L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Conversion of CD95 (Fas) Type II into Type I signaling by sub-lethal doses of cycloheximide

CD95 (Fas/Apo-1)-mediated apoptosis was shown to occur through two distinct pathways. One involves a direct activation of caspase-3 by large amounts of caspase-8 generated at the DISC (Type I cells). The other is related to the cleavage of Bid by low concentration of caspase-8, leading to the release of cytochrome c from mitochondria and the activation of caspase-3 by the cytochrome c/APAF-1/caspase-9 apoptosome (Type II cells). It is also known that the protein synthesis inhibitor cycloheximide (CHX) sensitizes Type I cells to CD95-mediated apoptosis, but it remains contradictory whether this effect also occurs in Type II cells. Here, we show that sub-lethal doses of CHX render both Type I and Type II cells sensitive to the apoptogenic effect of anti-CD95 antibodies but not to chemotherapeutic drugs. Moreover, Bcl-2-positive Type II cells become strongly sensitive to CD95-mediated apoptosis by the addition of CHX to the cell culture. This is not the result of a restraint of the anti-apoptotic effect of Bcl-2 at the mitochondrial level since CHX-treated Type II cells still retain their resistance to chemotherapeutic drugs. Therefore, CHX treatment is granting the CD95-mediated pathway the ability to bypass the mitochondria requirement to apoptosis, much alike to what is observed in Type I cells.

Systemic analysis of microarray data sets towards identifying genes regulating root development

Nitrogen and water are essential for plant growth and development. In this study, we designed experiments to produce gene expression data of poplar roots under nitrogen starvation and water deprivation conditions. We found low concentration of nitrogen led first to increased root elongation followed by lateral root proliferation and eventually increased root biomass. To identify genes regulating root growth and development under nitrogen starvation and water deprivation, we designed a series of data analysis procedures, through which, we have successfully identified biologically important genes. Differentially Expressed Genes (DEGs) analysis identified the genes that are differentially expressed under nitrogen starvation or drought. Protein domain enrichment analysis identified enriched themes (in same domains) that are highly interactive during the treatment. Gene Ontology (GO) enrichment analysis allowed us to identify biological process changed during nitrogen starvation. Based on the above analyses, we examined the local Gene Regulatory Network (GRN) and identified a number of transcription factors. After testing, one of them is a high hierarchically ranked transcription factor that affects root growth under nitrogen starvation. It is very tedious and time-consuming to analyze gene expression data. To avoid doing analysis manually, we attempt to automate a computational pipeline that now can be used for identification of DEGs and protein domain analysis in a single run. It is implemented in scripts of Perl and R.

Reproducibility of cerebral phenylalanine levels in patients with phenylketonuria determined by 1H-MR spectroscopy

The reproducibility of metabolite content determined by MR spectroscopy (MRS) is usually at best a few percent for the prominent singlets. When studying low-concentration metabolites, like phenylalanine (Phe), where tissue content can be <100 micromol/kg, better reproducibility is paramount-particularly in view of using MRS results for potential individual treatment advice. An optimized, targeted spectroscopy method was established at 1.5T and reproducibility was established in 21 patients with phenylketonuria (PKU) where three spectra were recorded in each of three independent sessions, two of which were in immediate succession to minimize physiologic variation. Intersession variation was found to be only 7 micromol/kg Phe for back-to-back repetition of sessions, in close agreement with the variation of 16 micromol/kg observed for single spectra within a session. Analysis of variance proved the individuality of the blood/brain Phe ratio-though this ratio seems to be influenced by physiologic factors that are not stable in time. The excellent reproducibility was achieved through optimization of various factors, including signal-to-noise ratio, repositioning, and prescan calibrations, but also by enforcing as much prior information as possible (e.g., lineshape and phase from reference scans, constant prior-knowledge-locked baseline). While the application of maximum general prior knowledge is a general method to reduce fluctuations, one should remember that it may introduce systematic errors.

[Cryptosporidiose (C. parvum) in a foal with diarrhea]

The protozoon parasite Cryptosporidium parvum is an important cause of diarrhea in farm animals, but it can also infect other animals and humans. In this case report, oocysts of Cryptosporidium spp. were microscopically detected by modified Ziehl-Neelsen staining in the feces of a 9 day old Arabian colt presented with yellowish, foul smelling, diarrhea and fever of 40 degrees C. PCR and sequencing of the isolate revealed C. parvum (bovine genotype). Hemato-chemical analysis of the foals blood revealed a marked hypogammaglobulinaemia (IgG 108mg/dl). The colt responded well to a supportive therapy and administration of plasma (until a gammaglobulin-concentration of 620 mg/dl was reached) and was released in good health from the clinic after 10 days. Follow-up testing for Cryptosporidium oocycsts remained negative. Cryptosporidiosis with life-threatening diarrhea is a rare diagnosis in foals in Switzerland. Immunodeficiency increases the risk for cryptosporidiosis. We hypothesize that the low concentration of gammaglobulins together with the weak INF-gamma response normally observed in young foals may have favored the clinical manifestation with diarrhea. Foals with diarrhea should be screened for cryptosporidia with specific tests.

Seismic anisotropy in the Morcles nappe shear zone: Implications for seismic imaging of crustal scale shear zones

Microstructures and textures of calcite mylonites from the Morcles nappe large-scale shear zone in southwestern Switzerland develop principally as a function of 1) extrinsic physical parameters including temperature, stress, strain, strain rate and 2) intrinsic parameters, such as mineral composition. We collected rock samples at a single location from this shear zone, on which laboratory ultrasonic velocities, texture and microstructures were investigated and quantified. The samples had different concentration of secondary mineral phases (< 5 up to 40 vol.%). Measured seismic P wave anisotropy ranges from 6.5% for polyphase mylonites (~ 40 vol.%) to 18.4% in mylonites with < 5 vol.% secondary phases. Texture strength of calcite is the main factor governing the seismic P wave anisotropy. Measured S wave splitting is generally highest in the foliation plane, but its origin is more difficult to explain solely by calcite texture. Additional texture measurements were made on calcite mylonites with low concentration of secondary phases (≤ 10 vol.%) along the metamorphic gradient of the shear zone (15 km distance). A systematic increase in texture strength is observed moving from the frontal part of the shear zone (anchimetamorphism; 280 °C) to the higher temperature, basal part (greenschist facies; 350–400 °C). Calculated P wave velocities become increasingly anisotropic towards the high-strain part of the nappe, from an average of 5.8% in the frontal part to 13.2% in the root of the basal part. Secondary phases raise an additional complexity, and may act either to increase or decrease seismic anisotropy of shear zone mylonites. In light of our findings we reinterpret the origin of some seismically reflective layers in the Grône–Zweisimmen line in southwestern Switzerland (PNR20 Swiss National Research Program). We hypothesize that reflections originate in part from the lateral variation in textural and microstructural arrangement of calcite mylonites in shear zones.

A rapid and efficient ion-exchange chromatography for Lu–Hf, Sm–Nd, and Rb–Sr geochronology and the routine isotope analysis of sub-ng amounts of Hf by MC-ICP-MS

The development and improvement of MC-ICP-MS instruments have fueled the growth of Lu–Hf geochronology over the last two decades, but some limitations remain. Here, we present improvements in chemical separation and mass spectrometry that allow accurate and precise measurements of 176Hf/177Hf and 176Lu/177Hf in high-Lu/Hf samples (e.g., garnet and apatite), as well as for samples containing sub-nanogram quantities of Hf. When such samples are spiked, correcting for the isobaric interference of 176Lu on 176Hf is not always possible if the separation of Lu and Hf is insufficient. To improve the purification of Hf, the high field strength elements (HFSE, including Hf) are first separated from the rare earth elements (REE, including Lu) on a first-stage cation column modified after Patchett and Tatsumoto (Contrib. Mineral. Petrol., 1980, 75, 263–267). Hafnium is further purified on an Ln-Spec column adapted from the procedures of Münker et al. (Geochem., Geophys., Geosyst., 2001, DOI: 10.1029/2001gc000183) and Wimpenny et al. (Anal. Chem., 2013, 85, 11258–11264) typically resulting in Lu/Hf < 0.0001, Zr/Hf < 1, and Ti/Hf < 0.1. In addition, Sm–Nd and Rb–Sr separations can easily be added to the described two-stage ion-exchange procedure for Lu–Hf. The isotopic compositions are measured on a Thermo Scientific Neptune Plus MC-ICP-MS equipped with three 1012 Ω resistors. Multiple 176Hf/177Hf measurements of international reference rocks yield a precision of 5–20 ppm for solutions containing 40 ppb of Hf, and 50–180 ppm for 1 ppb solutions (=0.5 ng sample Hf 0.5 in ml). The routine analysis of sub-ng amounts of Hf will facilitate Lu–Hf dating of low-concentration samples.

Effect of mannitol and cold treatments on phosphate uptake and protein phosphorylation in Lemna minor (L.) plants

This report is aimed at elucidating the effect of mannitol and cold treatments on P uptake and protein phosphorylation in Lemna minor plants. Duckweed p lants were incu bated in the presence of [32P]or [33P]Pi in half-strength phosphate deprived E-medium under constant light regime for 1.5 h. Total plant protein extracts (pellet and supernatant) were then prepared and subjected to IEF x SDS-PAGE. To analyse the effect of the stresses on P uptake and protein labelling, Lemna minor plants were preincubated with 0.1, 0.5 mol · L-1 mannitol and at 4°C respectively, for 4 hours, before adding labelled orthophosphate. The results show that the general protein phosphorylation (including LHCII) is related to the level of P uptake. Radioactive phosphate incorporation is stimulated by a low concentration of mannitol (0.1 mol · L-1) but reduced by 0.5 mol · L-1 mannitol and cold stress in planta. The labelling into proteins is affected neither when stresses were applied to the plants after incubation with labelled orthophosphate, nor after in vitro protein phosphorylation. This indicates that general protein kinase activities in vivo are strictly limited by P uptake. A marked accumulation of soluble hexoses (mainly sucrose, glucose, and fructose) is observed under imposed stress, suggesting that the inhibition of P uptake in response to hyperosmotic and cold stresses is mediated by sugar accumulation in situ. However, metabolisable sugars like glucose did not alter the entry of phosphate at concentrations of 0.5 mol · L-1, showing that the chemical nature of the osmoticum influences P uptake.

Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase $\alpha$ are the molecular targets for two metal ions, Zn$\sp{2+}$ and Cd$\sp{2+},$ and an anticancer drug, F-ara-ATP.^ Human DNA ligases were purified to homogeneity and their AMP binding domains were mapped. Although their AMP-binding domains are similar, there could be difference between the two ligases in their DNA binding domains.^ The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP.^ A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex.^ F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3$\sp\prime$-terminus of DNA nick by DNA polymerase $\alpha.$^ All steps of the DNA ligation reaction were inhibited by Zn$\sp{2+}$ and Cd$\sp{2+}$ in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn$\sp{2+}$ and Cd$\sp{2+}$ showed their contradictory effects on the fidelity of the reaction by human DNA polymerase $\alpha.$ Zn$\sp{2+}$ decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd$\sp{2+}$ increased the frequencies of both misinsertion and mispair extension at very low concentration. Our data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported. ^

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.

Rare earth elements determined in Antarctic ice by inductively coupled plasma

Inductively coupled plasma mass spectrometry (ICP-MS) is a suitable tool for multi-element analysis at low concentration levels. Rare earth element (REE) determinations in standard reference materials and small volumes of molten ice core samples from Antarctica have been performed with an ICP-time of flight-MS (ICP-TOF-MS) system. Recovery rates for REE in e.g. SPS-SW1 amounted to not, vert, similar ~103%, and the relative standard deviations were 3.4% for replicate analysis at REE concentrations in the lower ng/l range. Analyses of REE concentrations in Antarctic ice core samples showed that the ICP-TOF-MS technique meets the demands of restricted sample mass. The data obtained are in good agreement with ICP-Quadrupole-MS (ICP-Q-MS) and ICP-Sector Field-MS (ICP-SF-MS) results. The ICP-TOF-MS system determines accurately and precisely REE concentrations exceeding 5 ng/l while between 0.5 and 5 ng/l accuracy and precision are element dependent.

Geochemistry of surface and groundwaters from lower Tom Basin wetland, western Siberia

New data are reported on the major- and trace-component compositions of acidic and weakly acidic low-concentration wetland waters and other water types. Special attention was given to dissolved organic compounds: fulvic and humic acids, bitumens, and hydrocarbons. The first comprehensive data are presented for organic trace components in the wetland waters of western Siberia: alkanes, pentacyclic terpenoids, steranes, alkylbenzenes, naphthalenes, phenanthrenes, tetraarenes, etc.

