Evaluation of extractants for estimation of the phytoavailable trace metals in soils

Despite its environmental (and financial) importance, there is no agreement in the literature as to which extractant most accurately estimates the phytoavailability of trace metals in soils. A large dataset was taken from the literature, and the effectiveness of various extractants to predict the phytoavailability of Cd, Zn, Ni, Cu, and Pb examined across a range of soil types and contamination levels. The data suggest that generally, the total soil trace metal content, and trace metal concentrations determined by complexing agents (such as the widely used DTPA and EDTA extractants) or acid extractants (such as 0.1 M HCl and the Mehlich 1 extractant) are only poorly correlated to plant phytoavailability. Whilst there is no consensus, it would appear that neutral salt extractants (such as 0.01 M CaCl2 and 0.1 M NaNO3) provide the most useful indication of metal phytoavailability across a range of metals of interest, although further research is required.

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

Determination of the stability constants of mercury(II) complexes of mixed donor macrobicyclic encapsulating ligands

The reactions of mercury(II) with the mixed donor encapsulating ligands 3,6,16-trithia-6,11,19-triazabicyclo[6.6.6]icosane (AMN(3)S(3)sar) and 1-amino-8-methyl-6,19-dithia-3,10,13,16-tetraazabicyclo[6.6.6]icosane (AMN(4)S(2)sar) have been studied. NMR ligand-ligand competition experiments with the ligands 1,4,8,11-tetraazaeyclotetradecane ([14]aneN(4)), 1-thia-4,7,10-triazacyclododecane ([12]aneN(3)S) and ethylenediaminetetraacetic acid (EDTA) with AMN(3)S(3)sar and Hg(II) indicated that [14]aneN(4) would be an appropriate competing ligand for the, determination of the Hg(II) stability constant. Calculations indicated the ratio of concentrations of AMN3S3sar, [14]aneN(4) and Hg(II) required for the determination of the stability constant ranged from 1:1:1 to 1:5:1. Refinement of the titration curves yielded log(10)K[Hg(AMN(3)S(3)sar)](2+) = 17.7. A similar competition titration resulted in the determination of the stability constant for the AMN(4)S(2)sar system as log(10)K[Hg(AMN(4)S(2)sar)](2+) = 19.5. The observed binding constants for the mixed N/S donor systems and the hexaaza analogues sar (3,6,10,13,16,19-hexaazabicyclo [6.6.6]icosane) and diamsar (1,8-diamino-3,6,10,13,16,19 -hexazabicyclo [6.6.6] icosane (log(10)K-[Hg(diamsar)](2+) = 26.4; log(10)K[Hg(sar)](2+) = 28.1) differ by approximately ten orders of magnitude. The difference is ascribed not to a cryptate effect but to a mismatch in the Hg-N and Hg-S bond lengths in the N/S systems.

Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host, Pieris rapae

Polydnaviruses are essential for the survival of many Ichneumonoid endoparasitoids, providing active immune suppression of the host in which parasitoid larvae develop. The Cotesia rubecula bracovirus is unique among polydnaviruses in that only four major genes are detected in parasitized host ( Pieris rapae) tissues, and gene expression is transient. Here we describe a novel C. rubecula bracovirus gene (CrV3) encoding a lectin monomer composed of 159 amino acids, which has conserved residues consistent with invertebrate and mammalian C-type lectins. Bacterially expressed CrV3 agglutinated sheep red blood cells in a divalent ion-dependent but Ca2+-independent manner. Agglutination was inhibited by EDTA but not by biological concentrations of any saccharides tested. Two monomers of similar to14 and similar to17 kDa in size were identified on SDS-PAGE in parasitized P. rapae larvae. The 17-kDa monomer was found to be an N-glyscosylated form of the 14-kDa monomer. CrV3 is produced in infected hemocytes and fat body cells and subsequently secreted into hemolymph. We propose that CrV3 is a novel lectin, the first characterized from an invertebrate virus. CrV3 shows over 60% homology with hypothetical proteins isolated from polydnaviruses in two other Cotesia wasps, indicating that these proteins may also be C-type lectins and that a novel polydnavirus lectin family exists in Cotesia-associated bracoviruses. CrV3 is probably interacting with components in host hemolymph, resulting in suppression of the Pieris immune response. The high similarity of CrV3 with invertebrate lectins, as opposed to those from viruses, may indicate that some bracovirus functions were acquired from their hosts.

Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 mum ceramic membrane successfully recovered the granulocyte macrophagecolony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by infermenter Benzonase(R) digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and re-suspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations. (C) 2004 Wiley Periodicals Inc.

Chemical treatment of Escherichia coli .1. Extraction of intracellular protein from uninduced cells

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetetraacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. (C) 1997 John Wiley & Sons, Inc.

The reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) - Results of an international collaborative study

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is an autosomal dominant condition accounting for 2–5% of all colorectal carcinomas as well as a small subset of endometrial, upper urinary tract and other gastrointestinal cancers. An assay to detect the underlying defect in HNPCC, inactivation of a DNA mismatch repair enzyme, would be useful in identifying HNPCC probands. Monoclonal antibodies against hMLH1 and hMSH2, two DNA mismatch repair proteins which account for most HNPCC cancers, are commercially available. This study sought to investigate the potential utility of these antibodies in determining the expression status of these proteins in paraffin-embedded formalin-fixed tissue and to identify key technical protocol components associated with successful staining. A set of 20 colorectal carcinoma cases of known hMLH1 and hMSH2 mutation and expression status underwent immunoperoxidase staining at multiple institutions, each of which used their own technical protocol. Staining for hMSH2 was successful in most laboratories while staining for hMLH1 proved problematic in multiple labs. However, a significant minority of laboratories demonstrated excellent results including high discriminatory power with both monoclonal antibodies. These laboratories appropriately identified hMLH1 or hMSH2 inactivation with high sensitivity and specificity. The key protocol point associated with successful staining was an antigen retrieval step involving heat treatment and either EDTA or citrate buffer. This study demonstrates the potential utility of immunohistochemistry in detecting HNPCC probands and identifies key technical components for successful staining.

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUN(TM) beads and the whole blood Lyse/No-Wash protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 mul of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 It after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained. (C) 2003 Elsevier B.V. All rights reserved.

Urotensin-II-converting enzyme activity of furin and trypsin in human cells in vitro

Human urotensin-II (hU-II) is processed from its prohormone (ProhU-II) at putative cleavage sites for furin and serine proteases such as trypsin. Although proteolysis is required for biological activity, the endogenous urotensin-converting enzyme (UCE) has not been investigated. The aim of this study was to investigate UCE activity in cultured human cells and in blood, comparing activity with that of furin and trypsin. In a cell-free system, hU-II was detected by high-performance liquid chromatography-mass spectrometry after coincubating 10 muM carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37degreesC) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca2+ ions and supplemented with 2 mM EDTA at pH 7.0. hU-II was readily detected in the superperfusate of permeabilized epicardial mesothelial cells incubated with CTF-ProhU-II (3 h, 37degreesC), but it was only weakly detected in the superperfusate of intact cells. Conversion of CTF-ProhU-II to hU-II was attenuated in permeabilized cells using conditions found to inhibit furin activity. In a cell-free system, trypsin (0.05 mg/ml) cleaved CTF-ProhU-II to hU-II, and this was inhibited with 35 muM aprotinin. hU-II was detected in blood samples incubated with CTF-ProhU-II (3 h, 37degreesC), and this was also inhibited with aprotinin. The findings revealed an intracellular UCE in human epicardial mesothelial cells with furin-like activity. Aprotinin-sensitive UCE activity was detected in blood, suggesting that an endogenous serine protease such as trypsin may also contribute to proteolysis of hU-II prohormone, if the prohormone is secreted into the circulation.

Ecophysiology of the marine cyanobacterium, Lyngbya majuscula (Oscillatoriaceae) in Moreton Bay, Australia

Large blooms of the marine cyanobacterium Lyngbya majuscula in Moreton Bay, Australia (27 degrees 05'S, 153 degrees 08'E) have been re-occurring for several years. A bloom was studied in Deception Bay (Northern Moreton Bay) in detail over the period January-March 2000. In situ data loggers and field sampling characterised various environmental parameters before and during the L. majuscula bloom. Various ecophysiological experiments were conducted on L. majuscula collected in the field and transported to the laboratory, including short-term (2h) C-14 incorporation rates and long-term (7 days) pulse amplitude modulated (PAM) fluorometry assessments of photosynthetic capacity. The effects of L. majuscula on various seagrasses in the bloom region were also assessed with repeated biomass sampling. The bloom commenced in January 2000 following usual December rainfall events, water temperatures in excess of 24 degrees C and high light conditions. This bloom expanded rapidly from 0 to a maximum extent of 8 km(2) over 55 days with an average biomass of 210 g(dw)(-1) m(-2) in late February, followed by a rapid decline in early April. Seagrass biomass, especially Syringodium isoetifolium, was found to decline in areas of dense L. majuscula accumulation. Dissolved and total nutrient concentrations did not differ significantly (P > 0.05) preceding or during the bloom. However, water samples from creeks discharging into the study region indicated elevated concentrations of total iron (2.7-80.6 mu M) and dissolved organic carbon (2.5-24.7 mg L-1), associated with low pH values (3.8-6.7). C-14 incorporation rates by L. majuscula were significantly (P < 0.05) elevated by additions of iron (5 mu M Fe), an organic chelator, ethylenediaminetetra-acetic acid (5 mu M EDTA) and phosphorus (5 mu M PO4-3). Photosynthetic capacity measured with PAM fluorometry was also stimulated by various nutrient additions, but not significantly (P > 0.05). These results suggest that the L. majuscula bloom may have been stimulated by bioavailable iron, perhaps complexed by dissolved organic carbon. The rapid bloom expansion observed may then have been sustained by additional inputs of nutrients (N and P) and iron through sediment efflux, stimulated by redox changes due to decomposing L. majuscula mats. (c) 2004 Elsevier B.V. All rights reserved.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(H) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1muM of complexed Hg(II), and for inhibition of motility it was 0.05 muM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 muM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned. (C) 2004 Elsevier B.V. All rights reserved.

CEDIA (R) sirolimus assay compared with HPLC-MS/MS and HPLC-UV in transplant recipient specimens

The role of the therapeutic drug monitoring laboratory in support of immunosuppressant drug therapy is well established, and the introduction of sirolimus (SRL) is a new direction in this field. The lack of an immunoassay for several years has restricted the availability of SRL assay services. The recent availability of a CEDIA (R) SRL assay has the potential to improve this situation. The present communication has compared the CEDIA (R) SRL method with 2 established chromatographic methods, HPLC-UV and HPLC-MS/MS. The CEDIA (R) method, run on a Hitachi 917 analyzer, showed acceptable validation criteria with within-assay precision of 9.1% and 3.3%, and bias of 17.1% and 5.8%, at SRL concentrations of 5.0 mu g/L and 20 mu g/L, respectively. The corresponding between-run precision values were 11.5% and 3.3% and bias of 7.1% and 2.9% at 5.0 mu g/L and 20 mu g/L, respectively, The lower limit of quantification was found to be 3.0 mu g/L. A series of 96 EDTA whole-blood samples predominantly from renal transplant recipients were assayed by the 3 methods for comparison. It was found that the CEDIA (R) method showed a Deming regression line of CEDIA = 1.20 X HPLC-MS/MS - 0.07 (r = 0.934, SEE = 1.47), with a mean bias of 20.4%. Serial blood samples from 8 patients included in this evaluation showed that the CEDIA (R) method reflected the clinical fluctuations in the chromatographic methods, albeit with the variable bias noted. The CEDIA (R) method on the H917 analyzer is therefore a useful adjunct to SRL dosage individualization in renal transplant recipients.

Characterization of polyamine oxidase from the aquatic nitrogen-fixing fern Azolla imbricata

Biochemical properties of a polyamine oxidase (PAO; EC 1.5.3.3) purified from the aquatic nitrogen-fixing fern Azolla imbricata (Roxb.) Nak. were studied. The native molecular mass of the enzyme estimated by Sephadex G 200 get filtration was 66.2 kDa. SDS-PAGE gave a single protein band corresponding to a molecular mass of 65.5 kDa. The light yellow enzyme had absorption maxima at 278, 372 and 454 nm with 1 mol FAD per mole enzyme molecule as its cofactor. The PAO was active on both the triamine Spd and the tetraamine Spm as substrates. However, it was inactive on the diamines Put and Cad. It had a pH optimum of 6.5 for both Spd and Spm. The K-m(S) for Spd and Spm were 6.71 x 10(-2) and 1.13 x 10(-1) nM, respectively. Pre-incubation with 10 mM of K+ (KCl), Ca2(+) (CaCl2) or Mg2+ (MgCl2) had no effect on PAO activity. However, 10 mM Cu2+ (CuCl2), Mn2+ (MnCl2) and Fe2+ (FeSO4) inhibited enzyme activity by 37%, 43% and 58%, respectively. The metal chelator EDTA (10 mM), the carbonyl reagent hydroxylamine (0.5 mM) and the sulfhydryl reagent p-chloro-mercuribenzoate (0.5 mM) had no effect on PAO activity. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Ototoxicity and tolerance assessment of a TrisEDTA and polyhexamethylene biguanide ear flush formulation in dogs

Clinically healthy mixed breed dogs (n = 20) were used to determine if a Tris (tromethamine)-buffered test solution, Otinide((R)) (Trademark of Dermcare-Vet Pty-Ltd, Australia), containing disodium ethylenediamine tetraacetic acid (EDTA; 1.21 g/L) and polyhexamethylene biguanide (PHMB; 0.22 g/L) caused ototoxicity or vestibular dysfunction. The dogs were randomly assigned to either a control group (group A, n = 10) receiving saline, or a treatment group (group B, n = 10) receiving the test solution. Phase 1 of the study consisted of applying 5.0 mL of saline to both ears of the control group (group A) and 5 mL of test solution to both ears of the test group (group B), for 21 days. A bilateral myringotomy was then performed on each dog under deep sedation. Phase 2 of the study then consisted of applying 2.0 mL of the saline to both ears of the control group (group A) and 2.0 mL of the test solution to both ears of the test group (group B), for 14 days. Throughout the study, dogs were examined for clinical health, and underwent otoscopic, vestibular and auditory examinations. The auditory examinations included brainstem auditory evoked potential (BAEP) threshold and supra-threshold assessments using both click and 8 kHz tone burst stimuli. The absence of vestibular signs and effects on the BAEP attributable to the test solution suggested the test solution could be applied safely to dogs, including those with a damaged tympanic membrane.