Sequential Washing Research Articles

A Conserved Serine of Heterogeneous Nuclear Ribonucleoprotein L (hnRNP L) Mediates Depolarization-regulated Alternative Splicing of Potassium Channels

Molecular mechanisms of gene regulation underlying the activity-dependent long term changes of cellular electrical properties, such as those during memory, are largely unknown. We have shown that alternative splicing can be dynamically regulated in response to membrane depolarization and Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) activation, through special CaM kinase responsive RNA elements. However, proteins that mediate this regulation and how they are affected by CaMKIV are not known. Here we show that the regulation of the stress axis-regulated exon of the Slo1 potassium channel transcripts by membrane depolarization requires a highly conserved CaMKIV target serine (Ser-513) of the heterogeneous ribonucleoprotein L. Ser-513 phosphorylation within the RNA recognition motif 4 enhanced heterogeneous ribonucleoprotein L interaction with the CaMKIV-responsive RNA element 1 of stress axis-regulated exon and inhibited binding of the large subunit of the U2 auxiliary factor U2AF65. Both of these activities were abolished by a S513A mutation. Thus, through Ser-513, membrane depolarization/calcium signaling controls a critical spliceosomal assembly step to regulate the variant subunit composition of potassium channels.

Remediation of Heavy Metal Contamination in Calcareous Soil by Washing with Reagents: A Column Washing

This paper presents an evaluation of different dose of HCl and Na 2 EDTA for the removal of Cd and Pb contaminated calcareous soil in the column mode. The field soil contained high concentrations of DTPA-extractable Cd (12.82 mg kg -1 ) and Pb (105.38 mg kg -1 ). Both HCl and Na 2 EDTA were found to be effective on removing DTPA-extractable Cd, but low concentration of HCl was ineffective on removing DTPA-extractable Pb and the highest removal efficiency of DTPA-extractable Pb washed by optimal dose of Na 2 EDTA was only 45.39%. In order to effectively and economically remove both metals from the contaminated soil, a sequential use of the optimal dose of HCl followed by the optimal dose of Na2EDTA was tested and 87.3% and 73.2% removal efficiency were obtained for DTPA-extractable Cd and Pb, respectively. Compared to using single washing agent, the removal efficiencies of DTPA-extractable Cd and Pb for the sequential washing with HCl followed by Na 2 EDTA were increased more than 8.3% and 27.92%, respectively. Results of soil extractions experiments revealed that the predominant fraction of the removed Cd and Pb were Fe/Mn oxide fraction and carbonate fraction. Trace metals in leachate collected from sequential washing was successfully precipitated and the leachate was safe for discharge.

Simultaneous mobilization of macro- and trace elements (MTEs) and polycyclic aromatic hydrocarbon (PAH) compounds from soil with a nonionic surfactant and [ S, S]-ethylenediaminedisuccinic acid (EDDS) in admixture: PAH compounds

A laboratory study was conducted to assess the feasibility of a washing process with nonionic surfactant in combination with EDDS for the simultaneous mobilization of MTEs and PAH compounds from a field-contaminated soil. Unit processes consisting of complexometric extraction and surfactant-assisted mobilization were combined with reagent regeneration and detoxification steps to generate innocuous products. Thirty minutes of ultrasonic mixing of the soil with a combination of 20 mL L −1 surfactant suspension and a sparing quantity (2 mmol) of EDDS mobilized virtually all of the benzo[α] pyrene (B(a)P) and chrysene (Cry) and an appreciable portion of the burdens of Cd, Cr, Mn, Ni, Pb and Zn, lesser amounts of the As and Cu, but only small quantities of Al and Fe. Relative to individual reagents, combinations of surfactant (Brij98), with EDDS increased the recovery of B(a)P but seemingly did not influence Cry extraction efficiencies perceptibly. Nine sequential washes with the same initial dosage of mobilization aids decreased the quantities of both PAHs to levels in the soil that conformed to recommended maxima. What resulted was a soil that had been cleaned and a limited quantity of innocuous wash water.

Proteomic Profiling of S-acylated Macrophage Proteins Identifies a Role for Palmitoylation in Mitochondrial Targeting of Phospholipid Scramblase 3

S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.

