Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A.

Antiviral effects of persimmon extract on human norovirus and its surrogate, bacteriophage MS2.

UNLABELLED: Human noroviruses (NoVs) are the leading cause of gastroenteritis and foodborne illnesses worldwide. In this study, we investigated the effects of persimmon extract (PE) on NoV GII.4 and bacteriophage MS2. We also examined the relationship between the tannin content of PE and its antiviral effects to identify the active ingredient in PE. Different persimmon tannin (PT) solutions were prepared by mixing PE with different concentrations of bovine serum albumin. The antiviral efficacy of these solutions against NoV was evaluated by quantifying the amount of residual noroviral genome using a quantitative reverse transcription PCR (qRT-PCR) assay. The antiviral efficacy of PE against MS2 was examined with an infectivity assay (plaque assay). Solutions containing ≥ 0.11 mg/mL PT reduced the noroviral genome by more than 70.0% and the infectivity of MS2 by more than 2.5 log PFU/mL. However, the effects of PT on both viruses decreased markedly at a concentration of 0.08 mg/mL and solutions containing negligible PT had no antiviral activity. These results suggest that the PT component of PE inactivates NoV and MS2. Our results indicate that PE is a nontoxic antiviral agent effective against enteric viruses. PRACTICAL APPLICATION: Persimmon extract showed antiviral effects against NoV and bacteriophage MS2. Persimmon extract is suitable for use as an antiviral agent.

High hole mobility for a side-chain liquid-crystalline smectic polysiloxane exhibiting a nanosegregated structure with a terthiophene moiety.

Side-chain liquid-crystalline siloxane polymers bearing terthiophene moieties as mesogenic pendant groups have been synthesized. An alkenylterthiophene derivative was treated with poly(hydrogenmethylsiloxane) and poly(dimethylsiloxane-co-hydrogenmethylsiloxane)s in Me(2)SiO/MeHSiO ratios of 1:1 and 7:3, respectively, in the presence of the Karstedt catalyst, to produce pale yellow polymers. The degrees of introduction of the mesogenic unit were 100, 50, and 30%, respectively. The polymers exhibit ordered smectic phases at room temperature. The copolymers with dimethylsiloxane units form smectic phases as a consequence of nanosegregation between the mesogenic units and siloxane backbones with the alkylene spacers. Time-of-flight measurement reveals that the hole mobility exceeds 1×10(-2) cm(2) V(-1) s(-1) in the ordered smectic phase of the copolymer with a degree introduction of the mesogenic units of 50%. This value is comparable to that of the highly ordered mesophases of low-molecular-weight derivatives of phenylnaphthalene and terthiophene. Because of the segregation behavior induced by the flexible backbone, a closer molecular packing structure favorable for fast carrier transport may be formed in the smectic phase of the copolymer in spite of the low density of the mesogenic groups.

Motif-programmed artificial proteins mediated nucleation of octacalcium phosphate on titanium substrates.

We report the creation of motif-based artificial proteins that bind to titanium surfaces and mediate the deposition of crystalline calcium phosphate. These proteins enabled in aqua surface modification of titanium materials having an intricate structure used in medical applications.

A redox-switchable [2]rotaxane in a liquid-crystalline state.

Redox-driven mechanical movement, which has been achieved for a liquid-crystalline (LC) bistable [2]rotaxane in the LC phase, is accompanied by obvious electrochromism (electrochemically induced changes in color) of the material. The dumbbell-shaped LC [2]rotaxane with redox-active moieties, which interlocks with an ionic macrocycle, forms ordered redox-active condensed states.

mRNA display selection of a high-affinity, Bcl-X(L)-specific binding peptide.

