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1.
Russian Journal of Bioorganic Chemistry - We have studied the antimicrobial and hemolytic activity of synthetic antimicrobial peptides (SAMPs), i.e., Arg9Phe2 (P1-Arg), Lys9Phe2 (P2-Lys), and...  相似文献   

2.
In order to evaluate their antibacterial activities and toxicities, the cecropins–melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp’s 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect.  相似文献   

3.
PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state 15N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.The two antimicrobial peptides PGLa and magainin 2 (MAG2) from the African frog Xenopus laevis, which are active against Gram-positive and Gram-negative bacteria, show intriguing synergistic effects that are not yet well understood (1). Structural insights into this synergy may help in the development of a new antibiotic-combination therapy. Both peptides are known to form α-helices when bound to lipid bilayers (2–4). The orientation of such α-helices in a membrane can be readily determined from the 15N-NMR chemical shift in oriented lipid bilayers that are aligned with the sample normal parallel to the external magnetic field (5). If the 15N chemical shift is ∼90 ppm, the peptide lies flat on the membrane surface (in the so-called S-state). On the other hand, when the 15N chemical shift is ∼200 ppm, the peptide is fully inserted (the I-state) in a transmembrane alignment. For intermediate orientations, where the peptide is tilted (the T-state) with an angle of typically 30°–60° relative to the membrane normal, intermediate chemical shifts are expected, but the exact tilt angle cannot be determined from a single label in such cases (6). Using several selectively 2H- or 19F-labeled peptide analogs, more exact orientations can be obtained, since both the tilt and the azimuthal angles can be measured with high accuracy, and valuable information about dynamics also can be deduced (3,7–10).The orientation of PGLa and MAG2 in membranes has been extensively studied with solid-state NMR, and some clues about the synergistic mechanism have been observed. Notably, in DMPC/DMPG membranes, it has been shown that the peptides on their own are in the S-state or T-state, but when the peptides are mixed in a 1:1 molar ratio, PGLa changes to the I-state, whereas MAG2 stays on the membrane surface (1,11,12). Thus, in the mixed system, transmembrane pores, which would not be spontaneously formed by each peptide on its own, appear to be stable, which could be the basis for synergy. On the other hand, it was also reported that in POPC/POPG there is no change in orientation when PGLa and MAG2 are mixed, as both peptides stay always in the S-state (12). This observation was attributed to the greater hydrophobic thickness of the POPC/POPG bilayer, compared to the DMPC/DMPG bilayer, suggesting that the PGLa helix is so short that it can only insert into thin DMPC/DMPG membranes. However, we have recently shown for MSI-103, a designer-made antimicrobial peptide based on the PGLa sequence, that the orientation determined by 2H-NMR depends not on the bilayer thickness but rather on the intrinsic spontaneous curvature of the lipids (13). Accordingly, an insertion of PGLa and MAG2 into POPC/POPG should be prevented by the pronounced negative spontaneous curvature induced by the unsaturated acyl chains. However, a simple comparison of only two lipid systems does not yield an answer as to which of these two hypotheses is correct. Therefore, we have now collected data over a wide range of lipid systems, with systematic variations of acyl chain lengths (to address bilayer thickness) as well as chain saturations (to address lipid curvature) (see Table S1 in the Supporting Material). In this way, we found unambiguously that lipid curvature is also the decisive factor in the insertion of PGLa/MAG2.Here, 15N-NMR spectra of singly labeled peptides were recorded, and for each liquid-crystalline lipid system we prepared four oriented samples: 15N-MAG2 alone, 15N-MAG2 with PGLa, 15N-PGLa with MAG2, and 15N-PGLa alone. The total peptide/lipid molar ratio (P/L) was 1:50. 15N-NMR spectra are shown in Fig. 1, and the chemical shifts are listed in Fig. 2. The quality of each oriented sample was checked with 31P-NMR (see Fig. S1). Our previous detailed 2H- and 19F-NMR analysis of PGLa in DMPC and DMPC/DMPG showed that the helix realigns depending on the peptide concentration. Namely, at low concentration, PGLa is in an S-state with a tilt angle of ∼98° (3), but above a threshold concentration around P/L = 1:100, it flips into a tilted T-state with a tilt angle of ∼125° (9,14). In the presence of MAG2, it was found that PGLa inserts almost upright in an I-state with a tilt angle of ∼158° (11). The present 15N-NMR study confirms that in DMPC/DMPG (3:1), PGLa is in the I-state when mixed with MAG2, as indicated by the 15N chemical shift of 205 ppm. PGLa alone at P/L = 1:50 has a chemical shift of 116 ppm, which corresponds to a tilted orientation, as expected. MAG2 alone is found to be in the S-state (91 ppm), but when it is mixed with PGLa its signal moves to 105 ppm, indicating a small change in the alignment. MAG2 is, however, clearly not inserted like PGLa.Open in a separate windowFigure 115N-NMR spectra of 15N-labeled PGLa or MAG2, alone or in a synergistic 1:1 mixture with the other peptide, in differently oriented lipids. Powder spectra are shown in the top row. Red, green, and blue lines indicate chemical shifts associated with the S-, T-, and I-state orientations, respectively.Open in a separate windowFigure 2Schematic overview of the orientation of PGLa (red), MAG2 (green), and MSI-103 (orange (13)) in different lipids. The corresponding 