Efficacy of the International Embryo Transfer Society (IETS) washing procedure for rendering oocytes matured in vitro free of bovine viral diarrhea virus (BVDV)

To ensure the freedom of embryos from pathogenic agents prior to embryo transfer (ET), a specific sanitary washing procedure has been recommended by the International Embryo Transfer Society (IETS). In the present study, the efficacy of removing the bovine viral diarrhea virus (BVDV) from cumulus-free matured oocytes at the stage of extruded first polar body (N = 240) was evaluated, using the IETS-recommended 10 sequential wash procedure, after exposure in vitro to BVDV type 2 (strain PA-131, 1 × 105.2 TCID50/mL for 1 h). In general, the percentage of contaminated oocytes was reduced (P < 0.03) after the first two washes. Nevertheless, after 10 washes, approximately 20% of oocytes still remained infectious or contaminated with virus, as detected by the virus isolation test (VI) and quantitative reverse transcription PCR (qRT-PCR) of viral RNA (on average 13 copies/oocyte). Similarly, a higher percentage of positive washing fluid samples were detected in the first three washes (50–100%). The six subsequent washes had lower but variable proportions of fluid samples contaminated with infectious virus. We concluded that the standard washing procedure may not render all oocytes free from the infectious virus adhered to the zona pellucida (ZP), and application of an additional method of oocyte disinfection was warranted to ensure nontransmission of BVDV to recipients by embryos derived from infected oocytes.

154 EVALUATION OF THE EFFECTIVENESS OF TREATMENT WITH TRYPSIN IN MURINES EMBRYOS EXPERIMENTALLY EXPOSED TO BOVINE HERPES VIRUS TYPE-1 (BoHV-1) BY THE NESTED-PCR TECHNIQUE

The cryopreservation of bovine embryos has allowed an increase in their commercialization causing concern about the transmission of pathogens through the production and transfer of in vitro embryos. The International Society of Embryo Transfer (IETS) suggests the treatment of embryos with trypsin or antibiotics in alternating washes with culture medium in order to remove and/or inactivate infectious agents that may interfere with the final product. The aim of this study was to evaluate the effectiveness of trypsin treatment in the elimination and/or removal of bovine herpes virus type-1 (BoHV-1), Colorado strain, in murine embryos. Viral detection was made by n-PCR and cytopathic effect in Madin Darby bovine kidney (MDBK) cells. Six- to 8-wk-old female mice (Swiss) were superovulated and mated with fertile males of the same strain. After 24 h, the zygotes (n = 262) were divided into 3 groups: control group submitted to sequential wash (CSW), the group exposed to the virus (30 μL; 106.5 virus mL–1) and submitted to sequential wash (ESW), and the group exposed to the virus and submitted to the trypsin treatment (ETT). All the groups of zygotes and the last sequential wash drops were tested by nested-PCR and inoculated in MDBK cells to allow observation of cytopathic effects. All groups except for CSW showed positive results for the nested-PCR for both zygotes and for the last wash drops. A cytopathic effect was observed in all groups except for CSW, demonstrating the viability of the virus after treatment. These results demonstrated that trypsin treatment was not effective in eliminating or removal of BoHV-1. The need for such studies is fundamentally important when considering the potential risk of pathogen transmission by reproductive biotechnologies.

Establishment of an axenic culture of microcystin-producing Microcystis aeruginosa isolated from a Korean reservoir

In order to establish an axenic (bacteria-free) culture of Microcystis aeruginosa NIER 10039 isolated from a Korean reservoir, the culture was subjected to sequential treatment, including ultrasonication, washing, and addition of antibiotics. Three broad-spectrum antibiotics, namely, kanamycin, ampicillin, and imipenem, were applied separately in that order. Axenicity of the culture was confirmed by cultivation on bacterial media and observation under epifluorescence and scanning electron microscopes. We are the first to establish an axenic culture of a Microcystis strain isolated from Korean reservoirs and can be used in physiological and molecular studies to control toxic Microcystis blooms.

Aph-1 Associates Directly with Full-length and C-terminal Fragments of γ-Secretase Substrates

γ-Secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid β-peptide from the β-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the γ-secretase complex. Here, we provide evidence that both the β-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of γ-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an β-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical γ-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (β, αS, and αL), as they were all able to bind β-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for γ-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with γ-secretase substrates prior to their processing by the proteolytic complex.

