Expression,Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.     

Structure‐guided RP‐HPLC chromatography of diastereomeric α‐helical peptide analogs substituted with single amino acid stereoisomers

An α‐helical model peptide (Ac‐EAEKAAKE‐X‐EKAAKEAEK‐amide) was used as a template to examine the efficacy of conventional reversed‐phase high‐performance liquid chromatography (RP‐HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo‐Ile, d ‐Ile and d ‐allo‐Ile. We compared differences in peptide retention behavior on a C₈ column and a C₁₈ column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs. We also demonstrated the ability of RP‐HPLC to separate Ile‐ and allo‐Ile‐substituted analogs of a 26‐residue α‐helical antimicrobial peptide (AMP), with the substitution site towards the C‐terminus of the α‐helix. These peptides show different values of antibacterial activity and hemolytic activity, and different selectivity against bacteria and human cells. Our results underline the ability of RP‐HPLC to resolve even difficult diasteromeric peptide mixtures as well as its value in monitoring very subtle hydrophobicity changes in de novo‐designed AMP. Copyright © 2013 John Wiley & Sons, Ltd.     

蜂毒肽C末端片段的反序肽的抗菌活性和溶血活性

设计并合成了具有不同碱性氨基酸残基数和不同疏水性片段链长的基于Mel(12~26)的系列反序肽类似物.结果表明,反序肽的正电荷和疏水性对于抑菌活性都很重要,N端至少保留3个碱性氨基酸(正电荷>4)和C端的疏水性片段的链长至少为8个氨基酸残基的类似物具有较高的抑菌活性,具有较大的抑菌活性的最小反序肽类似物为具有11个氨基酸残基的RetroMel(13~23).这些反序肽的溶血活性都很小.     

Comparison on effect of hydrophobicity on the antibacterial and antifungal activities of α-helical antimicrobial peptides

HPRP-A1, a 15-mer α-helical cationic peptide, was derived from N-terminus of ribosomal protein L1 (RpL1) of Helicobacter pylori. In this study, HPRP-A1 was used as a framework to obtain a series of peptide analogs with different hydrophobicity by single amino acid substitutions in the center of nonpolar face of the amphipathic helix in order to systematically study the effect of hydrophobicity on biological activities of -helical antimicrobial peptides. Hydrophobicity and net charge of peptides played key roles in the biological activities of these peptide analogs; HPRP-A1 and peptide analogs with relative higher hydrophobicity exerted broad spectrum antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria and pathogenic fungi, but also showed stronger hemolytic activity; the change of hydrophobicity and net charge of peptides had similar effects with close trend and extent on antibacterial activities and antifungal activities. This indicated that there were certain correlations between the antibacterial mode of action and the antifungal mode of action of these peptides in this study. The peptides exhibited antimicrobial specificity for bacteria and fungi, which provided potentials to develop new antimicrobial drugs for clinical practices.     

Synthesis and Antibacterial Properties of Oligomeric Dehydrogenation Polymer from Lignin Precursors

The lignin precursors of coniferin and syringin were synthesised, and guaiacyl-type and guaiacyl-syringyl-type oligomeric lignin dehydrogenation polymers (DHP and DHP-GS) were prepared with the bulk method. The carbon-13 nuclear magnetic resonance spectroscopy showed that both DHP-G and DHP-GS contained β-O-4, β-5, β-β, β-1, and 5-5 substructures. Extraction with petroleum ether, ether, ethanol, and acetone resulted in four fractions for each of DHP-G (C₁₁–C₁₄) and DHP-GS (C₂₁–C₂₄). The antibacterial experiments showed that the fractions with lower molecular weight had relatively strong antibacterial activity. The ether-soluble fractions (C₁₂ of DHP-G and C₂₂ of DHP-GS) had strong antibacterial activities against E. coli and S. aureus. The C₁₂ and C₂₂ fractions were further separated by preparative chromatography, and 10 bioactive compounds (G₁–G₅ and GS₁–GS₅) were obtained. The overall antibacterial activities of these 10 compounds was stronger against E. coli than S. aureus. Compounds G₁, G₂, G₃, and GS₁, which had the most significant antibacterial activities, contained β-5 substructures. Of these, G₁ had the best antibacterial activity. Its inhibition zone diameter was 19.81 ± 0.82 mm, and the minimum inhibition concentration was 56.3 ± 6.20 μg/mL. Atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) showed that the antibacterial activity of G₁ was attributable to a phenylcoumarin dimer, while the introduction of syringyl units reduced antibacterial activity.     

