An ecotoxicological view on neurotoxicity assessment.

The numbers of potential neurotoxicants in the environment are raising and pose a great risk for humans and the environment. Currently neurotoxicity assessment is mostly performed to predict and prevent harm to human populations. Despite all the efforts invested in the last years in developing novel in vitro or in silico test systems, in vivo tests with rodents are still the only accepted test for neurotoxicity risk assessment in Europe. Despite an increasing number of reports of species showing altered behaviour, neurotoxicity assessment for species in the environment is not required and therefore mostly not performed. Considering the increasing numbers of environmental contaminants with potential neurotoxic potential, eco-neurotoxicity should be also considered in risk assessment. In order to do so novel test systems are needed that can cope with species differences within ecosystems. In the field, online-biomonitoring systems using behavioural information could be used to detect neurotoxic effects and effect-directed analyses could be applied to identify the neurotoxicants causing the effect. Additionally, toxic pressure calculations in combination with mixture modelling could use environmental chemical monitoring data to predict adverse effects and prioritize pollutants for laboratory testing. Cheminformatics based on computational toxicological data from in vitro and in vivo studies could help to identify potential neurotoxicants. An array of in vitro assays covering different modes of action could be applied to screen compounds for neurotoxicity. The selection of in vitro assays could be guided by AOPs relevant for eco-neurotoxicity. In order to be able to perform risk assessment for eco-neurotoxicity, methods need to focus on the most sensitive species in an ecosystem. A test battery using species from different trophic levels might be the best approach. To implement eco-neurotoxicity assessment into European risk assessment, cheminformatics and in vitro screening tests could be used as first approach to identify eco-neurotoxic pollutants. In a second step, a small species test battery could be applied to assess the risks of ecosystems.

The measurement of organically complexed FeII in natural waters using competitive ligand reverse titration.

Whilst there is increasing evidence for the presence of stabilized Fe(II) associated with organic matter in aquatic environments, the absence of a reliable method for determining Fe(II) speciation in solution has inhibited the study of this aspect of Fe biogeochemistry. A technique is described here for the determination of Fe(II) organic complexation in natural waters that is based on competitive ligand reverse titration and a model fit to experimental results, from which ligand concentration and a conditional stability constant can be obtained. Spectrophotometry was used to detect the Ferrozine (FZ) complex with reactive Fe(II), which in combination with a liquid waveguide capillary cell (LWCC) enabled high sensitivity and precision measurements of Fe(II) to be made. A series of samples was collected in the Itchen River in Southampton, UK to test the method at a wide range of salinities including river water. Levels of Fe(II) and total dissolved Fe were within previously reported values for this system. Fe(II) was found to occur organically complexed with values for K'(Fe(II)L) (conditional stability constant for Fe(II)-natural ligand complexes) of ≈8 at salinities between 0 and 21, whilst no measurable complexation was detected at a salinity of 31. This work demonstrates that spectrophotometry can be used in combination with ligand competition to investigate metal speciation in natural waters.

Rocuronium and cisatracurium-positive skin tests in non-allergic volunteers: determination of drug concentration thresholds using a dilution titration technique.

BACKGROUND: Muscle relaxants are believed to be responsible for 2/3 of the cases of anaphylactic reactions during anesthesia. This assumption is based mainly on positive skin tests obtained in individuals that have experienced anesthesia-related anaphylaxis. A positive skin test is supposed to be associated with mast cell degranulation of vasoactive amines. In the present study we tested the frequency of positive skin tests with two commonly used muscle relaxants, rocuronium and cisatracurium, in a selected group of volunteers with low potential for allergic reactions. METHODS: Thirty healthy volunteers without known allergy or previous exposure to muscle relaxants were studied. Low potential for allergic reactions was determined prior to inclusion in the study, using various allergy tests. Each individual was tested with intradermal and skin prick tests, and molar drug concentration thresholds for positive skin reactions were determined using a dilution titration technique. The presence or absence of mast cell degranulation was tested by electron microscopic investigation of skin biopsies obtained from positive and negative skin reactions. RESULTS: None of the volunteers had a positive skin prick test. More than 90% of the volunteers had a positive intradermal test with both rocuronium and cisatracurium. The highest molar drug concentration that was not associated with a positive intradermal test was 10(-6) M (rocuronium) and 10(-7) M (cisatracurium), equivalent to vial dilution 1 : 1000 for both drugs. In none of the volunteers was mast cell degranulation detected. CONCLUSION: Non-mast-cell-mediated positive intradermal skin reactions are frequently occurring with rocuronium and cisatracurium, even at vial dilution 1 : 1000. A clinically applicable test technique is needed that is able to separate positive skin tests associated with mast cell degranulation from non-mast-cell-mediated reactions.