274 ASSESSMENT OF TRYPSIN AND ANTIBIOTIC TREATMENT EFFECTIVENESS IN IN VITRO-MATURED OOCYTES EXPERIMENTALLY EXPOSED TO LEPTOSPIRA INTERROGANS SEROVAR GRIPPOTYPHOSA

Techniques of production and transfer of embryos is safe as long as it follows the control regulations defined by the manual of the International Embryo Transfer Society (IETS) for treating oocytes with trypsin, antibiotics, and TCM-199 medium. The aim of this work was to evaluate the effectiveness of treatments, established by IETS, in bovine oocytes experimentally exposed to Leptospira interrogans serovar Grippotyphosa and to assist implementation of quality control standards on in vitro embryo production. The oocytes were obtained through follicular puncture of ovaries derived from the slaughterhouse. They were selected and divided into 4 groups: the control group and groups exposed to 5, 10, and 30 μL of an L. interrogans strain at 4.7 × 105 μL-1; and 4 additional groups exposed to the same concentrations of another L. interrogans strain at 6.3 × 105 μL-1, in which the gene for virulence is not expressed. The groups were kept in maturation medium (TCM-199 medium, 0.5 (iLof FSH, 50IU mL-1 hCG, 1μL mL-1 17-βiestradiol) and incubated at 38°C, 5% CO2, and 95% humidity for 24 h. All the groups were separately subjected to the treatments with antibiotic, trypsin, and TCM-199 medium after maturation. The treatment involved 10 drops (each 200 μL), with 8 drops of TCM-199 medium and 2 drops of antibiotic (penicillin 10000 IU mL-1 and streptomycin 10 mg mL-1) or trypsin 0.25%; exposed to trypsin and antibiotic for 120 s. For the sequential washes, all drops contained TCM-199 medium. The analysis for presence of the pathogen by dark-field optical microscopy showed that in the groups exposed to L. interrogans and subjected to antibiotic washes, the effectiveness was 50% (100/200) for the group exposed to 5 μL, 40% (80/200) for that exposed to 10 μL, and 22.5% (45/200) for that exposed to 30 μL. We found the same results after the trypsin washes. After the washings with TCM-199 medium, the groups infected with 5 and 10 μL presented 100% of effectiveness; however, for the group infected with 30 μL, the washings were not effective. For the groups exposed to L. interrogans that did not express virulence, after the washings with antibiotics as well as with trypsin, the results showed no effectiveness in all of them (n = 200). Yet, after washings with TCM-199, the group exposed to 5 μL showed 28.5% (57/200) of effectiveness, whereas in those exposed to 10 and 30 μL, the medium washes were not effective. Complementary studies are being made with ultramicrotome cut and polymerase chain reaction for more reliable conclusions, to confirm the results. With such results, we conclude that the quality control regulations established by IETS for IVP could be reviewed and possibly redefined, because the effectiveness of the treatment may depend not only on the pathogen species, but also its virulence as well as its concentration and the action of the treatments on the type of pathogen. We thank Vitrocel/Embriolife for supporting the laboratory.

Soil remediation using a coupled process: soil washing with surfactant followed by photo-Fenton oxidation

In the present work the use of a coupled process, soil washing and photo-Fenton oxidation, was investigated for remediation of a soil contaminated with p, p′-DDT (DDT) and p, p′-DDE (DDE), and a soil artificially contaminated with diesel. In the soil washing experiments, Triton X-100 (TX-100) aqueous solutions were used at different concentrations to obtain wastewaters with different compositions. Removal efficiencies of 66% (DDT), 80% (DDE) and 100% (diesel) were achieved for three sequential washings using a TX-100 solution strength equivalent to 12 times the effective critical micelle concentration of the surfactant (12 CMC eff). The wastewater obtained was then treated using a solar photo-Fenton process. After 6 h irradiation, 99, 95 and 100% degradation efficiencies were achieved for DDT, DDE and diesel, respectively. In all experiments, the concentration of dissolved organic carbon decreased by at least 95%, indicating that residual concentration of contaminants and/or TX-100 in the wastewater was very low. The co-extraction of metals was also evaluated. Among the metals analyzed (Pb, Cr, Ni, Cu, Cd, Mn and Co), only Cr and Mn were detected in the wastewater at concentrations above the maximum value permitted by current Brazilian legislation. The effective removal of contaminants from soil by the TX-100 washing process, together with the high degradation efficiency of the solar photo-Fenton process, suggests that this procedure could be a useful option for soil remediation.

