Isolation and partial characterization of bacteria (Pseudoalteromonas sp.) with potential antibacterial activity from a marine costal environment from New Caledonia.

UNLABELLED: Marine bacteria are a rich source of bioactive metabolites. However, the microbial diversity of marine ecosystem still needs to be explored. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from various marine coastal environment of New Caledonia. We obtained 493 marine isolates from various environments and samples of which 63 (12.8%) presented an antibacterial activity against a panel of reference pathogenic strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis). Ten out of the most promising strains were cultured, fractionated and screened for antibacterial activity. Four of them (NC282, NC412, NC272 and NC120) showed at least an activity against reference and multidrug-resistant pathogenic strains and were found to belong to the genus Pseudoalteromonas, according to the 16S phylogenetic analysis. The NC282 strain does not belong to any described Pseudoalteromonas species and might be of interest for further chemical and biological characterization. These findings suggest that the identified strains may contribute to the discovery for new sources of antimicrobial substances to develop new therapies to treat infections caused by multidrug-resistant bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: With the constant increasing of bacterial resistance against known antibiotics in worldwide public health, it is now necessary to find new sources of antimicrobials. Marine bacteria from New Caledonia were isolated, tested for antibacterial activity and characterized to find new active molecules against multidrug-resistant bacteria. This study illustrates the diversity of the marine ecosystem with potent new bacteria species. Also the potential of marine bacteria as a rich source of bioactive molecule, for example antibiotics, is highlighted.

Clinical characteristics of molluscum contagiosum in children in a private dermatology practice in the greater Paris area, France: a prospective study in 661 patients.

BACKGROUND: Series regarding the clinical characteristics of molluscum contagiosum (MC) are retrospective and concern small defined populations seen in a hospital setting. METHODS: We prospectively studied patients under 15 years of age with MC seen in 25 private dermatology practices in the greater Paris area during 1 year. RESULTS: Six hundred fifty patients were included. The mean age was 6 years. Of the patients, 30.5% had had MC in the past, 53% had 5-20 MC, 43% had a history of atopic dermatitis (AD), 5% were using topical steroids, 25.5% had coexistent AD, 13% had giant MC (>5 mm), 21% had inflammatory MC, and 19% had MC surrounded by eczema. Bathing with siblings was associated with a higher number of MC. A history of AD was associated with the presence of MC surrounded by eczema. The use of topical steroids was associated with a higher risk of relapse. There was no relation between environmental factors (MC in surrounding people, going to the swimming pool, sports practice) and the number of lesions or between environmental factors and the number of recurrences. CONCLUSION: Atypical lesions are frequent. Bathing with siblings is associated with a higher number of MC. Topical steroid use increases the risk of relapses. AD prevalence is higher than in the general population.

Intraperitoneal linear polyethylenimine (L-PEI)-mediated gene delivery to ovarian carcinoma nodes in mice.

Linear polyethylenimine (L-PEI) is an efficient transfection agent for ovarian carcinoma cells in vitro and ex vivo. In the present work, we go a step further and evaluate the efficacy of L-PEI in human ovarian tumor nodes developed in mice. PEI/DNA complexes were administered intraperitoneally instead of intravenously to avoid sequestering of complexes in the lung and liver and to allow transfection of nonvascularized tumor nodes. Plasmid biodistribution was studied by PCR and gene expression was characterized using complementary luciferase and beta-galactosidase assays. Intraperitoneal (i.p.) injection of L-PEI/DNA complexes allowed the straightforward distribution of plasmid in the whole peritoneal cavity. Gene expression occurred in many organs, but tumor nodes appeared as preferential sites for transgene expression. The i.p. delivery route allowed repeated injections and administration of large amounts of DNA (up to 400 mug) without signs of toxicity, even for doses well beyond the intravenous lethal dose. Transgene expression was dose-dependent and transient. However, multiple injections allowed its persistence to increase. These results provide encouraging elements towards the development of PEI-based gene therapy protocols for the treatment of advanced stage ovarian carcinoma.

Gas chromatographic-mass spectrometric method for polycyclic aromatic hydrocarbon analysis in plant biota.

Using gas chromatography-mass spectrometry, a new method was developed for the identification and the quantification of polycyclic aromatic hydrocarbons (PAHs) in plants. This method was particularly optimised for PAH analyses in marine plants such as the halophytic species, Salicornia fragilis Ball et Tutin. The saponification of samples and their clean up by Florisil solid-phase extraction succeeded in eliminating pigments and natural compounds, which may interfere with GC-MS analysis. Moreover, a good recovery of the PAHs studied was obtained with percentages ranging from 88 to 116%. Application to the determination of PAH in a wide range of coastal halophytic plants is presented and validated the efficiency, the accuracy and the reproducibility of this method.

