Design of novel analogues of short antimicrobial peptide anoplin with improved antimicrobial activity

Currently, novel antibiotics are urgently required to combat the emergence of drug‐resistant bacteria. Antimicrobial peptides with membrane‐lytic mechanism of action have attracted considerable interest. Anoplin, a natural α‐helical amphiphilic antimicrobial peptide, is an ideal research template because of its short sequence. In this study, we designed and synthesized a group of analogues of anoplin. Among these analogues, anoplin‐4 composed of d ‐amino acids displayed the highest antimicrobial activity due to increased charge, hydrophobicity and amphiphilicity. Gratifyingly, anoplin‐4 showed low toxicity to host cells, indicating high bacterial selectivity. Furthermore, the mortality rate of mice infected with Escherichia coli was significantly reduced by anoplin‐4 treatment relative to anoplin. In conclusion, anoplin‐4 is a novel anoplin analogue with high antimicrobial activity and enzymatic stability, which may represent a potent agent for the treatment of infection. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthetic analogs of anoplin show improved antimicrobial activities

We present the antimicrobial and hemolytic activities of the decapeptide anoplin and 19 analogs thereof tested against methicillin‐resistant Staphylococcus aureus ATCC 33591 (MRSA), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), vancomycin‐resistant Enterococcus faecium (ATCC 700221) (VRE), and Candida albicans (ATCC 200955). The anoplin analogs contain substitutions in amino acid positions 2, 3, 5, 6, 8, 9, and 10. We use these peptides to study the effect of altering the charge and hydrophobicity of anoplin on activity against red blood cells and microorganisms. We find that increasing the charge and/or hydrophobicity improves antimicrobial activity and increases hemolytic activity. For each strain tested, we identify at least six anoplin analogs with an improved therapeutic index compared with anoplin, the only exception being Enterococcus faecium, against which only few compounds are more specific than anoplin. Both 2Nal⁶ and Cha⁶ show improved therapeutic index against all strains tested. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G¹LLKR⁵IKT⁸LL‐NH₂, it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly¹, Arg⁵, and Thr⁸ and lipophilic groups with different lengths in the N‐terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF⁵IKK⁸LL‐NH₂ exhibited high activity against Gram‐negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N‐terminus increased the antimicrobial activity against Gram‐positive and Gram‐negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α‐helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

A diphenyldiselenide derivative induces autophagy via JNK in HTB‐54 lung cancer cells

Symmetric aromatic diselenides are potential anticancer agents with strong cytotoxic activity. In this study, the in vitro anticancer activities of a novel series of diarylseleno derivatives from the diphenyldiselenide (DPDS) scaffold were evaluated. Most of the compounds exhibited high efficacy for inducing cytotoxicity against different human cancer cell lines. DPDS 2 , the compound with the lowest mean GI₅₀ value, induced both caspase‐dependent apoptosis and arrest at the G₀/G₁ phase in acute lymphoblastic leucemia CCRF‐CEM cells. Consistent with this, PARP cleavage; enhanced caspase‐2, ‐3, ‐8 and ‐9 activity; reduced CDK4 expression and increased levels of p53 were detected in these cells upon DPDS 2 treatment. Mutated p53 expressed in CCRF‐CEM cells retains its transactivating activity. Therefore, increased levels of p21^CIP1 and BAX proteins were also detected. On the other hand, DPDS 6 , the compound with the highest selectivity index for cancer cells, resulted in G₂/M cell cycle arrest and caspase‐independent cell death in p53 deficient HTB‐54 lung cancer cells. Autophagy inhibitors 3‐methyladenine, wortmannin and chloroquine inhibited DPDS 6 ‐induced cell death. Consistent with autophagy, increased LC3‐II and decreased SQSTM1/p62 levels were detected in HTB‐54 cells in response to DPDS 6 . Induction of JNK phosphorylation and a reduction in phospho‐p38 MAPK were also detected. Moreover, the JNK inhibitor SP600125‐protected HTB‐54 cells from DPDS 6 ‐induced cell death indicating that JNK activation is involved in DPDS 6 ‐induced autophagy. These results highlight the anticancer effects of these derivatives and warrant future studies examining their clinical potential.     