Thiols in coastal waters of the western North Sea and English Channel.

Thiols were determined in coastal waters of the western North Sea and English Channel. Detection was carried out on-board ship on-line by flow-analysis with detection by cathodic stripping voltammetry and calibration with thiourea. The thiol concentrations ranged from 0.70 to 3.60 nM (thiourea equivalents) and were found to vary over a relatively short distance perpendicular to the coast. Low concentrations in the area of greatest estuarine input (the Humber-Wash area) indicated that the thiols did not originate from low-salinity waters. Instead, variations in the thiol concentration were found to parallel those of chlorophyll. This correlation was confirmed by depth profiles which showed a trend for the thiols similar to that of chlorophyll. The data demonstrates that thiols are more widespread than anticipated, and that marine phytoplankton is an important source of the thiols. In view of their known ability to bind with metals, these data indicate that the thiols could be an important candidate to act as such ligands in the marine system. The measurements gave no evidence for the presence of sulfide in these waters which means that it must be present at less than 20% of the detected thiol levels.

Determination of picomolar levels of iron in seawater using catalytic cathodic stripping voltammetry.

A new procedure for the direct determination of picomolar levels of iron in seawater is presented. Cathodic stripping voltammetry (CSV) is preceded by adsorptive accumulation of the iron(III)-2,3-dihydroxynaphthalene (DHN) complex from seawater, containing 20 microM DHN at pH 8.0, onto a static mercury drop electrode, followed by reduction of the adsorbed species. The reduction current is catalytically enhanced by the presence of 20 mM bromate. Optimized conditions include a 60-s adsorption period at -0.1 V and a voltammetric scan using sampled dc modulation at 10 Hz. In these conditions, a detection limit of 13 pM iron in seawater was achieved which can be lowered further by extending the adsorption time to 300 s. The new catalytic CSV method is approximately 5 times more sensitive than existing CSV methods and was tested on samples from the Atlantic Ocean.

Determination of the complex stability of zinc with carbonic anhydrase in sea-water.

Carbonic anhydrase (CA) is inactive unless associated with zinc, with possible substitution by cobalt. In this work, the complexation of zinc by CA was determined in sea-water using cathodic stripping voltammetry (CSV) with ligand competition. The zinc was found to be released from the CA over a period of 3 h when equilibrated with a competing complexing ligand and the complex was re-formed with the CA when zinc was added. A value of 8.90+/-0.27 was found for logK'ZnCA where K'ZnCA is the conditional stability constant for the complex of Zn2+ with CA in pH 8 sea-water. A value for the molecular weight of CA was calculated from its equivalent ligand concentration (in nM) obtained by titrations with zinc at various CA concentrations (1-4 mg l(-1)). The value found (34740 g mol(-1)) for the molecular weight is consistent with values found previously by other methods (29000-31000 g mol(-1)) confirming that the stoichiometry of the complex between zinc and CA is 1:1. This work confirms that the zinc-CA complex is reversible and that the interaction between zinc and CA can be determined using CSV with ligand competition.

Effect of experience with spinal anaesthesia on the development of post-dural puncture complications.