Therapeutic bronchoscopy in a child with sand aspiration and respiratory failure from near drowning—case report and literature review

Foreign matter aspiration occurs relatively commonly in drowning and near-drowning events. In most cases, stomach contents are aspirated. Sand aspiration rarely occurs and there are no reported cases in children with near drowning. Limited data are available on clinical presentation and management of sand aspiration with accidental burial. We report a 3-year-old boy who nearly drowned while swimming in brackish waters and was found face down in sand. Sand aspiration was suspected when the child continued to have persistent wheezing and high ventilatory requirement despite intensive bronchodilator and corticosteroids therapy with an inability to wean after 4 days post-near-drowning event. Radiology was non-specific in the absence of sand bronchogram. Presence of sand in the airways was confirmed when a bronchoscopy was undertaken and sand seen in the bronchoalveolar lavage fluid. Sequential lung washing followed by exogenous surfactant administration (3 ml/kg) was undertaken and lead to significant improvement such that within 12 hr post-therapeutic lavage, his ventilatory requirements reduced substantially. The child was extubated 4 days post-lavage and on review 2 months post-event, was clinically well with airway resistance within normal predicted values measured on forced oscillatory spirometry (IOS).

A Highly Conserved Motif within the NH2-terminal Coiled-coil Domain of Angiopoietin-like Protein 4 Confers Its Inhibitory Effects on Lipoprotein Lipase by Disrupting the Enzyme Dimerization

Lipoprotein lipase (LPL) is a principal enzyme responsible for the clearance of chylomicrons and very low density lipoproteins from the bloodstream. Two members of the Angptl (angiopoietin-like protein) family, namely Angptl3 and Angptl4, have been shown to inhibit LPL activity in vitro and in vivo. Here, we further investigated the structural basis underlying the LPL inhibition by Angptl3 and Angptl4. By multiple sequence alignment analysis, we have identified a highly conserved 12-amino acid consensus motif that is present within the coiled-coil domain (CCD) of both Angptl3 and Angptl4, but not other members of the Angptl family. Substitution of the three polar amino acid residues (His(46), Gln(50), and Gln(53)) within this motif with alanine abolishes the inhibitory effect of Angptl4 on LPL in vitro and also abrogates the ability of Angptl4 to elevate plasma triglyceride levels in mice. The CCD of Angptl4 interacts with LPL and converts the catalytically active dimers of LPL to its inactive monomers, whereas the mutant protein with the three polar amino acids being replaced by alanine loses such a property. Furthermore, a synthetic peptide consisting of the 12-amino acid consensus motif is sufficient to inhibit LPL activity, although the potency is much lower than the recombinant CCD of Angptl4. In summary, our data suggest that the 12-amino acid consensus motif within the CCD of Angptl4, especially the three polar residues within this motif, is responsible for its interaction with and inhibition of LPL by blocking the enzyme dimerization.

DEVELOPMENT OF AN USER-FRIENDLY VIRTUAL ENVIRONMENT FOR DESIGN AND SIMULATION OF FOOD PROCESSING PLANTS: APPLICATION TO THE VALORIZATION PROCESS OF FISH SKINS TO OBTAIN GELATINES

ISHS IV International Symposium on Applications of Modelling as an Innovative Technology in the Agri-Food-Chain: Model-IT DEVELOPMENT OF AN USER-FRIENDLY VIRTUAL ENVIRONMENT FOR DESIGN AND SIMULATION OF FOOD PROCESSING PLANTS: APPLICATION TO THE VALORIZATION PROCESS OF FISH SKINS TO OBTAIN GELATINES

Evaluation of Trypsin Treatment on the Inactivation of Bovine Herpesvirus Type 1 onIn VitroProduced Pre-implantation Embryos

The aim of this study was to evaluate the efficiency of trypsin treatment on the inactivation of bovine herpesvirus type 1 (BoHV-1) on in vitro produced by fertilization and artificially infected bovine embryos. Bovine embryos on day 7 were exposed with 10 microl of BoHV-1, Los Angeles strain 10(7.5) TCID. These embryos and control embryos were divided in two groups: submitted to the sequential washes or to the trypsin treatment according to the International Embryo Transfer Society (IETS) guidelines. The embryos and the last washing drop of each group were used as inoculum to infect Madin Darby bovine kidney (MDBK) cells and submitted to nested PCR reaction using the primer that encodes the gene conserved region of virus glycoprotein gB. The data have shown that the control embryos and their last washing drop were negative. The exposed embryos that were treated with trypsin have shown positive results on the n-PCR and MDBK culture, and their last washing drop were negative. Our data have demonstrated that the trypsin treatment was not able to eliminate the BHV-1 of the embryos, suggesting an interaction between virus and embryo.

