Membrane translocation mechanism of the antimicrobial peptide buforin 2

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.     

A yield control approach to assess phytoavailability of Zn and Cu in irradiated,composted sewage sludges and composted manure in field experiments: I. Zinc

The threat of spreading diseases is a serious concern when organic wastes are applied to farmland. Irradiation and composting are effective methods to reduce pathogens. Field experiments were conducted to assess the influence of these pathogen-eliminating methods on plant availability of Zn in the wastes. Four organic wastes: digested and dewatered (DSS), irradiated (DISS), composted (DICSS) sewage sludge and composted livestock manure (CLM) were applied during two growing seasons at 10, 20, 30, and 40 t solids ha^–1 year^–1. Available N and K in the wastes were estimated and N and K fertilizers were added to the soil to equalize available N and K supply among treatments to avoid dilution of crop Zn concentration. A control treatment (CT) received fertilizers but no waste. Lettuce, snap beans and petunias were grown in 1990, and two cuts of lettuce were harvested in 1991. The influence of waste Zn application on crop Zn concentration was studied within approximately equal crop yields. Crop Zn concentration increased in all crops treated with DSS or DISS, and often reached a maximum at the 30 t ha^–1 rate of application, then slightly decreased at 40 t ha^–1. The response of crop Zn concentration to the amount of Zn applied in the wastes was best described by quadratic equations. Waste application also significantly increased soil Zn availability index, which was a function of DTPA (diethylenetriamine pentaacetic acid)-extractable soil Zn and soil pH. The index was highly correlated with crop Zn concentration. Although Zn concentration in DICSS was similar to those in DSS and DISS, Zn applied in DICSS did not increase crop Zn concentration or soil availability index. Composting reduced the availability of Zn. The similar concentrations of Zn in DSS and DISS in both years allowed the use of a paired t-test to determine the differences in crop Zn concentration caused by application of DSS and DISS. Zinc applied in DISS produced a higher Zn concentration in bean pods than Zn applied in DSS (t > T _0.05 at P < 0.02, df = 15), indicating that irradiation increased phytoavailability of Zn in the sludge. However, no similar effect was found in Zn concentrations in the two cuts of lettuce in 1991 or in soil Zn availability index.     

Properties of thiamin-binding proteins from sesame seed 2S albumins

Three thiamin-binding proteins (TBPs) from sesame ( Sesamum indicum L.) seeds (STBP-I, -II and -III) were characterized. Binding of thiamin to the three STBPs was inhibited by pyrithiamin, which did not inhibit the binding of thiamin to TBPs from other plant seeds. STBP-I alone bound 2-northiamin and hydroxyethylthiamin. Isoelectric points (pIs) of STBP-I and -II both were 7.5. The pI of STBP-III was 6.5. STBPs did not have immunological homology with TBPs from rice seeds and buckwheat seeds. On the other hand, the amino acid compositions of the small and large polypeptides isolated from STBP-I, -II and -III resembled each other. Both the polypeptides contained large amounts of Glu (or Gln) and Arg. The small polypeptides contained more Ser than the large polypeptides. The N-terminal amino acid sequences (the first 29 residues) of the small polypeptides were identified. The N-terminal amino acid sequences of the three STBPs were the same. The small polypeptides had homology to castor bean 2S albumin small subunit. These results showed that STBPs were part of a plant protein superfamily and that STBPs differed from the TBPs of other plant seeds as to the binding to thiamin-related compounds and immunological properties, and further, that STBP-I, -II and -III differed in the affinity for thiamin-related compounds and pI, indicating that STBP-I, -II and -III are isomers.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

S-myotrophin promotes the hypertrophy of skeletal muscle of mice in vivo

S-myotrophin is a newly discovered muscle growth factor. Effects of crude S-myotrophin injection on the growth and morphology of skeletal muscle of normal, ScN and mdx mice were investigated in the present study. Total dose of crude S-myotrophin was 100 microg (100 microg protein/ml x 50 microl x 20 times). In the case of normal mice (Sea:ddY), body weight and the weight of M. gluteus major of crude S-myotrophin injected mice was significantly heavier than that of control (PBS-injected) mice after 5 weeks' feeding. Antibody staining of laminin and dystrophin showed clear sarcolemmal and basement membrane structure surrounding each muscle fibre. The numbers of muscle fibres per 100 microm(2) was less in crude S-myotrophin-injected normal mice than in PBS-injected mice. Quite similar observations as in the case of normal mice were obtained in the case of ScN mice having heterogeneous gene of dystrophin. In the case of mdx mice, body weight and the weight of M. gluteus major of crude S-myotrophin injected mdx mice was significantly heavier than that of PBS-injected mdx mice. Antibody staining of laminin showed almost intact structure of the basement membrane containing laminin even in skeletal muscle of mdx mice subjected to crude S-myotrophin injection, while irregular and incompletely developed structure of muscle fibres or necrosis were observed in muscle fibres of PBS-injected mdx mice. In spite of crudeness of the preparation, the present animal experiments indicate that S-myotrophin has a strong growth promoting activity of muscle cells of normal and dystrophic mice.