BACKGROUND: This clinical study was conducted in order to investigate the effect of operator experience with spinal anaesthesia (SA) on development of postural post-dural puncture headache (PPDPH) and postoperative backache. METHODS: The study was a cohort study of the first 100 SA performed by each of 5 trainees in anaesthesiology at the very beginning of their training period. SA was conducted with assistance and guidance according to usual departmental practice. In each SA, data regarding level of puncture, needle size, number of punctures, use of introducer and infiltration anaesthesia were recorded. In addition, usual problems and complications connected with dural puncture were registered. A visual analogue scale was used to record how difficult the procedure was experienced by the trainees. Postoperatively, the patients were contacted by the same trainee, usually by telephone. A semi-structured interview was conducted where occurrence and duration of headache, backache and other complaints were recorded. Headache was classified as PPDPH or non-PPDPH, and intensity of the headache was registered using a numerical rating scale (NRS) from 0 to 10. RESULTS: Five hundred SA in 495 patients with a mean age of 61.3 years were included in the study. Of these, 394 patients were completely followed-up postoperatively; the main reason for the drop-out was patient-related factors such as advanced age and dementia. Headache occurred in 56 patients postoperatively. PPDPH was diagnosed in 33 and non-PPDPH occurred in 23 patients. Postoperative backache was experienced by 27 patients. No significant effect experience with SA could be found regarding the occurrence of postoperative complications; 16 compared to 17 patients with PPDPH were found in the first and the last half of patients. A marked inter-individual difference in the occurrence of PPDPH was found in the patients treated by the 5 trainees. CONCLUSIONS: We could not demonstrate an effect of experience and training on development of complications after SA with regard to PPDPH and backache.

Determination of cobalt in seawater by catalytic adsorptive cathodic stripping voltammetry.

A new procedure for the direct determination of picomolar levels of cobalt in seawater is presented. Cathodic stripping voltammetry is preceded by adsorptive accumulation of the cobalt-nioxime (cyclohexane-1,2-dione dioxime) complex from seawater containing 6 µM nioxime and 80 mM ammonia at pH 9.1, onto a hanging mercury drop electrode, followed by reduction of the adsorbed species. The reduction current is catalytically enhanced by the presence of 0.5 M nitrite. Optimized conditions for cobalt include a 30 s adsorption period at -0.7 V and a voltammetric scan using differential pulse modulation. According to the proposed reaction mechanism, dissolved Co(II) is oxidized to Co(III) upon addition of nioxime and high concentrations of ammonia and nitrite; a mixed Co(III)-ammonia-nitrite complex is adsorbed on the electrode surface; the Co(III) is reduced to Co(II) (complexed by nioxime) during the voltammetric scan, followed by its chemical reoxidation by the nitrite, initiating a catalytically enhanced current. A detection limit of 3 pM cobalt (at an adsorption period of 60 s) enables the detection of this metal in uncontaminated seawater using a very short adsorption time. UV digestion of seawater is essential, as part of the cobalt may occur strongly complexed by organic matter and rendered nonlabile. The method was applied successfully to the determination of the distribution of cobalt in the water column of the Mediterranean.

Restriction map of a 35-kb HLA fragment constructed by nested deletion 'drop-out' mapping.

An efficient method for generating detailed restriction maps of large cloned DNA segments is demonstrated. The mapping strategy entails comparing restriction fragments from a parent clone and from nested deletion derivatives of that clone. In a set of deletion plasmids of decreasing size, an individual fragment will be lost, or 'drop-out', according to its position in the cloned fragment. In this demonstration, nested deletions were generated in both directions in a 35-kb DNA segment from the human leukocyte antigen (HLA) region by intramolecular transposition of an engineered gamma delta (Tn1000) element present in a special 'deletion factory' cloning vector [Wang et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7874-7878]. Fifteen plasmids with deletions extending in one direction and eleven plasmids with deletions extending in the opposite direction were digested singly by each of four restriction enzymes. A total of 36 cleavage sites were mapped in the 35-kb HLA fragment. This drop-out approach using nested deletions provides a simple and efficient means of mapping restriction sites, genes and other features of interest in cosmid-sized cloned DNA segments or DNAs.

