Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis.

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3ß-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.

The effects of magainin 2-derived and rationally designed antimicrobial peptides on Mycoplasma pneumoniae.

Combating the spread of antimicrobial resistance (AMR) among bacteria requires a new class of antimicrobials, which desirably have a narrow spectrum because of their low propensity for the spread of AMR. Antimicrobial peptides (AMPs), which target the bacterial cell membrane, are promising seeds for novel antimicrobials because the cell membrane is essential for all cells. Previously, we reported the antimicrobial and haemolytic effects of a natural AMP, magainin 2 (Mag2), isolated from the skin of Xenopus laevis (the African clawed frog), four types of synthesised Mag2 derivatives, and three types of rationally designed AMPs on gram-positive and gram-negative bacteria. To identify novel antimicrobial seeds, we evaluated the effect of AMPs on Mycoplasma pneumoniae, which also exhibits AMR. We also evaluated the antimicrobial effects of an AMP, NK2A, which has been reported to have antimicrobial effects on Mycoplasma bovis, in addition to Mag2 and previously synthesised seven AMPs, on four strains of M. pneumoniae using colorimetric, biofilm, and killing assays. We found that three synthesised AMPs, namely 17base-Ac6c, 17base-Hybrid, and Block, had anti-M. pneumoniae (anti-Mp) effect at 8-30 µM, whereas others, including NK2A, did not have any such effect. For the further analysis, the membrane disruption activities of AMPs were measured by propidium iodide (PI) uptake assays, which suggested the direct interaction of AMPs to the cell membrane basically following the colorimetric, biofilm, and killing assay results. PI uptake assay, however, also showed the NK2A strong interaction to cell membrane, indicating unknown anti-Mp determinant factors related to the peptide sequences. Finally, we conclude that anti-Mp effect was not simply determined by the membrane disruption activities of AMPs, but also that the sequence of AMPs were important for killing of M. pneumoniae. These findings would be helpful for the development of AMPs for M. pneumoniae.

Rspo2 inhibits TCF3 phosphorylation to antagonize Wnt signaling during vertebrate anteroposterior axis specification.

The Wnt pathway activates target genes by controlling the ß-catenin-T-cell factor (TCF) transcriptional complex during embryonic development and cancer. This pathway can be potentiated by R-spondins, a family of proteins that bind RNF43/ZNRF3 E3 ubiquitin ligases and LGR4/5 receptors to prevent Frizzled degradation. Here we demonstrate that, during Xenopus anteroposterior axis specification, Rspo2 functions as a Wnt antagonist, both morphologically and at the level of gene targets and pathway mediators. Unexpectedly, the binding to RNF43/ZNRF3 and LGR4/5 was not required for the Wnt inhibitory activity. Moreover, Rspo2 did not influence Dishevelled phosphorylation in response to Wnt ligands, suggesting that Frizzled activity is not affected. Further analysis indicated that the Wnt antagonism is due to the inhibitory effect of Rspo2 on TCF3/TCF7L1 phosphorylation that normally leads to target gene activation. Consistent with this mechanism, Rspo2 anteriorizing activity has been rescued in TCF3-depleted embryos. These observations suggest that Rspo2 is a context-specific regulator of TCF3 phosphorylation and Wnt signaling.

Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence.

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

Highly synergistic antimicrobial activity of magainin 2 and PGLa peptides is rooted in the formation of supramolecular complexes with lipids.

Magainin 2 and PGLa are cationic, amphipathic antimicrobial peptides which when added as equimolar mixture exhibit a pronounced synergism in both their antibacterial and pore-forming activities. Here we show for the first time that the peptides assemble into defined supramolecular structures along the membrane interface. The resulting mesophases are quantitatively described by state-of-the art fluorescence self-quenching and correlation spectroscopies. Notably, the synergistic behavior of magainin 2 and PGLa correlates with the formation of hetero-domains and an order-of-magnitude increased membrane affinity of both peptides. Enhanced membrane association of the peptide mixture is only observed in the presence of phophatidylethanolamines but not of phosphatidylcholines, lipids that dominate bacterial and eukaryotic membranes, respectively. Thereby the increased membrane-affinity of the peptide mixtures not only explains their synergistic antimicrobial activity, but at the same time provides a new concept to increase the therapeutic window of combinatorial drugs.

Overexpression of Lin28a delays Xenopus metamorphosis and down-regulates albumin independently of its translational regulation domain.

