Cell fingerprint patterns using designed α-helical peptides to screen for cell-specific toxicity

We conducted cell-based cytotoxicity screening of a 101-membered α-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells.     

Antifungal thiopeptide cyclothiazomycin B1 exhibits growth inhibition accompanying morphological changes via binding to fungal cell wall chitin

Cyclothiazomycin B1 (CTB1) is an antifungal cyclic thiopeptide isolated from the culture broth of Streptomyces sp. HA 125-40. CTB1 inhibited the growth of several filamentous fungi including plant pathogens along with swelling of hyphae and spores. The antifungal activity of CTB1 was weakened by hyperosmotic conditions, and hyphae treated with CTB1 burst under hypoosmotic conditions, indicating increased cell wall fragility. CTB1-sensitive fungal species contain high levels of cell wall chitin and/or chitosan. Unlike nikkomycin Z, a competitive inhibitor of chitin synthase (CHS), CTB1 did not inhibit CHS activity. Although CTB1 inhibited CHS biosynthesis, the same result was also obtained with a non-specific proteins inhibitor, cycloheximide, which did not reduce cell wall rigidity. These results indicate that the primary target of CTB1 is not CHS, and we concluded that CTB1 antifungal activity was independent of this sole inhibition. We found that CTB1 bound to chitin but did not bind to β-glucan and chitosan. The results of the present study suggest that CTB1 induces cell wall fragility by binding to chitin, which forms the fungal cell wall. The antifungal activity of CTB1 could be explained by this chitin-binding ability.     

A novel β-loop scaffold of phage-displayed peptides for highly specific affinities

Loop peptides stabilized by two β-strands were used as a scaffold for a phage displayed peptide library. Affinity-based screening for insulin provided peptides, which showed affinity constants of 10(5) M(-1) order for insulin over 100 times greater than their affinity for the structurally similar insulin-like growth factor 1. The results suggested that the scaffold offers a powerful tool for generating and screening peptides as ligands for drugs and biologics.     

Structural elucidation of A-74528, an inhibitor for 2',5'-phosphodiesterase isolated from Streptomyces sp

A-74528 (1) is a metabolite of Streptomyces sp. discovered in the screening for 2',5'-oligoadenylate phosphodiesterase inhibitors. The planar structure of 1 was mainly elucidated by NMR techniques including natural abundance INADEQUATE, and the relative configuration and the conformation were elucidated by the analyses of NOEs and assessment of dihedral angles predicted by QUANTA/CHARMm computations and coupling constants. It was proved that 1 is a highly fused polyketide with a side-chain branching site that never appeared before from the nature.     

Interaction between aspterric acid and indole-3-acetic acid on reproductive growth in Arabidopsis thaliana

Application of indole-3-acetic acid (IAA) with a pollen growth inhibitor, aspterric acid (AA), results in the recovery of normal pollen development. In contrast, application of gibberellin (GA3) with AA do not induce normal pollen growth. In addition, application of different concentrations of IAA with AA shortens the period of growth from bolting to first flowering as compared to that treated with AA alone. Furthermore, stem length and number of flower bud treated with IAA and AA were similar to those of control. These results suggest, that IAA may play an important role in reproductive growth of A. thaliana.     

Anomalous reflection of gold applicable for a practical protein-detecting chip platform

A simple, convenient and label-free fiber optic detection system based on the characteristic property, 'anomalous reflection (AR)' of gold was developed and preliminary experiments showed that the AR signals were sensitive enough to monitor protein-peptide interactions on solid surfaces.     

Selection and Characterization of Nickel-Tolerant Tobacco Cells

Tobacco (Nicotiana tabacum L. cv. BY-2) cell lines tolerant to 700 M Ni in which unselected cells can not grow, were selected. The Ni-tolerant cells were also more tolerant to Co, but not to Cd than unselected cells. Ni concentrations in Ni-tolerant cells were always higher than those in medium. Since buthionine sulfoximine did not affect their Ni-tolerance, it is suggested that phytochelatins are not involved in Ni-tolerance of Ni-tolerant cells. On the other hand, histidine contents in Ni-tolerant and unselected cells, which were treated with Ni, were higher that those treated without Ni, and the degree of the elevation of histidine contents by Ni-treatment was higher in Ni-tolerant cells than in unselected cells. Additionally, exogenous histidine reduced the inhibitory effect of Ni on the growth of unselected cells. In addition, the cells that were tolerant to histidine-analogue, had higher contents of histidine and Ni-tolerance. These results suggest that histidine is involved in Ni-tolerance and the detoxification of Ni in symplast in Ni-tolerant cells.     

Alteration of the timing of implantation by in vivo gene transfer: delay of implantation by suppression of nuclear factor kappaB activity and partial rescue by leukemia inhibitory factor

Nuclear factor kappaB (NF-kappaB) is activated in the murine endometrium during implantation period [Am. J. Reprod. Immunol. 51 (2004) 16]. Transient transfection of IkappaBalpha mutant (IkappaBalphaM) cDNA into the mouse uterine cavity using hemagglutinating virus of Japan envelope vector suppressed uterine NF-kappaB activity less than half of that observed in control on days 3.5 and 4.5 p.c. IkappaBalphaM cDNA transfection led to significant delay of implantation. After IkappaBalphaM cDNA transfection, LIF mRNA expression in the uterus was significantly suppressed on days 3.5 and 4.5 p.c. Co-transfection of LIF cDNA with IkappaBalphaM cDNA in the uterus partially rescued the delay of implantation induced by suppression of NF-kappaB activity. Taken together, these findings indicate that NF-kappaB activation determines the timing of the implantation, at least in part, via control of LIF expression.     

Rational design of homogenous protein kinase assay platforms that allow both fluorometric and colorimetric signal readouts

Protein kinases play important roles in signaling pathways that regulate many cellular biological processes, including apoptosis, cell growth, and differentiation in response to extracellular stimuli. Design of homogenous protein kinase assay platforms including design of potent protein kinase substrates is essential for exploration of the phosphoproteome. Here, we describe a unique chromism-based assay (CHROBA) technique for the direct measurement of protein kinase activities. The CHROBA is a novel chemosensor system that produces signals based on the photochromic and thermodynamic properties of a spiropyran derivative incorporated into peptide substrates. The CHROBA technique for detecting protein kinase activities involves the following five steps: (i) phosphorylation, (ii) photobleaching of the reaction mixture, (iii) addition of ionic polymer(s), (iv) incubation in the dark, and (v) signal readout. This simple 'end-point' assay method allows quantitative measurements of protein kinase A, Src protein tyrosine kinase, c-Abl protein tyrosine kinase, and protein kinase Calpha activities even with excess ATP. Our results showed that spiropyran-containing peptide substrates with net charges between +2 and 0 are suitable for the present CHROBA method. This information should aid in the rational design of diverse protein kinase assay platforms. The present CHROBA technique can be adapted to a microplate format with both fluorometric and colorimetric readouts and would be useful for high-throughput drug discovery and analysis of the phosphoproteome.