Construction of a genomic DNA 'feature map' by sequencing from nested deletions: application to the HLA class I region.

We are applying a transposon-based approach for detecting and mapping features of special interest to construct 'feature maps' of currently uncharacterized portions of the human leukocyte antigen (HLA) complex on chromosome 6. Such feature maps should facilitate identifying regions for high resolution analysis. Here we describe the feature mapping of a 35 kb DNA fragment located between the HLA-C and HLA-E loci. This fragment was cloned into a transposon gamma delta-based cosmid vector designed for generating nested deletions in vivo. Seventy informative nested deletions extending into the cloned fragment were isolated, and DNA adjacent to the deletion endpoints was sequenced by fluorescent automated technology. These islands of DNA sequences constituted the foundation of the feature map, and (i) identified putative exons, (ii) determined the positions of Alu elements, (iii) determined the span of the keratinocyte-specific S gene, and (iv) localized evolutionarily conserved sequences. The construction of feature maps using this in vivo nested deletion-sequencing approach provides a rapid and efficient means to identify DNA regions that merit more detailed analysis.

Determination of cobalt in blood using cathodic stripping voltammetry.

A method was developed to determine low levels of cobalt in whole blood, serum and plasma. Samples of blood (0.2 ml) were mineralized at 160 degrees C in the presence of concentrated nitric acid. Residual organic matter was destroyed by digestion using ultraviolet irradiation after dilution with water. The cobalt concentration was determined by catalytic cathodic stripping voltammetry (CSV) preceded by adsorptive collection of cobalt complexed with diphenylglyoxime (DPG). The optimized analytical conditions for the CSV analysis included a DPG concentration of 0.5 mumol l-1, 0.05 mol l-1 ammonium chloride buffer (pH 9.3), 0.15 mol l-1 nitrite, a deposition potential of -0.75 V, an adsorption time of 30-120 s and a negative potential scan using the differential-pulse modulation. The limit of detection was 40 pg of cobalt in 0.2 ml of blood, which was limited by the blank level of cobalt in the reagents after purification.

Mycoplasma gallisepticum strain differentiation by arbitrary primer PCR (RAPD) fingerprinting.

We demonstrate here that the arbitrary primer polymerase-chain-reaction-based DNA fingerprinting method (also termed random amplified polymorphic DNA or RAPD) can be used to distinguish among strains of the avian pathogen Mycoplasma gallisepticum. Ten base oligonucleotide primers were used individually to prime DNA synthesis from genomic DNAs. Strain-specific arrays of DNA fragments were generated, which allowed us to identify and group isolates. Isolates of M. synoviae, M. gallinarum and M. iners yielded arrays of DNA fragments that differed markedly from those generated from the M. gallisepticum isolates using the same arbitrary primers. These results show that the RAPD fingerprinting method distinguishes genetically different strains of M. gallisepticum and indicates that it should be valuable for monitoring transmission of this pathogen.

Use of arbitrarily primed polymerase chain reaction analysis to type disease and carrier strains of Neisseria meningitidis isolated during a university outbreak.

Disease and carrier isolates of Neisseria meningitidis from two regions of the United States were typed by the arbitrarily primed polymerase chain reaction (PCR), or random amplified polymorphic DNA (RAPD), method. This technique generates strain-specific arrays of amplified DNA fragments using low-stringency PCR with single, arbitrarily chosen primers. Each of 3 disease isolates and 7 of 11 carrier isolates from an outbreak at the University of Connecticut were indistinguishable using each of 4 primers. In contrast, 22 other isolates (the remaining 4 carrier isolates plus 18 disease and carrier isolates from Connecticut, Illinois, and Missouri) were divided into 18 sets using the same 4 primers. This outcome supports the view that disease isolates from an outbreak may reflect sporadic invasive progression by a strain that also frequently causes asymptomatic colonization. Our results show that RAPD tests provide a sensitive and efficient means of distinguishing genetically different meningococcal strains and that they should facilitate clinical, epidemiologic, and population genetic studies of this important pathogen.