Treatment of mouse melanoma cells with phorbol 12-myristate 13-acetate counteracts mannosylerythritol lipid-induced growth arrest and apoptosis

Mannosylerythritol lipid (MEL), an extracellularglycolipid from yeast, induces the differentiation ofHL-60 promyelocytic leukemia cells towardsgranulocytes. We show here that MEL is also a potentinhibitor of the proliferation of mouse melanoma B16cells. Flow-cytometric analysis of the cell cycle ofMEL-treated B16 cells revealed the accumulation ofcells in the sub-G₀/G₁ phase, which is a hallmark ofcells undergoing apoptosis. Treatment of B16 cellsfor 24 h with phorbol 12-myristate 13-acetate (PMA),an activator of protein kinase C (PKC), did notinterfere with the growth and survival of the cells,but it effectively counteracted the MEL-induced growtharrest and apoptosis. The activity of PKC was reducedin B16 cells treated with MEL at a concentration atwhich MEL induced apoptosis. However, incubation withPMA in addition to MEL reversed this reduction in theactivity of PKC. These results suggest thatconverging signaling pathways are triggeredindependently by MEL and PMA and that the signalsmight both be mediated by PKC.     

Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach

Abstract. Objectives: Trabectedin (ET‐743, Yondelis^®) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)‐rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug‐induced cell cycle perturbations. Materials and Methods: An isogenic NER‐proficient cellular system (CHO‐AA8) and a NER‐deficient one (CHO‐UV‐96), lacking functional ERCC‐1, were studied. Flow cytometric assays showed progressive accumulation of cells in G₂ + M phase in NER‐proficient but not in NER‐deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. Results: Cells exposed to trabectedin during G₁ and G₂ + M first experienced a G₁ block, while those exposed in S phase were delayed in S and G₂ + M phases but eventually divided. In the presence of functional NER, exit from the G₁ block was faster; then, cells progressed slowly through S phase and were subsequently blocked in G₂ + M phase. This G₂ + M processing of trabectedin‐induced damage in NER‐proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. Conclusions: This might be due either to important damage left unrepaired by previous G₁ repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV‐96 cells repair mechanisms other than NER are activated both in G₁ and G₂ + M phases.     

Combination effect of PectaSol and Doxorubicin on viability, cell cycle arrest and apoptosis in DU-145 and LNCaP prostate cancer cell lines

The effect of PectaSol on Dox (Doxorubicin) cytotoxicity in terms of apoptosis and cell cycle changes in PCa (prostate cancer) cell lines (DU‐145 and LNCaP) has been investigated. Combination of PectaSol and Dox resulted in a viability of 29.4 and 32.6% (P<0.001) in DU‐145 and LNCaP cells. The IC₅₀ values decreased 1.5‐fold and 1.3‐fold in the DU‐145 and LNCaP cells respectively. In the DU‐145 cells, combination of PectaSol and Dox resulted in a reduction in p27 gene and protein expression (P<0.001). In LNCaP cells, this combination increased p53, p27 and Bcl‐2 expression. Treatment with both drugs in DU‐145 cells led to an increase in sub‐G₁ arrest (54.6% compared with 12.2% in Dox). In LNCaP cells, combination of the drugs led to an increased in G₂/M arrest (61.7% compared with 53.6% in Dox). Based on these findings, progressive cytotoxicity effect of Dox and PectaSol together rapidly induce cell death in DU‐145 through apoptosis and in LNCaP cells through cell cycle arrest (G₂/M arrest).     

