Peptides based on CcdB protein as novel inhibitors of bacterial topoisomerases

The ccd toxin-antitoxin system of the F plasmid encodes CcdB, a protein that poisons the essential Escherichia coli DNA gyrase, unique type IIA topoisomerase able to introduce negative supercoils into DNA. Based on CcdB structure, a series of linear peptide analogues were obtained by the solid-phase methodology. One of these peptides (CcdBET2) displayed inhibition of the supercoiling activity of bacterial DNA gyrase with a concentration required for complete inhibition (IC(100) = 10 mu M) lower than the wild type CcdB. For Topo IV, a second type IIA bacterial topoisomerase, CcdBET2 was better inhibited the relaxation activity with an IC100 of 5 mu M (wt CcdB > 10 mu M). The replacement of Gly, present in the three C-terminal amino acid residues, by Glu, abolished the capacity to inhibit the gyrase but not the Topo IV activities. These findings demonstrate that the mechanism by which CcdBET2 inhibits DNA gyrase is different of the mechanism by which inhibits Topo IV. Therefore, CcdBET2 is a new type II topoisomerase inhibitor with specificity for Topo IV. (C) 2008 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Buffalo cheese whey proteins, identification of a 24 kda protein and characterization of their hydrolysates: in vitro gastrointestinal digestion

Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

DNA binding, topoisomerase inhibition and cytotoxicity of palladium(II) complexes with 1,10-phenanthroline and thioureas.

International audienceMetallointercalators represent a promising alternative in cancer chemotherapy. We present herein DNA binding, topoisomerase inhibition and cytotoxic studies on a series of complexes of general formulae [Pd(phen)(tu∗)2]2+ incorporating the intercalator 1,10-phenanthroline and thiourea ligands (L = thiourea 1, N-methylthiourea 2, N,N′-dimethylthiourea 3). DNA-unwinding results showed that the complexes can induce the unwinding of the plasmid DNA. The binding constants (Kb) for the interaction of the complexes with SS-DNA were determined by UV spectroscopy. Competitive experiments with ethidium bromide (EB) were investigated by fluorescence spectroscopy and show that all the complexes were able to displace EB from the DNA–EB complex. The results suggest that they may interact with DNA by intercalation. Compounds were tested against human oral carcinoma cell line (KB), human breast cancer cell line (MCF7) and cisplatin-resistant human breast cancer cell line (MCF7-R) and showed good cytotoxic activity towards MCF7-R. Compounds 2 and 3 were also able to cause topo II inhibition

In Vitro Identification of Histatin 5 Salivary Complexes.

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein's function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein-protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein-protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance

Cationic Pd(II) complexes acting as topoisomerase II inhibitors: Synthesis, characterization, DNA interaction and cytotoxicity.

International audienceThe synthesis and characterization of the complexes [PdX(PPh3)(4-MeT)]X (4-MeT = 4-methyl-3-thiosemicarbazide; PPh3 = triphenylphosphine; X = Cl, Br, I, SCN) have been described. The complexes were evaluated for in vitro activity as cytotoxic agents on tumor cells and also as cathepsin B and topoisomerase I and II inhibitors

SDS-PAGE patterns for the H1 and H2 hydrolysates.

<p>In the both figures the Lane 1: molecular mass standards; Lane 2: treated bovine milk whey; Lane 3: treated buffalo milk whey. The lanes 4–12 are showing hydrolysates produced using the M1 method (Fig 5a) with incubation between 0–180 min, whereas the lanes 4–16 are showing hydrolysates produced using the M2 method (Fig 5b) with incubation between 0–1440 min.</p

Protein, lactose and fat concentrations of the <i>in natura</i> and treated buffalo milk whey.

<p>*reduction of lactose</p><p>**dialyzed, defatted and centrifuged</p><p>Protein, lactose and fat concentrations of the <i>in natura</i> and treated buffalo milk whey.</p

Relative concentration (nmol. L^-1) of the amino acids from buffalo milk whey before and after dialyzability.

<p>1 Hydrolysis with pepsin, trypsin, chymotrypsin and carboxypeptidase-A</p><p>2 Total of released amino acids by dialyzability (Dext + Dint) NH = non-hydrolyzed; H1 = low degree of hydrolysis; H2 = high degree of hydrolysis; GD = gastric digest; Dext = external intestinal digest, samples collected inside of the membrane; Dint = internal intestinal digest, samples collected outside of the membrane</p><p>3 Essential amino acids</p><p>4 Non-essential amino acids</p><p>Relative concentration (nmol. L^-1) of the amino acids from buffalo milk whey before and after dialyzability.</p

Amino acid sequence for β-Lg from <i>Bos taurus</i> (gi/229460).

<p>The tryptic peptides obtained for 24 kDa protein from buffalo cheese way were identical to the red sequence. It was obtained 37% of coverage.</p