Structure-function relationships of antimicrobial peptides

Antimicrobial peptides are ubiquitously produced throughout nature. Many of these relatively short peptides (6-50 residues) are lethal towards bacteria and fungi, yet they display minimal toxicity towards mammalian cells. All of the peptides are highly cationic and hydrophobic. It is widely believed that they act through nonspecific binding to biological membranes, even though the exact nature of these interactions is presently unclear. High-resolution nuclear magnetic resonance (NMR) has contributed greatly to knowledge in this field, providing insight about peptide structure in aqueous solution, in organic cosolvents, and in micellar systems. Solid-state NMR can provide additional information about peptide-membrane binding. Here we review our current knowledge about the structure of antimicrobial peptides. We also discuss studies pertaining to the mechanism of action. Despite the different three-dimensional structural motifs of the various classes, they all have similar amphiphilic surfaces that are well-suited for membrane binding. Many antimicrobial peptides bind in a membrane-parallel orientation, interacting only with one face of the bilayer. This may be sufficient for antimicrobial action. At higher concentrations, peptides and phospholipids translocate to form multimeric transmembrane channels that seem to contribute to the peptide's hemolytic activity. An understanding of the key features of the secondary and tertiary structures of the antimicrobial peptides and their effects on bactericidal and hemolytic activity can aid the rational design of improved analogs for clinical use.     

Design and synthesis of amphipathic antimicrobial peptides

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic alpha-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no alpha-helicity in buffer or liposomes, exhibits a modest degree of alpha-helical structure in the presence of the alpha-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an alpha-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.     

Modulation of magainin 2-lipid bilayer interactions by peptide charge

Magainin 2, an antimicrobial peptide from Xenopus skin, assumes an amphiphilic helix when bound to acidic phospholipids, forming a pore composed of a dynamic, peptide-lipid supramolecular complex [Matsuzaki et al. (1996) Biochemistry 35, 11361-11368]. Upon the disintegration of the pore, a fraction of the peptide molecules stochastically translocates across the bilayer (Matsuzaki, et al., 1995). In order to investigate the effects of peptide charge on the magainin 2-lipid bilayer interactions, we synthesized four magainin 2 analogs with different charges (0-6+). MG0: K10E, K11E, F12W-magainin 2. MG2+: K10E, F12W-magainin 2. MG4+: F12W-magainin 2. MG6+: F12W, E19Q-magainin 2 amide. An increase in charge resulted in a stronger binding of the peptide to the negatively charged membranes, suggesting that electrostatic attractions play a crucial role in the binding process. The helical stability in a trifluoroethanol/buffer mixture was decreased with increasing positive charge because of electrostatic repulsions between the closely spaced positive side chains, whereas the helicity in the lipid bilayer was much higher and appeared to be independent of the peptide charge. However, enhanced repulsions between the highly positively charged helices destabilized the pore. Therefore, the efficiency of the most basic peptide (MG6+) to translocate across the bilayer was the greatest by virtue of the short life span of its pore and the very tight membrane binding. The charge distribution of wild-type magainin 2 was found to be so designed as to exhibit the maximal lytic activity by simultaneously achieving a strong binding and a moderate pore stability.     

Self-assembly of designed antimicrobial peptides in solution and micelles

Hydrophobic interactions are responsible for stabilizing leucine zippers in peptides containing heptad repeats. The effects of substituting leucine by phenylalanine and alanine by glycine on the self-assembly of coiled-coils were examined in minimalist antimicrobial peptides designed to form amphipathic alpha-helices. The secondary structure of these peptides was monitored in solution and in diphosphocholine (DPC) micelles using circular dichroism spectroscopy. The leucine peptides (KLAKLAK)3 and (KLAKKLA)n (n = 3, 4) become alpha-helical with increasing concentrations of salt, peptide, and DPC. The aggregation state and equilibrium constant for self-association of the peptides were measured by sedimentation equilibrium. The glycine peptide (KLGKKLG)3 does not self-associate. The leucine peptides and phenylalanine peptides (KFAKFAK)3 and (KFAKKFA)n (n = 3, 4) are in a monomer-tetramer equilibrium in solution, with the phenylalanine zippers being 2-4 kcal/mol less stable than the equivalent leucine zippers. Thermodynamic parameters for the association reaction were calculated from the temperature dependence of the association constants. Leucine zipper formation has DeltaCp = 0, whereas phenylalanine zipper formation has a small negative DeltaCp, presumably due to the removal of the larger surface area of phenylalanine from water. Self-association of the peptides is coupled to formation of a hydrophobic core as detected using 1-anilino-naphthalene-8-sulfonate fluorescence. Carboxyfluorescein-labeled peptides were used to determine the aggregation state of (KLAKKLA)3 and (KLGKKLG)3 in DPC micelles. (KLAKKLA)3 forms dimers, and (KLGKKLG)3 is a monomer. Aggregation appears to correlate with the cytotoxicity of these peptides.     