Lignans from the Twigs of Euonymus alatus (Thunb.) Siebold and Their Biological Evaluation

A new sesquilignan, euonymolin A ( 1 ), and six known lignans, (?)‐de‐O‐methylmagnolin ( 2 ), (+)‐de‐O‐methylepimagnolin A ( 3 ), (+)‐syringaresinol ( 4 ), (+)‐pinoresinol ( 5 ), (+)‐medioresinol ( 6 ), and (+)‐lariciresinol 4′‐O‐β‐d ‐glucopyranoside ( 7 ), were isolated from the twigs of Euonymus alatus (Thunb .) Siebold (Celastraceae). The structures of the isolated compounds were elucidated based on spectroscopic analyses, including extensive 1D‐ and 2D‐NMR techniques, HR‐MS analysis and circular dichroism (CD) data, and the literature data. All of the isolated compounds were evaluated for antiproliferative activity against A549, SK‐OV‐3, SK‐MEL‐2, and HCT‐15 cell lines and inhibition of nitric oxide (NO) production in a lipopolysaccharide (LPS)‐activated BV2 cell line. All compounds showed cytotoxicity against the SK‐MEL‐2 cell line with IC₅₀ values of 23.24 – 48.14 μm and inhibited NO production in LPS‐activated BV‐2 cells with IC₅₀ values of 6.75 – 23.53 μm .     

Enantiomeric 9‐mer peptide analogs of protaetiamycine with bacterial cell selectivities and anti‐inflammatory activities

Protaetiamycine is an insect defensin, derived from the larvae of the beetle Protaetia brevitarsis. In our previous work, we designed 9‐mer peptide analogs of protaetiamycine, including 9Pbw2 (RLWLAIKRR‐NH₂), 9Pbw3 (RLWLAIWRR‐NH₂), and 9Pbw4 (RLWLAWKRR‐NH₂). 9Pbw2 and 9Pbw4 showed high antimicrobial activity without cytotoxicity, while 9Pbw3 with higher hydrophobicity compared to 9Pbw2 and 9Pbw4 showed high cytotoxicity as well as high antimicrobial activity (Shin et al., J. Pept. Sci. 2009; 15: 559–568). In this study, we investigated the anti‐inflammatory activities of 9Pbw2, 9Pbw3, and 9Pbw4 by quantitation of NO production in LPS‐stimulated RAW264.7 cells. The results showed that only 9Pbw3 has strong inhibition of NO production, implying that Trp⁷ as well as optimum level of hydrophobicity may play key roles in the anti‐inflammatory activity of 9Pbw3. In order to design potent anti‐inflammatory peptide with lower cytotoxicity as well as high stability from cleavage by protease compared to 9Pbw3, we synthesized 9Pbw3‐D , the all‐D ‐amino acid analog of 9Pbw3. 9Pbw3‐D showed less cytotoxicity against RAW264.7 cells as well as considerably stronger inhibition of NO production and inflammation‐induced cytokine production in LPS‐stimulated RAW264.7 cells than 9Pbw3. 9Pbw3‐D inhibited the gene expression of inflammatory‐induced cytokine significantly more than 9Pbw3 and showed high resistance to proteolytic digestion. Binding of 9Pbw3‐D with LPS caused higher enhancement of the FITC fluorescence as a result of its stronger interaction with LPS compared to that of 9Pbw3 and this result is in good agreement with their anti‐inflammatory activities. 9Pbw3‐D with higher anti‐inflammatory activity as well as lower cytotoxicity against mammalian cell compared to 9Pbw3 can be a potent noncytotoxic antibiotic candidates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Improving the biological activity of the antimicrobial peptide anoplin by membrane anchoring through a lipophilic amino acid derivative

The lipophilic amino acid, (S)-2-aminoundecanoic acid, was synthesized and incorporated at a number of specific positions within the peptide sequence of anoplin. These lipophilic anoplin analogs showed to be more active against Escherichia coli and Staphylococcus aureus compared to native anoplin, while the EC₅₀-value of hemolysis was at least one order of magnitude lower than the MIC values. This was in sharp contrast to the N-acylated anoplin derivative, where a gain in activity also led to a complete loss of selectivity. Thus, the incorporation of a lipophilic amino acid residue into anoplin enhanced the antimicrobial activity, while selectivity towards microbial membranes was retained.     