In Silico and In Vitro Structure–Activity Relationship of Mastoparan and Its Analogs

Antimicrobial peptides are an important class of therapeutic agent used against a wide range of pathogens such as Gram-negative and Gram-positive bacteria, fungi, and viruses. Mastoparan (MpVT) is an α-helix and amphipathic tetradecapeptide obtained from Vespa tropica venom. This peptide exhibits antibacterial activity. In this work, we investigate the effect of amino acid substitutions and deletion of the first three C-terminal residues on the structure–activity relationship. In this in silico study, the predicted structure of MpVT and its analog have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bonds. The secondary structure and the biological activity of six designed analogs are studied. The biological activity assays show that the substitution of phenylalanine (MpVT1) results in a higher antibacterial activity than that of MpVT without increasing toxicity. The analogs with the first three deleted C-terminal residues showed decreased antibacterial and hemolytic activity. The CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 40% 2,2,2-trifluoroethanol (TFE). In conclusion, the first three C-terminal deletions reduced the length of the α-helix, explaining the decreased biological activity. MpVTs show that the hemolytic activity of mastoparan is correlated to mean hydrophobicity and mean hydrophobic moment. The position and spatial arrangement of specific hydrophobic residues on the non-polar face of α-helical AMPs may be crucial for the interaction of AMPs with cell membranes.     

螺旋型抗菌肽的疏水性对抗细菌与抗真菌活性的影响比较

α-螺旋型多肽HPRP-A1由15个氨基酸残基组成,来源于幽门螺杆菌核糖体蛋白L1的N端.本研究以HPRP-A1为模板,在其非极性面中心通过单个氨基酸定点取代的方法,形成一系列疏水性不同的多肽类似物,系统地研究疏水性对α-螺旋型多肽生物活性的影响.结果显示,多肽疏水性及所带净电荷对多肽生物活性起着重要的作用;HPRP-A1及疏水性相对较高的多肽类似物具有较好的广谱抗菌活性（包括革兰氏阳性菌、革兰氏阴性菌及真菌）,但也有相对较高的溶血活性;多肽的疏水性与所带净电荷的变化对多肽抗细菌活性与抗真菌活性所产生的影响有着相似的变化趋势和程度.这意味着多肽与细菌的作用机制和多肽与真菌的作用机制存在一定的相关性.多肽对细菌和真菌的抗菌活性存在特异性,为设计出具有临床应用前景的抗菌肽药物奠定了基础.     

Bioactive Abietane-Type Diterpenoid Glycosides from Leaves of Clerodendrum infortunatum (Lamiaceae)

In this study, two previously undescribed diterpenoids, (5R,10S,16R)-11,16,19-trihydroxy-12-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-17(15→16),18(4→3)-diabeo-3,8,11,13-abietatetraene-7-one (1) and (5R,10S,16R)-11,16-dihydroxy-12-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-17(15→16),18(4→3)-diabeo-4-carboxy-3,8,11,13-abietatetraene-7-one (2), and one known compound, the C₁₃-nor-isoprenoid glycoside byzantionoside B (3), were isolated from the leaves of Clerodendrum infortunatum L. (Lamiaceae). Structures were established based on spectroscopic and spectrometric data and by comparison with literature data. The three terpenoids, along with five phenylpropanoids: 6′-O-caffeoyl-12-glucopyranosyloxyjasmonic acid (4), jionoside C (5), jionoside D (6), brachynoside (7), and incanoside C (8), previously isolated from the same source, were tested for their in vitro antidiabetic (α-amylase and α-glucosidase), anticancer (Hs578T and MDA-MB-231), and anticholinesterase activities. In an in vitro test against carbohydrate digestion enzymes, compound 6 showed the most potent effect against mammalian α-amylase (IC₅₀ 3.4 ± 0.2 μM) compared to the reference standard acarbose (IC₅₀ 5.9 ± 0.1 μM). As yeast α-glucosidase inhibitors, compounds 1, 2, 5, and 6 displayed moderate inhibitory activities, ranging from 24.6 to 96.0 μM, compared to acarbose (IC₅₀ 665 ± 42 μM). All of the tested compounds demonstrated negligible anticholinesterase effects. In an anticancer test, compounds 3 and 5 exhibited moderate antiproliferative properties with IC₅₀ of 94.7 ± 1.3 and 85.3 ± 2.4 μM, respectively, against Hs578T cell, while the rest of the compounds did not show significant activity (IC₅₀ > 100 μM).     

