Dense surface functionalization using peptides that recognize differences in organized structures of self-assembling nanomaterials

We obtained novel peptides that selectively bind to self-assembling peptide nanomaterials from a random peptide library displayed on phages. Affinity-dependent peptide screening gave phage clones displaying peptides with selective affinities against two kinds of highly networked nanofibers constructed of β-sheet peptides. Both peptide nanofibers have similar primary structures. Binding analyses of phage-displayed peptides by enzyme-linked immunosorbent assays demonstrated that the screened peptides specifically bind to the target nanofibers rather than to non-target nanofibers. Dot blot assays using chemically-synthesized peptides revealed that one screened peptide with the sequence Thr-Tyr-Leu-Pro-Trp-Pro-Ala has an affinity constant of 2.6 × 10(6) M(-1) against the target nanofibers. This affinity is 350 times greater than its affinity for non-target nanofibers and 90 times greater than its affinity for mixed nanofibers that contain 50% target nanofibers. These results suggest that this screened peptide can recognize organized fine-structures. Surface modification of the peptide nanofibers with the screened peptide demonstrated that the peptide specifically binds to discrete binding sites on the target nanofibers. Cell adhesion assays on the nanofibers by means of RGDS-conjugated screened peptides showed that the number of adhered cells is largely dependent on the amount of bound RGDS-peptide. These results suggest that screened peptides can recognize the organization of self-assembled peptide nanomaterials, and that the conjugation of functional groups to the peptides can be used to functionalize the target nanomaterials. Furthermore, simultaneous use of individual specific peptides that have specificities for nanomaterials can generate highly dense functionalized self-assembling peptide nanomaterials.     

Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways

Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55-protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.     

A rapid decrease in temperature induces latewood formation in artificially reactivated cambium of conifer stems

Background and Aims Latewood formation in conifers occurs during the later part of the growing season, when the cell division activity of the cambium declines. Changes in temperature might be important for wood formation in trees. Therefore, the effects of a rapid decrease in temperature on cellular morphology of tracheids were investigated in localized heating-induced cambial reactivation in Cryptomeria japonica trees and in Abies firma seedlings. Methods Electric heating tape and heating ribbon were wrapped on the stems of C. japonica trees and A. firma seedlings. Heating was discontinued when 11 or 12 and eight or nine radial files of differentiating and differentiated tracheids had been produced in C. japonica and A. firma stems, respectively. Tracheid diameter, cell wall thickness, percentage of cell wall area and percentage of lumen area were determined by image analysis of transverse sections and scanning electron microscopy. Key Results Localized heating induced earlier cambial reactivation and xylem differentiation in stems of C. japonica and A. firma as compared with non-heated stems. One week after cessation of heating, there were no obvious changes in the dimensions of the differentiating tracheids in the samples from adult C. japonica. In contrast, tracheids with a smaller diameter were observed in A. firma seedlings after 1 week of cessation of heating. Two or three weeks after cessation of heating, tracheids with reduced diameters and thickened cell walls were found. The results showed that the rapid decrease in temperature produced slender tracheids with obvious thickening of cell walls that resembled latewood cells. Conclusions The results suggest that a localized decrease in temperature of stems induces changes in the diameter and cell wall thickness of differentiating tracheids, indicating that cambium and its derivatives can respond directly to changes in temperature.     

Base pairing between hepatitis C virus RNA and microRNA 122 3' of its seed sequence is essential for genome stabilization and production of infectious virus

MicroRNA 122 (miR-122) facilitates hepatitis C virus (HCV) replication by recruiting an RNA-induced silencing complex (RISC)-like complex containing argonaute 2 (Ago2) to the 5' end of the HCV genome, thereby stabilizing the viral RNA. This requires base pairing between the miR-122 "seed sequence" (nucleotides [nt] 2 to 8) and two sequences near the 5' end of the HCV RNA: S1 (nt 22 to 28) and S2 (nt 38 to 43). However, recent reports suggest that additional base pair interactions occur between HCV RNA and miR-122. We searched 606 sequences from a public database (genotypes 1 to 6) and identified two conserved, putatively single-stranded RNA segments, upstream of S1 (nt 2 and 3) and S2 (nt 30 to 34), with potential for base pairing to miR-122 (nt 15 and 16 and nt 13 to 16, respectively). Mutagenesis and genetic complementation experiments confirmed that HCV nt 2 and 3 pair with nt 15 and 16 of miR-122 bound to S1, while HCV nt 30 to 33 pair with nt 13 to 16 of miR-122 at S2. In genotype 1 and 6 HCV, nt 4 also base pairs with nt 14 of miR-122. These 3' supplementary base pair interactions of miR-122 are functionally important and are required for Ago2 recruitment to HCV RNA by miR-122, miR-122-mediated stabilization of HCV RNA, and production of infectious virus. However, while complementary mutations at HCV nt 30 and 31 efficiently rescued the activity of a 15C,16C miR-122 mutant targeting S2, similar mutations at nt 2 and 3 failed to rescue Ago2 recruitment at S1. These data add to the current understanding of miR-122 interactions with HCV RNA but indicate that base pairing between miR-122 and the 5' 43 nt of the HCV genome is more complex than suggested by existing models.     

Regulation of a proteinaceous elicitor-induced Ca2+ influx and production of phytoalexins by a putative voltage-gated cation channel, OsTPC1, in cultured rice cells

Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.     

BMP2, BMP4, and their receptors are expressed in the differentiating muscle tissues of mouse embryonic tongue

To investigate the role of bone morphogenetic proteins (BMPs) in the differentiation process of skeletal muscle, we analyzed the in vivo expression of BMP2 and BMP4, of BMP receptors (BMPR) IA, IB, and II, and of activin receptors (ActR) IA, II, and IIB in mouse tongue muscle between embryonic day 11 (E11) and E17. The mRNA expression levels for BMP2 were 5-fold to 11-fold greater than those for BMP4 between E13 and E17 (P < 0.05-0.01). Expression of the BMP2, BMPRIB, ActRIA, ActRII, and ActRIIB proteins was first observed at E13. Expression of BMP2 and BMPRIB was detected in the whole area of the differentiating muscle tissues identified by immunostaining for fast myosin heavy chain (fMHC), but that of ActRIA, ActRII, and ActRIIB was detected only in the peripheral area of the differentiating muscle tissues. In the E15 tongue, all of the BMPs, BMPRs, and ActRs studied herein were expressed in the whole area of the differentiating muscle tissues identified by immunostaining for fMHC. These results suggest that BMPs play a role in the differentiation of tongue muscle tissues at E15 but have little or no effect at E13. This study was supported in part by grants-in-aid for funding scientific research (no. 16591871 to A.Y.) from the Bio-ventures and High-Technology Research Center of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan.