Marginal zinc deficiency increases oxidative DNA damage in the prostate after chronic exercise

Approximately 12% of Americans do not consume the recommended level of zinc and could be at risk for marginal zinc deficiency. Zinc functions in antioxidant defense and DNA repair and could be important for prostate health. We hypothesized that marginal zinc deficiency sensitizes the prostate to oxidative stress and DNA damage. Rats were fed a zinc-adequate (ZA; 30 mg Zn/kg) or marginally zinc-deficient (MZD; 5–6 mg Zn/kg) diet for 6 weeks. MZD increased p53 and PARP expression but no change in 8-hydroxy-2′-deoxyguanosine levels was detected. To examine the susceptibility to exogenous oxidative stress, rats fed a ZA or MZD diet were assigned to exercising (EXE) or sedentary (SED) groups for 9 weeks. MZD or EXE alone did not affect oxidative DNA damage in the prostate; however, combined MZD + EXE increased DNA damage in the dorsolateral lobe. PARP and p53 expression was not further induced with MZD + EXE, suggesting that MZD interferes with DNA repair responses to stress. Finally, the addition of phytase to the MZD diet successfully restored zinc levels in the prostate and decreased DNA damage back to ZA levels. Overall, this study suggests that marginal zinc deficiency sensitizes the prostate to oxidative stress and demonstrates the importance of maintaining optimal zinc nutrition in physically active populations.     

Diverse climate sensitivity of Mediterranean tree-ring width and density

Understanding long-term environmental controls on the formation of tree-ring width (TRW) and maximum latewood density (MXD) is fundamental for evaluating parameter-specific growth characteristics and climate reconstruction skills. This is of particular interest for mid-latitudinal environments where future rates of climate change are expected to be most rapid. Here we present a network of 28 TRW and 21 MXD chronologies from living and relict conifers. Data cover an area from the Atlantic Ocean in the west to the Mediterranean Sea in the east and an altitudinal gradient from 1,000 to 2,500 m asl. Age trends, spatial autocorrelation functions, carry-over effects, variance changes, and climate responses were analyzed for the individual sites and two parameter-specific regional means. Variations in warm season (May–September) temperature mainly control MXD formation (r = 0.58 to 0.87 from inter-annual to decadal time-scales), whereas lower TRW sensitivity to temperature remains unstable over space and time.     

Discovery of a novel series of potent S1P1 agonists

The discovery of a novel series of S1P1 agonists is described. Starting from a micromolar HTS positive, iterative optimization gave rise to several single-digit nanomolar S1P1 agonists. The compounds were able to induce internalization of the S1P1 receptor, and a selected compound was shown to be able to induce lymphopenia in mice after oral dosing.     

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI^q carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.The use of fluorescent proteins as probes has proven to be a vital tool in elucidating spatial and temporal patterns of gene expression, protein localization, and protein-protein interactions in vivo. In addition to well-known contributions to cell biology and biochemistry, the use of fluorescent proteins to label bacteria has provided insight into complex host-pathogen interactions and routes of entry (14, 23, 29).The zebrafish has become an attractive model for in vivo infection studies. As an optically clear organism at the embryo and larval developmental stages, zebrafish are amenable to visualization during challenge with fluorescently labeled pathogens. In addition, fertilization in the zebrafish occurs ex utero, thereby simplifying infection by either static immersion or microinjection. The visualization of infection in situ can be conducted on the whole embryo using confocal or wide-field epifluorescence microscopy, an approach not possible in other model systems. To date, several zebrafish infection models have been established employing both fish and mammalian pathogens, including Edwardsiella tarda (21), E. ictaluri (14, 23), Staphylococcus aureus (16), Streptococcus pyogenes and S. iniae (18), and Pseudomonas aeruginosa (4). Illustrating the value of fluorescently labeled bacteria in studies of microbial pathogenesis, Van der Sar and colleagues established the zebrafish as a model for Salmonella enterica serovar Typhimurium infections using DsRed-labeled bacteria (30). Similarly, O''Toole and colleagues made use of a green fluorescent protein (GFP)-labeled strain of Vibrio (Listonella) anguillarum to elucidate the natural route of invasion of this fish pathogen (20). However, obstacles to the construction of fluorescently labeled microbes have prevented many from taking advantage of this technique to permit the real-time in situ analysis of infection. These obstacles include the nontransformable nature of many pathogens and the toxicity of RFP expression from high-copy-number plasmids.Recently, two transgenic lines of zebrafish that express GFP in cells of the myeloid lineage have been developed. Lawson et al. described a transgenic (Tg) zebrafish that utilizes the protooncogene fli1 promoter to drive the expression of enhanced GFP (EGFP) (15). The fli1 gene is preferentially expressed in endothelial and hematopoietic cells. Intended for the study of vascular development, Tg(fli1::EGFP) zebrafish not only have fluorescent endothelial cells but also macrophages (15), making this zebrafish line attractive to those who study innate immunity. It has been shown that zebrafish macrophages possess strong phagocytic properties and migrate to sites of bacterial infection (13). In 2006, Renshaw and his colleagues engineered a novel Tg(mpo::GFP) zebrafish that expresses GFP under the control of the neutrophil-specific myeloperoxidase promoter (22). In this study, they characterized the GFP-labeled neutrophils, noting their capability of chemotaxis at wound sites (22). Given the recent progress in zebrafish genetics with regard to these GFP-expressing transgenic lines, there was a clear need for a simple method for the RFP labeling of Gram-negative bacteria. Such a method would facilitate further studies of microbial pathogenesis in the zebrafish and could further elucidate phagocyte (GFP labeled)-pathogen (RFP labeled) interactions, kinetics of bacterial clearance, and mechanisms of phagocyte migration to sites of bacterial infection.For the purposes of this study, we employed dimeric (d) and monomeric (m) red fluorescent protein variants d-Tomato, td-Tomato, m-Orange, and m-Cherry, which were constructed by Shaner et al. (25). These variants were chosen based on combinations of their high relative brightness and decreased RFP maturation times compared to those of the original RFPs (25). We describe here a simple method for the RFP labeling of Gram-negative bacteria.     

