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.

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.

Cloning, sequencing and expression of the two genes encoding the mitochondrial single-stranded DNA-binding protein in Xenopus laevis.

In Xenopus laevis the single-stranded DNA binding protein imported into the mitochondria consists of two highly related polypeptides. The establishment of the genomic nucleotide sequences reveals that they are encoded by two different genes, XLSSB1 and XLSSB2. The deduced amino acid sequence is identical to the direct amino acid sequence determined by Edman degradation of the mitochondrial polypeptides [Ghrir. R., Lecaer, J.P., Dufresne, C. and Gueride, M. (1991) Primary structure of the two variants of Xenopus laevis mtSSB, a mitochondrial DNA binding protein. Arch. Biochem. Biophys. 291, 395-400]. Both genes are organized in seven exons and six introns, the sequence of the peptide leader is interrupted by an intervening sequence (intron 2). The exon/intron junctions are in exactly conserved positions, splitting the same codon. A high level of identity is observed between corresponding introns of the two genes over part or most of their lengths. Structural features of intronic sequences reveal multiple rearrangements and exchanges during the evolution of X. laevis species. A CCAAT box and the potential regulatory elements NRF-2 and Sp 1 are observed in the 5'-flanking region of both genes. During oogenesis, XLSSB gene expression is correlated with the replicative activity of the mitochondrial DNA.

Ecthyma gangrenosum in a kidney transplant recipient with Pseudomonas septicemia.

This report describes a renal transplant recipient in whom Pseudomonas septicemia and ecthyma gangrenosum developed within days of renal transplantation. Microscopic skin sections showed perivascular bacillary invasion. Pseudomonas organisms were cultured and microscopically visualized in sections from the transplanted kidney. Although cultures from the donor kidney preservation perfusate fluid showed no growth, Pseudomonas aeruginosa was found in the recipient's urine, blood, and peritoneal fluid. The recipient's course was complicated by septic shock, cardiopulmonary arrest, coma, and extensive skin lesions; but his condition improved with appropriate antibiotic therapy, wound debridement, and an aggressive rehabilitative program. He is now a candidate for retransplantation. This is the first known case of ecthyma gangrenosum in a renal transplant recipient.

Synthesis and structure-activity relationships of a novel class of 5-lipoxygenase inhibitors. 2-(Phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans: the development of L-656,224.

The synthesis of a series of 2-(phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans and their inhibitory effects against leukotriene biosynthesis and 5-lipoxygenase activity in vitro are described. Many compounds in this series were found to be potent inhibitors of LTB4 production by human polymorphonuclear leukocytes with IC50 values ranging from 7 to 100 nM. Structure-activity relationships of the series are presented. Within this series, 2-[(4'-methoxyphenyl)methyl]-4-hydroxy-3-methyl-5-propyl-7-chlorobenz ofuran (L-656,224) showed extremely potent activity, inhibiting leukotriene biosynthesis in intact human leukocytes (IC50 = 11 nM), as well as the 5-lipoxygenase reaction catalyzed by cell-free preparations from rat leukocytes (IC50 = 36 nM), human leukocytes (IC50 = 0.4 microM), and the purified enzyme from porcine leukocytes (IC50 = 0.4 microM). The compound also shows oral activity in a number of animal models in vivo.

Development of 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (L-670,630) as a potent and orally active inhibitor of 5-lipoxygenase.

Leukotrienes are potent biological mediators of allergic and inflammatory diseases and are derived from arachidonic acid through the action of the 5-lipoxygenase. In this study, the syntheses and comparative biological activities of three series of 2,3-dihydro-2,6-disubstituted-5-benzofuranols with various substituents on position 3 are described. Compounds from each series were evaluated for their ability to inhibit the production of leukotriene B4 (LTB4) in human peripheral blood polymorphonuclear (PMN) leukocytes and the 5-lipoxygenase reaction in cell-free preparations from rat PMN leukocytes. The structure-activity relationships of each series in vitro and in vivo are presented. The bioavailability, metabolism, and toxicity profile of each series are discussed. The series with no substituent at position 3 was the most potent and among the compounds in that series 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (46, L-670,630) was chosen for further development.

Selective protection and relative importance of the carboxylic acid groups of zaragozic acid A for squalene synthase inhibition.

