Single giant unilamellar vesicle method reveals effect of antimicrobial peptide magainin 2 on membrane permeability

It is thought that magainin 2, an antimicrobial peptide, acts by binding to lipid membranes. Recent studies using a suspension of large unilamellar vesicles (LUVs) indicate that magainin 2 causes gradual leakage from LUVs containing negatively charged lipids. However, the details of the characteristics of the membrane permeability and the mechanism of pore formation remain unclear. In this report, we investigated the interaction of magainin 2 with single giant unilamellar vesicles (GUVs) composed of a dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol mixture (50% DOPG/50% DOPC GUVs) containing the fluorescent dye, calcein, by phase contrast, fluorescence microscopy using the single GUV method. Low concentrations (3-10 microM) of magainin 2 caused the rapid leakage of calcein from single GUVs but did not disrupt the liposomes or change the membrane structure, showing directly that magainin 2 forms membrane pores through which calcein leaked. The rapid leakage of calcein from a GUV started stochastically, and once it began, the complete leakage occurred rapidly (6-60 s). The fraction of completely leaked GUV, P(L), increased with time and also with an increase in magainin 2 concentration. Shape changes in these GUVs occurred prior to the pore formation and also at lower concentrations of magainin 2, which could not induce the pore formation. Their analysis indicates that binding of magainin 2 to the external monolayer of the GUV increases its membrane area, thereby raising its surface pressure. The addition of lysophosphatidylcholine into the external monolayer of GUVs increased P(L). On the basis of these results, we propose the two-state transition model for the pore formation.     

Quantitative analysis of helix-coil transition of block copolypeptide, Glu12-Ala12, by combined use of CD and NMR spectroscopy

To investigate helix-coil transition mechanisms, conformations of Glu12-Ala12, EA, in aqueous solution have been studied in detail over the pH range from 2 to 8 and the temperature range from 20 to 60 degrees C using CD and NMR spectroscopy. The 750-MHz NMR spectra displayed excellent dispersion of the backbone amide proton signals, and permitted essentially complete sequence-specific resonance assignments. These assignments, together with short- and medium-range nuclear Overhauser effect (NOE) constraints and coupling constants, enable us to analyze conformational characteristics of all the residues in the EA peptide individually. A combined use of CD and NMR techniques reveals that the EA peptide assumes a stable alpha-helix from Glu12 to Ala19 in 0.1 M NaCl solution at 20 degrees C above pH 7. The alpha-helix is getting longer as decreasing pH. Below pH 4, the peptide assumes the longest alpha-helix from Glu3 to Ala23. The important observation of the present study is that the helix-coil transition occurs stepwise, residue by residue, from both the N- and C-termini of the alpha-helix. No conformational equilibrium between the helical and random-coil states is detected for the residues in the central region of the alpha-helix. Quantitative analysis of temperature-induced helix-to-coil transitions at various pHs provides a pH-independent residual enthalpy change delta H(r) = 0.95 kcal res(-1). Similar values have been reported for a 50-residue alanine-rich peptide (1.2 kcal res(-1)), poly-L-glutamate (1.1 kcal res(-1)), poly-L-lysine (1.1 kcal res(-1)), and poly-L-alanine (0.86 kcal res(-1)). Those investigations, along with our present result, suggest that delta H(r) is mainly determined by the transformation of the backbone associated with the disruption of the intramolecular hydrogen bond. These results should increase our understanding of the helix-coil transition.     

Ascorbic acid promotes osteoclastogenesis from embryonic stem cells

Ascorbic acid (AA) is known to regulate cell differentiation; however, the effects of AA on osteoclastogenesis, especially on its early stages, remain unclear. To examine the effects of AA throughout the process of osteoclast development, we established a culture system in which tartrate-resistant acid phosphate (TRAP)-positive osteoclasts were induced from embryonic stem cells without stromal cell lines. In this culture system, the number of TRAP-positive cells was strongly increased by the addition of AA during the development of osteoclast precursors, and reducing agents, 2-mercaptoethanol, monothioglycerol, and dithiothreitol, failed to substitute for AA. The effect of AA was stronger when it was added during the initial 4 days during the development of mesodermal cells than when it was added during the last 4 days. On day 4 of the culture period, AA increased the total cell recovery and frequency of osteoclast precursors. Magnetic cell sorting using anti-Flk-1 antibody enriched osteoclast precursors on day 4, and the proportion of Flk-1-positive cells but not that of platelet-derived growth factor receptor alpha-positive cells was increased by the addition of AA. These results suggest that AA might promote osteoclastogenesis of ES cells through increasing Flk-1-positive cells, which then give rise to osteoclast precursors.     