Synthetic peptides corresponding to the beta-hairpin loop of rabbit defensin NP-2 show antimicrobial activity

Mammalian defensins, a class of antibacterial peptides, are composed of 29-35 amino acids with six cysteines which form three disulfide bonds. Structural studies indicate a triple stranded beta-sheet structure with a well defined beta-hairpin loop at the C-terminal region. It is demonstrated in this report that 18 and 26 residue synthetic peptides corresponding to the beta-hairpin region, constrained by a single disulfide bond, have potent antimicrobial activity without hemolytic activity. Circular dichroism spectroscopy indicates that the single S-S bridge appears to constrain the peptides to a beta-structure. Peptides corresponding to the beta-hairpin region of defensins could thus be attractive candidates as therapeutic agents as well as good model compounds for investigation of the various physiological actions of defensins.     

Epithelial antimicrobial peptides: review and significance for oral applications

Epithelial tissues provide the first line of defense between an organism and the environment. Disruption of this barrier leads to bacterial invasion and subsequent inflammation. This is precisely the situation existing in the human oral cavity, where tissues are constantly exposed to a variety of microbial challenges that can lead to bacterially induced periodontal diseases, and to infections of the oral mucosa by bacteria, fungi, and viruses. With the recent discoveries of host-derived peptide antibiotics in mammalian mucosal epithelium, a new line of investigation is emerging to test the hypothesis that one class of these peptides, called "beta-defensins", functions to protect the host against microbial pathogenesis at these critical, confrontational sites. In that light, impairment of beta-defensin activity has recently been implicated in chronic bacterial infections in cystic fibrosis patients. The first direct evidence of expression of defensin peptides in the oral mucosa was the identification of a novel epithelial beta-defensin in mammalian tongue. It was shown to be upregulated in inflammation, suggesting that it participates in host defense. It is theorized that epithelial cell-derived antimicrobial peptides function to keep the natural flora of micro-organisms in a steady state in different niches such as the skin, the intestines, the airway, the endocervix, and the mouth. There is now evidence indicating that normal gingival epithelial cells and tissues express two beta-defensins, hBD-1 and the newly described hBD-2. In addition, a cathelin-class antimicrobial peptide, designated LL-37 and found in human neutrophils, is also expressed in skin and gingiva. It is highly likely that these and/or other epithelial antimicrobial peptides play an important role in determining the outcome of the host-pathogen interaction at the oral mucosal barrier, and that they may have important future applications in antibiotic treatment.     

Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity

Novel alpha-helical antimicrobial peptides have been devised by comparing the N-terminal sequences of many of these peptides from insect, frog and mammalian families, extracting common features, and creating sequence templates with which to design active peptides. Determination of the most frequent amino acids in the first 20 positions for over 80 different natural sequences allowed the design of one peptide, while a further three were based on the comparison of the sequences of alpha-helical antimicrobial peptides derived from the mammalian cathelicidin family of precursors. These peptides were predicted to assume a highly amphipathic alpha-helical conformation, as indicated by high mean hydrophobic moments. In fact, circular dichroism experiments showed clear transitions from random coil in aqueous solution to an alpha-helical conformation on addition of trifluoroethanol. All four peptides displayed a potent antibacterial activity against selected gram-positive and gram-negative bacteria (minimum inhibitory concentrations in the range 1-8 microM), including some antibiotic resistant strains. Permeabilization of both the outer and cytoplasmic membranes of the gram-negative bacterium, Escherichia coli, by selected peptides was quite rapid and a dramatic drop in colony forming units was observed within 5 min in time-killing experiments. Permeabilization of the cytoplasmic membrane of the gram-positive bacterium, Staphylococcus aureus, was instead initially quite slow, gathering speed after 45 min, which corresponds to the time required for significant inactivation in time-killing studies. The cytotoxic activity of the peptides, determined on several normal and transformed cell lines, was generally low at values within the minimum inhibitory concentration range.     

Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.     

Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides

Cathelicidins are a novel family of antimicrobial peptide precursors from mammalian myeloid cells. They are characterized by a conserved N-terminal region while the C-terminal antimicrobial domain can vary considerably in both primary sequence and length. Four cathelicidins, proBac5, proBac7, prododecapeptide and proBMAP-28, have been concurrently purified from bovine neutrophils, using simple and rapid methodologies. The correlation of ES-MS data from the purified proteins with their cDNA-deduced sequences has revealed several common features of their primary sequence, such as the presence of N-terminal 5-oxoproline (pyroglutamate) residues and two disulfide bridges in a 1-2, 3-4 arrangement. The N-terminal domains of the cathelicidins present one or two Asp-Pro bonds, which are particularly acid-labile in proBac5 and proBac7, but stable in prododecapeptide. This suggests that the spatial organization around these bonds may vary in different cathelicidins, and favour hydrolysis in some cases. An unexpected feature of the prododecapeptide is that it exists as dimers formed by three possible combinations of its two isoforms. The isolation of a truncated, monomeric form of this protein, lacking the cysteine-containing antimicrobial dodecapeptide, indicates that dimerization occurs via disulfide bridge formation at the level of the C-terminal domain and that the dodecapeptide is likely released as a dimer from its precursor. Sequence-based secondary structure predictions and CD results indicate for cathelicidins a 30-50% content of extended conformation and <20% content of alpha-helical conformation, with the alpha-helical segment placed near the N-terminus. Finally, similarity searching and topology-based structure prediction underline a significant sequential and structural similarity between the conserved N-terminal domain of cathelicidins and cystatin-like domains, placing this family within the cystatin superfamily. When assayed against cathepsin L, unlike the potent cystatin inhibitors, three of the four cathelicidins show only a poor inhibitory activity (Ki = 0.6-3 microM).     

Radiolabelled antimicrobial peptides for imaging of infections: a review

Risk factors suggestive of relatively late exposure to EBV have been consistently associated with Hodgkin's disease (HD) in younger adults. In addition, evidence of EBV infection has been found in the Reed-Sternberg cells themselves in about one-third to one-half of all HD cases. However, no study yet published has correlated these childhood social environment risk factors with the presence of EBV in Hodgkin's tumor cells. We examined whether EBV-positive HD occurs in those patients whose childhood environment would predispose them to relatively late exposure to EBV. The study population consisted of 102 cases of mixed cellularity (MC; n = 25) or nodular sclerosing (n = 77) HD. Samples that tested positive for either EBV-encoded RNA or latent membrane protein or both were considered EBV-positive. Of the 102 cases, 83 completed a questionnaire regarding childhood social environment. The association with EBV-positivity was estimated by the odds ratio (OR) with 95% confidence intervals (CI). Twenty-two percent of the cases were EBV-positive. These cases were more likely to be MC (OR, 6.2; CI, 2.3-16.3) and male (OR, 3.4; CI, 1.3-9.0). History of infectious mononucleosis (IM) was not predictive of EBV-positivity, with only 3 of 14 such patients being EBV-positive (P = 0.82). Contrary to our hypothesis, no association between EBV and childhood environment risk factors was identified. The association of EBV with MC histology and male gender agrees with previous reports. The most intriguing finding was the dissociation between IM history and EBV-positivity, in that almost all of the cases with a history of IM were EBV-negative.     

Detection of anionic antimicrobial peptides in ovine bronchoalveolar lavage fluid and respiratory epithelium

Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.     