Bcl-X(L), an antiapoptotic member of the Bcl-2 family, is a mitochondrial protein that inhibits activation of Bax and Bak, which commit the cell to apoptosis, and it therefore represents a potential target for drug discovery. Peptides have potential as therapeutic molecules because they can be designed to engage a larger portion of the target protein with higher specificity. In the present study, we selected 16-mer peptides that interact with Bcl-X(L) from random and degenerate peptide libraries using mRNA display. The selected peptides have sequence similarity with the Bcl-2 family BH3 domains, and one of them has higher affinity (IC(50)=0.9 microM) than Bak BH3 (IC(50)=11.8 microM) for Bcl-X(L) in vitro. We also found that GFP fusions of the selected peptides specifically interact with Bcl-X(L), localize in mitochondria, and induce cell death. Further, a chimeric molecule, in which the BH3 domain of Bak protein was replaced with a selected peptide, retained the ability to bind specifically to Bcl-X(L). These results demonstrate that this selected peptide specifically antagonizes the function of Bcl-X(L) and overcomes the effects of Bcl-X(L) in intact cells. We suggest that mRNA display is a powerful technique to identify peptide inhibitors with high affinity and specificity for disease-related proteins.

In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments.

To what extent has alternative splicing contributed to the evolution of protein-function diversity? We previously constructed a pool of block-deletion mutants of the human estrogen receptor alpha ligand binding domain by random multi-recombinant PCR. Here we performed iterative in vitro selection of GTP-binding proteins by using the library of mRNA-displayed proteins and GTP-affinity chromatography combined with quantitative real-time PCR. We obtained a novel GTP-binding protein with moderate affinity and substrate-specificity. The results of our in vitro simulation imply that alternative splicing may have contributed substantially to the diversification of protein function during evolution.

Directional BMP-2 for functionalization of titanium surfaces.

Efficient immobilization of biomacromolecules on material surfaces is a key to development in areas of regenerative medicine and tissue engineering. However, strong and irreversible immobilization of cytokines on surfaces often diminishes their biological functionality. A destructive hydrophobic interaction between the material surface and the biomolecule may underlie this inactivation. Alternatively, dissociation of the cytokine from the material may be necessary for signal transduction. Here we propose a new method for immobilizing cytokines on material surfaces: a material-binding artificial peptide is used to mediate reversible interaction between the cytokine and the material surface. We created artificial proteins that contained three copies of a Ti-binding motif, and fused them to the N-terminal of BMP-2. The engineered BMP-2 showed reversible binding to Ti surfaces and induced BMP signaling activity. When a hydrophobic protein devoid of the Ti-binding motif was fused to BMP-2, the protein tightly bound to Ti surfaces but showed little BMP activity, confirming the importance of the mode of immobilization.

Direct transformation from amorphous to crystalline calcium phosphate facilitated by motif-programmed artificial proteins.

An animal's hard tissue is mainly composed of crystalline calcium phosphate. In vitro, small changes in the reaction conditions affect the species of calcium phosphate formed, whereas, in vivo, distinct types of crystalline calcium phosphate are formed in a well-controlled spatiotemporal-dependent manner. A variety of proteins are involved in hard-tissue formation; however, the mechanisms by which they regulate crystal growth are not yet fully understood. Clarification of these mechanisms will not only lead to the development of new therapeutic regimens but will also provide guidance for the application of biomineralization in bionanotechnology. Here, we focused on the peptide motifs present in dentin matrix protein 1 (DMP1), which was previously shown to enhance hydroxylapatite (HAP) formation when immobilized on a glass substrate. We synthesized a set of artificial proteins composed of combinatorial arrangements of these motifs and successfully obtained clones that accelerated formation of HAP without immobilization. Time-resolved static light-scattering analyses revealed that, in the presence of the protein, amorphous calcium phosphate (ACP) particles increased their fractal dimension and molecular mass without increasing their gyration radii during a short period before precipitation. The protein thus facilitated reorganization of the internal structure of amorphous particles into ordered crystalline states, i.e., the direct transformation of ACP to HAP, thereby acting as a nucleus for precipitation of crystalline calcium phosphate. Without the protein, the fractal dimension, molecular mass, and gyration radii of ACP particles increased concurrently, indicating heterogeneous growth transformation.