Chemistry that allows selective modification of the carboxylic acid groups of the squalene synthase inhibitor zaragozic acid A (1) was developed and applied to the synthesis of compounds modified at the 3-,4-,5-,3,4-,3,5-, and 4,5-positions. A key step in this procedure is the selective debenzylation by transfer hydrogenolysis in the presence of other olefinic groups. These compounds were tested in the rat squalene synthase assay and in vivo mouse model. Modification at C3 retains significant enzyme potency and enhances oral activity, indicating that C3 is not essential for squalene synthase activity. Modification at C4 and C5 results in significant loss in enzyme activity. In contrast, substitution at C3 or C4 enhances in vivo activity. Furthermore, disubstitution at the C3 and C4 positions results in additive in vivo potency.

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.

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.

Separation of craniopagus Siamese twins using cardiopulmonary bypass and hypothermic circulatory arrest.

Occipitally joined craniopagus Siamese twins were separated with the use of cardiopulmonary bypass and hypothermic circulatory arrest. The 7-month-old infants shared a large sagittal venous sinus that precluded conventional neurosurgical approach because of risk of exsanguination and air embolism. After craniotomy and preliminary exposure of the sinus, each twin underwent sternotomy and total cardiopulmonary bypass with deep hypothermia. Hypothermic circulatory arrest allowed safe division and subsequent reconstruction of the sinus remnants. Several unusual problems were encountered, including transfusion of a large blood volume from one extracorporeal circuit to the other through the common venous sinus, deleterious warming of the exposed brain during circulatory arrest, and thrombosis of both pump oxygenators. Both infants survived, although recovery was complicated in each by neurologic injury, cranial wound infection, and hydrocephalus. This case demonstrates the valuable supportive role of cardiopulmonary bypass and hypothermic circulatory arrest in the management of complex surgical problems of otherwise inoperable patients.

The use of sclerotherapy for treatment of postmastectomy wound seromas.

Five patients who developed seromas following mastectomy with lymph node dissection were treated with aspiration of the seromas and instillation of a sclerosant solution containing tetracycline. All seromas resolved promptly without infection, flap necrosis, or recurrence. The technique is recommended for treatment of this postoperative complication.

Achalasia of the cardia associated with pulmonary sarcoidosis.

The case of a patient with sarcoidosis and symptoms suggestive of achalasia cardia is presented. Because of increasingly severe dysphagia, cardiomyotomy was performed and biopsy specimens of the esophageal wall were obtained. Light and electron microscopy revealed lesions of the nerves in Auerbach's plexus consisting of an inflammatory process and demyelinization of the nerve fibers. Since the operation, the patient has been swallowing without difficulty. Although previous reports have described dysphagia in patients with sarcoidosis, presumably secondary to mechanical compression by adjacent lymph nodes or infiltration of the esophageal wall by sarcoid granulomata, this report documents for the first time that dysphagia can also be caused by direct involvement of the innervation of the esophagus by sarcoidosis.

Can infected prosthetic grafts be salvaged with rotational muscle flaps?

The conventional approach to prosthetic graft infection, including graft removal and extraanatomic reconstruction, conveys a substantial risk of limb loss and death and mandates more innovative solutions. From January 1985 to January 1989 eight rotational muscle flaps were performed on four men and three women with prosthetic graft infection who ranged in age from 42 to 79 years (mean, 67 years). The grafts involved included aortofemoral (three patients), femoropopliteal (two patients), femorofemoral (one patient), and subclavian-carotid-carotid (one patient) and were composed of Dacron (five grafts) or polytetrafluorethylene (two grafts). Infections were in the groin in six patients and in the neck in the other patient; all patients had anastomotic exposure. Clinical presentations included abscess/purulent drainage (four patients), anastomotic hemorrhage (two patients), and anastomotic false aneurysm (one patient) and was associated with fever and/or leukocytosis in all patients. Positive bacterial cultures were obtained from all patients. Rotational muscle flaps performed included rectus abdominis (five grafts), pectoralis major (one graft), gracilis (one graft), tensor fascia lata (one graft); in two patients, rotational muscle flaps were performed after failed local sartorius muscle transfer. No major complications of the RMF procedures were encountered. One patient died 4 months after the operation of complications of ischemic colitis/perforation. The other six patients were discharged with completely healed wounds. One patient developed recurrent infection 12 months after the rotational muscle flap procedure; five (83%) patients have been followed for 12 to 51 months (mean, 27 months) without evidence of recurrent infection. These preliminary results suggest that rotational muscle flaps are a safe and effective treatment for prosthetic graft infection, even when local sartorius muscle coverage has failed.

Anatomic and clinical considerations of an internal mammary artery harvest.

