliorative effect with Ca2+. These results indicate that the primary target of proton toxicity may be linked to a disturbance of the stability in the pectic polysaccharide network, where calcium plays a key role in plant roots.     

Selective secretion of free glycine,a neutralizer against a plant defense chemical,in the digestive juice of the privet moth larvae

The larva of the privet moth, Brahmaea wallichii (Brahmaeidae) is a specialist feeder of the privet tree, Ligustrum obtusifolium (Oleaceae). A very high concentration (50 mM or 0.4%) of free glycine, found in the digestive juice of the larvae, works as a neutralizer against the very strong protein-denaturing activity of privet leaves that is caused by oleuropein, an iridoid that functions in chemical defense. Concentration of free glycine was high in the anterior region of the midgut lumen and low in the posterior region. To examine if some glycine-specific secretion mechanism exists, injection experiments were performed using (15)N-labeled amino acids. When 13 &mgr;mol (1 mg) of (15)N-glycine was injected into hemolymph of fifth instar larvae of B. wallichii, a high concentration of (15)N (5 mM or 75 &mgr;g/g midgut content) was detected in the anterior parts of the midgut lumen 1 h after injection. (15)N-NMR data indicated at least 60% of the (15)N found in midgut lumen existed as (15)N-glycine. Approximately, 25% of the injected (15)N-glycine was estimated to have moved from the hemolymph to the midgut lumen. In contrast, no (15)N was detected in the midgut lumen when 13 &mgr;mol of (15)N-labeled alanine, lysine and glutamate were injected into hemolymph. Glycine was the only amino acid whose concentration was higher in the midgut lumen (50 mM) than in the hemolymph (22 mM). These data suggest the existence of some active and glycine-specific secretory mechanism in the midgut of B. wallichii.