Mercury uptake by halophytes in response to a long-term contamination in coastal wetland salt marshes (northern Adriatic Sea).

Mercury (Hg) distribution in saltmarsh sediments and in three selected halophytes (Limonium narbonense, Sarcocornia fruticosa and Atriplex portulacoides) of a wetland system (Marano and Grado Lagoon, Italy) following a contamination gradient in sediments was investigated. The Hg uptake was evaluated at the root system level by calculating the enrichment factor (EF) and in the aboveground tissues by means of the translocation factor (TF). The related methylmercury (MeHg) concentrations in the halophytes were also investigated with regard to the location of the sites and their degree of contamination. Hg concentration in halophytes seemed poorly correlated both with the total Hg in rhizo-sediments and with the specific plant considered, supporting the evidence that the chemico-physical parameters of sediments could significantly affect metal availability for plants. Hg concentrations in roots increased with depth and were 20-fold higher than content measured in related rhizo-sediments (high EF). A low content of Hg is translocated in aboveground tissues (very low TF values), thus highlighting a kind of avoidance strategy of these halophytes against Hg toxicity. MeHg values were comparable between the two sites and among species, but the translocation from below- to aboveground plant tissues was more active.

Clickable Cisplatin Derivatives as Versatile Tools to Probe the DNA Damage Response to Chemotherapy.

Cisplatin induces DNA crosslinks that are highly cytotoxic. Hence, platinum complexes are frequently used in the treatment of a broad range of cancers. Efficiency of cisplatin treatment is limited by the tumor-specific DNA damage response to the generated lesions. We reasoned that better tools to investigate the repair of DNA crosslinks induced by cisplatin would therefore be highly useful in addressing drug limitations. Here, we synthesized a series of cisplatin derivatives that are compatible with click chemistry, thus allowing visualization and isolation of DNA-platinum crosslinks from cells to study cellular responses. We prioritized one alkyne and one azide Pt(II) derivative, Pt-alkyne-53 and Pt-azide-64, for further biological characterization. We demonstrate that both compounds bind DNA and generate DNA lesions and that the viability of treated cells depends on the active DNA repair machinery. We also show that the compounds are clickable with both a fluorescent probe as well as biotin, thus they can be visualized in cells, and their ability to induce crosslinks in genomic DNA can be quantified. Finally, we show that Pt-alkyne-53 can be used to identify DNA repair proteins that bind within its proximity to facilitate its removal from DNA. The compounds we report here can be used as valuable experimental tools to investigate the DNA damage response to platinum complexes and hence might shed light on mechanisms of chemoresistance.

POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells.

Polymerase theta (POLθ) is an error-prone DNA polymerase whose loss is synthetically lethal in cancer cells bearing breast cancer susceptibility proteins 1 and 2 (BRCA1/2) mutations. To investigate the basis of this genetic interaction, we utilized a small-molecule inhibitor targeting the POLθ polymerase domain. We found that POLθ processes single-stranded DNA (ssDNA) gaps that emerge in the absence of BRCA1, thus promoting unperturbed replication fork progression and survival of BRCA1 mutant cells. A genome-scale CRISPR-Cas9 knockout screen uncovered suppressors of the functional interaction between POLθ and BRCA1, including NBN, a component of the MRN complex, and cell-cycle regulators such as CDK6. While the MRN complex nucleolytically processes ssDNA gaps, CDK6 promotes cell-cycle progression, thereby exacerbating replication stress, a feature of BRCA1-deficient cells that lack POLθ activity. Thus, ssDNA gap formation, modulated by cell-cycle regulators and MRN complex activity, underlies the synthetic lethality between POLθ and BRCA1, an important insight for clinical trials with POLθ inhibitors.

Knowledge Gaps around Smoking and Nicotine among U.S. Military Veterans Who Smoke and Implications for Practice.

The present study describes the knowledge about smoking and nicotine among a sample of current Iraq-/Afghanistan-era veterans who smoke (N = 117). A majority of participants had knowledge regarding general risks of smoking and benefits of nicotine replacement therapy. However, many participants underestimated their personal cardiovascular and cancer risk as a smoker. Many participants also inaccurately believed that nicotine causes cancer and that nicotine medications work by making one physically sick if used while smoking. These beliefs could lead to reluctance to use nicotine replacement therapy. Discussion of findings offers potential solutions in the form of patient education as well as emphasis on training healthcare providers training on best practices for patient education (beyond simple advice to quit). More nuance and detail in patient education may facilitate increased knowledge about smoking and nicotine among U.S. military veterans with the ultimate goal of increasing cessation rates.