An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an approximately 58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of approximately 3.6 microg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed alpha-chitin binding activity with Kd approximately 0.64 microm and Bmax approximately 4.68 micromol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an approximately 8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

Genetic Recombinant Expression and Characterization of Human Augmenter of Liver Regeneration

To establish a highly effective prokaryotic recombinant expression system for human augmenter of liver regeneration (hALR) and to characterize the recombinant hALR both in vitro and in vivo. ALR cDNA was synthesized and inserted into expression vector pET28a+, the recombinant plasmid was transformed into BL21, and expression of hALR was induced by IPTG. Recombinant hALR (rhALR) was purified by sequential detergent wash, enterokinase (EK) digestion, gel-filtration, and chelating chromatography. The rhALR was identified by SDS-PAGE, immunoblotting, MALDI-TOF-MS, and N-terminal sequencer. Cell proliferative effect of rhALR on human hepatocytes was analyzed by MTT. The protective effect of rhALR on liver function was observed on CCl(4)-induced intoxicated mice. Recombinant expression plasmid of ALR [pET28(a+)-hALR] was confirmed by restriction enzyme digestion and DNA sequencing. The expressed rhALR constituted 30% of total bacterial protein. Molecular weight was 15,029 for monomer and 30,136 for dimer by mass determination. N-terminal was M-R-T-Q-Q, exactly the same as anticipated for hALR. The purified protein migrating at about 15 KD showed excellent antigenicity in immunoblotting. The rhALR also showed a strong stimulative effect on hepatocyte proliferation. ALT and AST levels, liver histological structure, as well as the survival rate of CCl(4)-intoxicated mice were significantly improved when rALR was administrated at 40 microg/kg or 200 microg/kg. The rhALR is successfully expressed highly effectively with anticipated MW, N-terminal, and antigenicity. It could play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and improving liver function in CCl(4)-intoxicated mice.

New practical algorithm for modelling analyte recovery in bioanalytical reversed phase and mixed-mode solid phase extraction

Solid phase extraction (SPE) is a widely used method for sample cleanup and sample concentration in bioanalytical sample preparation. A few methods to model the retention behaviour on SPE cartridges have been described previously but they are either not applicable to ionised species or are not suitable when using multiple wash and elution steps with solvents differing in volume, modifier concentration and acidity. Furthermore, these models were not applied to mixed mode SPE sorbents. In order to overcome these limitations a new SPE modelling algorithm was proposed. The retention behaviour was determined directly on the SPE cartridge by connecting the cartridge online with an HPLC system using a simple but suitable device that was developed and described. The results from these online experiments were used to model the elution behaviour using a quadratic retention function combined with an exponentially modified Gaussian peak shape model to predict analyte recovery under different wash and elution conditions. The validity of the proposed algorithm was tested using practical SPE experiments with an aqueous test mixture as well as with spiked human plasma. Different sequential wash and elution steps were performed using solvents differing in volume and composition. The predicted band shape and recoveries in each collected step were in good agreement with the results obtained from practical experiments. The proposed algorithm is very useful for the description of the SPE behaviour of the analytes on the actual used SPE cartridge and can be used in structural and automated SPE method development.

Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

Construction of Escherichia coli dnaK-deletion mutant infected by λDE3 for overexpression and purification of recombinant GrpE proteins

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. coli dnaK-deletion mutant. The E. coli ΔdnaK52 mutant was infected by λDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.

Hair analysis for cocaine: Factors in laboratory contamination studies and their relevance to proficiency sample preparation and hair testing practices

Hair samples were contaminated by rubbing with cocaine (COC) followed by sweat application, multiple shampoo treatments and storage. The samples were then washed with isopropanol for 15 min, followed by sequential aqueous washes totaling 3.5 h. The amount of drug in the last wash was used to calculate a wash criterion to determine whether samples were positive due to use or contamination. Analyses of cocaine and metabolites were done by LC/MS/MS. These procedures were applied to samples produced by a U.S. government-sponsored cooperative study, in which this laboratory participated, and to samples in a parallel in-house study. All contaminated samples in both studies were correctly identified as contaminated by cutoff, benzoylecgonine (BE) presence, BE ratio, and/or the wash criterion. A method for determining hair porosity was applied to samples in both studies, and porosity characteristics of hair are discussed as they relate to experimental and real-world contamination of hair, preparation of proficiency survey samples, and analysis of unknown hair samples.