Faecal contamination of echinoderms: first report of heavy Escherichia coli loading of sea urchins from a natural growing area.

UNLABELLED: Although little evidence existed to support that view, European countries and in particular France, have regarded echinoderms, including sea urchins, as low risk in terms of feacal contamination. It is hypothesized that the sea urchins mode of feeding, which is based on grazing and differs from bivalve molluscs, would prevent it from concentrating high levels of Escherichia coli. Here, we monitored E. coli levels in sea urchins (Paracentrotus lividus) and in filter-feeder mussels (Mytillus galloprovincialis), collected concurrently from the same natural area over a 1-year period to verify this assumption. Sea urchins were collected on the seafloor, whereas mussels were collected from the water column at a depth of 4 m. Our results showed heavy bacterial loading of sea urchins in a natural growing environment. Moreover, we highlighted that E. coli contamination of sea urchins could, in certain conditions, be higher than those detected in filter-feeding mussels collected at the same location. Finally, the results showed a significant correlation between rainfall and E. coli concentrations in sea urchins, suggesting that the bacterial safety of sea urchin could be linked to the quality of the surrounding water. SIGNIFICANCE AND IMPACT OF THE STUDY: The European regulation requires competent authorities to monitor the sanitary status of shellfish, including live echinoderms, through faecal indicator organisms. In the French Mediterranean, sea urchin production is significant. Until now, as no data showed significant E. coli contamination levels, no monitoring programs focused on this species. This study demonstrates that sea urchins are more vulnerable to faecal contamination than previously hypothesized, especially during heavy rainfall. In consequence, the European authority general approach to microbiological management of shellfish should be applied to sea urchins.

The EVI5 TBC domain provides the GTPase-activating protein motif for RAB11.

The human EVI5 gene was originally isolated through its involvement with a constitutional chromosome translocation in a patient with stage 4S neuroblastoma. Recently, it has been shown that EVI5 is a centrosomal protein in interphase cells, which relocalizes to the midbody during late phases of mitosis. Disruption of its function leads to incomplete cell division and the formation of multinucleate cells. The EVI5 protein contains a TBC (TRE2/BUB/CDC16 homology) motif located in the N-terminal region. Proteins containing a TBC domain have been shown in some cases to act as GTPase-activating proteins (GAPs) and function through the interaction with Rab-like small G proteins. Despite the identification of over 50 TBC-containing proteins, and over 70 Rab-like proteins, only three combinations have been shown to have Rab/GAP activity to date. In this study, using linear ion trap mass spectroscopy, we have demonstrated that EVI5 exists in a protein complex with Rab11. Further, using a specific Rab-binding assay, we have shown that EVI5 preferentially interacts with the guanosine triphosphate-bound form of Rab11, and in a GAP activity assay, we have confirmed that EVI5 functions as a GAP for the Rab11 GTPase.

Mitochondrial upstream promoter sequences modulate in vivo the transcription of a gene in yeast mitochondria.

An in vivo study of the importance of the length and/or structures of sequences upstream of a mitochondrial promoter was undertaken in Saccharomyces cerevisiae. Short tandem mtDNA repeats were introduced upstream of the COX2 gene. Our data show that its expression is modulated by the sequence located over 200 bp upstream of the promoter. A deletion decreases the level of transcripts to about 50%. The initial level can be recovered by a fill-in AT-rich sequence or partially by the presence of a long repeat tract; on the contrary, a smaller number of copies tends to intensify the effect of the deletion. These results show that the length and base composition upstream of mitochondrial promoter are involved in vivo in the modulation of the gene expression.

Propofol metabolites in man following propofol induction and maintenance.