The RNA polymerase II carboxy-terminal domain: links to a bigger and better 'holoenzyme'?

The largest subunit of eukaryotic RNA polymerase II has an unusual tandemly repeated heptapeptide sequence at its carboxyl terminus. The function of this evolutionarily conserved carboxy-terminal domain is unknown; however, new evidence links it to the formation of a large multi-component RNA polymerase II complex possessing enhanced transcriptional initiation properties. The existence of a pre-assembled RNA polymerase II 'holoenzyme' in the cell calls into question the long held view of transcription initiation as an ordered multi-step process.

Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein.

We have used protein-blotting and protein affinity chromatography to demonstrate that each of the two glutamine-rich activation domains of the human transcription factor Sp1 can bind specifically and directly to the C-terminal evolutionarily conserved domain of the human TATA box-binding protein (TBP). These activation domains of Sp1 also bind directly to Drosophila TBP but bind much less strongly to TBP from the yeast Saccharomyces cerevisiae. The abilities of the Sp1 activation domains to interact directly with the TBPs of various species correlate well with their abilities to activate transcription in extracts derived from the same species. We also show that a glutamine-rich transcriptional activating region of the Drosophila protein Antennapedia binds directly to TBP in a species-specific manner that reflects its ability to activate transcription in vivo. These results support the notion that TBP is a direct and important target of glutamine-rich transcriptional activators.

Large-scale functional analysis using peptide or protein arrays.

The array format for analyzing peptide and protein function offers an attractive experimental alternative to traditional library screens. Powerful new approaches have recently been described, ranging from synthetic peptide arrays to whole proteins expressed in living cells. Comprehensive sets of purified peptides and proteins permit high-throughput screening for discrete biochemical properties, whereas formats involving living cells facilitate large-scale genetic screening for novel biological activities. In the past year, three major genome-scale studies using yeast as a model organism have investigated different aspects of protein function, including biochemical activities, gene disruption phenotypes, and protein-protein interactions. Such studies show that protein arrays can be used to examine in parallel the functions of thousands of proteins previously known only by their DNA sequence.

Cystic fibrosis transmembrane conductance regulator: expression and helicity of a double membrane-spanning segment.

The gene responsible for cystic fibrosis encodes a membrane protein--the 1480-residue cystic fibrosis transmembrane conductance regulator (CFTR)--in which membrane-based CF-phenotypic mutants alter pore structure and/or impair ion transport. We report the preparation in milligram quantities and conformational characterization of a polypeptide comprised of CFTR transmembrane (TM) segments 3-4, a putative 'helical hairpin' portion of the CFTR TM1-6 domain. The TM segment 3-4 of CFTR was expressed in E. coli as a fusion protein linked to the C-terminus of His-tagged thioredoxin. Nickel chelate affinity chromatography, followed by release from the carrier by digestion with thrombin protease, gave free CFTR(TM3-4). Monitoring of the folding properties and conformational state(s) of the TM3-4 polypeptide using circular dichroism spectroscopy indicated a partial alpha-helical conformation in aqueous buffer, with up to 30% increase in alpha-helical content observed in membrane-mimetic environments.

Immunogenicity of immunoliposomes.

Multilamellar immunoliposomes were prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH), sphingomyelin (SPH) and biotinylated dipalmitoylphosphatidylethanolamine (PEB) in the molar ratio of 1:1:1:0.1 with surface linked avidin-biotinylated sheep (anti-mouse IgG) IgG (AV-sIgGB) or GK1.5 monoclonal rat (antimouse L3T4 antigen) IgG (AV-GK1.5B). The ability of these immunoliposomes to induce antibody responses against AV, sIgG or GK1.5 was determined. GK1.5B and sIgGB elicited a low-level antibody response (5-10 microgram/ml serum) after i.v. immunization and boosting. Liposomes (1 mumol) containing GK1.5B or sIgGB were more effective than free GK1.5B or sIgGB in eliciting antibodies (20-30 and 100-120 micrograms/ml serum, respectively). Liposomal AV mixed with either sIgG or GK1.5 gave antibody levels comparable to immunization with free GK1.5B or sIgGB. Liposomes with surface AV-sIgGB or AV-GK1.5B elicited antibodies against AV and high levels against GK1.5 or sIgG. Immunoliposomes possessing surface AV-sIgGB or AV-GK1.5B were eliminated from the circulation of normal mice relatively slowly (T1/2 15.5 and 30 min): in contrast, liposomal AV-sIgGB or AV-GK1.5B was rapidly eliminated from the circulation of immunized mice (T1/2 4.5 and 4.0 min). These results demonstrate that liposomes with surface IgG (immunoliposomes) are immunogenic, and that repeated administration elicits anti-IgG antibodies that result in a significant reduction in blood circulation residence times.