Ultrastructural study of the bacillary, granular and mucoid proboscidial gland cells of Riseriellus occultus (Nemertini, Heteronemertini)

Junoy, J., Montalvo, S., Roldán, C. and García‐Corrales, P. 2000. Ultrastructural study of the bacillary, granular and mucoid proboscidial gland cells of Riseriellus occultus (Nemertini, Heteronemertini). — Acta Zoologica (Stockholm) 81 : 235–242. The ultrastructure of six types (G₅‐G₁₀) of proboscidial gland cells whose cell necks emerge independently on the epithelium surface is analysed and compared with data from other nemerteans. These types differ in cytological features, as well as in the morphology of their respective secretory granules. Secretory granules of the types G₅ and G₆ have a bacillary shape, and differ from each other based on their contents and dimensions. Secretory granules of the types G₇ and G₈ are spherical to ovoid; type G₈ gland cells are monociliated, and their secretory granules contain a paracrystalline material. Types G₉ and G₁₀ gland cells are typically goblet‐shaped; secretory granules in the type G₉ have a spherical shape, contain a homogeneous electron dense material and maintain their individuality, whereas those of the G₁₀ type are elongate and have fibrillar contents, showing a tendency to fuse before they are extruded. The mucus sheet of the proboscis is responsible for lubrication of its epithelial surface. Secretion products of type G₁₀ gland cells form the background substance of this mucus, and those of the G₅ type confer stickiness to it. Type G₉ gland cells could provide the toxic component to the mucus, and type G₇ and G₈ gland cells could be concerned with the production of enzymatic secretions.     

Effect of melphalan and hyperthermia on cell cycle progression and cyclin B1 expression in human melanoma cells

We have investigated the effect of mild hyperthermia (42°C) on the cytotoxic activity of a 1 h melphalan exposure in human melanoma cell lines. Hyperthermia did not affect cell growth of any culture, but it increased, to a different extent, melphalan cytotoxicity in all cell lines, with a reduction in the IC₅₀ of 1.7 to 2.6-fold. Flow cytometric analysis showed that in normal temperature conditions melphalan caused S phase cell accumulation, which was evident only at 24 h in JR8, M14 and 2/21 cell lines and was still persistent at 72 h in 2/60 cells. Moreover, in all cell lines, the delay in S phase was paralleled, or followed, by an accumulation of cells in G₂+ M, which was transient in JR8 and M14 cells and persisted until 72 h in 2/21 and 2/60 melanoma clones. Hyperthermia caused a stabilization and prolongation of melphalan induced G₂+ M accumulation in JR8 and M14 cells. Conversely, in 2/21 and 2/60 clones, cell cycle perturbations induced by the drug were similar under normothermic or hyperthermic conditions. Specifically, in JR8, for which the maximum enhancement by hyperthermia on melphalan cytotoxicity was observed, cell accumulation in G₂+ M was still present 120 h after treatment. The accumulation was accompanied by an inhibition in the G₂ - M transition, as demonstrated by the significant reduction in the mitotic index of cells exposed to combined treatment compared to controls. Moreover, a bivariate distribution of cells stained for DNA and cyclin B₁ showed that, following melphalan and hyperthermia treatment, the fraction of cyclin B₁-expressing cells paralleled the fraction of G₂+ M phase cells, thus indicating that the inability of cells to enter mitosis was not ascribable to a reduction of cyclin B₁ expression. On the whole, our results indicate that hyperthermia can stabilize the G₂ accumulation induced by melphalan in human melanoma cells. Such a stabilization could contribute to the enhancement of melphalan cytotoxicity by heat, even though a strict correlation was not observed between the magnitude and persistence of the cell cycle perturbations and the extent of melphalan activity.     

Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells

Certain antitumor agents have recently been extracted from the roots of Salvia miltiorrhiza Bunge. The diterpene derivative, tanshinone IIA, possesses cytotoxic activity against several human carcinoma cell lines. It also inhibits invasion and metastasis of cancer cells. In the present study, we isolated tanshinone IIA from S. miltiorrhiza, and it exhibited strong growth inhibition against human cervical cancer cells in dose‐ and time‐dependent manners with a 50% cell growth inhibition value of 2.5 μg/mL (8.49 μM). Flow cytometric analysis of cell cycle progression revealed that G₂/M arrest was initiated after a 24 h exposure to the drug. It also resulted in DNA fragmentation and degradation of poly (ADP‐ribose) polymerase indicating that tanshinone IIA may be a potential antitumor agent. Furthermore, we performed a comprehensive proteomic analysis to survey global protein changes induced by tanshinone IIA treatment on HeLa cells. Significant changes in the levels of cytoskeleton proteins as well as stress‐associated proteins were observed. Immunoblot analysis and immunofluorescence staining were used to confirm the levels of protein expression. Overexpression of the vimentin rescued these tanshinone IIA‐induced events. Computational docking methods indicated that tanshinone IIA could stably bind to the β‐subunit of the microtubule protein. An interaction network analysis of these 12 proteins using MetaCore? software suggested that tanshinone IIA treatment regulated the expressions of proteins involved in apoptotic processes, spindle assembly, and p53 activation, including vimentin, Maspin, α‐ and β‐tubulin, and GRP75. Taken together, our results suggest that tanshinone IIA strongly inhibited the growth of cervical cancer cells through interfering in the process of microtubule assembly, leading to G₂/M phase arrest and sequent apoptosis. The success of this large‐scale effort was assessed by a bioinformatics analysis of proteins through predictions of protein domains and possible functional roles. The possible contributions of these proteins to the cytotoxicity of tanshinone IIA provide potential opportunities for the development of cancer therapeutics.     

Cell proliferation and cell cycle alterations in oesophageal p53‐mutated cancer cells treated with cisplatin in combination with photodynamic therapy

Objectives: The major goal of anti‐cancer therapies is selective destruction of tumour cells with minimum side effects on normal cells. Towards this aim, combination of different therapeutic modalities has been evaluated for improving control of neoplastic diseases and quality of life for the patient. Photodynamic therapy (PDT) is a procedure for treatment of various types of cancer, but its combination with other established treatments has not been evaluated in detail. We have used KYSE‐510 cells from a human oesophageal carcinoma as an in vitro model to investigate whether cisplatin (CDDP) could be combined with PDT to increase cell death with respect to single treatments. Materials and methods: p53‐mutated KYSE‐510 cells were treated with CDDP alone or in combination with PDT. Analyses of cell viability, cell cycle progression and apoptosis induction were carried out at specific times after treatments. Results: Decrease in cell viability, cell cycle arrest at the G₂/M‐ and S‐phases boundary, and apoptosis induction were observed after single and combined treatments. Conclusions: Our results show that low CDDP doses (0.25–1 μm ) induce cell mortality and cell cycle perturbation, which were more evident when given in combination with PDT, but in contrast to work of other authors no synergistic activity was found. Apoptosis occurred via intrinsic pathways in treated cells, although it did not represent the predominant mode of cell death.     

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D₄ receptor (D₄R) activation promotes light adaptation and suppresses the light‐sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light‐ and D₄R‐signaling pathways converge on the type 1 Ca²⁺/calmodulin‐stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D₄R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D₄R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D₄R, cyclic AMP levels in the dark‐adapted retina are significantly lower compared to wild‐type retina and are unresponsive to light. These changes in Drd4?/? mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity in retinal membranes compared with wild‐type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild‐type mice treated chronically with a D₄R antagonist, L‐745870. Thus, activation of D₄R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross‐talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.     