In an effort to understand the perceived correlation of internal mammary artery harvesting and wound healing difficulties in the inferior margins of the sternotomy incision, we showed the cutaneous vascular perfusion in the sternal and xiphoid areas by India ink injection studies in cadavers. With these studies, we demonstrated an inherent paucity of nutrient supply to the inferior sternum and xiphoid area. The classic internal mammary artery harvest further compromises the blood supply to these areas. We believe that limiting the most inferior dissection of the internal mammary artery and not including the distal bifurcation leaves intact the lateral musculophrenic nutrient supply to the inferior sternum and xiphoid area and to the ipsilateral abdominal rectus muscle. These guidelines will help to prevent ischemic complications of this area and may aid in reconstruction. If the bifurcation is harvested, we believe that the removal of the avascular xiphoid cartilage at the time of the initial bypass procedure may eliminate this as a potential septic focus.

Suppression of amygdala kindling with massed stimulation: effect of noradrenaline antagonists.

Afterdischarge (AD) triggered by brief, daily stimulation of the amygdala progressively increases in complexity and duration and, over days, develops into generalized convulsions. This progression, called kindling, is delayed by noradrenaline (NA). When brief stimulation of the amygdala occurs too frequently (massed), there is a suppression of AD growth and little evidence of kindling. Previously we showed that depletion of NA before massed amygdala stimulation prevented the suppression of AD growth described above, and readily precipitated generalized seizures. In the present report, we examined the role of NA in maintaining this suppression of AD growth, after it was well established. We showed that suppression of AD development during the first 15 massed stimulations (interstimulus interval of 5 min) was reduced by subsequent injection of the NA alpha 2 antagonist, yohimbine, with most rats exhibiting occasional generalized convulsions. Conversely, rats exposed to the beta antagonist, propranolol, like controls, not only showed suppressed AD growth, but also elevated AD thresholds. Three weeks later, only a small positive transfer to daily kindling was observed in all groups. We conclude that alpha 2 NA receptors help maintain suppression of AD growth induced by massed stimulation of the amygdala, while beta receptors provide only a small proepileptic influence. These results and those from the 'rapid' kindling model (Lothman et al., Brain Research, 360 (1985) 83-91) are compared, and related to NA receptor subtype variations in the amygdala and hippocampus.

Three-dimensional computed tomographic reconstructions of intracranial meningiomas.

In a series of four patients with intracranial meningiomas, three-dimensional computed tomographic (CT) reconstructions were a useful diagnostic and surgical adjunct. Three-dimensional images are created from standard CT data by a boundary-detecting computer software program. Three-dimensional images of tumor invading or adjacent to the bony calvarium are projected about the x, y, and z axes. Axial and sagittal sections delineate the lesions. The images created allow a surgical view of the meningiomas in three-dimensional space and demonstrate the relationship of these masses to the skull. The use of three-dimensional reconstructions in craniofacial surgery and in neurosurgery is reviewed.

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.

Differential sensitivity of various temporal lobe structures in the rat to kindling and status epilepticus induction.

Using focal brain stimulation (kindling), discrete seizures can be triggered from many neuroanatomic sites with varying degrees of facility. From several of these sites, protracted seizures or status epilepticus (SE) also can be triggered. To date, no comparison has been made between different brain sites in their sensitivity both to kindling and to SE development. In this report, we have compared the kindling profiles of three amygdala nuclei, namely the basal (BL), central (CE), and medial (ME) nuclei, to the adjacent piriform (PIR) and perirhinal (PRH) cortices. In addition, three weeks following kindling, the susceptibility of each kindled site to status epilepticus (SE) was assessed by exposing the site to 60 min of electrical stimulation. We observed that (a) during the course of daily kindling, the afterdischarge threshold dropped progressively and significantly in all structures, (b) the rate of kindling in the PRH and PIR cortices and the CE amygdala was significantly faster than either the BL or ME amygdala, (c) when discrete convulsions were triggered, the latency to forelimb clonus in the PRH cortex and CE amygdala was significantly shorter than the other three structures, and (d) despite being slower to kindle than most other sites, stimulation of the BL nucleus most readily triggered SE. The kindling data suggest that discharges triggered from the PRH and CE more readily access motor systems supporting limbic convulsions than discharges triggered from the BL, ME nuclei or the PIR cortex. On the other hand, the SE data indicate that the mechanisms and circuits associated with the development of discrete kindled seizures are not identical to those associated with the induction of limbic SE.