15N-NMR chemical shifts (in ppm) of the spectra in Fig. 1 are indicated beneath each peptide.In thin DLPC bilayers (12 carbon atoms in the chains) we see a behavior similar to that in DMPC (14 carbons), even though the exact chemical shifts are slightly different. PGLa alone is in the T-state, but in combination with MAG2, it flips into the I-state. MAG2, on the other hand, stays in the S-state with and without PGLa. In DPPC bilayers (16 carbons) also, the behavior is similar. PGLa alone is in the T-state but flips into the I-state in the presence of MAG2. MAG2 is slightly more tilted than in DMPC, but it never reaches the I-state.In contrast, in unsaturated lipids, both peptides are always in the S-state, both alone and in the presence of the synergistic partner. In POPC/POPG (9:1), the 15N chemical shifts of both PGLa and MAG2, alone and in the 1:1 mixture, are between 84 and 89 ppm, clearly indicating a flat alignment on the bilayer surface. As there are no changes in chemical shift with or without the other peptide, this could indicate that there are no interactions between them, in contrast to the situation in saturated lipids. Also, in thin DMoPC bilayers (with 14 carbon atoms and a double bond), the chemical shifts of all samples show that both peptides remain always in the S-state, whether alone or mixed.These results clearly demonstrate that the hydrophobic membrane thickness is not a critical factor for the insertion of PGLa in the presence of MAG2. In DMoPC (thinner than DMPC), there is no insertion, whereas in DPPC (thicker than POPC) insertion occurs. On the other hand, the results fully support the lipid-curvature hypothesis, which states that peptides remain on the surface in membranes composed of lipids with a negative spontaneous curvature, but are more easily tilted or inserted when the lipids have a positive spontaneous curvature (13).In a special lipid mixture, POPE/POPG/TOCL (72:23:5), often used to mimic the composition of the inner membrane of Escherichia coli (15), the result is practically the same as in POPC/POPG (9:1). Also here, chemical shifts of ∼84 ppm indicate that PGLa and MAG2 are always in the S-state, both alone and as a mixture. This behavior is in accordance with the curvature hypothesis, since PE and CL both have a strong negative curvature. On the other hand, when lyso-MPC is added to DMPC to increase the positive curvature, the chemical shift of MAG2 increases to 117 ppm, indicating a more tilted orientation in the membrane with enhanced curvature compared to DMPC or DMPC/DMPG, both with and without PGLa. PGLa alone gives a somewhat larger chemical shift but stays in the T-state, whereas PGLa together with MAG2 flips again into the I-state.We can now compare the results presented here with those from our previous study on the related peptide MSI-103 (13) to find strong correlations. Fig. 2 gives an overview of all results, illustrating the peptide orientations in the different lipid systems. PGLa on its own behaves just like MSI-103 and assumes the same S-state or T-state in the same systems, in full accordance with the lipid-curvature hypothesis. MAG2 alone behaves similarly but seems to have a higher concentration threshold to flip from the S-state to the T-state. In DMPC and DMPC/DMPG, where PGLa is already in the T-state, MAG2 is still in the S-state at P/L = 1:50. However, at P/L = 1:10 (Fig. S2), MAG2 has also reached the T-state. Since MAG2 is charged at both termini, whereas PGLa and MSI-103 are amidated and thus uncharged on the C terminus, it is indeed expected that MAG2 should not start to tilt as easily as PGLa or MSI-103. The polar sector of MAG2 is also larger (Fig. S3).When PGLa and MAG2 are mixed 1:1, their behavior correlates well with that of the individual peptides. In systems where PGLa and MSI-103 are in the S-state, the mixture of PGLa and MAG2 also remains in the S-state. Only when PGLa alone prefers the T-state does it get fully pushed into the I-state by the presence of MAG2. Thus, the model of MAG2-assisted insertion of PGLa proposed previously (12), which suggested that MAG2 would facilitate a thinning of the membrane such that PGLa would be able to insert into it, cannot be correct. We can instead conclude that only lipid systems that encourage peptide insertion per se show the MAG2-induced I-state of PGLa. The relationship between lipid shape and the tendency of peptides to insert into the membrane, as previously discussed (13), is illustrated in Fig. S4. Interestingly, common bacterial lipids like PE and CL have a negative spontaneous curvature and should thus not support peptide insertion and stable pores. However, pores could still be transient in native membranes, or other components like membrane proteins could influence the overall spontaneous curvature.In conclusion, we propose several criteria that encourage a peptide to insert from the surface-bound S-state more deeply into the membrane (i.e., into a T-state or I-state): 1), positive lipid spontaneous curvature, which is enhanced by large headgroups and ordered lipid chains (due to saturation, but also found at low temperatures close to the gel-to-liquid-crystalline phase transition); 2), a narrow polar sector and uncharged termini of the peptide; and 3), the presence of another peptide. The other peptide might have an indirect effect by changing the membrane properties via crowding. However, for PGLa/MAG2, a distinct synergistic activity has been demonstrated, indicating more specific interactions between these two peptides. The present 15N-NMR analysis shows that the two partner peptides are not aligned side-by-side as a dimer. Further solid-state NMR distance measurements will be required to clarify their detailed mode of assembly.  相似文献   

