Novel Gemini vitamin D(3) analogs have potent antitumor activity

The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.     

Amyloid aggregation of lysozyme: The synergy study of red wine polyphenols

The amyloidoses are diseases associated with nonnative folding of proteins and characterized by the presence of protein amyloid aggregates. The ability of quercetin, resveratrol, caffeic acid, and their equimolar mixtures to affect amyloid aggregation of hen egg white lysozyme in vitro was detected by Thioflavin T fluorescence assay. The anti‐amyloid activities of tested polyphenols were evaluated by the median depolymerization concentrations DC₅₀ and median inhibition concentrations IC₅₀. Single substances are more efficient (by at least one order) in the depolymerization of amyloid aggregates assay than in the inhibition of the amyloid formation with IC₅₀ in 10^?4 to 10^?5M range. Analyzed mixture samples showed synergic or antagonistic effects in both assays. DC₅₀ values ranged from 10^?5 to 10^?8M and IC₅₀ from 10^?5 to 10^?9M, respectively. We observed that certain mixtures of studied polyphenols can synergistically inhibit production of amyloids aggregates and are also effective in depolymerization of the aggregates. Synergic or antagonistic effects of studied mixtures were correlated with protein–small ligand docking studies and AFM results. Differences in these activities could be explained by binding of each polyphenol to a different amino acid sequence within the protein. Our results indicate that synergic/antagonistic anti‐amyloid effects of studied mixtures depend on the selective binding of polyphenols to the known amyloidogenic sequences in the lysozyme chain. Our findings of the effective reduction of amyloid aggregation of lysozyme by polyphenol mixtures in vitro are of the utter physiological relevance considering the bioavailability and low toxicity of tested phenols. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Two Trichosporon species isolated from Central-European mygalomorph spiders (Araneae: Mygalomorphae)

Trichosporon (Dikarya: Basidiomycota) is a genus of anamorphic yeasts typically associated with soil and water, although many species are causative agents of diseases in animals and man. Here we provide the first compelling evidence that spiders can be occasionally colonized by at least two Trichosporon species. Trichosporon dulcitum (Berkhout) Weijman 1979 was isolated from the exoskeleton of purse-web spider Atypus piceus, while Trichosporon porosum (Stautz) Middelhoven, Scorzetti & Fell 2001 was isolated from the exoskeleton of purse-web spider Atypus affinis. Both of the species were identified based on DNA sequence analysis of the host specimens displaying macroscopic signs of the superficial white mycosis on their exoskeleton. Only two specimens with macroscopic signs of superficial yeast growth were identified among the 125 individuals of A. affinis, A. piceus and Atypus muralis examined that were collected at various sites throughout the Czech Republic. The consistent burrow microclimate, uninterrupted occupancy of the single burrow for several subsequent years, and presence of prey remnants in the burrow below the purse-web may play a role in the course of infection of the mygalomorphs examined. The phylogenetic relationships of Trichosporon species are analyzed, concluding that association with invertebrates clusters predominantly among four groups of closely related species in independent Trichosporon clades.     

Inheritance of cleft palate in Italy. Evidence for a major autosomal recessive locus

Although several studies have demonstrated familial aggregation of nonsyndromic cleft palate (CP), the mode of inheritance still remains uncertain. We report the results of complex segregation analysis performed in families of 357 consecutive newborns affected with nonsyndromic CP (i.e., CP not a component feature of malformation syndrome, sequence or association), and registered in the North East Italy and Emilia Romagna congenital malformation registries in the period 1981–1993. This sample, based on a large number of consecutive births, in a well-defined geographical area, with quality control to detect associated anomalies and malformation syndromes, is independent of the number of affected subjects in the family and of CP severity, fitness, and survival. We have analyzed, using the mixed model, the whole sample of nonsyndromic CP, including isolated (i.e., without other anomalies) CP (CPI) and CP associated with at least one other anomaly (CPA), for which a diagnosis of malformation syndrome was not possible. When nonsyndromic CP (including CPA) are considered in the analysis, there is no heterogeneity between CPA and CPI nor between CP including hard palate (CPH) and CP of the soft palate only (CPS). POINTER and COMDS programs cannot discriminate between alternative genetic models; only the hypothesis of non-genetic transmission is rejected. The COMDS analysis two-locus model, which indicates that a modifier locus (or loci) operates in addition to a single major locus (SML), does not show evidence of better fit than SML, polygenic, and multifactorial models. When the severity parameter (defined as CPH and CPS) is added, CPI and CPA show heterogeneity. Eventually, when the analysis is limited to CPI and includes information on severity, a recessive SML, with low penetrance and determining CPH, provides a significant best fit. To have defined a genetic model for CPI and provided evidence for SML inheritance suggests that genetic linkage studies could be implemented. This conclusion is in agreement with previous studies which showed a significant association between alleles of transforming growth factor alpha and CP only in humans, and that single recessive genes may play a crucial role during palatogenesis in mice as well as in Brittany spaniels. Application of the candidate genes to human CPH families could reveal whether these genes are involved. Received: 16 December 1996 / Accepted: 16 March 1997     

Human Rap1 modulates TRF2 attraction to telomeric DNA

More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1–TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1–TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.     

Effect of chlorophenols on the membrane lipids of bacterial cells

Chlorophenols, widespread soil and water contaminants and often degradation products of some pesticides, are a potential stress factor for survival of environmental bacteria. The effect of pentachlorophenol (PCP) and 2,4-chlorophenol (2,4-CP) on the growth, amount of lipid, and fatty acid composition in the membrane lipids was examined in a strain of the bacterium Kocuria varians, able to degrade chlorophenols. The index of fatty acid unsaturation in two main membrane lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased in the presence of chlorophenols. Transformation of stearic acid into oleic acid was significantly increased by PCP addition only in PE, but conversion of oleic acid into linoleic acid was blocked by PCP and 2,4-CP in both PC and PE. This observation may indicate that while Δ⁹ desaturase was sensitive mainly to 2,4-CP, activity of Δ¹² desaturase was inhibited by both PCP and 2,4-CP.     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.     

Heavy metal accumulation of Danube river aquatic plants — indication of chemical contamination

In this paper, the ecological status of a section of the Danube River flowing through Serbia from Bezdan to Djerdap was evalutated. Using the chemical composition of water, sediment samples from the littoral zone and dominant aquatic macrophytes, the level of chemical pollution was ascertained. Chemical analyses of the water and sediment indicated that the tributaries flowing into the Danube significantly influenced the chemical load of the water and as a direct consequence, the sediment. The concentration of heavy metals including Cu, Mn and Cd found in plants of the Potamogeton genus, further indicated significant chemical pollution, establishing a clear link between the chemical composition of plant tissues and the chemical composition of water and sediment. This paper therefore describes how the chemical composition of aquatic plants can be used as a reliable indicator for heavy metal pollution of aquatic ecosystems.