Effects of exogenous melatonin on body mass and thermogenesis in red-backed vole (Eothenomys miletus) between Kunming and Dali regions.

Melatonin (MEL) is an indole hormone synthesized and secreted by the pineal gland at night, which is involved in the regulation of body mass and thermogenesis in small mammals. To test the effects of exogenous MEL on body mass and thermogenic ability in two different red-backed vole (Eothenomys miletus) populations from two different regions (Kunming [KM] and Dali [DL]) with different annual variation in climatic variables, such as temperature, sunshine and rainfall. we traced the changes of energy balance in E. miletus from KM and DL, which were placed at 25 ± 1°C with photoperiod of 12 L:12 D, intraperitoneal injection of MEL was performed daily for 28 days. The results showed that body mass and food intake were significantly decreased, while resting metabolic rate (RMR) and nonshivering thermogenesis (NST) were significantly increased after MEL injection; Contents of total protein, mitochondrial protein, the activities of cytochrome C oxidase (COX) and α-glycerophosphate oxidase (α-PGO) in liver and brown adipose tissue (BAT) were enhanced; the activity of thyroxin 5'-deiodinase (T4 5'-DII) and uncoupling protein 1 (UCP1) in BAT were also increased. Serum leptin, triiodothyronine (T3 ) levels and T3 /T4 ratio were significantly increased, thyroxine (T4 ) levels was significantly decreased. Moreover, body mass and food intake in E. miletus from KM were higher than those from DL, but RMR and NST were lower than those from DL. Changes of body mass, food intake and thermogenic activity of KM were higher than those of DL when exposed to injection of MEL, indicating that E. miletus in KM were more sensitive to MEL. Furthermore, MEL was involved in the regulation of body mass and thermogenesis in E. miletus between KM and DL.

Metabonomics of white adipose tissue and brown adipose tissue in Tupaia belangeri during cold acclimation.

In the present study, liquid chromatography-mass spectrometer (LC-MS) was used to perform untargeted metabolomics analysis of white adipose tissue (WAT) and brown adipose tissue (BAT) in Tupaia belangeri during cold acclimation. Differences in biochemical composition between WAT and BAT were compared. Clarifying how the two adipose tissues respond to the lower temperature in terms of metabolomics, which elucidate the metabolic process and energy homeostasis regulation mechanism in T. belangeri. The results showed that there were 34, 59 and 20 differential metabolites in the WAT, BAT and WAT compared with BAT, respectively. WAT and BAT had significant differences in various metabolic pathways such as sugar metabolism, amino acid metabolism, lipid metabolism, and nucleotide metabolism, which were closely related to the different biological roles of the two tissues. Increasing the concentrations of intermediate products of tricarboxylic acid (TCA) cycle, pyruvic acid, and phosphoenolpyruvic acid (PEP) in WAT and increasing the metabolites in TCA cycle, glyoxylate and dicarboxylate metabolism pathways in BAT, likely to increase the thermogenic capacity in T. belangeri in response to cold stress. There were more differential metabolic pathways in BAT during cold acclimation than that of in WAT. Moreover, compared to WAT, BAT responds to cold stress by adjusting the concentration of nucleotide metabolites.

Evidence for the 'rate-of-living' hypothesis between mammals and lizards, but not in birds, with field metabolic rate.

Longevity, an important life-history trait, is determined by extrinsic and/or intrinsic causing mortality. Here, we used body mass (BM), field metabolic rate (FMR), longevity, and female maturity data reported from 300 amniote species to test whether 1) longevity was related to BM, FMR and female maturity, and 2) FMR, female maturity, or both, had a direct effect on longevity and whether an indirect effect of FMR on female maturity improved model fit. The results showed that BM was positively correlated with longevity and FMR, but negatively correlated with mass-specific FMR (mFMR) in amniotes. Phylogenetic confirmatory path analysis showed that, in the best model, longevity had a direct negative correlation with mFMR in lizards, and an indirect negative correlation with mFMR through female maturity in mammals. However, longevity had a direct positive correlation with mFMR in birds. Furthermore, longevity was positively correlated with female maturity in endotherms (birds and mammals) but weakly correlated with female maturity in ectotherms (lizards). Thus, our results are consistent with the life-history theory and the "rate-of-living" hypothesis in lizards and mammals but not support them in birds.

Metabolomics of Eothenomys miletus from five Hengduan Mountains locations in summer.

