Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.     

Synthesis of structurally simplified analogues of aplidinone A,a pro-apoptotic marine thiazinoquinone

The synthesis of analogues of aplidinone A (7), a prenylated quinone isolated from the Mediterranean ascidian Aplidium conicum, has been performed. This work not only allowed confirming the structural assignment of aplidinone A, previously made with the support of GIAO shielding calculations, but, above all, made a series of structurally related quinone derivatives (compounds 8–13 and the natural metabolite) available for a screening in vitro for cytotoxic and pro-apoptotic activity and for SAR studies. The study evidenced one of the synthetic analogues (11) as a potent cytotoxic and pro-apoptotic agent against several tumor cell lines which also inhibits the TNFα-induced NF-κB activation in a human leukemia T cell line. This exemplifies the potential of a natural product to qualify as lead structure for medicinal chemistry campaigns, affording simplified analogues with better bioactivity and easier to synthesize.     

Role of 2′,6′-dimethyl-l-tyrosine (Dmt) in some opioid lead compounds

Here we evaluated how the interchange of the amino acids 2′,6′-dimethyl-l-tyrosine (Dmt), 2′,6′-difluoro-l-tyrosine (Dft), and tyrosine in position 1 can affect the pharmacological characterization of some reference opioid peptides and pseudopeptides. Generally, Dft and Tyr provide analogues with a similar pharmacological profile, despite different pK_a values. Dmt/Tyr(Dft) replacement gives activity changes depending on the reference opioid in which the modification was made. Whereas, H-Dmt-Tic-Asp1-Bid is a potent and selective δ agonist (MVD, IC₅₀ = 0.12 nM); H-Dft-Tic-Asp1-Bid and H-Tyr-Tic-Asp1-Bid are potent and selective δ antagonists (pA₂ = 8.95 and 8.85, respectively). When these amino acids are employed in the synthesis of deltorphin B and its Dmt¹ and Dft¹ analogues, the three compounds maintain a very similar δ agonism (MVD, IC₅₀ 0.32–0.53 nM) with a decrease in selectivity relative to the Dmt¹ analogue. In the less selective H-Dmt-Tic-Gly1-Bid the replacement of Dmt with Dft and Tyr retains the δ agonism but with a decrease in potency. Antagonists containing the Dmt-Tic pharmacophore do not support the exchange of Dmt with Dft or Tyr.     

A 50 Hz sinusoidal magnetic field does not damage MG-63 three-dimensional tumor spheroids but induces changes in their invasive properties

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.     

Cytotoxic and antiproliferative effects induced by a non terpenoid polar extract of A. indica seeds on 3T6 murine fibroblasts in culture

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. Its composition is very complex and the oil exhibits a number of biological activities. The most studied component is the terpenoid azadirachtin which is used for its insecticidal and putative antimicrobial properties. In this report we investigate the biological activity of partially purified components of the oil obtained from A. indica. We show that the semi-purified fractions have moderate to strong cytotoxicity. However, this is not attributable to azadirachtin but to other active compounds present in the mixture. Each fraction was further purified by appropriate extraction procedures and we observed a differential cytotoxicity in the various sub-fractions. This led us to investigate the mode of cell death. After treatment with the oil fractions we observed positivity to TUNEL staining and extensive internucleosomal DNA degradation both indicating apoptotic death. The anti-proliferative properties of the neem oil-derived compounds were also assayed by evaluation of the nuclear PCNA levels (Proliferating Cell Nuclear Antigen). PCNA is significantly reduced in cells treated with a specific fraction of neem oil. Finally, our results strongly suggest a possible involvement of the mitochondrial pathway in the apoptotic death.     

Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels

The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na(v) channels.     

Identification of Single Nucleotide Polymorphisms in the Nicastrese Goat and Sardinia Sheep Mannose-Binding Lectin

This study was undertaken to detect polymorphisms in the goat and sheep mannose-binding lectin encoding gene (MBL2) and to explore allelic variability of this gene in these two species. The analysis and comparison of the sequences obtained from sheep showed 13 polymorphic sites, six in the promoter and seven in exon 1, four of which were of the missense type. In the goats, 12 polymorphic sites were detected, five intronic, five in the promoter, and one exonic. The exon site was responsible for an amino acid change. Mutations detected at the MBL2 locus in the sheep are of particular interest, being potentially responsible for the alterations of gene expression. A population survey involved 102 ewes of the Sardinian breed and 218 goats of the Nicastrese breed, all reared in southern Italy.