Functional synergism between putative gamma-aminobutyrate-containing neurons and pyramidal neurons in prefrontal cortex

The responses of putative gamma-aminobutyratergic interneurons (fast-spiking) and pyramidal (regular-spiking) cell pairs were compared in monkeys performing visual and memory-guided oculomotor tasks. Both fast- and regular-spiking neurons had similar receptive fields, indicating that gamma-aminobutyratergic interneurons carry a specific informational signal, as opposed to providing nonspecific modulation. However, the responses of the pairs were inverted and the timing of excitatory and inhibitory responses appeared to be phased, a property consistent with gamma-aminobutyrate-mediated shaping of receptive fields. These observations (i) provide evidence that interneurons and pyramidal cells can be differentiated in vivo and (ii) begin to elucidate the role of gamma-aminobutyratergic mechanisms in cognition.     

Conformational and electrostatic similarity between polyprotonated and Ca(2+)-bound mu-opioid peptides

PS-6 unleaded gasoline (UG) and methyl tert-butyl ether (MTBE), an UG additive, with long-term exposure at high concentrations increased liver tumors selectively in female mice. PS-6 UG is a liver tumor promoter in N-nitrosodiethylamine-initiated female mice and produces short-term effects potentially relevant to its tumor promoting ability. The new formulation of UG (91-01) and MTBE were evaluated for similar short-term effects in mouse liver. Mice were exposed to 7814 ppm MTBE, 2014 ppm 91-01 UG, or 2028 ppm PS-6 UG vapor for 3 or 21 days, 6 hr/day, 5 day/week. Relative liver weights increased and uterine weights decreased in MTBE-, 91-01 UG-, and PS-6 UG-exposed mice. Because the decrease in relative uterine weight is suggestive of hormonal modulation, we evaluated the effects of MTBE, 91-01 UG, and PS-6 UG in vivo on hepatic 17-beta estradiol metabolism in vitro. Gavage treatment with either blend of UG and with MTBE increased estrogen metabolism in isolated mouse hepatocytes. Hepatic microsomal P450 activity was assessed by 7-pentoxyresorufin-O-dealkylase (PROD) and 7-ethoxyresorufin-O-deethylase (EROD) activities. Similar increases in P450 content and PROD and EROD activities were observed in all exposed mice as compared to controls. No hepatoxicity was observed in any treatment group. The hepatic labeling index, as measured by the incorporation of 5-bromo-2'-deoxyuridine, was increased in all exposed mice at 3 days but not 21 days, indicating that MTBE and 91-01 UG are also hepatic mitogens. These data demonstrate that a newer blend of UG and the UG additive MTBE elicit short-term effects similar to those of PS-6 UG. Given that these effects are potentially related to tumor promotion and the general lack of genotoxic activity, MTBE and 91-01 UG may exhibit tumor promoting activity similar to that seen with PS-6 UG. Since the liver is under multihormonal control, the increase in hepatic estrogen metabolism and uterine effects supports a potential role for endocrine modulations in both MTBE-and UG-induced hepatocarcinogenesis.     

Evaluation of the synergism between ketorolac and morphine in the treatment of postoperative pain

BACKGROUND: The aim of the study is to determine what concentration of ketorolac and morphine administered together i.v. achieve best synergic effect between NSAID antiinflammatory and opioids analgesic properties. DESIGN: Randomized comparative study was carried out on 180 patients, ASA II-IV, undergoing major general surgery, in an University Clinic. METHODS: Postoperative pain therapy by i.v. PCA: group 1 morphine 0.75 mg.ml + ketorolac 0.75 mg.ml; group 2 morphine 0.50 mg.ml + ketorolac 1.50 mg.ml; group 3 morphine 0.25 mg.ml + ketorolac 1.50 mg.ml; in saline solution. Initial bolus: 2 ml. Continuous infusion 1.5 ml.h. Demand bolus: 0.2 ml. Lockout time: 30 minutes. Evaluations included: pain intensity (T0, T3, T18); total amount of infused drugs (T18); number of valid demands and attempts (T18); amount of autoadministered analgesic drugs in percent of highest available amount (T18); side effects (T18); patient's judgment. DATA ANALYSIS: ANOVA and Student's "t"-test. RESULTS: A statistically significant reduction of pain intensity was found after 3 and 18 hours in the three groups, no differences were found among the groups. Group 2 required an amount of autoadministered drugs significantly lower than other groups. Rare side effects. Patient's judgment was generally positive. CONCLUSIONS: Results suggest a greater synergetic effect between morphine and ketorolac in concentrations used in group 2.     