Climatic characteristics of Hengduan Mountains region were diverse, and Eothenomys miletus was a native species throughout this region. To investigate adaptive strategies of E. miletus to environmental factors in different locations in this region, five locations were selected, including Deqin (DQ), Xianggelila (XGLL), Lijiang (LJ), Jianchuan (JC) and Ailaoshan (ALS). Then, body mass, visceral organ masses, and serum and liver metabolomes of E. miletus from each location were examined. The results showed that body mass was significantly different among these five sites. Liver mass was lower in ALS than in other locations. PLS-DA analysis, metabolite tree maps and heat maps of serum and liver metabolites showed that samples from DQ and XGLL clustered together, as did the samples from LJ, JC and ALS. Serum concentrations of lipid and amino acid metabolites, concentrations of TCA cycle intermediates, lipid metabolites and amino acid metabolites in livers from DQ and XGLL were higher than those from other three regions. However, the concentrations of glycolytic metabolites were lower in DQ and XGLL. All these results indicated that E. miletus adapts to changes in environmental temperature and altitude of this region by adjusting body mass and serum and liver metabolite concentrations.

Respiratory Capacity and Reserve Predict Cell Sensitivity to Mitochondria Inhibitors: Mechanism-Based Markers to Identify Metformin-Responsive Cancers.

Metformin has been extensively studied for its impact on cancer cell metabolism and anticancer potential. Despite evidence of significant reduction in cancer occurrence in diabetic patients taking metformin, phase II cancer trials of the agent have been disappointing, quite possibly because of the lack of molecular mechanism-based patient stratification. In an effort to identify cancers that are responsive to metformin, we discovered that mitochondria respiratory capacity and respiratory reserve, which vary widely among cancer cells, correlate strongly to metformin sensitivity in both the in vitro and in vivo settings. A causal relationship between respiratory function and metformin sensitivity is demonstrated in studies in which we lowered respiratory capacity by either genetic knockdown or pharmacologic suppression of electron transport chain components, rendering cancer cells more vulnerable to metformin. These findings led us to predict, and experimentally validate, that metformin and AMP kinase inhibition synergistically suppress cancer cell proliferation.

Role of thermal physiology and bioenergetics on adaptation in tree shrew (Tupaia belangeri): the experiment test.

Ambient conditions, as temperature and photoperiod, play a key role in animals' physiology and behaviors. To test the hypothesis that the maximum thermal physiological and bioenergetics tolerances are induced by extreme environments in Tupaia belangeri. We integrated the acclimatized and acclimated data in several physiological, hormonal, and biochemical markers of thermogenic capacity and bioenergetics in T. belangeri. Results showed that T. belangeri increased body mass, thermogenesis capacity, protein contents and cytochrome c oxidase (COX) activity of liver and brown adipose tissue in winter-like environments, which indicated that temperature was the primary signal for T. belangeri to regulate several physiological capacities. The associated photoperiod signal also elevated the physiological capacities. The regulations of critical physiological traits play a primary role in meeting the survival challenges of winter-like condition in T. belangeri. Together, to cope with cold, leptin may play a potential role in thermogenesis and body mass regulation, as this hormonal signal is associated with other hormones. The strategies of thermal physiology and bioenergetics differs between typical Palearctic species and the local species. However, the maximum thermal physiology and bioenergetic tolerance maybe is an important strategy to cope with winter-like condition of T. belangeri.

Inhibition of Isoprenylcysteine Carboxylmethyltransferase Induces Cell-Cycle Arrest and Apoptosis through p21 and p21-Regulated BNIP3 Induction in Pancreatic Cancer.

Pancreatic cancer remains one of the most difficult to treat human cancers despite recent advances in targeted therapy. Inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that posttranslationally modifies a group of proteins including several small GTPases, suppresses proliferation of some human cancer cells. However, the efficacy of ICMT inhibition on human pancreatic cancer has not been evaluated. In this study, we have evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition. In these cells, ICMT suppression inhibited proliferation and induced apoptosis. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both a small-molecule inhibitor and shRNA-targeting ICMT. Mechanistically, we found that, in sensitive pancreatic cancer cells, ICMT inhibition induced mitochondrial respiratory deficiency and cellular energy depletion, leading to significant upregulation of p21. Furthermore, we characterized the role of p21 as a regulator and coordinator of cell signaling that responds to cell energy depletion. Apoptosis, but not autophagy, that is induced via p21-activated BNIP3 expression accounts for the efficacy of ICMT inhibition in sensitive pancreatic cancer cells in both in vitro and in vivo models. In contrast, cells resistant to ICMT inhibition demonstrated no mitochondria dysfunction or p21 signaling changes under ICMT suppression. These findings not only identify pancreatic cancers as potential therapeutic targets for ICMT suppression but also provide an avenue for identifying those subtypes that would be most responsive to agents targeting this critical enzyme. Mol Cancer Ther; 16(5); 914-23. ©2017 AACR.