4.
The aim of the study was to compare the preservative effectiveness of plant extracts (Matricaria chamomilla, Aloe vera, Calendula officinalis) and essential oils (Lavandulla officinalis, Melaleuca alternifolia, Cinnamomum zeylanicum) with methylparaben in cosmetic emulsions against skin microflora during 2 months of application by volunteers. Cosmetic emulsions with extracts (2.5 %), essential oils (2.5 %), methylparaben (0.4 %) or placebo were tested by 40 volunteers during 2 months of treatment. In order to determine microbial purity of the emulsions, the samples were taken after 0, 2, 4, 6 and 8 weeks of application. Throughout the trial period it was revealed that only cinnamon oil completely inhibited the growth of bacteria, yeast and mould, as compared to all other essential oils, plant extracts and methylparaben in the tested emulsions. This result shows that cinnamon oil could successfully replace the use of methylparaben in cosmetics, at the same time ensuring microbiological purity of a cosmetic product under its in-use and storage conditions.  相似文献   

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PurposeThe displacement hypothesis predicts that physical activity and media use compete in adolescents; however, findings are inconsistent. A more differentiated approach at determining the co-occurrence of physical activity and media use behaviors within subjects may be warranted. The aim of this study was to determine the co-occurrence of physical activity and media use by identifying clusters of adolescents with specific behavior patterns including physical activity in various settings (school, sports club, leisure time) and different types of media use (watching TV, playing console games, using PC / Internet).MethodsCross-sectional data of 2,083 adolescents (11–17 years) from all over Germany were collected between 2009 and 2012 in the Motorik-Modul Study. Physical activity and media use were self-reported. Cluster analyses (Ward’s method and K-means analysis) were used to identify behavior patterns of boys and girls separately.ResultsEight clusters were identified for boys and seven for girls. The clusters demonstrated that a high proportion of boys (33%) as well as girls (42%) show low engagement in both physical activity and media use, irrespective of setting or type of media. Other adolescents are engaged in both behaviors, but either physical activity (35% of boys, 27% of girls) or media use (31% of boys and girls) predominates. These adolescents belong to different clusters, whereat in most clusters either one specific setting of physical activity or a specific combination of different types of media predominates.ConclusionThe results of this study support to some extent the hypothesis that media use and physical activity compete: Very high media use occurred with low physical activity behavior, but very high activity levels co-occurred with considerable amounts of time using any media. There was no evidence that type of used media was related to physical activity levels, neither setting of physical activity was related to amount of media use in any pattern.  相似文献   