Multiple Mechanisms of the Synthesized Antimicrobial Peptide TS against Gram-Negative Bacteria for High Efficacy Antibacterial Action In Vivo

The emergence of drug-resistant bacteria emphasizes the urgent need for novel antibiotics. The antimicrobial peptide TS shows extensive antibacterial activity in vitro and in vivo, especially in gram-negative bacteria; however, its antibacterial mechanism is unclear. Here, we find that TS without hemolytic activity disrupts the integrity of the outer bacterial cell membrane by displacing divalent cations and competitively binding lipopolysaccharides. In addition, the antimicrobial peptide TS can inhibit and kill E. coli by disintegrating the bacteria from within by interacting with bacterial DNA. Thus, antimicrobial peptide TS’s multiple antibacterial mechanisms may not easily induce bacterial resistance, suggesting use as an antibacterial drug to be for combating bacterial infections in the future.     

Identification of the Primary Structure of Selenium-Containing Polysaccharides Selectively Inhibiting T-Cell Proliferation

We previously described the biosynthesis, isolation, and immunosuppressive activity of the selenium-containing polysaccharide fraction isolated from the mycelial culture of Lentinula edodes. Structural studies have shown that the fraction was a protein-containing mixture of high molar mass polysaccharides α- and β-glucans. However, which of the components of the complex fraction is responsible for the immunosuppressive activity non-typical for polysaccharides of fungal origin has not been explained. In the current study, we defined four-polysaccharide components of the Se-containing polysaccharide fraction determined their primary structure and examined the effect on T- and B-cell proliferation. The isolated Se-polysaccharides, α-1,4-glucan (M_w 2.25 × 10⁶ g/mol), unbranched β-1,6-d-glucan, unbranched β-1,3-d-glucan and β-1,3-branched β-1,6-d-glucan (M_w 1.10 × 10⁵ g/mol), are not typical as components of the cell wall of L. edodes. All are biologically active, but the inhibitory effect of the isolated polysaccharides on lymphocyte proliferation was weaker, though more selective than that of the crude fraction.     

Quinol-containing ligands enable high superoxide dismutase activity by modulating coordination number,charge, oxidation states and stability of manganese complexes throughout redox cycling