Forkhead box protein O3 transcription factor negatively regulates autophagy in human cancer cells by inhibiting forkhead box protein O1 expression and cytosolic accumulation.

FoxO proteins are important regulators in cellular metabolism and are recognized to be nodes in multiple signaling pathways, most notably those involving PI3K/AKT and mTOR. FoxO proteins primarily function as transcription factors, but recent study suggests that cytosolic FoxO1 participates in the regulation of autophagy. In the current study, we find that cytosolic FoxO1 indeed stimulates cellular autophagy in multiple cancer cell lines, and that it regulates not only basal autophagy but also that induced by rapamycin and that in response to nutrient deprivation. These findings illustrate the importance of FoxO1 in cell metabolism regulation independent of its transcription factor function. In contrast to FoxO1, we find the closely related FoxO3a is a negative regulator of autophagy in multiple cancer cell lines, a previously unrecognized function for this protein, different from its function in benign fibroblast and muscle cells. The induction of autophagy by the knockdown of FoxO3a was found not to be mediated through the suppression of mTORC1 signaling; rather, the regulatory role of FoxO3a on autophagy was determined to be through its ability to transcriptionally suppress FoxO1. This complicated interplay of FoxO1 and FoxO3a suggests a complex checks- and balances-relationship between FoxO3a and FoxO1 in regulating autophagy and cell metabolism.

Variations in thermal physiology and energetics of the tree shrew (Tupaia belangeri) in response to cold acclimation.

Variations in environmental factors instigate significant changes in the physiology and behavior of animals, necessary for their survival. The present study investigated the hypothesis that ambient temperature is a cue capable of inducing changes in body mass, energy intake, and thermogenic capacity. Moreover, the current study determined the potential role of leptin in regulating adaptive thermogenesis in tree shrews (Tupaia belangeri). The tree shrew was chosen as the experimental animal as they inhabit a wide area of Asia and must acclimatize to the cold. Animals were subjected to either 5° C over 28 days to simulate cold acclimation, or maintained under the original climate of room temperature. At 28 days cold-acclimatized shrews had increased body mass by 9.41 g compared to controls. The increase in body mass was found primarily to be due to growth of the digestive organs, combined with significantly increased food intake. Under cold acclimation, uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) was significantly elevated, while serum leptin concentration was significantly depressed below control levels. Serum leptin concentration was negatively correlated with body mass, energy intake, and thermogenic capacity during cold acclimation. In summary, these findings indicate that tree shrews adjust energy intake, thermogenic capacity, and body reserves in response to the cold, and further suggest that circulating leptin may act as a key signaling protein to regulate these adaptations.

A role for Rac3 GTPase in the regulation of autophagy.

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

Cell selectivity and mechanism of action of short antimicrobial peptides designed from the cell-penetrating peptide Pep-1.

Pep-1-K (PK) is a good cell-selective antimicrobial peptide designed from cell-penetrating peptide Pep-1. To develop novel short antimicrobial peptides with higher cell selectivity and shorter length compared with PK, several PK analogs were designed by the deletion, addition and/or substitution of amino acids. Among these analogs, PK-12-KKP (KKPWWKPWWPKWKK) showing the sequence and structure homology with a Trp/Pro-rich natural antimicrobial peptide, indolicidin (IN), displayed a 20-fold higher cell selectivity as compared to IN. Circular dichroism analysis revealed that PK-12-KKP adopts a folded structure combined with some portions of unordered structure. PK-12-KKP selectively binds to negatively charged bacterial membrane-mimetic vesicles, and its high phospholipid selectivity corresponds well with its high cell selectivity. Moreover, it showed very weak potential in depolarization of the cytoplasmic membrane of Staphylococcus aureus at 8 microM (4x minimal inhibitory concentration) and dye leakage from negatively charged liposomes. These results suggest that the ultimate target of our designed PK-12-KKP maybe the intracellular components (e.g. protein, DNA or RNA) rather than the cytoplasmic membranes. Collectively, our designed short Trp/Pro-rich peptide, PK-12-KKP, appears to be an excellent candidate for future development as a novel antimicrobial agent.

Effects of dimerization of the cell-penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action.

The cell-penetrating peptide Tat (48-60) (GRKKRRQRRRPPQ) derived from HIV-1 Tat protein showed potent antibacterial activity (MIC: 2-8 microM). To investigate the effect of dimerization of Tat (48-60) analog, [Tat(W): GRKKRRQRRRPWQ-NH(2)], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di-Tat(W)-C and di-Tat(W)-K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial-membrane-mimicking egg yolk L-alpha-phosphatidylethanolamine/egg yolk L-alpha-phosphatidyl-DL-glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane-targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents.