Geochemistry and structural formulas of heavy minerals from sands and sandstones of ODP Leg 126 samples

Lower Oligocene to Pleistocene volcaniclastic sands and sandstones recovered around the Izu-Bonin Arc during Ocean Drilling Program Leg 126 were derived entirely from Izu-Bonin Arc volcanism. Individual grains consist of volcanic glass, pumice, scoria, basaltic or andesitic fragments, plagioclase, pyroxene, and minor olivine and hornblende. In Pliocene-Pleistocene samples plagioclase and heavy minerals in the volcaniclastic sands and sandstones are present in the following abundances: plagioclase > orthopyroxene > clinopyroxene > pigeonite > olivine. In contrast, plagioclase and heavy minerals found in Oligocene-Miocene samples occur in the following order: plagioclase > clinopyroxene > orthopyroxene > hornblende. The low concentration of Al, Ti, and Cr in calcium-rich clinopyroxenes in Oligocene to Holocene sediments suggests that the sources of the volcaniclastic detritus were nonalkalic igneous rocks. There are, however, some distinctive differences in the chemical composition of pyroxene between the Pliocene-Pleistocene and Oligocene-Miocene volcaniclastic sands and sandstones. Orthopyroxene belongs to the hypersthene-ferrohypersthene series (Fe-rich) in Pliocene-Pleistocene sediments, and the bronzitehypersthene series (Mg-rich) in Oligocene-Miocene sediments. Clinopyroxene is characterized by augite and pigeonite in Pliocene-Pleistocene sediments, and by the diopside-augite series in Oligocene-Miocene sediments. Mineral assemblages and mineral chemistry of the volcaniclastic sands and sandstones reflect those of the volcanic source rocks. Therefore, the observed changes in mineralogy record the historical change in volcanism of the Izu-Bonin Arc. The mineralogy is consistent with the geochemistry of the volcaniclastic sands and sandstones and the geochemistry of forearc volcanic rocks of the Izu-Bonin Arc since the Oligocene.

Geochemistry of reduction haloes in ODP Holes 135-834A and 135-837A

The sediment column overlying basement in the Lau Basin consists of a sequence of volcaniclastic turbidites interbedded with hemipelagic clayey nannofossil mixed sediments, overlain in turn by a sequence of hemipelagic clayey nannofossil oozes containing sporadic calcareous turbidites. The clayey nannofossil oozes and mixed sediments are pervasively stained by hydrothermally derived iron and manganese oxyhydroxides. Sharply defined, lighter colored bands occur in the hemipelagic sediments, immediately beneath some (but by no means all) volcaniclastic and calcareous turbidites. These are identified as reduction haloes, of a type previously identified in quite different turbidite/pelagic sequences. The haloes are attributed to the burial of labile surficial Corg by turbidites, followed by the remineralization of this Corg with Mn and Fe oxyhydroxides as electron acceptors. The resultant characteristic Mn and Fe concentration/depth profiles are described, and a model is proposed for their development. The color alteration of the halo is ascribed to the removal of Mn oxyhydroxides, because, although the Fe content fluctuates through the haloes, this does not appear to affect their color. Other elements (Co, Cu, and Ni) are also at low concentration levels in the haloes like Mn, consistent with remobilization and migration out of the halo section, although the profile shapes are not identical with those of Mn. The behavior of V is distinctive in that it appears to have migrated into the haloes to be enriched there. Haloes are unlikely to form if turbidite emplacement is erosive and removes the near-surface layer, which generally is the most fluid part of the sediment and contains the highest levels of reactive Corg to drive the reduction process. Conversely, the presence of a halo implies that emplacement of the overlying turbidite did not significantly erode the pre-existing sediment/water interface.

Isotopic and micropaleontological record of sediments of the Baffin Bay

The oxygen and carbon isotopic compositions of the planktonic foraminifer, Neogloboquadrina pachyderma (sinistral), were determined at 20-cm intervals through the 'composite' top ~ 22 m of sediments at ODP Site 645 (Holes 645B, 645C, 645F, and 645G) and at 10-cm intervals through a 9-m piston core (85-027-016) collected during the Hudson site survey. Quantitative analyses of palynomorphs, notably dinocysts, and of planktonic foraminifers were performed. Organic and nitrogen contents and isotopic composition of nitrogen and carbon in organic matter also were determined. These data provide a high-resolution record of changes that occurred in surface-water masses during the last glacial cycle in Baffin Bay. The basin experienced low planktonic productivity during most of the late Pleistocene, either from dilution in surface water by meltwater discharges from the surrounding ice-sheet or from the presence of a relatively dense sea-ice cover. Peaks of meltwater discharge are indicated by d18O values as low as about 1.5 per mil, correlative d13C- d18O shifts, low concentration of planktonic foraminifers, high concentrations of glacially reworked pre-Quaternary palynomorphs, and low-salinity dinocyst assemblages. As a whole, d18O values ranging between 4.5 and 2.5 per mil allow the establishment of an 18O stratigraphy spanning isotopic stages 5 to 1. Because of the poor core recovery, the general paucity of microflora and microfauna, and the possible occurrence of slumping or debris flow at Site 645, further interpretation remains problematic.