7.
The subsite structure of Thermoactinomyces vulgaris α-amylase was estimated from its action mode and rate parameters of hydrolysis on maltooligosaccharides. These results led to the conclusion that this α-amylase has six subsites with the catalytic site located between the third and fourth subsites from the non-reducing end side. Subsite affinities were calculated to be 0.38, 5.46, 2.72 and 0.23 kcal/mol for subsites 1, 2, 5 and 6, respectively, and the sum of the affinities of subsite 3 and 4 to be ?3.41 kcal/mol. The unique action mode of this α-amylase on various substrates was interpreted in terms of the subsite structure.  相似文献   

8.
The association between podoplanin and ezrin in the process of odontogenic tumors invasion has been suggested, but was not studied yet. Our purpose was to investigate the relationship between podoplanin and ezrin expressions in the odontogenic epithelium of ameloblastomas. Forty-seven ameloblastomas were analyzed by immunohistochemistry using anti-podoplanin and anti-ezrin antibodies. The expressions of both proteins were evaluated using a score method and the comparison and association between these proteins were verified, respectively, by Wilcoxon Signed-Rank test and by Spearman’s rank correlation coefficient, using a statistical significance level of 0.05. The majority of tumors (87.2%) exhibited strong membranous expression of podoplanin in the peripheral cells. Cytoplasmic expression of ezrin in the peripheral cells of ameloblastomas was stronger than its membranous expression. No statistically significant correlation was observed between podoplanin and ezrin. However, there was statistically significant difference between membranous podoplanin and membranous ezrin expressions, between cytoplasmic podoplanin and membranous ezrin expressions, and between cytoplasmic podoplanin and cytoplasmic ezrin expressions. There was no statistical difference between membranous podoplanin and cytoplasmic ezrin expressions. These results suggest a synergistic role of both proteins in the process of invasion of ameloblastomas.Key words: Odontogenic tumor, ameloblastoma, podoplanin, ezrin, adhesion molecule  相似文献   

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The pathogenesis of Alzheimer’s disease (AD) is associated with the aggregation of amyloid-β (Aβ) peptides into toxic aggregates with β-sheet character. In a previous computational study, we showed that pristine single-walled carbon nanotubes (SWCNTs) can inhibit the formation of β-sheet-rich oligomers in the central hydrophobic core fragment of Aβ (Aβ16–22). However, the poor solubility of SWCNTs in water hinders their use in biomedical applications and nanomedicine. Here, we investigate the influence of hydroxylated SWCNT, a water-soluble SWCNT derivative, on the aggregation of Aβ16–22 peptides using all-atom explicit-water replica exchange molecular dynamics simulations. Our results show that hydroxylated SWCNTs can significantly inhibit β-sheet formation and shift the conformations of Aβ16–22 oligomers from ordered β-sheet-rich structures toward disordered coil aggregates. Detailed analyses of the SWCNT-Aβ interaction reveal that the inhibition of β-sheet formation by hydroxylated SWCNTs mainly results from strong electrostatic interactions between the hydroxyl groups of SWCNTs and the positively charged residue K16 of Aβ16–22 and hydrophobic and aromatic stacking interactions between SWCNTs and F19 and F20. In addition, our atomic force microscopy and thioflavin T fluorescence experiments confirm the inhibitory effect of both pristine and hydroxylated SWCNTs on Aβ16–22 fibrillization, in support of our previous and present replica exchange molecular dynamics simulation results. These results demonstrate that hydroxylated SWCNTs efficiently inhibit the aggregation of Aβ16–22; in addition, they offer molecular insight into the inhibition mechanism, thus providing new clues for the design of therapeutic drugs against amyloidosis.  相似文献   

12.

Introduction

Regular physical activity has been shown to reduce cardiovascular disease risk in the general population. While smaller studies in specified groups (highly trained versus untrained individuals) indicate a certain dose-dependent effect of physical activity on the reduction of carotid stiffness (an indicator of subclinical vascular disease), it is unclear whether this association is present in a representative sample. Thus, we investigated this question cross-sectionally in participants from the population-based Swiss Cohort Study on Air Pollution And Lung and Heart Diseases In Adults (SAPALDIA).

Methods

Self-reported total, moderate and vigorous physical activity and distensibility as a measure of local arterial stiffness among 1636 participants aged 50 to 81 years without clinically manifest diseases were evaluated. Mixed regression models were used to examine associations of physical activity intensity with distensibility.

Results

Vigorous physical activity, but not total nor moderate physical activity, was significantly associated with increased distensibility (= reduced carotid stiffness) in univariate analyses (percent change in the geometric mean and 95% confidence interval per 1 standard deviation increment in vigorous physical activity = 2.54 (0.69; 4.43), p<0.01; in total physical activity = 1.62 (-0.22; 3.50), p = 0.08; in moderate physical activity = 0.70 (-1.12; 2.56), p = 0.45). These associations disappeared when we additionally adjusted for age.