Antimicrobial and cytolytic activities and plausible mode of bactericidal action of the cell penetrating peptide penetratin and its lys-linked two-stranded peptide.

The cell penetrating peptide, penetratin (RQIKIWFQNRRMKWKK-NH2) showed potent antimicrobial activity (MIC: 0.5-4 microM) without any cytotoxicity against mammalian cells. This study investigated the effect of linking together two peptide chains of penetratin on antimicrobial and cytolytic activities and plausible mode of bactericidal action. Two-stranded penetratin was prepared by a simultaneous solid-phase synthesis of the two strands of a single lysine residue attached to the solid support. Two-stranded penetratin markedly increased cytolytic activity against human erythrocytes and NIH-3T3 mouse fibroblast cells without a significant effect on antimicrobial activity. This finding suggested that penetratin is active as a monomer to bacterial cells but as an oligomer to mammalian cells. Circular dichroism analysis revealed that the alpha-helical content of the membrane-bound penetratin was unaffected by two-stranded Lys-linkage. Penetratin had very weak ability in the depolarization of membrane potential of intact Staphylococcus aureus cells and the fluorescent dye leakage of calcein-entrapped negatively charged bacterial membrane-mimicking vesicles. In contrast, two-stranded penetratin significantly caused membrane depolarization and dye leakage. These results suggest that the two-stranded penetratin induces a significant change in its mode of bactericidal action from the intracellular-target mechanism to the membrane-targeting mechanism.

Cell selectivity of an antimicrobial peptide melittin diastereomer with D-amino acid in the leucine zipper sequence.

Melittin (ME), a linear 26-residue non-cell-selective antimicrobial peptide, displays strong lytic activity against bacterial and human red blood cells. To design ME analogue with improved cell selectivity, we synthesized a melittin diastereomer (ME-D) with D-amino acid in the leucine zipper sequence (Leu-6, Lue-13 and Ile-20). Compared to ME, ME-D exhibited the same or 2-fold higher antibacterial activity but 8-fold less hemolytic activity. Circular dichroism analysis revealed that ME-D has much less alpha-helical content in alpha-helical content in the presence of zwitterionic EYPC/cholesterol (10 : 1, w/w) liposomes compared to negatively charged EYPE/EYPG (7 : 3, w/w) liposomes. The blue shift of the fluorescence emission maximum of ME-D in zwitterionic EYPC/ cholesterol (10 : 1, w/w) liposomes was much smaller than in negatively charged EYPE/EYPG (7 : 3, w/w) liposomes. These results suggested that the improvement in therapeutic index/cell selectivity of ME-D is correlated with its less permeability to zwitterionic membranes.

Cathelicidin-derived Trp/Pro-rich antimicrobial peptides with lysine peptoid residue (Nlys): therapeutic index and plausible mode of action.

Recently, we designed a novel cell-selective antimicrobial peptide (TPk) with intracellular mode of action from Pro --> Nlys (Lys peptoid residue) substitution in a noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptide, tritrpticin-amide (TP; VRRFPWWWPFLRR-NH(2)) (Biochemistry 2006; 45: 13007-13017). In this study, to elucidate the effect of Pro --> Nlys substitution on therapeutic index and mode of action of other noncell-selective cathelicidin-derived Trp/Pro-rich antimicrobial peptides and develop novel short antimicrobial peptides with high cell selectivity/therapeutic index, we synthesized Nlys-substituted antimicrobial peptides, TPk, STPk and INk, in which all proline residues of TP, symmetric TP-analogue (STP; KKFPWWWPFKK-NH(2)) and indolicidin (IN; ILPWKWPWWPWRR-NH(2)) were replaced by Nlys, respectively. Compared to parent Pro-containing peptides (TP, STP and IN), Nlys substituted peptides (TPk, STPk and Ink) had 4- to 26-fold higher cell selectivity/therapeutic index. Parent Pro-containing peptides induced a significant depolarization of the cytoplasmic membrane of intact Staphylococcus aureus at their MIC, whereas Nlys-substituted antimicrobial peptides did not cause visible membrane depolarization at their MIC. These results suggest that the antibacterial action of Nlys-substituted peptides is probably not due to the disruption of bacterial cytoplasmic membranes but the inhibition of intracellular components. Taken together, our results showed that Pro --> Nlys substitution in other noncell-selective Trp/Pro-rich antimicrobial peptides such as STP and IN as well as TP can improve the cell selectivity/therapeutic index and change the mode of antibacterial action from membrane-disrupting to intracellular targeting. In conclusion, our findings suggested that Pro --> Nlys substitution in noncell-selective Trp/Pro-rich antimicrobial peptides is a promising method to develop cell-selective antimicrobial peptides with intracellular target mechanism.

