Genetic diversity of two African and sixteen South American populations determined on the basis of six hypervariable loci

A total of 582 individuals (1,164 chromosomes) from two African, eight African-derived South American, five South American Amerindian, and three Brazilian urban populations were studied at four variable number of tandem repeat (VNTR) and two short tandem repeat (STR) hypervariable loci. These two sets of loci did not show distinct allele profiles, which might be expected if different processes promoted their molecular differentiation. The two African groups showed little difference between them, and their intrapopulational variation was similar to those obtained in the African-derived South American communities. The latter showed different degrees of interpopulation variability, despite the fact that they presented almost identical average degrees of non-African admixture. The F_ST single locus estimates differed in the five sets of populations, probably due to genetic drift, indicating the need to consider population structure in the evaluation of their total variability. A high interpopulational diversity was found among Amerindian populations in relation to Brazilian African-derived isolated communities. This is probably a consequence of the differences in the patterns of gene flow and genetic drift that each of these semi-isolated groups experienced. Am J Phys Anthropol 109:425–437, 1999. © 1999 Wiley-Liss, Inc.     

Preservation by freezing of potentially probiotic strains ofLactobacillus rhamnosus

The aim of this work was to detect the best conditions to preserve by freezing potentially probiotic strains ofLactobacillus rhamnosus isolated from food. Four strains isolated from Parmigiano Reggiano cheese, the commercial strainLactobacillus GG and the type strain ATCC 7469T were used in the present study. Two different pre-incubation times (5 and 24 h), three protective media (Skim milk, Skim milk plus glucose and MRS plus glycerol) and two storage temperatures (?20 and ?80 °C) were used for a preservation period of 90 days. A sensible loss of survival of the strains was detected and the acidifying activity decreased depending on the different factors analysed. Moreover, plate counts performed in MRS plus bile salts evidenced that a considerable percentage of cells suffers damages deriving from cold. This study showed that the growth phase of the cells plays an important role for the resistance to the storage by freezing. Finally, Skim milk had the best protective action, showing the highest activity at ?80 °C.     

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE), Part II: Development of inhibitors with broad spectrum,Gram-negative antibacterial activity

The structurally related bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as prime candidates for the development of broad spectrum antibacterial agents. However, GyrB/ParE targeting antibacterials with spectrum that encompasses robust Gram-negative pathogens have not yet been reported. Using structure-based inhibitor design, we optimized a novel pyrrolopyrimidine inhibitor series with potent, dual targeting activity against GyrB and ParE. Compounds were discovered with broad antibacterial spectrum, including activity against Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli. Herein we describe the SAR of the pyrrolopyrimidine series as it relates to key structural and electronic features necessary for Gram-negative antibacterial activity.     

Influence of allosteric effectors on the heme conformation of dromedary ferrous nitrosylhemoglobin detected by XANES spectroscopy

The changes of the Fe heme-active site conformation of dromedary (Camelus dromedarius) nitrosylhemoglobin (HbNO) induced by inositol hexakisphosphate (IHP) and chlofibric acid (CFA) have been studied by using X-ray absorption near-edge structure (XANES) spectroscopy. Structural information has been determined by multiple scattering analysis of the Fe K-edge XANES spectra. The proximal histidine is found to move away from iron centers by about 0.4 Angstrom on the average over the four hemes upon binding of CFA or stoichiometric amount of IHP. In molar excess of polyanion or in the simultaneous presence of IHP, CFA and chloride, the proximal histidine moves back to a position very close to that observed in pure buffer; yet, the structure modulation induced by the allosteric effectors is not completely reversible. Such findings parallel with the functional properties and the spectroscopic (e.g., EPR and absorbance) characteristics of HbNO.     

