Adding diversity to ruthenium (II)-arene anticancer (RAPTA) compounds via click chemistry: the influence of hydrophobic chains

The application of click chemistry to develop libraries of organometallic ruthenium-arene complexes with potential anticancer properties has been investigated. A series of ruthenium-imidazole-triazole complexes, with hydrophobic tails, were prepared from a common precursor via click chemistry. The tail could be attached to the ligand prior to coordination to the ruthenium complex were screened for cytotoxicity in tumourigenic and non-tumourigenic cell lines, and while the compounds were only moderately cytotoxic, good selectivity for tumourigenic cells were abserved.

Lack of directed V alpha 14-J alpha 281 rearrangements in NK1+ T cells.

NK1.1+ T cells are an unusual subset of TCR alpha beta cells distinguished by their highly restricted V beta repertoire and predominant usage of an invariant V alpha 14-J alpha 281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant alpha chain, we have analyzed V alpha 14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1+ T cells. As expected a high proportion (17/20) of the hybrids had rearranged V alpha 14 to J alpha 281. However, V alpha 14-J alpha 281 rearrangements always occurred on only one chromosome and were accompanied by other V alpha-J alpha rearrangements (not involving V alpha 14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of V alpha 14-J alpha 281 rearrangements in NK1.1+ T cells.

Constraints on the upper Permian to upper Triassic marine carbon isotope curve : case studies from the Tethys

Secular variations of the seawater carbon isotopic composition provide evidence for paleoceanographic and paleoclimatic changes and may serve for chemiostratigraphic correlations. The present study aimed to improve the current knowledge on the Upper Permian and Triassic segment of the Phanerozoic marine carbon isotope curve, whose Triassic part was poorly constrained by previous studies. Profiles of inorganic carbon isotopes are provided for sections from Himalaya (Salt Range, Kashmir, Spiti and Nepal), Oman and North Dobrogea (Romania) on the basis of whole-rock carbonate analysis. The data acquired, together with a literature compilation confirmed that most of the Upper Permian is characterized by high δ13C values (averaging +40/00) but failed to detect a positive excursion as suggested by recent compilations. In the light of these observations, the large drop in δ13C values associated with the end-Permian mass extinction appears to be driven by a sudden transfer of previously stocked 13C depleted carbon, rather than by the overturn of a Late Permian stratified ocean. The Triassic data-set outlines significant secular variations. The best documented is a carbon isotope positive excursion just across the Lower-Middle Triassic boundary, globally developed since it was detected in various paleogeographic settings. It is interpreted to reflect variations in surface ocean chemistry, possibly related to increased primary productivity, at times when the biotic recovery after the end-Permian mass-extinction began to accelerate significantly and when a sharp rise in seawater δ34S values occurred globally. Strontium isotope data obtained from well preserved biogenic phosphates allow a refinement of the Middle Triassic segment of the seawater strontium isotope curve and show a major inflexion point of the seawater strontium isotope curve also near the Lower Triassic - Middle Triassic boundary. These facts suggest that the transition from the Early to the Middle Triassic was a time of revolutionary global change which represented an important step in the evolution of Mesozoic marine environments. A tentative carbon isotope curve for the Upper Permian to Upper Triassic time interval is proposed. Its major features are: ? high but constant δ13C values during the Late Permian ? a sharp drop in δ13C values in the latest Permian ? subsequent recovery of δ13C values ? a short-lived positive excursion across the Early-Middle Triassic boundary ? a gradual rise in δ13C values starting in the Late Ladinian or in the Early Carnian It is foreseen that these fluctuations of the carbon isotope curve may serve as chronostratigraphic markers and further assist in the correlation of Permian and Triassic carbonate deposits.

Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

Calibration of surface contamination monitors for the detection of iodine incorporation in the thyroid gland.