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action.

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.

Solution structure and cell selectivity of piscidin 1 and its analogues.

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure that was isolated from the mast cells of hybrid striped bass [Silphaduang, U., and Noga, E. J. (2001) Nature 414, 268-269]. Pis-1 is not selective for bacterial versus mammalian cells. In the present study, to develop novel antibiotic peptides with selectivity for bacterial cells, we examined the effect of substituting two glycine residues, Gly8 and Gly13, with Ala or Pro on this peptide's structure and biological activities. The bacterial cell selectivity of the peptides decreased in the following order: Gly-->Pro analogues > Gly-->Pro/Ala analogues > Pis-1 > Gly-->Ala analogues. The antimicrobial and hemolytic activities and abilities to permeabilize the model phospholipid membranes were higher for Pis-1 with Gly or Pro at position 8 than for its counterparts with either Gly or Pro at position 13. We determined the tertiary structure of Pis-1 and its analogues in the presence of SDS micelles by NMR spectroscopy. We found that Pis-1 has an alpha-helical structure from Phe2 to Thr21. Also, Pis-1 AA (Gly8, Gly13-->Ala8, Ala13) with higher antibacterial and hemolytic activity than Pis-1 has a stable alpha-helical structure from Phe2 to Thr21. Pis-1 PG (Gly-->Pro8) with bacterial cell selectivity has a hinge structure at Pro8, which provides flexibility in piscidin, followed by a three-turn helix from Val10 to Gly22 in the C-terminal region. Taken together, our results demonstrate that the conformational flexibility provided by introduction of a Pro at position 8, coupled with the primary anchoring of phenylalanines and histidines in the N-terminus to the cell membrane and the optimal length of the C-terminal amphipathic alpha-helix, are the critical factors that confer antibacterial activity and bacterial cell selectivity to Pis-1 PG. Pis-1 PG may be a good candidate for the development of a new drug with potent antibacterial activity but without cytotoxicity.

Solution structures and biological functions of the antimicrobial peptide, arenicin-1, and its linear derivative.

Arenicin-1 (AR-1) is a novel antimicrobial peptide that was isolated from coelomocytes of the marine polychaeta lugworm Arenicola marina and shown to contain a single disulfide bond between Cys3 and Cys20, forming an 18-residue ring [Ovchinnikova, T. V. et al., FEBS Lett 2004, 577, 209-214]. To determine the role of this disulfide bond, we synthesized AR-1 (RWCVYAYVRVRGVLVRYRRCW) and its linear derivative, arenicin-1-S (AR-1-S: RWSVYAYVRVRGVLVRYRRSW). Activity assays revealed that AR-1-S is somewhat less active against bacterial cells than AR-1. Both peptides were very hydrophobic, and displayed cytotoxicity against human red blood cells. Analysis of the tertiary structures of AR-1 and AR-1-S by NMR spectroscopy disclosed that AR-1 has two-stranded antiparallel beta-sheet structures with amphipathicity, while AR-1-S displays a random coil structure in DMSO. Our biological data for AR-1 and AR-1-S indicate that the hydrophobic-hydrophilic balance, disulfide bridge, and the amphipathic beta-sheet structure of the peptides play important roles in their biological activities. Elucidation of the structure of AR-1 and its derivative should facilitate the design of novel non-cytotoxic peptide antibiotics with potent antibacterial activities.

Effects of Pro --> peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide.

To investigate the effect of Pro --> peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --> Nphe (TPf) or Pro --> Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --> Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --> Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.

Improvement of bacterial cell selectivity of melittin by a single Trp mutation with a peptoid residue.

To design melittin (ME) analogues that are not cytotoxic against mammalian cells but which possessing potent antimicrobial activity, we synthesized a ME analogue (ME-w) in which the Trp-19 residue of ME was replaced by a Trp-peptoid residue (Nhtrp). ME-w exhibited similar antimicrobial activity compared to ME against the tested six bacteria and C. albicans. However, it was much less cytotoxic against the hRBCs and HeLa and NIH-3T3 cells than ME. Tryptophan fluorescence and CD spectra revealed that the Trp-19 --> Nhtrp substitution in ME contributed to a much lower helical assembly in an aqueous environment and structural flexibility and exterior localization to zwitterionic membrane which modulates its selectivity toward bacterial cells.