BACKGROUND: The pharmacokinetics of propofol in man is characterized by a rapid metabolic clearance linked to glucuronidation of the parent drug to form the propofol-glucuronide (PG) and sulfo- and glucuro-conjugation of hydroxylated metabolite via cytochrome P450 to produce three other conjugates. The purpose of this study was to assess the urine metabolite profile of propofol following i.v. propofol anaesthesia in a Caucasian population. METHODS: The extent of phase I and phase II metabolism of propofol was studied in 18 female and 17 male patients after an anaesthesia induced and maintained for at least 4 h with propofol. The infusion rates (mg kg(-1) h(-1)) of propofol were (mean (SD)) 4.1 (1.0) and 4.5 (1.3) for males and females, respectively. Urine was collected from each patient for the periods 0-4, 4-8, 8-12, and 12-24 h after the start of propofol administration. In a preliminary study, the three main glucuro-conjugated metabolites were isolated from urine and characterized by magnetic resonance spectroscopy. The quantification of these metabolites for the different collection periods was then performed by a HPLC-UV assay. RESULTS: Total recovery of propofol in the metabolites studied amounts to 38%, of which 62% was via the PG metabolite and 38% via cytochrome P-450. This percentage is significantly higher than that previously reported from patients after a bolus dose of propofol. Extreme values for PG (0-24 h period) were included from 73 to 49%. There was no significant difference between female and male patients in the metabolite ratio. CONCLUSIONS: We conclude that the extent of hydroxylation in propofol metabolism was higher than in previous findings after administration of anaesthetic doses of propofol. Moreover, the ratio between hydroxylation and glucuronidation of propofol is subject to an inter-patient variability but this does not correlate with the dose of propofol. However, the variation of the metabolite profile observed in the present report does not seem to indicate an extended role of metabolism in pharmacokinetic variability.

Fortum stability in different disposable infusion devices by pyridine assay.

The stability of ceftazidime in 5% dextrose injection and 0.9% sodium chloride injection when stored in a different disposable infusion device was determined. Solutions of ceftazidime 40 mg/ml were used to fill the drug administration devices. Stability was determined for both 5% dextrose injection and 0.9% sodium chloride injection solutions at 37 degrees C in four disposable infusion devices. Ceftazidime and its mean degradation product, pyridine, were simultaneously assayed in triplicate by a stability-indicating high-performance liquid chromatographic (HPLC) method. This method was simple, sensitive (limit of quantitation (LOQ), 2 ng injected for both compounds), rapid (run time was 7 min) and precise (mean recovery was 100.5+/-2.9 and 103.6+/-1.9% for pyridine and ceftazidime, respectively). The ceftazidime stability in the 5% dextrose solution was lower than in the 0.9% sodium chloride solution. When stored at 37 degrees C in a disposable infusion device, the stability of the ceftazidime is included in large hourly range, depending strongly on the manufacturer. The stability of ceftazidime exceed 19 h in none studied cases. The pyridine formed in 24 h was in the range of 100-400 mg depending on devices and infusions.

Stability of sufentanil in human plasma samples.

The stability of sufentanil in human plasma kept under various storage conditions was investigated. Extraction was performed using solid phase extraction with new mixed-mode cation exchange Oasis MCX columns; quantification was carried out using gas chromatography equipped with a mass spectrometry detector. When plasma was left at 4 degrees C in nonsilanized tubes, concentrations of sufentanil decreased significantly during the first hour. In plasma samples kept at -25 degrees C for 8 hours in nonsilanized glass tubes, a significant decrease of sufentanil concentrations was found, with an average loss of 10.1% of the initial concentration. A significant decrease occurred when plasma was kept in silanized glass tubes for 12 hours at -25 degrees C. The current study emphasizes the importance of sampling and storage conditions for an accurate determination of sufentanil concentration in plasma.

Reduced enzymatic activity of glucokinase after affinity labeling: results from spectrophotometry and electrospray ionization mass spectrometry.

Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.

A review of latest research findings on the health promotion properties of tea.

Important progress has been made in the past five years concerning the effects of green and black tea on health. Experimentation with new accurate tools provide useful information about the metabolism of tea components in the body, their mode of action as antioxidants at the cellular level and their protective role in the development of cancer, cardiovascular disease and other pathologies. The use of tea components as nutraceuticals and functional foods are also discussed.

New sensitive assay of vancomycin in human plasma using high-performance liquid chromatography and electrochemical detection.