In Switzerland, individuals exposed to the risk of activity intake are required to perform regular monitoring. Monitoring consists in a screening measurement and is meant to be performed using commonly available laboratory instruments. More particularly, iodine intake is measured using a surface contamination monitor. The goal of the present paper is to report the calibration method developed for thyroid screening instruments. It consists of measuring the instrument response to a known activity located in the thyroid gland of a standard neck phantom. One issue of this procedure remains that the iodine radioisotopes have a short half-life. Therefore, the adequacy and limitations to simulate the short-lived radionuclides with so-called mock radionuclides of longer half-life were also evaluated. In light of the results, it has been decided to use only the appropriate iodine sources to perform the calibration.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Organometallic cages as vehicles for intracellular release of photosensitizers.

Water-soluble metalla-cages were used to deliver hydrophobic porphin molecules to cancer cells. After internalization, the photosensitizer was photoactivated, significantly increasing the cytotoxicity in cells. During the transport, the photosensitizer remains nonreactive to light, offering a new strategy to tackle overall photosensitization, a limitation often encountered in photodynamic therapy.

Cell surface expression of a functional rubella virus E1 glycoprotein by addition of a GPI anchor.

Rubella virus (RV) envelope glycoproteins E1 and E2 are targeted to the Golgi as heterodimers. While E2 contains a transmembrane Golgi retention signal, E1 is arrested in a pre-Golgi compartment in the absence of E2, and appears to require heterodimerization in order to reach the Golgi. Various forms of E1 with deletions in the ectodomain or lacking the cytoplasmic (CT) and transmembrane (TM) domains, as well as the 29 C-terminal amino acid residues of the ectodomain were also retained intracellularly. We therefore investigated the possibility of targetting E1 to the plasma membrane by addition of a glycosylphosphatidylinositol (GPI) anchor. We found that E1GPI was transported to the cell surface where it retained the hemadsorption activity characteristic of the wild-type E1/E2 heterodimer. Furthermore, coexpression of a mammalian GPI-specific phospholipase D (GPI-PLD) resulted in the release of E1GPI and in constitutive expression of a soluble form of E1. This study thus demonstrates that the GPI anchor has a dominant effect over the E1 pre-Golgi retention signal and that E1 is sufficient for hemadsorption.

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Anchoring of an immunogenic Plasmodium falciparum circumsporozoite protein on the surface of Dictyostelium discoideum.

The circumsporozoite protein (CSP), a major antigen of Plasmodium falciparum, was expressed in the slime mold Dictyostelium discoideum. Fusion of the parasite protein to a leader peptide derived from Dictyostelium contact site A was essential for expression. The natural parasite surface antigen, however, was not detected at the slime mold cell surface as expected but retained intracellularly. Removal of the last 23 amino acids resulted in secretion of CSP, suggesting that the C-terminal segment of the CSP, rather than an ectoplasmic domain, was responsible for retention. Cell surface expression was obtained when the CSP C-terminal segment was replaced by the D. discoideum contact site A glycosyl phosphatidylinositol anchor signal sequence. Mice were immunized with Dictyostelium cells harboring CSP at their surface. The raised antibodies recognized two different regions of the CSP. Anti-sporozoite titers of these sera were equivalent to anti-peptide titers detected by enzyme-linked immunosorbent assay. Thus, cell surface targeting of antigens can be obtained in Dictyostelium, generating sporozoite-like cells having potentials for vaccination, diagnostic tests, or basic studies involving parasite cell surface proteins.