BACKGROUND: Lin28 regulates stem cell biology and developmental timing. At the molecular level Lin28 inhibits the biogenesis of the micro RNA let-7 and directly controls the transcription and translation of several genes. In Xenopus, Lin28 overexpression delays metamorphosis and affects the expression of genes of the thyroid hormone (TH) axis. The TH carrier albumin, synthesized by the liver, is down-regulated in limbs and tail after Lin28 overexpression. The molecular mechanisms underlying the interaction between Lin28, let-7, and the hypothalamus-pituitary-thyroid gland (HPT) axis are unknown. RESULTS: We found that precursor and mature forms of let-7 increase during Xenopus metamorphosis. In the liver, lin28b is down-regulated and albumin is up-regulated during metamorphosis. Overexpression of a truncated form of Lin28a (Lin28aΔC), which has been shown not to interact with RNA helicase A to regulate translation, delays metamorphosis, indicating that the translational regulation domain is not required to inhibit the HPT axis. Importantly, both full length Lin28a and Lin28aΔC block the increase of albumin mRNA in the liver independently of changes in TH signaling. CONCLUSIONS: These results suggest that Lin28 delays metamorphosis through regulation of let-7 and that the decrease of the TH carrier albumin is one of the early changes after Lin28 overexpression.

Molecular and functional characterization of a short-type peptidoglycan recognition protein, PGRP-S in the amphibian Xenopus laevis.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the function of frog PGRPs. In this study, a short-type PGRP (termed Xl-PGRP-S) gene was identified in the African clawed frog, Xenopus laevis. The predicted protein of Xl-PGRP-S contains several structural features known in PGRPs, including a typical PGRP domain and two closely spaced conserved cysteines. Xl-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in muscle. As a typical PRR, Xl-PGRP-S is inducible after peptidoglycan (PGN) stimulation, and has an ability to bind PGN. In addition, Xl-PGRP-S has been proven to have Zn2+-dependent amidase activity and antibacterial activity against Edwardsiella tarda. The present study represents the first discovery on the function of frog PGRPs, thus contributing to a better understanding of the functional evolution of PGRPs in early tetrapods.

Grafted Polymer Coatings Enhance Fouling Inhibition by an Antimicrobial Peptide on Reverse Osmosis Membranes.

Bacterial biofilms that are formed on surfaces are highly detrimental to many areas of industry and medicine. Seawater desalination by reverse osmosis (RO) suffers from biofilm growth on the membranes (biofouling), which limits its widespread use because biofouling decreases water permeance and necessitates module cleaning and replacement, leading to increased economic and environmental costs. Antimicrobial peptides (AMPs) bound covalently to RO membranes inhibit biofilm growth and might delay membrane biofouling. Here we examined how various hydrophilic membrane coatings composed of zwitterionic, neutral, positively charged, and poly(ethylene glycol) (PEG)-grafted polymers affected the biocidal activity and the biofilm inhibition of a covalently bonded AMP on RO membranes. AMP magainin-2 was linked by the copper-catalyzed azide-alkyne cycloaddition reaction to a series of RO membranes that were grafted with different methacrylate polymers. Surface characterization by infrared spectroscopy, X-ray photoelectron spectroscopy, and water drop contact angle gave evidence of successful RO modifications, and zeta potential analysis reflected the increase in surface charge due to the linked, positively charged peptide. All AMP-modified membranes inhibited Pseudomonas aeruginosa growth compared to unmodified membranes, and the grafted methacrylic polymers did not significantly interfere with the peptide activity. On the other hand, membranes coated with zwitterionic and other acrylate polymers including AMP attachment inhibited biofilm growth more than either the AMP or the polymer coating alone. This enhancement led to ∼20% less biofilm biovolume on the membrane surfaces. The combination of antimicrobial coatings with polymer coatings known to resist fouling might aid future designs of surface coatings susceptible to biofilm growth.

Antibacterial and Antibiofilm Activity and Mode of Action of Magainin 2 against Drug-Resistant Acinetobacter baumannii.

Antimicrobial peptides (AMPs) are promising therapeutic agents for treating antibiotic-resistant bacterial infections. Previous studies showed that magainin 2 (isolated from African clawed fogs Xenopus laevis) has antimicrobial activity against gram-positive and gram-negative bacteria. The present study was conducted to investigate the antibacterial activity of magainin 2 against Acinetobacter baumannii. Magainin 2 showed excellent antibacterial activity against A. baumannii strains and high stability at physiological salt concentrations. This peptide was not cytotoxic towards HaCaT cells and showed no hemolytic activity. Biofilm inhibition and elimination were significantly induced in all A. baumannii strains exposed to magainin 2. We confirmed the mechanism of magainin 2 on the bacterial outer and inner membranes. Collectively, these results suggest that magainin 2 is an effective antimicrobial and antibiofilm agent against A. baumannii strains.