A method using reversed-phase high-performance liquid chromatography with electrochemical detection for the analysis of vancomycin in human plasma was developed. Chromatographic conditions included an octadecyl column, a mobile phase of acetonitrile-sodium phosphate buffer (pH 7) (12:88), a total run time of 12 min, and coulometric electrochemical detection at +700 mV. Linear detector response was found in the range 5-100 microg ml(-1) after a 1:80 dilution or from 0.5 to 50 microg ml(-1) after a 1:20 dilution of the samples. In both cases the correlation coefficient (r) of the calibration curve standard was better than 0.995. Vancomycin determination was based on a denaturation of plasma proteins with methanol, then a dilution with mobile phase was performed. Recovery of vancomycin from plasma was 103.1+/-3.9%, and no interference from commonly used drugs or endogenous compounds was observed. A significant correlation was shown with the EMIT assay (r=0.92, P<0.001) using clinical samples from children. This HPLC technique is simple, sensitive, rapid, precise, selective and requires only 100 microl of plasma for completion.

Detection of new propofol metabolites in human urine using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry techniques.

Using hyphenated analytical techniques, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), a study on minor propofol metabolites in human urine was conducted. These techniques allowed identification of two new phase I metabolites (2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol). In addition, their four corresponding conjugates (three glucuronides and one sulphate) were detected. Thus in human urine at least eight conjugate metabolites are produced, derived from four different aglycones (propofol; 2, 6-diisopropyl-1,4-quinol; 2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol).

Verrucous malformations: their presentation and management.

Verrucous hemangiomas are a distinct subset of vascular malformations that have not been described extensively in the plastic surgery literature. They are characterized by reactive epidermal acanthosis, papillomatosis, hyperkeratosis, and extension into the subcutaneous tissues. In response to injury, infection, or subtotal resection, they enlarge and become increasingly keratotic. In light of contemporary definitions of hemangiomas and malformations, the authors recommended that these lesions be renamed verrucous malformations. The authors review their evaluation and treatment of 6 patients with this lesion and offer an algorithm that emphasizes excision over ablative therapy. Of the 6 patients, 3 patients had failed either cryotherapy or laser removal by a nonsurgeon, with a consequent increase in lesion size and discomfort. The lesions were all subsequently excised. Because of the size and location of the verrucous malformations, staged removal was required in 3 patients. Patients have been followed for as long as 7 years. A single recurrence was controlled with reexcision. Excision of verrucous malformations, rather than laser ablation or cryosurgery, is supported by the authors' favorable results.

The psychological burden of peanut allergy as perceived by adults with peanut allergy and the parents of peanut-allergic children.

BACKGROUND: Peanut-allergic patients are affected by a condition which forces them and their families to exercise extreme dietary vigilance and experience constant uncertainty throughout their lives. OBJECTIVE: To compare the quality of life and family relations of children and adults with a peanut allergy to that of children and adults with a rheumatological disease. METHODS: Patients with a confirmed diagnosis of peanut allergy or a rheumatological disease completed (for children less than 18 years, by proxy) self-report questionnaires regarding the impact of their condition on their quality of life and family relations. A vertical visual analogue scale and the Impact on Family Questionnaire (IFQ) served as outcome measures. RESULTS: One hundred and fifty-three peanut-allergic children were compared with 69 children with a rheumatological disease while 37 peanut-allergic adults were compared with 42 adults with a rheumatological disease. The parents of peanut-allergic children, compared to the parents of children with a rheumatological disease, reported that their children had significantly more disruption in their daily activities. Furthermore, the parents of peanut-allergic children reported more impairment in the familial-social dimension of the IFQ. Conversely, adults with a chronic rheumatological disease reported more disruption in their family relations than peanut-allergic adults. CONCLUSION: Given the considerable disruption in daily activities and family relations reported by the parents of peanut-allergic children, accurate diagnosis of peanut allergy is essential. Our work should make health care professionals dealing with children with confirmed peanut allergy more aware of the support that these families may require. Furthermore, we hope to motivate food industries to offer more 'peanut free' products to decrease the dietary restrictions of these patients while minimizing their potential for accidental ingestion.

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs.

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.

The design and development of an integrated natural products screening database.

We designed and developed NEXUS--a new natural products screening database and related suite of software applications--to utilize the spectacular increases in assay capacity of the modern high throughput screening (HTS) environment. NEXUS not only supports seamless integration with separate HTS systems, but supports user-customized integration with external laboratory automation, particularly sample preparation systems. Designed and developed based on a detailed process model for natural products drug discovery, NEXUS comprises two integrated parts: (1) a single schema of Oracle tables and callable procedures and functions, and (2) software "front-ends" to the database developed using Microsoft Excel and Oracle Discovery/2000. Many of the back-end processing functions were written in Programming Language/Structured Query Language (PL/SQL) to provide an Application Programmer's Interface, which allows end users to create custom applications with little input from information technology professionals.

