Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH₂) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O₂) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.     

Novel antimicrobial peptides from the venom of the eusocial bee Halictus sexcinctus (Hymenoptera: Halictidae) and their analogs

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH₂ (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys–His-Ile-Leu-Lys-NH₂ (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic α-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, α-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.     

Synthesis of α-carboxyphosphinopeptides derived from norleucine

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.     

Intra-sex dimorphism in crayfish females

A unique cycle of female form alternation has been revealed in an experimental population of Orconectes limosus during a year-long observation. Significant cyclic changes observed in chelae length, width, and robustness, as well as in abdomen width, demonstrated a form alternation similar to that in conspecific males. Small females alternate between sexually active and sexually inactive forms with a short time interval between successive molts as well as different growth patterns of some body parts. Form alternation efficiently produces larger chelae, abdomen, and body dimensions, especially the molt to form I (sexually active). Larger females that undergo only a single annual molt do not alter between forms and are sexually active. They grow slowly and lose chelae robustness. The cycle of form alternation, consisting of two molts per year, may facilitate the effective utilization of resources to increase the size of body parts important to survival and reproduction.     

Maintenance of an endemic lineage of brown trout (Salmo trutta) within the Duero river basin

The Iberian Peninsula contains diverse populations of freshwater fish, with major river basins comprising differentiated biogeographic units. The Duero River flows through the North‐Western Iberian Peninsula and is one of the most important rivers within the Iberian glacial refuge. Brown trout (Salmo trutta) populate this whole basin, and studies using both allozyme and microsatellite loci have detected a geographically sorted distribution of genetic variation in this species. In this work, sequences of the mitochondrial control region obtained from 299 brown trout from the Duero River were compared with other Iberian and European datasets. Two differentiated haplotype groups were detected inside the Duero River basin. One of them was related to the Atlantic (AT) lineage that is present in Northern European populations, whereas the other comprised an unique group that was restricted to the inner region of the basin. The amount of divergence of this Duero group from the other brown trout populations studied is consistent with a new trout lineage (Duero, DU) that is endemic to this river basin and that diverged from other Atlantic populations during the Pleistocene. The distribution of the DU and AT quaternary lineages in the Duero River was consistent with the ichthyological pattern described in the basin that originated during the Miocene–Pliocene. Evidence of selective processes that favour the haplotypes of the DU lineage may explain this discrepancy.     

Indoloditerpenes from an algicolous isolate of Aspergillus oryzae

Two new indoloditerpene derivatives asporyzin A (1) and asporyzin B (2), one new indoloditerpene asporyzin C (3), and three known related indoloditerpenes JBIR-03 (4), emindole SB (5), and emeniveol (6) were isolated from an endophytic fungus Aspergillus oryzae, isolated from the marine red alga Heterosiphonia japonica. Their structures were unambiguously established by spectroscopic techniques. In addition, all the isolates were evaluated preliminarily for insecticidal and antimicrobial activities in order to probe into their chemical defensive function. Compound 4 was more active against brine shrimp than the others, and 3 possessed potent activity against Escherichia coli.     

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC₅₀ values below 1 μM were selected. Out of them, four compounds strongly inhibited the enzyme with IC₅₀ values lying in a range of 11–45 nM. These most potent compounds might be bi-substrate analogues.     

Evaluation of secondary amide replacements in a series of CCR5 antagonists as a means to increase intrinsic membrane permeability. Part 1: Optimization of gem-disubstituted azacycles

Replacement of a secondary amide with an N-acyl or N-sulfonyl gem-disubstituted azacyle in a series of CCR5 antagonists led to the identification of compounds with excellent in vitro HIV antiviral activity and increased intrinsic membrane permeability.     

BROTHER OF FT AND TFL1 (BFT), a member of the FT/TFL1 family,shows distinct pattern of expression during the vegetative growth of Arabidopsis

Transition to the flowering stage is precisely controlled by a few classes of regulatory molecules. BROTHER OF FT AND TFL1 (BFT) is a member of FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family, an important class of flower development regulators with unidentified biochemical function. BFT has a TFL1-like activity and plays a role in axillary inflorescence development. To elucidate the expression pattern of BFT, we analyzed the subcellular localization and conditional expression of BFT in this study. We generated 35S::BFT:GFP plants to investigate the subcellular localization of BFT protein. 35S::BFT:GFP plants showed late flowering, similarly as did 35S::BFT plants. BFT:GFP fusion protein was localized in the nucleus and the plasma membrane, which was different from the localization pattern of FT and TFL1. BFT expression was induced by abiotic stress conditions. ABA, drought, and osmotic stress treatments induced BFT expression, whereas cold, salt, and heat stress conditions did not, suggesting that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions. The induction pattern of BFT was different from those of other FT/TFL1 family genes. Our studies indicated that BFT showed a distinct expression pattern from its homologous genes during the vegetative growth in Arabidopsis.Key words: flowering time, flowering locus T, terminal flower 1, brother of FT and TFL1, abiotic stress, subcellullar localizationThe FLOWERING LOCUS T (FT)/TERMINAL FLOWER 1 (TFL1) family is a small gene family whose members play a pivotal role in flower development in Arabidopsis. The family includes FT, TFL1, TWIN SISTER OF FT (TSF), Arabidopsis thaliana CENTRORADIALIS homologue (ATC), MOTHER OF FT AND TFL1 (MFT) and BROTHER OF FT AND TFL1 (BFT).^{3,5,6,9,15,17} FT is a floral promoter that integrates signal inputs from various pathways that regulate flowering time in Arabidopsis.^5,6 TFL1 plays an antagonistic role to that of FT, functioning as a floral inhibitor. Unlike FT, TFL1 also plays an important role in controlling plant architecture by regulating the expression of LEAFY (LFY) and APETALA1 (AP1), two important floral meristem identity genes in the shoot apical meristem (SAM).^3,7 TSF regulates flowering by a mechanism similar to that of FT, although a lesion in TSF does not have an apparent effect on the determination of flowering time. MFT has a weak FT-like activity.¹⁷ ATC acts as a floral repressor, and its role is similar to that of TFL1.⁹ Finally, BFT has a TFL1-like activity, in spite of its amino acid homology to FT,^2,4,16 and functions redundantly with TFL1 in inflorescence meristem development in Arabidopsis.¹⁶ Although genetic studies elucidated an intricate role of the FT/TFL1 family genes, not much is known about the expression pattern of the remaining members except FT and TFL1. Here, we report that BFT expression showed a distinct pattern from its homologous genes during the vegetative phase. BFT:GFP fusion protein was detected in the nucleus and the plasma membrane. BFT expression was induced by abiotic stress conditions, distinct from other FT/TFL1 family genes, raising the possibility that BFT plays a role in regulating flowering time and inflorescence structure under drought conditions.