Gastroprotective effect and cytotoxicity of natural and semisynthetic labdane diterpenes from Araucaria araucana resin

The resin of the tree Araucaria araucana (Araucariaceae) is used by the Mapuche Amerindians in southern Chile and Argentina to treat ulcers and has been shown to display a gastroprotective effect in animal models. A study was undertaken to isolate, identify and assess the gastroprotective effect of the resin constituents and its semisynthetic derivatives as well as to evaluate the cytotoxicity of the products in cell cultures. Eleven diterpenes (ten labdane and a pimarane) were isolated from a resin sample collected in Chile. The labdane derivatives 15-acetoxylabd-8(17)-en-19-ol as well as 15,19-diacetoxylabd-8(17)-en are reported for the first time as natural products. Six diterpenes previously described from other plant sources are reported for the first time for the A. araucana resin. The structure of all compounds was elucidated by spectroscopic means. Some 24 diterpenes isolated/prepared in amounts over 10 mg were evaluated for gastroprotective effects in the ethanol/HCl-induced ulcer model in mice at 100 mg/kg. The highest gastroprotective activities were provided by 15-hydroxyimbricatolal, 15-acetoxyimbricatolal, 15-acetoxylabd-8(17)-en-19-oic acid methyl ester and 15-acetoxy-19-labdanoic acid, all of them being as active as the reference drug lansoprazole at 20 mg/kg. The cytotoxicity of 30 diterpenes as well as lansoprazole was assessed towards human lung fibroblasts (MRC-5) and 26 compounds were evaluated on the human gastric epithelial cell line AGS by means of the neutral red uptake assay. A concentration-dependent cell viability inhibition was found with IC50 values ranging from 27 up to > 1000 microM. The relationship between the cytotoxicity data and lipophilicity of the products is also discussed.     

Essential role of non-canonical Wnt signalling in neural crest migration

Migration of neural crest cells is an elaborate process that requires the delamination of cells from an epithelium and cell movement into an extracellular matrix. In this work, it is shown for the first time that the non-canonical Wnt signalling [planar cell polarity (PCP) or Wnt-Ca2+] pathway controls migration of neural crest cells. By using specific Dsh mutants, we show that the canonical Wnt signalling pathway is needed for neural crest induction, while the non-canonical Wnt pathway is required for neural crest migration. Grafts of neural crest tissue expressing non-canonical Dsh mutants, as well as neural crest cultured in vitro, indicate that the PCP pathway works in a cell-autonomous manner to control neural crest migration. Expression analysis of non-canonical Wnt ligands and their putative receptors show that Wnt11 is expressed in tissue adjacent to neural crest cells expressing the Wnt receptor Frizzled7 (Fz7). Furthermore, loss- and gain-of-function experiments reveal that Wnt11 plays an essential role in neural crest migration. Inhibition of neural crest migration by blocking Wnt11 activity can be rescued by intracellular activation of the non-canonical Wnt pathway. When Wnt11 is expressed opposite its normal site of expression, neural crest migration is blocked. Finally, time-lapse analysis of cell movement and cell protrusion in neural crest cultured in vitro shows that the PCP or Wnt-Ca2+ pathway directs the formation of lamellipodia and filopodia in the neural crest cells that are required for their delamination and/or migration.     

Maturation of phage T7 involves structural modification of both shell and inner core components

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.     

Investigation of clonal distribution and persistence of Salmonella Senftenberg in the marine environment and identification of potential sources of contamination

Salmonella Senftenberg was detected in the coastal areas of Galicia (NW Spain) in 1998, where it remained the predominant serovar for the next four years. Although the overall incidence of this serovar in the zone was lower than 1%, contamination by Salmonella serovar Senftenberg was located in very specific areas of the Ría de Arousa, where it persisted for more than five years. A total of 60 Salmonella serovar Senftenberg isolates, originating from surveillance activities in marine environments, was subjected to molecular characterization by pulsed-field gel electrophoresis (PFGE). PFGE analysis of the marine isolates allowed the differentiation of three main PFGE types, which contained the majority of the isolates, each type showing a specific spatial distribution in the coastal waters. The most prevalent pulse types persisted for more than four years, emphasizing their capacity to adapt and survive in marine environments. Using PFGE analysis, marine isolates were compared with Salmonella serovar Senftenberg isolates from neighbouring mussel-processing facilities and to other epidemiologically unrelated isolates from human, animal and feed sources. Comparison of the restriction patterns showed that indistinguishable PFGE types were present in the isolates from mussel-processing facilities and their surrounding marine areas, suggesting that the mussel processing is the main source for contamination with Salmonella Senftenberg in these marine environments. A molecular fingerprinting relationship was established between three shellfish isolates and a human isolate, which could be considered as preliminary evidence of infection caused by Salmonella Senftenberg associated with molluscan consumption.     

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 g–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.     

Effect of human chorionic gonadotrophin supplementation during different culture periods on in vitro maturation of canine oocytes

The IVM of canine oocytes is characterized by low rates of metaphase II. The objective of this study was to evaluate the effects of hCG on meiotic development of canine oocytes for culture periods up to 96 h. Oocytes were collected after ovariohysterectomy. Only oocytes >110 microm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. For IVM, the COCs were cultured in TCM-199+10% fetal calf serum, without (medium A control) or supplement with 10 IU/mL of hCG (medium B), or with a combination of both media (treatment B/A). The COCs were randomly allocated into three groups. The first and second groups were cultured in either medium A or B, respectively for 24, 48, 72, and 96 h. Oocytes of the third group (treatment B/A) were incubated in medium with hCG (medium B) the first 48 h and then transferred to medium without hCG (medium A) for an additional 24 or 48 h. The proportion of COCs with cumulus cell expansion was also evaluated before fixation. Oocytes were stained with propidium iodide prior to nuclear assessment (with epifluorescence microscopy). COCs with cumulus expansion were evident after 48 h of culture. The proportion of COCs with cumulus expansion was higher (P<0.05) for media containing hCG (B or B/A) than for meda lacking hCG (A); this difference was maintained for 72 and 96 h in culture. In media A, B and B/A, 23.3, 31.7 and 29.5%, respectively, of oocytes were at metaphase II after 72 h, with 20.7, 33.1 and 43.4% at this stage after 96 h. The advancement of meiosis was directly proportional to the time of incubation; the highest percentage (P<0.05) of oocytes at metaphase II was observed after 96 h of culture when 10 IU/mL hCG was present for only the first 48 h of culture.     

Differential phospholipid binding by site 3 and site 4 toxins. Implications for structural variability between voltage-sensitive sodium channel domains

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.