The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.     

Preparation and antimicrobial assessment of 2-thioether-linked quinolonyl-carbapenems

This reports the synthesis and in vitro antimicrobial properties of a series of 2-thioether-linked quinolonyl-carbapenems. Although the title compounds exhibited broad spectrum activity, the MICs were generally higher than those observed for selected benchmark carbapenems, quinolonyl-penems, and quinolones. Enzyme assays suggested that the title compounds are potent inhibitors of penicillin binding proteins and inefficient inhibitors of bacterial DNA-gyrase. Uptake studies indicated that the new compounds are not substrates for the norA encoded quinolone efflux pump.     

In vitro synergism between 5-fluorouracil and natural beta interferon in human colon carcinoma cells

The combination of 5-fluorouracil (FUra) plus IFN-beta was studied in vitro using a human colon carcinoma cell line, HCT-8. Continuous exposure to high concentrations of IFN-beta is cytotoxic to these cells (ED50, 600 +/- 50 IU/ml). A strong synergism (P < 0.002) was observed when a short-term (1-h), high-concentration exposure to fluoropyrimidine (300 or 1000 microM) was followed by IFN-beta given continuously. In fact, the mean ratio between the expected (product of the survival of each agent alone) and the observed clonogenic cell survival rates of the combination was 3.4 (range, 2.4-4.9). Longer exposures to the fluoropyrimidine (24 h, 7 days) produced less than additive effects with IFN-beta, indicating strong schedule dependency for this synergism. The mechanism of interaction was studied at four levels. First, thymidylate synthase (TS) activity, inhibition, and recovery were measured by an in situ assay in cells treated with FUra, IFN-beta, and their combination. When the prolonged infusion of IFN-beta followed a 1-h exposure to FUra, the observed TS inhibition and recovery, at each time point, were very similar to the expected values, indicating that the interactions between these drugs at the level of TS are not the determinant of the synergism. Second, cell cycle analysis was done. During the continuous exposure to IFN-beta, a significant accumulation of HCT-8 cells in S-phase was observed at 24, 48, and 72 h compared to untreated controls (P = 0.003); however, under the same experimental conditions producing synergy in the clonogenic assay, no significant cell cycle perturbations were produced by the combination of FUra followed by IFN-beta compared to those caused by each agent alone. Third, using the alkaline elution test, we also demonstrated that the synergism is not due to enhanced FUra-induced DNA single-strand break frequency in high molecular weight DNA. Finally, nucleic acid incorporation experiments, using tritiated FUra, showed that the cytokine, given continuously (300 IU/ml), enhanced the amount of FUra incorporated into nucleic acids 24 h after a 1-h exposure to 300 and 1000 microM of FUra. The median percentage of enhancement values were 31.6 +/- 11.5%,m for the lower drug concentration and 18. 4 +/- 8.1% for the higher drug concentration tested. These results suggest that the mechanism of this synergism may be related to the ability of IFN-beta to promote the incorporation of intracellular FUra metabolites into nucleic acids and/or to inhibit the cleavage of FUra nucleotides from RNA/DNA.     

Ranatuerins: antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana

Nine peptides, termed ranatuerins 1-9, with antimicrobial activity towards Staphylococcus aureus, were isolated from an extract of the skin of the adult American bullfrog, Rana catesbeiana. In common with other cytolytic peptides from Ranid frogs, (e.g. ranalexin, gaegurins, brevinins), ranatuerins 1 and 4 contain an intramolecular disulfide bridge forming a heptapeptide ring whereas in ranatuerins 2 and 3 the disulfide bridge forms a hexapeptide ring. The structurally related ranatuerins 5-9 comprise 12 - 14 amino acids and show sequence similarity towards the hemolytic peptides A1 and B9 previously isolated from the skin of Rana esculenta. Of the peptides purified, ranatuerin 1 (SMLSVLKNLGKVGLG FVACKINKQC) showed the broadest spectrum of antimicrobial action with inhibitory activity against S. aureus, Escherichia coli and Candida albicans.