[Investigation in a slaughter house and processing of pork meat. Repetitive task work and osteo-articular and musculotendinous pathology of the upper limbs]. / Indagine su un'azienda di macellazione e lavorazione di carni suine. Lavoro con compiti ripetitivi e patologia osteo-articolare e muscolotendinea degli arti superiori.

The investigation concerned 47 workers (43 males and 4 females), whose average age was 41.5 years and average length of service 12 years. The aim of the study was to quantify the presence in an abattoir and meat processing plant of risk factors represented by repetitive movements requiring the use of force, and to describe the work-related musculo-skeletal disorders (WMSDs) of the upper limbs found in a group of workers exposed to such risk factors. The work was found to feature high speeds and very particular operations which, for most of the workers, required highly repetitive actions often associated with the use of force. Almost all the tasks had duration cycles of less than 30 seconds and a medium-high rate of actions/minute (from 20 to 60), with peak rates reached in the boning operations; the postural involvement was also considerable, particularly for the right wrist. The amount of force employed-calculated as a percentage of the Maximum Voluntary Contraction-averaged 50%. With very few exceptions, there were no significant pauses during the cycle. The group displayed a high prevalence of pain and paraesthesia and joint disorders, particularly in the over 35 age groups; statistically significant differences emerged with respect to the data from a matched population sample given the same anamnestic and clinical protocol. The group also exhibited significantly different Carpal Tunnel Syndromes with respect to the control population: 7 right-hand CTSs and five left-hand. CTS affected two out of every three women aged over 35 and 3 out of every 23 men over 35. The authors discuss the results in the light of previous studies and of the definition of CTS. The study concludes that investigating and preventing WMSDs in the meat processing industry is a justified, albeit very difficult, task whilst the protection afforded by current legislation appears to be most inadequate.

A laboratory-incubated redox oscillation experiment to investigate Hg fluxes from highly contaminated coastal marine sediments (Gulf of Trieste, Northern Adriatic Sea).

Mercury (Hg) mobility at the sediment-water interface was investigated during a laboratory incubation experiment conducted with highly contaminated sediments (13 µg g(-1)) of the Gulf of Trieste. Undisturbed sediment was collected in front of the Isonzo River mouth, which inflows Hg-rich suspended material originating from the Idrija (NW Slovenia) mining district. Since hypoxic and anoxic conditions at the bottom are frequently observed and can influence the Hg biogeochemical behavior, a redox oscillation was simulated in the laboratory, at in situ temperature, using a dark flux chamber. Temporal variations of several parameters were monitored simultaneously: dissolved Hg (DHg) and methylmercury (MeHg), O2, NH4 (+), NO3 (-) + NO2 (-), PO4 (3-), H2S, dissolved Mn(2+), dissolved inorganic and organic carbon (DIC and DOC). Under anoxic conditions, both Hg (665 ng m(2) day(-1)) and MeHg (550 ng m(2) day(-1)) fluxed from sediments into the water column, whereas re-oxygenation caused concentrations of MeHg and Hg to rapidly drop, probably due to re-adsorption onto Fe/Mn-oxyhydroxides and enhanced demethylation processes. Hence, during anoxic events, sediments of the Gulf of Trieste may be considered as an important source of DHg species for the water column. On the contrary, re-oxygenation of the bottom compartment mitigates Hg and MeHg release from the sediment, thus acting as a natural "defence" from possible interaction between the metal and the aquatic organisms.

MEC1-dependent phosphorylation of Rad9p in response to DNA damage.

