The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples.     

Incidence and seroprevalence of canine ehrlichiosis in the Medjez El Bab region (northwestern Tunisia during 1994, 1995 and 1996

A sero-epidemiological survey, realized in the Medjez El Bab region (North-West of Tunisia), has concerned 180 dogs which status has been determined during the study. The animals were identified, then underwent an annual blood sampling during three successive years, in order to search for antibodies against E. canis and E. chaffeensis by indirect immunofluorescence. The results show that, in all sero-positive dogs, the levels of antibodies against E. canis were higher than those against E. chaffeensis. The sero-prevalence of E. canis was 42.8%, 50% and 48.9%, in 1994, 1995 and 1996, respectively, and was higher than that against E. chaffeensis during the three year studies. The incidence of E. canis infection was 12.6% during the three years whereas E. chaffeensis infection did not exceed 4.7%.     

Deciphering the mechanisms of homeostatic plasticity in the hypothalamo-neurohypophyseal system--genomic and gene transfer strategies

The hypothalamo-neurohypophyseal system (HNS) is the specialised brain neurosecretory apparatus responsible for the production of a peptide hormone, vasopressin, that maintains water balance by promoting water conservation at the level of the kidney. Dehydration evokes a massive increase in the regulated release of hormone from the HNS, and this is accompanied by a plethora of changes in morphology, electrical properties and biosynthetic and secretory activity, all of which are thought to facilitate hormone production and delivery, and hence the survival of the organism. We have adopted a functional genomic strategy to understand the activity dependent plasticity of the HNS in terms of the co-ordinated action of cellular and genetic networks. Firstly, using microarray gene-profiling technologies, we are elucidating which genes are expressed in the HNS, and how the pattern of expression changes following physiological challenge. The next step is to use transgenic rats to probe the functions of these genes in the context of the physiological integrity of the whole organism.     

Microarray analysis reveals interleukin-6 as a novel secretory product of the hypothalamo-neurohypophyseal system

Physiological activation of the hypothalamo-neurohypophyseal system (HNS) by dehydration results is a massive release of vasopressin (VP) from the posterior pituitary. This is accompanied by a functional remodeling of the HNS. In this study we used cDNA arrays in an attempt to identify genes that exhibit differential expression in the hypothalamus following dehydration. Our study revealed nine candidate genes, including interleukin-6 (IL-6) as a putative novel secretory product of HNS worthy of further analysis. In situ hybridization and immunocytochemistry confirmed that IL-6 is robustly expressed in the supraoptic (SON) and the paraventricular (PVN) nuclei of the hypothalamus. By double staining immunofluorescence we showed that IL-6 is largely co-localized with VP in the SON and PVN. In situ hybridization, immunocytochemistry, and Western blotting all revealed IL-6 up-regulation in the SON and PVN following dehydration, thus validating the array data. The same dehydration stimulus resulted in an increase in IL-6 immunoreactivity in the axons of the internal zone of the median eminence and a marked reduction in IL-6-like material in the posterior pituitary gland. We thus suggest that IL-6 takes the same secretory pathway as VP and is secreted from the posterior pituitary following a physiological stimulus.     

Purification of turkey pancreatic phospholipase A2

Turkey pancreatic phospholipase (TPP) has been purified from delipidated pancreases. The purification included ammonium sulfate fractionation, acidic (pH 5) treatment, followed by sequencial column chromatographies on DEAE-cellulose, Sephadex G-75, and reverse phase high pressure liquid chromatography. The purified enzyme was found to be a monomeric protein with molecular mass of 14 kDa. The optimal activity was measured at pH 8 and 37 degrees C using egg yolk emulsion as substrate. Our results show that the enzyme (TPP) was not stable for 1 h at 60 degrees C, and that bile salt and Ca2+ were required for the expression of the purified enzyme. The sequence of the N-terminal amino acids of the purified enzyme shows a very close similarity between TPP and all other known pancreatic phospholipases.     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

Lead and cadmium concentrations in seawater and algae of the Tunisian coast

Both lead and cadmium are toxic trace metals, even in very weak concentrations. The aim of this study was to estimate lead and cadmium pollution in various sites of the Tunisian coast and to verify the possibility of modification of the algae bioconcentration power according to water physico-chemical conditions. Our study concerned 99 samples of algae and 99 samples of seawater, taken in different sites of the Tunisian littoral. The analysis was realized by atomic absorption spectrophotometry (oven graphite). In algae, Sfax site presented the highest concentrations of lead when Sousse site showed the lowest ones. In seawater, the most amounts of lead were observed in Bizerte, Mahdia and Sfax sites, and those of cadmium in Bizerte and Medenine coasts. Bizerte's coast seems to be the most exposed zone to pollution. Indeed, the intensification of sea traffic may take place on this pollution because hydrocarbons derived from petroleum contain some tetraethylic lead characterised by its great toxicity. Sousse's region is the least polluted zone; it might be due to the development of tourism and a strict regulation of pollution in this district.     

Cell Wall and Cytoplasmic Isozymes of Radish beta-Fructosidase Have Different N-Linked Oligosaccharides

When 36-hour-old dark grown radish seedlings are transferred to far-red light, there is a decrease in cytoplasmic β-fructosidase (βF) and an increase in cell wall βF compared to the dark controls. Cytoplasmic and cell wall-bound β-fructosidase are both glycoproteins and exhibit high antigenic similarities, but differ according to charge heterogeneity and carbohydrate microheterogeneity. Growth of radish seedlings in the presence of tunicamycin results in a partial inhibition of βF glycosylation but nonglycosylated βF still accumulates in the cell wall under far-red light. Thus, glycosylation is not necessary for intracellular transport, for correct targetting, or for wall association of an active βF. The nonglycosylated cytoplasmic and cell wall βF forms have the same relative molecular mass but glycosylated forms have different oligosaccharide side-chains, with respect to size and susceptibility to α-mannosidase and endoglycosidase D digestion. The oligosaccharides of both forms are partly removed by endoglycosidase H when βF is denatured. Isoelectric focusing analysis of βF shows that the cell wall-associated isozymes are more basic than the cytoplasmic isozymes, and that the charge heterogeneity also exists within a single plant. A time course of changes in βF zymograms shows a far red light stimulation of the appearance of the basic forms of the enzyme. However, the more basic cell wall specific βF forms are not present when N-glycosylation is prevented with tunicamycin. These results indicate that cytoplasmic and cell wall βF probably have common precursor polypeptides and basic cell wall forms arise via processing events which are tunicamycin sensitive.