Hiroshi Yamaguchi

Effect of α, α-dialkyl amino acids on the protease resistance of peptides

A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of α,α-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1′ positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1′ and P2 positions resulted in complete resistance.

Second transmembrane domain of human uncoupling protein 2 is essential for its anion channel formation.

Uncoupling proteins (UCP) are known to transport anions, such as Cl−, in addition to H+ transport. Although H+ transport by UCP is clearly involved in thermogenesis, the mechanism of its anion transport is not clearly understood. In this study, we examined the anion channel characteristics of the six individual helical transmembrane (TM) domains of the human UCP2. The second TM domain peptide (TM2) forms multi‐state channels by assemblies of conductive oligomers. Furthermore, the TM2 exhibited voltage‐dependent anion channels with properties comparable to those of UCP1 chloride channel. However, the other five TM peptides did not form UCP1‐like channels. Moreover, an analog of TM2 in which two Arg residues were substituted by Ala residues did not form stable channels, implying the significance of Arg residues for anion transport. These results suggest that the anion channel structure of UCP2 protein is oligomeric and the second TM domain is essential for the voltage‐dependence of this anion channel.

In vitro and in vivo evaluation of the efficacy of bovine colostrum against human rotavirus infection.

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6–7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.

Modifications on amphiphilicity and cationicity of unnatural amino acid containing peptides for the improvement of antimicrobial activity against pathogenic bacteria

The widespread natural sources‐derived cationic peptides have been reported to reveal bacterial killing and/or growth‐inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug‐resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib‐Arg‐Aib‐Ala sequences, showed strong antibacterial activity against both Gram‐negative and Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug‐resistant Pseudomonas aeruginosa, known as proteases‐secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib‐derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices. Copyright

Structure-Function Relationship of Gramicidin-Alamethicin Hybrid Analogs

Gramicidin is a 16-meric linear peptide, which is composed of alternating L- and D-amino acid residues and forms dimeric β6.4-helical structures in biological membranes. Alamethicin is an Aib-containing peptide composed of 20 amino acids and consists of a dominantly α-helical N-terminal and a central kinked region. Both peptides are potently antimicrobial and form ion channel structures in cell membranes. We have previously reported of Aib-containing analogs of gramicidin (GAA and GBA) [1]. Both analogs formed stable helical structures in both organic and aqueous systems, and in phospholipid vesicles [1]. Moreover, GBA showed a multi-state channel-type activity, similar to that of alamethicin, in phospholipid bilayers [2].

Effect of environment on the stability of helical Aib-model peptides

It is well known that peptides containing Aib residues form αand/or 3 10 helical structures leading to voltage-dependent ion channel in lipid membranes. We have previously reported the synthesis of Aib-containing gramicidin analogs [1,2] and model-peptides, Ac-(Aib–Lys–Aib–Ala)n -NH2 (n = 1–5, BKBA-N , N denotes the number of residues). Among the Aib-peptides, the longer peptide, BKBA-20 may aggregate and form alamethicin-like barrel-stave conformations in phospholipid bilayers [2]. In the present study, to characterize the structures and ion-selectivities of the Aib-peptides containing charged or aromatic residue, we have synthesized novel Aib-peptides containing Glu, Ser, Gly and Trp residues (Figure 1). The conformational stabilities of the synthesized peptides in aqueous and phospholipid membrane environments have been investigated by means of CD spectroscopy.