Bentho-planktonic evidence from the Austrian Alps for a decline in sea-surface carbonate production at the end of the Triassic

A high-resolution micropalaeontological study, combined with geochemical and sedimentological analyses was performed on the Tiefengraben, Schlossgraben and Eiberg sections (Austrian Alps) in order to characterize sea-surface carbonate production during the end-Triassic crisis. At the end-Rhaetian, the dominant calcareous nannofossil Prinsiosphaera triassica shows a decrease in abundance and size and this is correlated with a increase in delta O-18 and a gradual decline in delta C-13(carb) values. Simultaneously, benthic foraminiferal assemblages show a decrease in diversity and abundance of calcareous taxa and a dominance of infaunal agglutinated taxa. The smaller size of calcareous nannofossils disturbed the vertical export balance of the biological carbon pump towards the sea-bottom, resulting in changes in feeding strategies within the benthic foraminiferal assemblages from deposit feeders to detritus feeders and bacterial scavengers. These micropalaeontological data combined with geochemical proxies suggest that changes in seawater chemistry and/or cooling episodes might have occurred in the latest Triassic, leading to a marked decrease of carbonate production. This in turn culminated in the quasi-absence of calcareous nannofossils and benthic foraminifers in the latest Triassic. The aftermath (latest Triassic earliest Jurassic) was characterised by abundance peaks of ``disaster'' epifaunal agglutinated foraminifera Trochammina on the sea-floor. Central Atlantic Magmatic Province (CAMP) paroxysmal activity, superimposed on a major worldwide regressive phase, is assumed to be responsible for a deterioration in marine palaeoenvironments. CAMP sulfuric emissions might have been the trigger for cooling episodes and seawater acidification leading to disturbance of the surface carbonate production at the very end-Triassic.

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Surface-sampling and analysis of TATP by swabbing and gas chromatography/mass spectrometry

The method of sample recovery for trace detection and identification of explosives plays a critical role in several criminal investigations. After bombing, there can be difficulties in sending big objects to a laboratory for analysis. Traces can also be searched for on large surfaces, on hands of suspects or on surfaces where the explosive was placed during preparatory phases (e.g. places where an IED was assembled, vehicles used for transportation, etc.). In this work, triacetone triperoxide (TATP) was synthesized from commercial precursors following reported methods. Several portions of about 6 mg of TATP were then spread on different surfaces (e.g. floors, tables, etc.) or used in handling tests. Three different swabbing systems were used: a commercial swab, pre-wetted with propan-2-ol (isopropanol) and water (7:3), dry paper swabs, and cotton swabs wetted with propan-2-ol. Paper and commercial swabs were also used to sample a metal plate, where a small charge of about 4 g of TATP was detonated. Swabs were sealed in small glass jars with screw caps and Parafilm® M and sent to the laboratory for analysis. Swabs were extracted and analysed several weeks later by gas chromatography/mass spectrometry. All the three systems gave positive results, but wetted swabs collected higher amounts of TATP. The developed procedure showed its suitability for use in real cases, allowing TATP detection in several simulations, including a situation in which people wash their hands after handling the explosive.

Rapid profiling of intact glucosinolates in Arabidopsis leaves by UHPLC-QTOFMS using a charged surface hybrid column.

INTRODUCTION: The analysis of glucosinolates (GS) is traditionally performed by reverse-phase liquid chromatography coupled to ultraviolet detection after a time-consuming desulphation step, which is required for increased retention. Simpler and more efficient alternative methods that can shorten both sample preparation and analysis are much needed. OBJECTIVE: To evaluate the feasibility of using ultrahigh-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) for the rapid profiling of intact GS. METHODOLOGY: A simple and short extraction of GS from Arabidopsis thaliana leaves was developed. Four sub-2 µm reverse-phase columns were tested for the rapid separation of these polar compounds using formic acid as the chromatographic additive. High-resolution QTOFMS was used to detect and identify GS. RESULTS: A novel charged surface hybrid (CSH) column was found to provide excellent retention and separation of GS within a total running time of 11 min. Twenty-one GS could be identified based on their accurate mass as well as isotopic and fragmentation patterns. The method was applied to determine the changes in GS content that occur after herbivory in Arabidopsis. In addition, we evaluated its applicability to the profiling of other Brassicaceae species. CONCLUSION: The method developed can profile the full range of GS, including the most polar ones, in a shorter time than previous methods, and is highly compatible with mass spectrometric detection.