N- and C-terminal fragments of a globular protein constructed by elongation of modules as a units associated for functional complementation.

We have been interested in partially folded proteins with marginal stability and activity, because they have a potential to be mature proteins by artificial evolution. A module is defined as a contiguous peptide chain forming a compact region in a globular protein. Modules may be used as building blocks to create partially folded proteins. Barnase, a ribonuclease consisting of 110 amino acids, has been divided into six modules (M1-M6), four peptide fragments, M12 (1-52), M123 (1-73), M1234 (1-88) and M12345 (1-98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 (1-98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 (1-98) with M56 (89-110) showed weak but distinct RNase activity. Unfolded M12345 (1-96) was constructed by removal of two residues from the C-terminus of M12345 (1-98). The mixture of M12345 (1-96) with M56 (89-110) also showed RNase activity. Further, the interaction endowed M12345 (1-96) with conformational stability. We propose that N- and C-terminal fragments obtained by successive elongation of modules would interact to be a complex with marginal stability and activity, which would be used for creating a mature complex by artificial evolution.

Towards the creation of novel proteins by block shuffling.

We have been investigating the creation of novel proteins by means of block shuffling, where the term block refers to an amino acid sequence that corresponds to particular features of proteins, such as secondary structures, modules, functional motifs, and so on. Block shuffling makes it possible to explore the global sequence space, which is not feasible with conventional methods, such as DNA shuffling or family shuffling. To investigate what properties are required for the building blocks, we have analyzed the foldability and enzymatic activity of barnase mutants obtained by permutation of modules or secondary structure units. This reconstructive approach indicated that secondary structure units with mutual long-range interactions are more suitable than modules as building blocks, at least in the case of barnase. The results also suggested that proteins in evolutionarily intermediate states are created by block shuffling, and such proteins have the potential to be evolved into mature globular proteins. For the construction of combinatorial protein libraries, we have developed random multi-recombinant PCR (RM-PCR), which can combine different DNA fragments without homologous sequences. The libraries can be utilized for in vitro selection using in vitro virus (mRNA display) or stable (DNA display), which have also been developed in our laboratory. In this review article, we summarize our strategy to create novel proteins by block shuffling and review key literature in the field. Possible applications of the block shuffling strategy are also discussed.

Foldability, enzymatic activity, and interacting ability of barnase mutants obtained by permutation of secondary structure units.

Barnase, a well-characterized ribonuclease, has been decomposed into six modules (M1-M6) or secondary structure units (S1-S6). We have studied the foldability and activity of the barnase mutants obtained by permutation of the four internal modules (M2-M5) or secondary structure units (S2-S5) to investigate whether permutation of these building blocks is a useful way to create foldable and/or functional proteins. In this study, we found that one of the secondary structure unit mutants was expressed in Escherichia coli only when His102 was substituted by alanine, which is a catalytic residue of wild-type barnase. This mutant (S2354H102A) had ordered conformations, which unfolded cooperatively during urea-induced unfolding experiments. S2354H102A interacted with other barnase mutants to show a distinct RNase activity, although its own activity was quite weak. This interaction was specific, because S2354H102A interacted with only barnase mutants having His 102 and certain orders of the secondary structure units giving a distinct RNase activity. These results suggest that secondary structure units permuted in barnase mutants maintain their intrinsic "interacting ability" that is used for the folding of wild-type barnase, and the units can form certain conformations that complement those of the appropriate counterparts. Seven of 23 secondary structure unit mutants and only 2 of 23 module mutants had RNase activity. On the basis of the results of analyses of foldability and RNase activity of the mutants performed in this and previous studies, we conclude that secondary structure units are more suitable than modules as building blocks to create novel foldable and/or functional proteins in the case of barnase.