Inversions and deletions generated by a mini-gamma delta (Tn1000) transposon.

Intramolecular transposition by an engineered derivative of the transposon gamma delta (Tn1000) is described. This 1-kb element contains inverted repeats of the 40 bp of the delta end of gamma delta, bracketing a kan gene, but it contains no resolution site. Transposition was analyzed in two plasmids; one contained two contraselectable (conditional lethal) genes (thyA and sacB) adjacent to the mini-gamma delta element in a 13.0-kb pBR322/pUC-based two-component plasmid (a heterodimer), and the other contained a different contraselectable gene (strA [rpsL]) in a 13.2-kb three-component plasmid (a heterotrimer). Selection for loss of function of a single contraselectable gene yielded inversions and deletions. Each inversion plasmid was 1 kb larger than the parent plasmid: it had a second copy of mini-gamma delta inserted in the contraselected gene, with that copy plus the intervening segment inverted, and the 5-bp target site duplicated. Each deletion plasmid was smaller than the parent plasmid and had a deletion that extended from one transposon end into or through the contraselected gene for distances of up to 9.4 kb. The frequencies of deletions versus inversions ending in a single target gene were similar, although overall, deletions outnumbered inversions because deletions, but not inversions, into sites beyond the contraselected gene inactivate it. This work also demonstrates that thyA (which encodes thymidylate synthetase) is a useful contraselectable marker.

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.

pDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo.

We describe a transposon gamma delta-containing cosmid cloning vector, pDUAL (previously called pJANUS), and demonstrate an efficient strategy for isolating nested deletions in both large-scale and small-scale DNA sequencing efforts. This "deletion factory" strategy takes advantage of the ability of gamma delta (Tn1000) to generate deletions that extend from an end of the transposon into adjacent DNA when gamma delta transposes to new sites in the same DNA molecule. pDUAL contains the contraselectable (conditional lethal) sacB+ (sucrose sensitivity) and strA+ (streptomycin sensitivity) genes just outside each end of an engineered gamma delta and selectable kan+ (Kanr) and tet+ (Tetr) genes between the cloning site and sacB and strA, respectively. Selection on sucrose tetracycline medium yields deletions that extend from one gamma delta end for various distances into the cloned DNA, while selection on streptomycin kanamycin medium yields comparable deletions in the other direction. Both types of deletions are recoverable because the essential plasmid replication origin is embedded in the gamma delta component and is thereby retained in each deletion product. Pilot experiments with pDUAL clones showed that deletion end points can be mapped or selected by plasmid size and that both DNA strands of any single clone can be accessed for sequencing by using a pair of universal primers specific for sequences that are just interior to the gamma delta ends.

Redesigning, implementing and integrating Escherichia coli genome software tools with an object-oriented database system.

This paper reports our exploratory work to redesign, implement and integrate a collection of genome software tools with an object-oriented database system. Our software tools deal with genome data from Escherichia coli K-12, a bacterium that has been studied intensively and provides richer data sets than any other living organism. The object-oriented DBMS used for the integration is ONTOS, a commercial object-oriented system from Ontologic Inc. This redesign and implementation task was performed in two steps. First, C programs were converted into C++, and then the C++ version programs were modified and integrated with an object-oriented modeling of the data to form an ONTOS database application. The first step helps us develop a conceptual view for a DBMS-independent object-oriented construct. The second step elucidates what additional DBMS-dependent modification steps are needed to provide persistency to the objects. Examples are included to illustrate steps of the redesign and implementation. Overall, the outcome of this project demonstrates that programs and data can be successfully integrated with an object-oriented database, while providing the objects with persistency and shareability. This paper includes discussions using concrete examples on what advantage the object-oriented database approach provides over the relational database approach.