Tanaidacea (Crustacea) of the north-east Atlantic: the subfamily Pseudotanainae (Pseudotanaidae) and the family Nototanaidae

Sixteen species of the tanaidacean subfamily Pseudotanainae and three of the family Nototanaidae have been recorded from shallow and deep waters in the north-east Atlantic. Six new species of Pseudotanais , and the new genera Mystriocentrus, Parapseudotanais and Bathytanaissus are described. The species Pseudotanais affinis is re-described, and a cladistic analysis of the 'affinis' species-group is presented. Pseudotanais is a eurybathic genus and individuals of this taxon are common in the region. The Nototanaidae is poorly represented in the deep-sea, and the present record is the first for the Northern Hemisphere.     

Tanaidacea (Crustacea) of the north-east Atlantic: the subfamily Pseudotanainae (Pseudotanaidae) and the family Nototanaidae

Sixteen species of the tanaidacean subfamily Pseudotanainae and three of the family Nototanaidae have been recorded from shallow and deep waters in the north-east Atlantic. Six new species of Pseudotanais, and the new genera Mystriocentrus, Parapseudotanais and Bathytanaissus are described. The species Pseudotanais affinis is re-described, and a cladistic analysis of the ‘affinis’ species-group is presented. Pseudotanais is a eurybathic genus and individuals of this taxon are common in the region. The Nototanaidae is poorly represented in the deep-sea, and the present record is the first for the Northern Hemisphere.     

Analysis of N,N-diethyl-m-toluamide in porcine skin perfusates using solid-phase extraction disks and reversed-phase high-performance liquid chromatography

N,N-Diethyl-m-toluamide (DEET) is frequently used as an insect repellent by military and civilian populations. Because dermal exposure has resulted in several cases of DEET toxicosis, there is a need to rapidly and reliably determine DEET concentrations in biological matrices. An improved method for the analysis of DEET was developed for determining transdermal diffusion of low levels of DEET following application to an in vitro porcine skin flow-through diffusion cell system. The technical improvement involved the use of disk solid-phase extraction (SPE) instead of packed-bed SPE. The disk SPE method required small volumes of preconditioning, wash, and elution solvent (0.5-1 ml) to extract DEET from perfusate samples containing bovine serum albumin (BSA). The limit of quantitation (LOQ) was estimated as 0.08 micro g/ml DEET and recoveries from BSA media samples spiked with DEET ranged from 90.1 to 117% with relative standard deviation (RSD) ranging from 2.0 to 13.1%. This method was used to analyze perfusate samples from skin (n=4) topically exposed to DEET-ethanol formulations. The data from these analyses determined that DEET permeability in porcine skin was 2.55 x 10(-5)+/-0.54 x 10(-5) cm/h.     

The composition of the caudal skeleton of teleosts (Actinopterygil: Osteichthyes)

The diural caudal skeleton of teleostean actinopterygians develops phylogeneticaily and ontogenetically from a polyural skeleton. The reduction of the polyural anlage to four, three, two or fewer centra in the adult caudal skeleton takes different pathways in different genera (e.g. compare Elops and Albula) and groups of teleosts. As a result, ural centra are not homologous throughout the teleosts. By numbering the ural centra in a homocercal tail in polyural fashion, one can demonstrate these and the following differences. The ventral elements (hypurals) always occur in sequential series, whereas the dorsal elements (epurals and uroneurals) may alter like the ural centra. The number of epurals, five or four in fossil primitive teleosts, is reduced in other primitive and advanced teleosts, but the same epurals are not always lost. The number of uroneurals, seven in fossil teleosts, is reduced in living teleosts, but it has not been demonstrated that the first uroneural is always derived from the neural arch of the same ural centrum. The landmark in the homocercal tail is the preural centrum I which can be identified by (1) bifurcation of the caudal artery and vein in its ventral element, the parhypural, (2) its position directly caudal to the preural centrum (PU2) which supports the lowermost principal caudal ray with its haemal spine, (3) carrying the third hypaxial element ventral to the course of arteria and vena pinnalis, and (4) by carrying the first haemal spine (parhypural) below the dorsal end of the ventral cartilage plate. The study of the development of the vertebral column reveals that teleosts have different patterns of centrum formation. A vertebral centrum is a complete or partial ring of mineralized, cartilaginous or bony material surrounding at least the lateral sides of the notochord. A vertebral centrum may be formed by arcocentrum alone, or arcocentral arcualia and chordacentrum, or arco-, chorda- and autocentrum, or arcocentral arcualia and autocentrum. This preliminary research demonstrates that a detailed ontogenetic interpretation of the vertebral centra and of the caudal skeleton of different teleosts may be useful tools for further interpretations of teleostean interrelationships.     

Synthesis of [(18)O(2)]valproic acid and its use as an internal standard for the quantitative measurement by gas chromatography-electron ionization mass spectrometry

A specific method for the quantitative determination of valproic acid in human plasma is presented. Valproate was extracted from acidified plasma by hexane extraction and converted to its trimethylsilyl derivative without sample concentration. The derivatives were analyzed without any further purification. Using gas chromatography-electron ionization mass spectrometry, diagnostic useful fragment ions at m/z 201 and 205 were obtained for valproic acid and [(18)O(2)]valproic acid internal standard, respectively. [(18)O(2)]Valproic acid was synthesized from unlabeled valproate by acid-catalyzed exchange reaction in H(2)(18)O. The method was validated in the expected concentration range of a pharmacokinetic study. Thus, calibration graphs were linear within a range of 0.47-120 microgram/ml plasma. Intra-day precision was 2.29% (0.47 microgram/ml), 2.93% (4 microgram/ml), 3.22% (20 microgram/ml) and 4.40% (80 microgram/ml), inter-day variability was found to be 1.49% (0.47 microgram/ml), 3.79% (20 microgram/ml), 2.74% (40 microgram/ml) and 3.03% (80 microgram/ml). Inter-day accuracy showed deviations of 1.94% (0.47 microgram/ml), 0.53% (4 microgram/ml), -0.32% (20 microgram/ml) and 0.06% (80 microgram/ml). The method is rugged and robust and has been applied to the batch analysis of valproate during pharmacokinetic profiling of the drug.     