X-ray absorption spectroscopy studies of the adducts formed between cytotoxic gold compounds and two major serum proteins

Gold metallodrugs form a class of promising antiproliferative agents showing a high propensity to react with proteins. We exploit here X-ray absorption spectroscopy (XAS) methods [both X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS)] to gain insight into the nature of the adducts formed between three representative gold(I, III) metallodrugs (i.e., auranofin, [Au(2,2′-bipyridine)(OH)₂](PF₆), Aubipy, and dinuclear [Au₂(6,6′-dimethyl-2,2′-bipyridine)₂(μ-O)₂](PF₆)₂, Auoxo6) and two major plasma proteins, namely, bovine serum albumin (BSA) and human serum apotransferrin (apoTf). The following metallodrug–protein systems were investigated in depth: auranofin/apoTf, Aubipy/BSA, and Auoxo6/apoTf. XANES spectra revealed that auranofin, upon protein binding, conserves its gold(I) oxidation state. Protein binding most probably takes place through release of the thiosugar ligand and its subsequent replacement by a thiol (or a thioether) from the protein. This hypothesis is independently supported by EXAFS results. In contrast, the reactions of Aubipy with serum albumin and of Auoxo6 with serum apoTf invariantly result in gold(III) to gold(I) reduction. Gold(III) reduction, clearly documented by XANES, is accompanied, in both cases, by release of the bipyridyl ligands; for Auoxo6 cleavage of the gold–gold dioxo bridge is also observed. Gold(III) reduction leads to formation of protein-bound gold(I) species, with deeply modified metal coordination environments, as evidenced by EXAFS. In these adducts, the gold(I) centers are probably anchored to the protein through nitrogen donors. In general, these two XAS methods, i.e., XANES and EXAFS, used here jointly, allowed us to gain independent structural information on metallodrug/protein systems; detailed insight into the gold oxidation state and the local environment of protein-bound metal atoms was achieved in the various cases.     

Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer

Therapies targeting estrogen receptor α (ERα, encoded by ESR1) have transformed the treatment of breast cancer. However, large numbers of women relapse, highlighting the need for the discovery of new regulatory targets modulating ERα pathways. An siRNA screen identified kinases whose silencing alters the estrogen response including those previously implicated in regulating ERα activity (such as mitogen-activated protein kinase and AKT). Among the most potent regulators was lemur tyrosine kinase-3 (LMTK3), for which a role has not previously been assigned. In contrast to other modulators of ERα activity, LMTK3 seems to have been subject to Darwinian positive selection, a noteworthy result given the unique susceptibility of humans to ERα+ breast cancer. LMTK3 acts by decreasing the activity of protein kinase C (PKC) and the phosphorylation of AKT (Ser473), thereby increasing binding of forkhead box O3 (FOXO3) to the ESR1 promoter. LMTK3 phosphorylated ERα, protecting it from proteasomal degradation in vitro. Silencing of LMTK3 reduced tumor volume in an orthotopic mouse model and abrogated proliferation of ERα+ but not ERα- cells, indicative of its role in ERα activity. In human cancers, LMTK3 abundance and intronic polymorphisms were significantly associated with disease-free and overall survival and predicted response to endocrine therapies. These findings yield insights into the natural history of breast cancer in humans and reveal LMTK3 as a new therapeutic target.     

Synthesis, structural characterization, formation constants and in vitro cytotoxicity of phenanthroline and imidazolidine-2-thione copper(II) complexes

The synthesis, crystal structures, physicochemical properties and complex formation constants of [Cu(phen)₂(L)](ClO₄)₂ complexes, where phen is 1,10-ortho-phenanthroline and L is a series of substituted imidazolidine-2-thione, have been studied. Single crystal X-ray diffraction revealed a distorted trigonal-bipyramidal geometry for all the molecules. The complex formation constants were determined in nonaqueous media by spectrophotometric measurements. Testing copper(II) complexes in mouse neuroblastoma N2a cells persistently infected with the 22L strain of the scrapie prion protein (22L-N2a) resulted in high cytotoxicity but no antiprion activity at nontoxic doses.