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex‐specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G₁/G₀ arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours. Results. In order to unravel the mechanisms utilized by germ cells to achieve and maintain the correct cell cycle states, we analysed the expression of a large number of cell cycle genes in purified germ cells across the crucial time of sex differentiation. Our results revealed common signalling for both XX and XY germ cell survival involving calcium signalling. A robust mechanism for apoptosis and checkpoint control was observed in XY germ cells, characterized by p53 and Atm (ataxia telangiectasia mutated) expression. Additionally, a member of the retinoblastoma family and p21 were identified, linking these factors to XY germ cell G₁/G₀ arrest. Lastly, in XX germ cells we observed a down‐regulation of genes involved in both G₁‐ and G₂‐phases of the cell cycle consistent with their entry into meiosis. Conclusion. The present study has provided a detailed analysis of cell cycle gene expression during fetal germ cell development and identified candidate factors warranting further investigation in order to understand cases of aberrant cell cycle control in these specialized cells.     

PMS‐1077, a PAF antagonist,induced differentiation of HL‐60 cells with its novel activity

The cell differentiation‐inducing effect of 2‐N,N‐diethylaminocarbonyloxymethyl‐1 ‐diphenylmethyl‐4‐(3,4,5‐trimethoxybenzoyl) piperazine, hydrochloride (PMS‐1077) was determined in human leukaemic HL‐60 cells with profiling of cell proliferation, analysis of cell cycling, characterization of expression of various CD molecules and determination of phagocytotic activity of differentiated HL‐60 cells. After treatment with PMS‐1077, HL‐60 cells exhibited a decreased cell viability during which cell cycle was arrested in G₀‐/G₁‐phase. Flow cytometric analysis showed CD11b and CD14 were up‐regulated, whereas CD15 was unaffected. Together with the finding that PMS‐1077‐treated HL‐60 cells exhibited activities of differentiation by examining their ability of phagocytosing latex beads, an antiproliferative effect and a differentiation‐inducing role were determined for PMS‐1077 in HL‐60 cells.     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G₂/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G₁/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G₁ phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

Role of voltage‐gated potassium channels in the fate determination of embryonic stem cells

Embryonic stem cells (ESCs) possess two unique characteristics: self‐renewal and pluripotency. In this study, roles of voltage‐gated potassium channels (K_v) in maintaining mouse (m) ESC characteristics were investigated. Tetraethylammonium (TEA⁺), a K_v blocker, attenuated cell proliferation in a concentration‐dependent manner. Possible reasons for this attenuation, including cytotoxicity, cell cycle arrest and differentiation, were examined. Blocking K_v did not change the viability of mESCs. Interestingly, K_v inhibition increased the proportion of cells in G₀/G₁ phase and decreased that in S phase. This change in cell cycle distribution can be attributed to cell cycle arrest or differentiation. Loss of pluripotency as determined at both molecular and functional levels was detected in mESCs with K_v blockade, indicating that K_v inhibition in undifferentiated mESCs directs cells to differentiate instead of to self‐renew and progress through the cell cycle. Membrane potential measurement revealed that K_v blockade led to depolarization, consistent with the role of K_v as the key determinant of membrane potential. The present results suggest that membrane potential changes may act as a “switch” for ESCs to decide whether to proliferate or to differentiate: hyperpolarization at G₁ phase would favor ESCs to enter S phase while depolarization would favor ESCs to differentiate. Consistent with this notion, S‐phase‐synchronized mESCs were found to be more hyperpolarized than G₀/G₁‐phase‐synchronized mESCs. Moreover, when mESCs differentiated, the differentiation derivatives depolarized at the initial stage of differentiation. This investigation is the first study to provide evidence that K_v and membrane potential affect the fate determination of ESCs. J. Cell. Physiol. 224:165–177, 2010 © 2010 Wiley‐Liss, Inc.