Reactivity assays previously suggested that two quinol-containing MRI contrast agent sensors for H₂O₂, [Mn(H2qp1)(MeCN)]²⁺ and [Mn(H4qp2)Br₂], could also catalytically degrade superoxide. Subsequently, [Zn(H2qp1)(OTf)]⁺ was found to use the redox activity of the H2qp1 ligand to catalyze the conversion of O₂˙⁻ to O₂ and H₂O₂, raising the possibility that the organic ligand, rather than the metal, could serve as the redox partner for O₂˙⁻ in the manganese chemistry. Here, we use stopped-flow kinetics and cryospray-ionization mass spectrometry (CSI-MS) analysis of the direct reactions between the manganese-containing contrast agents and O₂˙⁻ to confirm the activity and elucidate the catalytic mechanism. The obtained data are consistent with the operation of multiple parallel catalytic cycles, with both the quinol groups and manganese cycling through different oxidation states during the reactions with superoxide. The choice of ligand impacts the overall charges of the intermediates and allows us to visualize complementary sets of intermediates within the catalytic cycles using CSI-MS. With the diquinolic H4qp2, we detect Mn(iii)-superoxo intermediates with both reduced and oxidized forms of the ligand, a Mn(iii)-hydroperoxo compound, and what is formally a Mn(iv)-oxo species with the monoquinolate/mono-para-quinone form of H4qp2. With the monoquinolic H2qp1, we observe a Mn(ii)-superoxo ↔ Mn(iii)-peroxo intermediate with the oxidized para-quinone form of the ligand. The observation of these species suggests inner-sphere mechanisms for O₂˙⁻ oxidation and reduction that include both the ligand and manganese as redox partners. The higher positive charges of the complexes with the reduced and oxidized forms of H2qp1 compared to those with related forms of H4qp2 result in higher catalytic activity (k_cat ∼ 10⁸ M⁻¹ s⁻¹ at pH 7.4) that rivals those of the most active superoxide dismutase (SOD) mimics. The manganese complex with H2qp1 is markedly more stable in water than other highly active non-porphyrin-based and even some Mn(ii) porphyrin-based SOD mimics.

Manganese complexes with polydentate quinol-containing ligands are found to catalyze the degradation of superoxide through inner-sphere mechanisms. The redox activity of the ligand stabilizes higher-valent manganese species.     

Oxy-Polybrominated Diphenyl Ethers from the Indonesian Marine Sponge,Lamellodysidea herbacea: X-ray,SAR, and Computational Studies

Polybrominated diphenyl ether (PBDE) compounds, derived from marine organisms, originate from symbiosis between marine sponges and cyanobacteria or bacteria. PBDEs have broad biological spectra; therefore, we analyzed structure and activity relationships of PBDEs to determine their potential as anticancer or antibacterial lead structures, through reactions and computational studies. Six known PBDEs (1–6) were isolated from the sponge, Lamellodysdiea herbacea; ¹³C NMR data for compound 6 are reported for the first time and their assignments are confirmed by their theoretical ¹³C NMR chemical shifts (RMSE < 4.0 ppm). Methylation and acetylation of 1 (2, 3, 4, 5-tetrabromo-6-(3′, 5′-dibromo-2′-hydroxyphenoxy) phenol) at the phenol functional group gave seven molecules (7–13), of which 10, 12, and 13 were new. New crystal structures for 8 and 9 are also reported. Debromination carried out on 1 produced nine compounds (1, 2, 14, 16–18, 20, 23, and 26) of which 18 was new. Debromination product 16 showed a significant IC₅₀ 8.65 ± 1.11; 8.11 ± 1.43 µM against human embryonic kidney (HEK293T) cells. Compounds 1 and 16 exhibited antibacterial activity against Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae with MID 0.078 µg/disk. The number of four bromine atoms and two phenol functional groups are important for antibacterial activity (S. aureus and K. pneumoniae) and cytotoxicity (HEK293T). The result was supported by analysis of frontier molecular orbitals (FMOs). We also propose possible products of acetylation and debromination using analysis of FMOs and electrostatic charges and we confirm the experimental result.     

净电荷对螺旋型抗癌肽生物活性的影响

以高活性两亲性α-螺旋型阳离子抗癌肽A12L/A20L(多肽P)为模板, 在其亲水面进行氨基酸定点取代, 获得了一系列带有不同净电荷的多肽类似物, 研究了净电荷对螺旋型抗癌肽生物活性的影响. 结果表明, 抗癌肽净电荷的改变对其溶血活性影响较小(最大差异为2倍), 而对抗癌活性和选择性的影响显著(最大差异为10倍). 抗癌肽P的净电荷最适范围为+7到+8, 分子间静电排斥作用的最佳数目为3~5个, 高于或低于此范围, 其抗癌活性和选择性均明显降低. 与人的正常细胞相比, 负电性的癌细胞膜对于抗癌肽的净电荷变化更敏感, 表明两亲性螺旋型抗癌肽针对癌细胞与正常细胞表现出良好的选择特异性.     