The planning and establishment of a sample preparation laboratory for drug discovery.

Nature has always been a productive source of new drugs. With the advent of high-throughput screening, it has now become possible to rapidly screen large sample collections. In addition to seeking greater diversity from natural product sources (micro-organisms, plants, etc.), fractionation of the crude extracts prior to screening is becoming a more important part of our efforts. As sample preparation protocols become more involved, automation can help to achieve and maintain a desired sample throughput. To address the needs of our screening program, two robotic systems were designed. The first system processes crude extracts all the way to 96-well plates, containing solutions suitable for screening in biological and biochemical assays. The system can dissolve crude extracts, fractionate them on solid-phase extraction cartridges, dry and weigh each fraction, re-dissolve them to a known concentration, and prepare mother plates. The second system replicates mother plates into a number of daughter plates.

FAST and SLOW amygdala kindling rat strains: comparison of amygdala, hippocampal, piriform and perirhinal cortex kindling.

In our companion paper, we selectively bred offspring of a Long Evans Hooded and Wistar rat cross for either fast or slow rates of amygdala kindling (Racine et al., 1999. Development of kindling-prone and kindling resistant rats: Selective breeding and electrophysiological studies, Epilepsy Res. 35, 183-195). Within 10 generations, there was no overlap in the distribution of kindling rates between these newly developed FAST and SLOW kindling strains. In the present report, we compared the local excitability, kindling rates, and convulsion profiles of kindling sites in either the amygdala, dorsal hippocampus, piriform cortex or perirhinal cortex in the two strains. Local excitability, measured as the local afterdischarge (AD) threshold and its duration, showed varied effects between structures and strains. Before kindling, the AD threshold was lower in the FAST than the SLOW rats in the hippocampus, piriform and perirhinal cortices, but not the amygdala (the selection structure). Also, the duration of the AD threshold duration was significantly longer in the FAST than in the SLOW rats in all structures, except the CA1 hippocampus. Most of these differences were maintained after kindling. Kindling itself was significantly faster in the FAST compared with the SLOW rats in all structures; however, the different structural kindling rates showed proportional differences between strains that were about five times different in the amygdala compared with only about two times different in the hippocampus. This suggested a selection bias for the amygdala and its networks. As in other rat strains, the fastest kindling rates were seen in the perirhinal cortex followed by the piriform cortex, amygdala and hippocampus in both FAST and SLOW rats. Other important differences between strains and structures occurred in the stage-5 convulsion profiles, including latency to forelimb clonus, clonus duration and duration of associated local afterdischarges. The differences in kindling profiles between strains and structures were discussed with respect to possible underlying mechanisms, significance for epileptogenesis, and impact on other normal behaviours.

High-resolution electrospray ionization Fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus Stearothermophilus.

Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2). Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies. Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein. Electrospray FTMS provides evidence of a solution phase dimer. In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein. The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.

A series of non-quinoline cysLT1 receptor antagonists: SAR study on pyridyl analogs of Singulair.

The structure-activity relationship of a series of styrylpyridine analogs of MK-0476 (montelukast, Singulair) is described. This work has led to the identification of a number of potent and orally active cysLT1 receptor (LTD4 receptor) antagonists including 2ab (L-733,321) as an optimized candidate.

Metabolic profiles of montelukast sodium (Singulair), a potent cysteinyl leukotriene1 receptor antagonist, in human plasma and bile.

Montelukast sodium [1-([(1(R)-(3-(2-(7-chloro-2-quinolinyl)-(E)- ethenyl)phenyl)-3-(2-(1-hydroxy-1-methylethyl)phenyl)propyl)thio]methyl)cyclopropylacetic acid sodium salt] (MK-476, Singulair) is a potent and selective antagonist of the cysteinyl leukotriene (Cys-LT1) receptor and is under investigation for the treatment of bronchial asthma. To assess the metabolism and excretion of montelukast, six healthy subjects received single oral doses of 102 mg of [14C]montelukast, and the urine and feces were collected. Most of the radioactivity was recovered in feces, with