Detection and determination of reticuline and N-methylcoculaurine in the Annonaceae family using liquid chromatography-tandem mass spectrometry

In Guadeloupe, the French West Indies, there is a high incidence of atypical parkinsonism or progressive supranuclear palsy, and all of the investigated patients had taken herbal tea or tropical fruits of the Annonaceae family. Local inhabitants consume the fruits, and also drink tea made from the leaves. In the present study, we used liquid chromatography-tandem mass spectrometry (LC/MS/MS) with multiple reaction monitoring (MRM) to detect low-molecular-weight neurotoxic benzylisoquinoline derivatives in the Annonaceae family. We detected reticuline and N-methylcoculaurine in every Annona muricata sample examined, except for pulp and seed. They were not detected in sweetsop fruits. Norreticuline was not detected in any sample. These three compounds were toxic to SH-SY5Y neuroblastoma cells and inhibited mitochondrial respiratory complex I. It is possible that uptake of the benzylisoquinoline derivatives reticuline and N-methylcoculaurine and their accumulation in the brain may be related to the pathogenesis of the local endemic disease.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

Site-specific fluorescent derivatization and liquid chromatographic-mass spectrometric characterization of long R(3) IGF-I for bioanalytical applications

Recombinant Long R(3) IGF-I was derivatized with fluorescein isothiocyanate (FITC) at a single location by careful selection of reaction conditions (i.e. pH, and FITC/protein amino group ratio). High-performance liquid chromatography (LC) and electrospray mass spectrometry (MS) were used to confirm the extent of fluorescein conjugation. The protein conjugate was isolated and subjected to cyanogen bromide (CNBr) cleavage, followed by LC-MS to determine the site of modification. The isolated species of Long R(3) IGF-I-FITC was labeled at the N-terminal Met residue. Recognition of this fluorescent analog by monoclonal anti-IGF-I was preserved, indicating its potential for immunodiagnostic applications.     

Proteomics of Staphylococcus aureus--current state and future challenges

This paper presents a short review of the proteome of Staphylococcus aureus, a gram-positive human pathogen of increasing importance for human health as a result of the increasing antibiotic resistance. A proteome reference map is shown which can be used for future studies and is followed by a demonstration of how proteomics could be applied to obtain new information on S. aureus physiology. The proteomic approach can provide new data on the regulation of metabolism as well as of the stress or starvation responses. Proteomic signatures encompassing specific stress or starvation proteins are excellent tools to predict the physiological state of a cell population. Furthermore proteomics is very useful for analysing the size and function of known and unknown regulons and will open a new dimension in the comprehensive understanding of regulatory networks in pathogenicity. Finally, some fields of application of S. aureus proteomics are discussed, including proteomics and strain evaluation, the role of proteomics for analysis of antibiotic resistance or for discovering new targets and diagnostics tools. The review also shows that the post-genome era of S. aureus which began in 2001 with the publication of the genome sequence is still in a preliminary stage, however, the consequent application of proteomics in combination with DNA array techniques and supported by bioinformatics will provide a comprehensive picture on cell physiology and pathogenicity in the near future.     

Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm

Long-chain fatty acids can transfer passively across mammalian cell membranes. However, under physiological conditions of low fatty acid to albumin ratios in the circulation, the major fraction of uptake appears to be mediated by a saturable, protein-facilitated component. A simple diffusion process becomes significant at high molar ratios of fatty acid to albumin as the concentration of free fatty acid in solution is increased. Identification of the mammalian membrane fatty acid transporter(s) has been the focus of active investigation by several research groups. In this review we discuss three candidate proteins: FABPm, FAT/CD36 and FATP which have been cloned and are currently being characterized. Recent evidence arguing for an important role of the fatty acid transport step in general metabolism and linking these proteins to physiologic or metabolic abnormalities is described.     

Rapid quantification of the delta-opioid receptor selective enkephalin DPDPE in canine cerebrospinal fluid by liquid chromatography--mass spectrometry

An atmospheric pressure ionization liquid chromatographic-mass spectrometric assay was developed and validated for the determination of D-penicillamine(2,5) enkephalin (DPDPE) in cerebrospinal fluid (CSF) from dog. DPDPE and internal standard (D-Ala(2),D-Leu(5) enkephalin=DADLE) were isolated from CSF by reversed-phase C(18) solid-phase extraction with ZipTip micro-cartridges. Aliquots of extracted eluate were injected onto an Agilent Zorbax SB C(18) column (30 x 2.2 mm; 3.5 microm) at a flow-rate of 0.4 ml/min. The isocratic mobile phase of methanol-10 mM ammonium formate (pH 3) (75:25, v/v) was then diverted to waste for 45 s after injection, after which time flow was directed to the single quadrupole mass spectrometer. DPDPE was detected by positive mode selected ion monitoring. Standard curves were linear (r(2)> or =0.991) over the concentration range 1-1000 ng/ml. The efficiency of extraction recovery was greater than 97%, and the intra-assay and inter-assay precisions were within 9% relative standard deviation. DPDPE and the internal standard were stable in the injection solvent at 4 degrees C for at least 48 h. The assay was applied to the pharmacokinetic study of intrathecal DPDPE administration in the dog animal model.