Targeting antioxidant pathways with ferrocenylated N-heterocyclic carbene supported gold(i) complexes in A549 lung cancer cells

Ferrocene containing N-heterocyclic carbene (NHC) ligated gold(i) complexes of the type [Au(NHC)₂]⁺ were prepared and found to be capable of regulating the formation of reactive oxygen species (ROS) via multiple mechanisms. Single crystal X-ray analysis of bis(1-(ferrocenylmethyl)-3-mesitylimidazol-2-ylidene)-gold(i) chloride (5) and bis(1,3-di(ferrocenylmethyl)imidazol-2-ylidene)-gold(i) chloride (6) revealed a quasi-linear geometry around the gold(i) centers (i.e., the C–Au–C bond angle were measured to be ∼177° and all the Au–C_carbene bonds distances were in the range of 2.00 (7)–2.03 (1) Å). A series of cell studies indicated that cell proliferation inhibition and ROS generation were directly proportional to the amount of ferrocene contained within the [Au(NHC)₂]⁺ complexes (IC₅₀ of 6 < 5 < bis(1-benzyl-3-mesitylimidazol-2-ylidene)-gold(i) chloride (4)). Complexes 4–6 were also confirmed to inhibit thioredoxin reductase as inferred from lipoate reduction assays and increased chelatable intracellular zinc concentrations. RNA microarray gene expression assays revealed that 6 induces endoplasmic reticulum stress response pathways as a result of ROS increase.     

Expression of Hybrid Peptide EF-1 in Pichia pastoris,Its Purification,and Antimicrobial Characterization

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni²⁺ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.     

New Flavone C-Glycosides from Scleranthus perennis and Their Anti-Collagenase Activity

Three new flavone glycosides, one known flavone glycoside, and the phenolic derivative apiopaenonside were isolated and identified from the ethyl acetate fraction of the aerial parts of Scleranthus perennis. The planar structures were elucidated through extensive analysis of UV-Vis, IR, and ¹H NMR and ¹³C NMR spectral data, including the 2D techniques COSY, HSQC, and HMBC, as well as ESI mass spectrometry. The isolated compounds were established as 5,7,3′-trihydroxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (1), 5,7,3′-trihydroxy-4′-methoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (2), 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-glucoside (3), 5,7-dihydroxy-3′-methoxy-4′-acetoxyflavone-8-C-β-d-xylopyranoside-2′′-O-(4′′′-acetoxy)-glucoside (4), and apiopaenonside (5). Moreover, all isolated compounds were evaluated for anti-collagenase activity. All compounds exhibited moderate inhibitory activity with IC₅₀ values ranging from 36.06 to 70.24 µM.     

Hemolytic and Antimicrobial Activities Differ Among Saponin-rich Extracts From Guar, Quillaja, Yucca, and Soybean

Hemolytic and antibacterial activities of eight serial concentrations ranged from 5-666 µg/mL of saponin-rich extracts from guar meal (GM), quillaja, yucca, and soybean were tested in 96-well plates and read by enzyme-linked immunosorbent assay plate-well as 650 nm. Hemolytic assay used a 1% suspension of chicken red blood cells with water and phosphate buffered saline as positive and negative controls, respectively. Antibacterial activity against Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli were evaluated using ampicillin and bacteria without saponin-rich extract as positive and negative controls, respectively. The 100% MeOH GM and commercial quillaja saponin-rich extracts were significantly the highest in both hemolytic and antibacterial activities against all bacteria at the same concentration tested. Soybean saponin-rich extract had no antibacterial activity against any of the bacteria at the concentrations tested while yucca saponin-rich extract had no antibacterial activity against the gram-negative bacteria at the concentrations tested. GM and quillaja saponin-rich extracts were hemolytic, while yucca and soybean saponin-rich extracts were not hemolytic at the concentrations tested. No saponin-rich extract source had antibacterial activity against S. typhimurium or E. coli at the concentrations tested. Both GM and quillaja saponin-rich extracts exhibited antibacterial activity against S. aureus. Saponin-rich extracts from different plant sources have different hemolytic and antibacterial activities.     