Efficacy of designer K11 antimicrobial peptide (a hybrid of melittin, cecropin A1 and magainin 2) against Acinetobacter baumannii-infected wounds.

Due to emergence of multidrug resistance in pathogens, the attention of the scientific community is now directed towards strengthening the reservoir of antimicrobial compounds. Prior to in vivo studies, the interaction and penetration of a hybrid peptide K11 in bacterial cells using confocal microscopy was assessed which was observed as early as 10 min after incubation with the peptide. Cell lysis along with leakage of cytoplasmic content was confirmed by electron microscopy. To evaluate the in vivo performance of the peptide, it was contained in carbopol hydrogel. Efficacy of the hydrogel formulation was then evaluated against Acinetobacter baumannii-infected wounds using a murine excision model. Treatment resulted in restoration of body weight, complete clearance of infection from the wound by day 7 and 99% wound enclosure by day 21, in contrast to the persistence of infection and 70% wound enclosure in the infected group. Further, this treatment resulted in a 2.6-fold decrease in the levels of malondialdehyde along with a 4.5-fold increase in the levels of catalase on day 3. Appearance of normal histo-architecture was observed in the treatment group. Based on these results, the peptide hydrogel can be exploited in future as one of the strategies for developing a topical anti-infective therapeutic agent.

Lipidation and conformational constraining for prolonging the effects of peptides: Xenopus glucagon-like peptide 1 analogues with potent and long-acting hypoglycemic activity.

The main disadvantages of glucagon-like peptide 1 (GLP-1) are its rapid degradation and excretion. These bottlenecks can be overcome by lipidation or other structural modification. The aim of this study was to design a series of long-acting GLP-1 analogues based on our previously discovered Xenopus GLP-1 analogs (1-3). The structure-activity relationship around lipidated 1-3 derivatives (1a-3l) with respect to in vitro potency as well as protraction was firstly explored. Compound 3g was selected for further modification. The Gly2 of 3g was replaced with Aib2, and a lactam constraint between Glu16 and Lys20 (i to i + 4) was introduced to further improve the in vivo activity and stability, affording compound 4. The receptor activation capability and in vitro stability of 4 were better than 3g and liraglutide. In addition, the hypoglycemic and insulinotropic activity of 4 was significantly better than liraglutide in db/db mice. Moreover, the enhanced in vitro stability of 4 translated into improved in vivo pharmacokinetic profiles and a prolonged antidiabetic duration. Administration of 4 twice daily for one week in diet-induced obese mice caused a significant decrease in food intake, body fat and body weight. The five-week treatment study on db/db mice of 4 further demonstrated the therapeutic effects of 4 on body weight, HbA1c and glucose tolerance. These preclinical studies demonstrate the therapeutic potential of 4 for type 2 diabetes and obesity. The present study also suggests that combined lipidation and conformational constraint strategy has potential to be used for improving the therapeutic properties of peptides.

Lithocholic Acid-Based Peptide Delivery System for an Enhanced Pharmacological and Pharmacokinetic Profile of Xenopus GLP-1 Analogs.

GLP-1 analogs suffer from the main disadvantage of a short in vivo half-life. Lithocholic acid (LCA), one of the four main bile acids in the human body, possesses a high albumin binding rate. We therefore envisioned that a LCA-based peptide delivery system could extend the half-life of GLP-1 analogs by facilitating the noncovalent binding of peptides to human serum albumin. On the basis of our previously identified Xenopus GLP-1 analogs (1-3), a series of LCA-modified Xenopus GLP-1 conjugates were designed (4a-4r), and the bioactivity studies of these conjugates were performed to identify compounds with balanced in vitro receptor activation potency and plasma stability. 4c, 4i, and 4r were selected, and their LCA side chains were optimized to further increase their stability, affording 5a-5c. Compound 5b showed a more increased albumin affinity and prolonged in vitro stability than that of 4i and liraglutide. In db/ db mice, 5b exhibited comparable hypoglycemic and insulinotropic activity to liraglutide and semaglutide. Importantly, the enhanced albumin affinity of 5b resulted in a prolonged in vivo antidiabetic duration. Finally, chronic treatment investigations of 5b demonstrated the therapeutic effects of 5b on HbA1c, body weight, blood glucose, and pancreatic endocrine deficiencies on db/ db mice. Our studies revealed 5b as a promising antidiabetic candidate. Furthermore, our study suggests the derivatization of Xenopus GLP-1 analogs with LCA represents an effective strategy to develop potent long-acting GLP-1 receptor agonists for the treatment of type 2 diabetes.