In budding yeast, DNA damage can activate a checkpoint surveillance system controlled by the RAD9, RAD53, and MEC1 genes, resulting in a delay in cell cycle progression. Here, I report that DNA damage induces rapid and extensive phosphorylation of Rad9p in a manner that correlates directly with checkpoint activation. This response is dependent on MEC1, which encodes a member of the evolutionarily conserved ATM family of protein kinases, and on gene products of the RAD24 epistasis group, which have been implicated in the recognition and processing of DNA lesions. Since the phosphorylated form of Rad9p appears capable of interacting stably with Rad53p in vivo, this phosphorylation response likely controls checkpoint signaling by Rad9p.

Enhanced antibody response to liposome-associated protein antigens: preferential stimulation of IgG2a/b production.

The effect of liposome encapsulation on the antibody response to bovine serum albumin (BSA), human carcinoembryonic antigen (CEA) and sheep IgG (sIgG) has been determined in the mouse. Dipalmitoylphosphatidylcholine/dimyristolylphosphatidylglycerol liposomes (10:1 molar ratio; 1 mumol) containing BSA, CEA or sIgG induced significant levels of IgG antibodies after one injection, and enhanced the proportion of IgG2a/2b to IgG1 on subsequent boost injection. The IgG antibody titre induced by liposomal antigen was 100-400-fold greater than immunization with antigen alone. Immunization with antigen and the water-soluble adjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; 50 micrograms) resulted in antibody titres intermediate between free and liposomal antigen. MDP did not enhance the proportion of IgG2a/2b to IgG1. Incorporation of the lipid soluble MDP derivative MDP-glycerol dipalmitate (MDP-GDP; 10 micrograms) liposomes containing protein antigens resulted in higher titres and enhanced IgG2b isotype expression. Analysis of serum IgG antibody-isotype levels after immunization and boost with BSA/MDP showed that the half-life of IgG2a/2b and IgG3 was significantly less than that of IgG1. Liposomal encapsulation resulted in longer IgG2a/2b and IgG3 half-lives, especially when MDP-GDP was present in the liposome. These results demonstrate that, whereas MDP preferentially stimulates IgG1 antibodies, liposomes elicit high levels of IgG2a/2b isotypes with significantly longer serum half-lives.

Promoter-dependent photocross-linking of the acidic transcriptional activator E2F-1 to the TATA-binding protein.

Sequence-specific transcriptional activators, such as the human factor E2F-1, increase the rate of initiation of transcription by RNA polymerase II, possibly by contacting one or more of the RNA polymerase II-associated general initiation factors. One candidate target of transactivators is the TATA-binding protein (TBP), which, when bound to a promoter, nucleates the formation of a preinitiation complex. Previous studies using affinity chromatography techniques have shown that the activation domains of certain activators, including the acidic activation domain of E2F-1, can interact with TBP in the absence of DNA. Using a site-directed photoaffinity cross-linking approach, we demonstrate here that the activation domain of the chimeric activator LexA-E2F-1 can be cross-linked to TBP when both factors are bound to a transcriptionally responsive RNA polymerase II promoter. Mutations within the activation domain of LexA-E2F-1 that impaired its ability to activate transcription in vitro were found to reduce cross-linking of LexA-E2F-1 to TBP. The association of initiation factor TFIIB with the TBP-promoter complex did not preclude this promoter-dependent cross-linking to LexA-E2F-1; however, this cross-linking was promoter-independent. In contrast, TFIIA strongly inhibited the promoter-dependent cross-linking of LexA-E2F-1 to TBP. These results directly demonstrate that acidic activators such as E2F-1 can interact with TBP during the earliest stages in the assembly of an RNA polymerase II preinitiation complex.

A novel yeast protein influencing the response of RNA polymerase II to transcriptional activators.

A sensitive in vitro crosslinking technique using a photoactive derivative of the chimeric activator LexA-E2F-1 was used to identify yeast proteins that might influence the response of RNA polymerase II to transcriptional activators. We found that a novel yeast protein, Xtc1p, could be covalently crosslinked to the activation domain of LexA-E2F-1 when this derivatized activator was bound to DNA upstream of an activator-responsive RNA polymerase II promoter. Because affinity chromatography experiments showed that Xtc1p also bound directly and specifically to the activation domains of E2F-1, the viral activator VP16, and the yeast activator Gal4p and copurified with the RNA polymerase II holoenzyme complex, Xtc1p may modulate the response of RNA polymerase II to multiple activators. Consistent with this notion, yeast strains deleted for the XTC1 gene exhibited pleiotropic growth defects, including temperature sensitivity, galactose auxotrophy, and a heightened sensitivity to activator overexpression, as well as an altered response to transcriptional activators in vivo.