The Antifungal Action Mode of N-Phenacyldibromobenzimidazoles

Our study aimed to characterise the action mode of N-phenacyldibromobenzimidazoles against C. albicans and C. neoformans. Firstly, we selected the non-cytotoxic most active benzimidazoles based on the structure–activity relationships showing that the group of 5,6-dibromobenzimidazole derivatives are less active against C. albicans vs. 4,6-dibromobenzimidazole analogues (5e–f and 5h). The substitution of chlorine atoms to the benzene ring of the N-phenacyl substituent extended the anti-C. albicans action (5e with 2,4-Cl₂ or 5f with 3,4-Cl₂). The excellent results for N-phenacyldibromobenzimidazole 5h against the C. albicans reference and clinical isolate showed IC₅₀ = 8 µg/mL and %I = 100 ± 3, respectively. Compound 5h was fungicidal against the C. neoformans isolate. Compound 5h at 160–4 µg/mL caused irreversible damage of the fungal cell membrane and accidental cell death (ACD). We reported on chitinolytic activity of 5h, in accordance with the patterns observed for the following substrates: 4-nitrophenyl-N-acetyl-β-d-glucosaminide and 4-nitrophenyl-β-d-N,N′,N″-triacetylchitothiose. Derivative 5h at 16 µg/mL: (1) it affected cell wall by inducing β-d-glucanase, (2) it caused morphological distortions and (3) osmotic instability in the C. albicans biofilm-treated. Compound 5h exerted Candida-dependent inhibition of virulence factors.     

Selective Ruthenium Labeling of the Tryptophan Residue in the Bee Venom Peptide Melittin

Melittin is a membrane‐active peptide from bee venom with promising antimicrobial and anticancer activity. Herein we report on a simple and selective method for labeling of the tryptophan residue in melittin by the organometallic fragment [(C₅H₅)Ru]⁺ in aqueous solution and in air. Ruthenium coordination does not disturb the secondary structure of the peptide (as verified by 2D NMR spectroscopy), but changes the pattern of its intermolecular interactions resulting in an 11‐fold decrease of hemolytic activity. The high stability of the organometallic conjugate allowed the establishment of the biodistribution of the labeled melittin in mice by inductively coupled plasma MS analysis of ruthenium.     

Effect of Kaempferol and Its Glycoside Derivatives on Antioxidant Status of HL-60 Cells Treated with Etoposide

Kaempferol is a well-known antioxidant found in many plants and plant-based foods. In plants, kaempferol is present mainly in the form of glycoside derivatives. In this work, we focused on determining the effect of kaempferol and its glycoside derivatives on the expression level of genes related to the reduction of oxidative stress—NFE2L2, NQO1, SOD1, SOD2, and HO-1; the enzymatic activity of superoxide dismutases; and the level of glutathione. We used HL-60 acute promyelocytic leukemia cells, which were incubated with the anticancer drug etoposide and kaempferol or one of its three glycoside derivatives isolated from the aerial parts of Lens culinaris Medik.—kaempferol 3-O-[(6-O-E-caffeoyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P5), and kaempferol 3-O-[(6-O-E-feruloyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P7). We showed that none of the tested compounds affected NFE2L2 gene expression. Co-incubation with etoposide (1 µM) and kaempferol (10 and 50 µg/mL) leads to an increase in the expression of the HO-1 (9.49 and 9.33-fold at 10 µg/mL and 50 µg/mL, respectively), SOD1 (1.68-fold at 10 µg/mL), SOD2 (1.72-fold at 10–50 µg/mL), and NQO1 (1.84-fold at 50 µg/mL) genes in comparison to cells treated only with etoposide. The effect of kaempferol derivatives on gene expression differs depending on the derivative. All tested polyphenols increased the SOD activity in cells co-incubated with etoposide. We observed that the co-incubation of HL-60 cells with etoposide and kaempferol or derivative P7 increases the level of total glutathione in these cells. Taken together, our observations suggest that the antioxidant activity of kaempferol is related to the activation of antioxidant genes and proteins. Moreover, we observed that glycoside derivatives can have a different effect on the antioxidant cellular systems than kaempferol.