Novel antimicrobial peptide CPF-C1 analogs with superior stabilities and activities against multidrug-resistant bacteria.

As numerous clinical isolates are resistant to most conventional antibiotics, infections caused by multidrug-resistant bacteria are associated with a higher death rate. Antimicrobial peptides show great potential as new antibiotics. However, a major obstacle to the development of these peptides as useful drugs is their low stability. To overcome the problem of the natural antimicrobial peptide CPF-C1, we designed and synthesized a series of analogs. Our results indicated that by introducing lysine, which could increase the number of positive charges, and by introducing tryptophan, which could increase the hydrophobicity, we could improve the antimicrobial activity of the peptides against multidrug-resistant strains. The introduction of d-amino acids significantly improved stability. Certain analogs demonstrated antibiofilm activities. In mechanistic studies, the analogs eradicated bacteria not just by interrupting the bacterial membranes, but also by linking to DNA, which was not impacted by known mechanisms of resistance. In a mouse model, certain analogs were able to significantly reduce the bacterial load. Among the analogs, CPF-9 was notable due to its greater antimicrobial potency in vitro and in vivo and its superior stability, lower hemolytic activity, and higher antibiofilm activity. This analog is a potential antibiotic candidate for treating infections induced by multidrug-resistant bacteria.

Amphibian (Xenopus laevis) tadpoles and adult frogs mount distinct interferon responses to the Frog Virus 3 ranavirus.

Infections of amphibians by Frog Virus 3 (FV3) and other ranavirus genus members are significantly contributing to the amphibian declines, yet much remains unknown regarding amphibian antiviral immunity. Notably, amphibians represent an important step in the evolution of antiviral interferon (IFN) cytokines as they are amongst the first vertebrates to possess both type I and type III IFNs. Accordingly, we examined the roles of type I and III IFNs in the skin of FV3-challenged amphibian Xenopus laevis) tadpoles and adult frogs. Interestingly, FV3-infected tadpoles mounted type III IFN responses, whereas adult frogs relied on type I IFN immunity. Subcutaneous administration of type I or type III IFNs offered short-term protection of tadpoles against FV3 and these type I and type III IFNs induced the expression of distinct antiviral genes in the tadpole skin. Moreover, subcutaneous injection of tadpoles with type III IFN significantly extended their survival and reduced FV3 dissemination.

Physicochemical and biological characterizations of Pxt peptides from amphibian (Xenopus tropicalis) skin.

Pxt peptides (Pxt-1 through Pxt-12) have been isolated from amphibian, Xenopus tropicalis Pxt-related peptides (Pxt-2, Pxt-5, Pxt-12, reverse Pxt-2, reverse Pxt-5 and reverse Pxt-12) with significant foaming properties were further characterized. In the physicochemical experiments, all Pxt-related peptides formed significant amphiphilic α-helices in 50% 2,2,2-trifluoroethanol by circular dichroism measurements. Among Pxt-related peptides, both Pxt-5 and reverse Pxt-5 were the most effective in reducing their surface tensions. Moreover, Pxt-2, Pxt-5 and reverse Pxt-5 produced constant surface tensions above their critical association concentrations, suggesting the micelle-like assemblies. In the biological experiments, Pxt-5 possessed the most potent hemolytic activity, while reverse Pxt-5 exhibited the most remarkable gene expression of interleukin 8 and heme oxygenase 1 and the most potent cytotoxicity in HaCaT cells. In contrast, Pxt-12 and reverse Pxt-12 were much weaker in antimicrobial assays for Gram-negative bacteria, Gram-positive bacteria and yeasts, as well as in hemolytic, cell viability and cytotoxicity assays in HaCaT cells. All Pxt-related peptides exhibited about 20-50% of the total cellular histamine release at 10(-5) M, as well as mastoparan and melittin in mast cells. Real-time polymerase chain reaction analysis confirmed the gene expressions of Pxt-5 in testis and Pxt-12 in muscle, in addition to skin, while Pxt-2 was only in skin.

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro.

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFß, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.

Peptoid-Substituted Hybrid Antimicrobial Peptide Derived from Papiliocin and Magainin 2 with Enhanced Bacterial Selectivity and Anti-inflammatory Activity.