Dynamic interaction of DNA damage checkpoint protein Rad53 with chromatin assembly factor Asf1.

The evolutionarily conserved yeast checkpoint protein kinase Rad53 regulates cell cycle progression, transcription, and DNA repair in response to DNA damage. To uncover potential regulatory targets of Rad53, we identified proteins physically associated with it in vivo using protein affinity purification and tandem mass spectrometry. Here we report that Rad53 interacts in a dynamic functional manner with Asf1, a chromatin assembly factor recently shown to mediate deposition of acetylated histones H3 and H4 onto newly replicated DNA. Biochemical and molecular genetic studies suggest that Asf1 is an important target of the Rad53-dependent DNA damage response and that Rad53 may directly regulate chromatin assembly during DNA replication and repair.

Characterization of the interaction between the acidic activation domain of VP16 and the RNA polymerase II initiation factor TFIIB.

Contact between a transcriptional activator and one or more components of the RNA polymerase II transcription initiation machinery is generally believed important for activators to function. Several different molecular targets have been suggested for direct contact by herpes simplex virus virion protein VP16, including the general initiation factor TFIIB. In this report we have used several strategies to critically assess this interaction between VP16 and TFIIB. Affinity columns of VP16 bound TFIIB activity from HeLa cell extracts and the binding was reduced by mutations in the activation domain of VP16. In assays of direct binding, VP16 bound recombinant human TFIIB but not Drosophila or yeast TFIIB. Unlike binding from an extract, however, we found that the interaction between VP16 and recombinant human TFIIB was not affected by mutations in VP16 that reduce transactivation. Point mutations within human TFIIB that reduce transactivation by VP16 have been shown to reduce VP16 binding, but we show here that these same mutations critically affect both the important TBP-TFIIB interaction and the ability of TFIIB to support activator-independent basal transcription in vitro. Taken together our results suggest more evidence is needed to support the notion that TFIIB is a functionally important target for the activator VP16.

Apolipoprotein A-I promotes the formation of phosphatidylcholine core aldehydes that are hydrolyzed by paraoxonase (PON-1) during high density lipoprotein oxidation with a peroxynitrite donor.

High density lipoprotein (HDL) is rich in polyunsaturated phospholipids that are sensitive to oxidation. However, the effect of apolipoprotein A-I and paraoxonase-1 (PON-1) on phosphatidylcholine oxidation products has not been identified. We subjected native HDL, trypsinized HDL, and HDL lipid suspensions to oxidation by the peroxynitrite donor, 3-morpholinosydnonimine. HDL had a basal level of phosphatidylcholine mono- and di-hydroperoxides that increased to a greater extent in HDL, compared with either trypsinized HDL or HDL lipid alone. Phosphatidylcholine core aldehydes, which were present in small amounts, increased 10-fold during oxidation of native HDL, compared with trypsinized HDL (p = 0.004), and 4-fold compared with HDL lipid suspensions (p = 0.0021). In addition, the content of lysophosphatidylcholine increased 300% during oxidation of native HDL, but only 80 and 25%, respectively, during oxidation of trypsinized HDL and HDL lipid suspensions. Phosphatidylcholine isoprostanes accumulated in comparable amounts during the oxidation of all three preparations. Incubation of apolipoprotein A-I with 1-palmitoyl-2-linoleoyl glycerophosphocholine proteoliposomes in the presence of 3-morpholinosydnonimine or apoAI with phosphatidylcholine hydroperoxides resulted in a significant increase in phosphatidylcholine core aldehydes with no formation of lysophosphatidylcholine. We propose that apolipoprotein A-I catalyzes a one-electron oxidation of alkoxyl radicals. Purified PON-1 hydrolyzed phosphatidylcholine core aldehydes to lysophosphatidylcholine. We conclude that, upon HDL oxidation with peroxynitrite, apolipoprotein AI increases the formation of phosphatidylcholine core aldehydes that are subsequently hydrolyzed by PON1.

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.