Diverse effects on the native β-sheet of the human prion protein due to disease-associated mutations

Prion diseases are fatal neurodegenerative disorders that involve the conversion of the normal cellular form of the prion protein (PrP(C)) to a misfolded pathogenic form (PrP(Sc)). There are many genetic mutations of PrP associated with human prion diseases. Three of these point mutations are located at the first strand of the native β-sheet in human PrP: G131V, S132I, and A133V. To understand the underlying structural and dynamic effects of these disease-causing mutations on the human PrP, we performed molecular dynamics of wild-type and mutated human PrP. The results indicate that the mutations induced different effects but they were all related to misfolding of the native β-sheet: G131V caused the elongation of the native β-sheet, A133V disrupted the native β-sheet, and S132I converted the native β-sheet to an α-sheet. The observed changes were due to the reorientation of side chain-side chain interactions upon introducing the mutations. In addition, all mutations impaired a structurally conserved water site at the native β-sheet. Our work suggests various misfolding pathways for human PrP in response to mutation.     

Ribosomal Protein Genes RPS10 and RPS26 Are Commonly Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in ∼30%–50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.     

Molecular enzymology of lipoxygenases

Lipoxygenases (LOXs) are lipid peroxidizing enzymes, implicated in the pathogenesis of inflammatory and hyperproliferative diseases, which represent potential targets for pharmacological intervention. Although soybean LOX1 was discovered more than 60 years ago, the structural biology of these enzymes was not studied until the mid 1990s. In 1993 the first crystal structure for a plant LOX was solved and following this protein biochemistry and molecular enzymology became major fields in LOX research. This review focuses on recent developments in molecular enzymology of LOXs and summarizes our current understanding of the structural basis of LOX catalysis. Various hypotheses explaining the reaction specificity of different isoforms are critically reviewed and their pros and cons briefly discussed. Moreover, we summarize the current knowledge of LOX evolution by profiling the existence of LOX-related genomic sequences in the three kingdoms of life. Such sequences are found in eukaryotes and bacteria but not in archaea. Although the biological role of LOXs in lower organisms is far from clear, sequence data suggests that this enzyme family might have evolved shortly after the appearance of atmospheric oxygen on earth.     

Influence of pH on the human prion protein: insights into the early steps of misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. Conversion from the normal cellular form (PrP^C) or recombinant PrP (recPrP) to a misfolded form is pH-sensitive, in that misfolding and aggregation occur more readily at lower pH. To gain more insight into the influence of pH on the dynamics of PrP and its potential to misfold, we performed extensive molecular-dynamics simulations of the recombinant PrP protein (residues 90-230) in water at three different pH regimes: neutral (or cytoplasmic) pH (∼7.4), middle (or endosomal) pH (∼5), and low pH (<4). We present five different simulations of 50 ns each for each pH regime, amounting to a total of 750 ns of simulation time. A detailed analysis and comparison with experiment validate the simulations and lead to new insights into the mechanism of pH-induced misfolding. The mobility of the globular domain increases with decreasing pH, through displacement of the first helix and instability of the hydrophobic core. At middle pH, conversion to a misfolded (PrP^Sc-like) conformation is observed. The observed changes in conformation and stability are consistent with experimental data and thus provide a molecular basis for the initial steps in the misfolding process.     

Epithelial stem cells: turning over new leaves

Most epithelial tissues self-renew throughout adult life due to the presence of multipotent stem cells and/or unipotent progenitor cells. Epithelial stem cells are specified during development and are controlled by epithelial-mesenchymal interactions. Despite morphological and functional differences among epithelia, common signaling pathways appear to control epithelial stem cell maintenance, activation, lineage determination, and differentiation. Additionally, deregulation of these pathways can lead to human disorders including cancer. Understanding epithelial stem cell biology has major clinical implications for the diagnosis, prevention, and treatment of human diseases, as well as for regenerative medicine.