Antimicrobial peptides (AMPs) are important components of the host innate immune system. Papiliocin is a 37-residue AMP purified from larvae of the swallowtail butterfly Papilio xuthus. Magainin 2 is a 23-residue AMP purified from the skin of the African clawed frog Xenopus laevis. We designed an 18-residue hybrid peptide (PapMA) incorporating N-terminal residues 1-8 of papiliocin and N-terminal residues 4-12 of magainin 2, joined by a proline (Pro) hinge. PapMA showed high antimicrobial activity but was cytotoxic to mammalian cells. To decrease PapMA cytotoxicity, we designed a lysine (Lys) peptoid analogue, PapMA-k, which retained high antimicrobial activity but displayed cytotoxicity lower than that of PapMA. Fluorescent dye leakage experiments and confocal microscopy showed that PapMA targeted bacterial cell membranes whereas PapMA-k penetrated bacterial cell membranes. Nuclear magnetic resonance experiments revealed that PapMA contained an N-terminal α-helix from Lys(3) to Lys(7) and a C-terminal α-helix from Lys(10) to Lys(17), with a Pro(9) hinge between them. PapMA-k also had two α-helical structures in the same region connected with a flexible hinge residue at Nlys(9), which existed in a dynamic equilibrium of cis and trans conformers. Using lipopolysaccharide-stimulated RAW264.7 macrophages, the anti-inflammatory activity of PapMA and PapMA-k was confirmed by inhibition of nitric oxide and inflammatory cytokine production. In addition, treatment with PapMA and PapMA-k decreased the level of ultraviolet irradiation-induced expression of genes encoding matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in human keratinocyte HaCaT cells. Thus, PapMA and PapMA-k are potent peptide antibiotics with antimicrobial and anti-inflammatory activity, with PapMA-k displaying enhanced bacterial selectivity.

Influence of hydrophobic residues on the activity of the antimicrobial peptide magainin 2 and its synergy with PGLa.

Magainin 2 (MAG2) and PGLa are two related antimicrobial peptides found in the skin of the African frog Xenopus laevis with a pronounced synergistic activity, which act by permeabilizing bacterial membranes. To probe the influence of hydrophobic peptide-lipid and peptide-peptide interactions on the antimicrobial activity and on synergy, the sequence of MAG2 was modified by replacing single amino acids either with a small alanine or with the stiff and bulky hydrophobic 3-(trifluoromethyl)-L-bicyclopent-[1.1.1]-1-ylglycine side chain. The minimum inhibitory concentration of 14 MAG2 analogs was strongly influenced by these single substitutions: the antimicrobial activity was consistently improved when the hydrophobicity was increased on the hydrophobic face of the amphiphilic helix, while the activity decreased when the hydrophobicity was reduced. The synergy with PGLa, on the other hand, was rather insensitive to mutations of hydrophobic residues. It thus seems that the antimicrobial effect of MAG2 on its own depends strongly on the hydrophobicity of the peptide, while the synergy with PGLa does not depend on the overall hydrophobicity of MAG2.

Erythropoietin protects red blood cells from TRAIL1-induced cell death during red blood cell transition in Xenopus laevis.

In anuran amphibians, larval red blood cells (RBCs) are replaced by adult-type RBCs during metamorphosis. We previously showed that tumor necrosis factor-related apoptosis-inducing ligand 1 (TRAIL1) induces apoptosis in larval-, but not adult-type RBCs in Xenopus laevis. We also found that protein kinase C (PKC) activation is involved in establishing resistance to TRAIL1-induced apoptosis in adult-type RBCs. Here, we investigated whether erythropoietin (EPO), which induces PKC activation in mammalian erythroblasts, is involved in the RBC transition in X. laevis. RT-PCR analysis revealed that epo mRNA was upregulated in the lung, from the metamorphic climax (stage 60) onward. In an RBC culture system, EPO pretreatment significantly attenuated the TRAIL1-induced death of larval- and adult-type RBCs isolated from tadpoles and adults, probably due partly to PKC activation. In samples from froglets undergoing RBC transition, which included both larval- and adult-type RBCs, EPO exhibited a stronger protective effect on the adult-type than the larval-type RBCs. Newly differentiated RBCs isolated from tadpoles treated with a hemolytic reagent were more resistant to TRAIL1-induced cell death than non-treated controls. These results suggest that EPO functions to protect adult-type RBCs from TRAIL1-induced cell death during RBC transition, and that the protective effect might decrease as RBCs age.

Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes.

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼ 100-160 nm in diameter after 60-90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10-500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.