Conclusion

After adjustment for the most important confounders and risk factors, we found no evidence for an association of physical activity with carotid stiffness in the general middle aged to elderly population.  相似文献   

13.

To form silver nanoparticles by reduction from metal ions in the presence of a reducing agent, D-glucose, a water-soluble derivative of chitosan, succinyl-chitosan, was used as a polymer matrix at room temperature. The synthesis of silver nanoparticles can also be carried out without a reducing agent by thermal activation of the system using an alkali (NaOH) as an accelerator. The presence of silver nanoparticles in the obtained colloidal solutions was judged by the appearance of an absorption band in the electron plasmon resonance spectra (?max = 417 nm). It has been shown that the use of an additional component, polyethylene oxide, in a macromolecular system makes it possible to obtain small silver nanoparticles (1–3 nm). The results of in vitro studies of the antimicrobial activity of the obtained colloidal solutions containing silver nanoparticles confirm that a decrease in the size of silver nanoparticles leads to an expansion of the spectrum of antibacterial activity of strains of gram-positive and gram-negative bacteria (B. subtilis ATCC 6633, S. aureus 209P, E. coli ATCC 25922) and to the manifestation of a pronounced antifungal action in relation to A. niger INA 00760.

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Spencer  A. N. 《Hydrobiologia》1991,216(1):565-571
A family of peptides with the carboxy-terminus Arg-Phe-amide has been localized to specific subpopulations of neurons in every cnidarian species examined. These neurons are typically sensory in character or are associated with smooth muscle. Although a transmitter role for these peptides has been suggested for anthozoans at neuromuscular synapses, no such evidence is available for hydrozoans. Instead there is evidence that RF-amides can be modulators of neuronal activity which takes the form of a biphasic (inhibitory then excitatory) response in vivo, while in vitro only the inhibitory response is seen. Voltage clamp studies of identified motor neurons showed large, transitory outward currents when Pol-RF-amide peptide was applied.  相似文献   

16.
Atomic-level structural information on αB-Crystallin (αB), a prominent member of the small heat-shock protein family, has been a challenge to obtain due its polydisperse oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on an ∼ 580-kDa human αB assembled from 175-residue 20-kDa subunits. An ∼ 100-residue α-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and Cβ resonances have been obtained for residues 64-162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-crystallin domain have similar chemical shifts in both solid-state and solution-state, indicating similar structures for the domain in its isolated and oligomeric forms. Sites of intersubunit interaction are identified from chemical shift differences that cluster to specific regions of the α-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) 1H-15N heteronuclear single quantum coherence spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer-monomer transition over the pH range 7.5-6.8. This steep pH-dependent switch may be important for αB to function optimally (e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia, which is accompanied by acidosis).  相似文献   

17.
Hauswirth et al. (1968) proposed that epinephrine acts on iKK2 by adding its own positive charge to the external membrane surface near the iKK2 channel. This hypothesis was tested by using noncationic compounds, theophylline and R07-2956, which mimicked epinephrine's effects on pacemaker activity and on iKK2. In maximally effective doses, theophylline or R07-2956 occluded the effect of epinephrine, indicating a shared final common mechanism. Since theophylline and R07-2956 are noncationic at pH 7.4, the common mechanism cannot be a direct change in external surface charge. On the contrary, epinephrine does not interfere with the voltage shift produced by La+++, which is thought to modify the external surface charge. The results argue against the original hypothesis but leave open the possibility that an alteration in internal surface charge generates the observed voltage shift. The potency of theophylline and R07-2956 as phosphodiesterase inhibitors suggests that the final common mechanism begins with the elevation of intracellular cyclic AMP, leading to a saturable process which limits the voltage shift's magnitude. This hypothesis is used to generate dose-response curves describing the combined effects of epinephrine and theophylline, and these are compared with experimental data.  相似文献   

18.
The frequency of epileptic seizures was observed in a controlled therapeutic trial on 23 epileptic inpatients before and after treatment with vitamin D2 or placebo in addition to anticonvulsant drugs. The number of seizures was reduced during treatment with vitamin D2 but not with placebo. The effect was unrelated to changes in serum calcium or magnesium. The results may support the concept that epileptics should be treated prophylactically with vitamin D.  相似文献   

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