Identification of the N- and C-terminal substrate binding segments of ferredoxin-NADP+ reductase by NMR

Ferredoxin-NADP(+) reductase (FNR) catalyzes the reduction of NADP(+) through the formation of an electron transfer complex with ferredoxin. To gain insight into the interaction of this enzyme with substrates at both ends of the polypeptide chain, we performed NMR analyses of a 314-residue maize leaf FNR with a nearly complete assignment of the backbone resonances. The chemical shift perturbation upon formation of the complex indicated that a flexible N-terminal region of FNR contributed to the interaction with maize ferredoxin, and an analysis of N-terminally truncated mutants of FNR confirmed the importance of this region for the binding of ferredoxin. Comparison between the spectra of FNR in the NADP(+)- and inhibitor-bound states also revealed that the nicotinamide moiety of NADP(+) was accessible to the C-terminal Tyr314. We propose that the formation of the catalytic competent complex of FNR and substrates is achieved through the interaction of the N- and C-terminal segments with ferredoxin and NADP(+), respectively. Since the ends of the polypeptide chain act as flexible regions of proteins, they may contribute to the search of a larger space for a binding partner and to the opening of active sites.     

An infrared spectroscopy study of acid stability and thermal unfolding process of granulocyte-colony stimulating factor

Temperature-dependent (25-80 degrees C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5-2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the alpha-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5-2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1.     

Skewed X-inactivation in cloned mice

In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P<0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders.     

Effects of the dietary supplements, activated charcoal and copper chlorophyllin, on urinary excretion of trimethylamine in Japanese trimethylaminuria patients

Trimethylaminuria (TMAU) is a metabolic disorder characterized by the inability to oxidize and convert dietary-derived trimethylamine (TMA) to trimethylamine N-oxide (TMAO). This disorder has been relatively well-documented in European and North American populations, but no reports have appeared regarding patients in Japan. We identified seven Japanese individuals that showed a low metabolic capacity to convert TMA to its odorless metabolite, TMAO. The metabolic capacity, as defined by the concentration of TMAO excreted in the urine divided by TMA concentration plus TMAO concentration, in these seven individuals ranged from 70 to 90%. In contrast, there were no healthy controls examined with less than 95% of the metabolic capacity to convert TMA to TMAO. The intake of dietary charcoal (total 1.5 g charcoal per day for 10 days) reduced the urinary free TMA concentration and increased the concentration of TMAO to normal values during charcoal administration. Copper chlorophyllin (total 180 mg per day for 3 weeks) was also effective at reducing free urinary TMA concentration and increasing TMAO to those of concentrations present in normal individuals. In the TMAU subjects examined, the effects of copper chlorophyllin appeared to last longer (i.e., several weeks) than those observed for activated charcoal. The results suggest that the daily intake of charcoal and/or copper chlorophyllin may be of significant use in improving the quality of life of individuals suffering from TMAU.     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Identification of the primary electron donor in PS II of the Chl d-dominated cyanobacterium Acaryochloris marina

The primary electron donor of photosystem (PS) II in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina was confirmed by delayed fluorescence (DF) and further proved by pigment contents of cells grown under several light intensities. The DF was found only in the Chl a region, identical to Synechocystis sp. PCC 6803, and disappeared following heat treatment. Pigment analyses indicated that at least two Chl a molecules were present per each two pheophytin a molecules, and these Chl a molecules are assigned to P(D1) and P(D2). These findings clearly indicate that Chl a is required for water oxidation in PS II.