Evidence for bioaccumulation of PAHs within internal shoot tissues by a halophytic plant artificially exposed to petroleum-polluted sediments.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants of natural and anthropic origins. Despite their poor water solubility, they can be taken up and bioaccumulated by plants. This study was aimed at determining whether the PAHs present in sediments artificially polluted by heavy fuel oil are transferred to shoots of a coastal and edible plant, Salicornia fragilis Ball et Tutin. Bioaccumulation was quantified after a one-week exposure to sediments polluted with 0.2%, 2% and 20% fuel oil (w/w) and over a six-week monitoring at 0.2%. Quantification by GC-MS of PAH amounts in plants and sediments evidenced a bioaccumulation in the shoots by a soil-to-plant transfer through the root system. This bioaccumulation depended on the duration of exposure and on the substratum contamination. PAHs distributions in plants and sediments looked alike with a predominance of low- and medium-weight hydrocarbons. Moreover, high-weight PAHs were also detected in the upper part of plants.

Effet paradoxal du type d'excision sur la prise et le délai de cicatrisation des greffes expansées pour le traitement des brûlures aiguës: a propos de 1129 cas.

Split-thickness meshed skin graft is frequently used in the treatment of acute burns. We studied the effect of the type of basement preparation on graft intake and healing time. We retrospectively analysed 1,129 meshed grafts used in the treatment of acute burns between 1995 and 2005. Intake was significantly better after avulsion (82%) than after tangential excision (75%). Intake was better if avulsion was performed before day 7 (83% vs. 73%). A trend for better intake after tangential excision was seen when performed between day 7 and 21. Healing time was significantly shorter after tangential excision. These results show the paradox that avulsion favours graft intake but delays healing time, contrary to tangential excision.

The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells.

The seriousness of ovarian cancer, which is related to the observed link between recurrency and cell cycle control defect, prompted us to explore the effect of ectopic expression of the cdk inhibitor p21(cip1/waf1) on ovarian carcinoma chemosensitivity. The transfection of p21(cip1/waf1) cDNA into SKOV3 and OVCAR3 cells led to reduction of tumor cell growth, enhanced susceptibility to cisplatin-induced apoptosis, and abolition of recurrency after cisplatin exposure. p21(cip1/waf1) gene transfer allowed a marked reduction of the cisplatin concentration needed to erradicate the tumor cell population. These results suggest exploring the possible use of p21(cip1/waf1) as an adjunctive to conventional chemotherapy.

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells.

One herbicide (isoproturon), two fungicides (carbendazim and chlorothalonil) and etoposide (an effective antitumor agent used as a positive control), were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary (CHOK1) cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis (SCGE) assay and the chromosomal aberration (CA) test in absence of noticeable cytotoxicity. With the SCGE assay, a clear induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 microM concentration range. In the CA test, chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observed with the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations in the concentration range of 25 microM to 100 microM, and isoproturon did not induce any significant increase in CA. In conclusion, chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, did not induce DNA strand breaks in the SCGE assay.

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.

Microbial communities and exopolysaccharides from Polynesian mats.

Microbial mats present in two shallow atolls of French Polynesia were characterized by high amounts of exopolysaccharides associated with cyanobacteria as the predominating species. Cyanobacteria were found in the first centimeters of the gelatinous mats, whereas deeper layers showing the occurrence of the sulfate reducers Desulfovibrio and Desulfobacter species as determined by the presence of specific biomarkers. Exopolysaccharides were extracted from these mats and partially characterized. All fractions contained both neutral sugars and uronic acids with a predominance of the former. The large diversity in monosaccharides can be interpreted as the result of exopolymer biosynthesis by either different or unidentified cyanobacterial species.

Discrimination of allied species within the genus Turbinaria (Fucales, Phaeophyceae) using HRMAS NMR spectroscopy.

A novel chemotaxonomical method based on 1D (1)H HRMAS NMR spectroscopy is being tested for taxonomical purposes. This powerful technique allowed us to discriminate between specimens belonging to two sister species of Turbinaria, which are difficult to tell apart using only morphological characters. Based on spectra analysis, the results allowed us to successfully group the specimens according to their species. Thus, the efficiency of HRMAS NMR spectroscopy for the discrimination of algal species and for the pre-screening of potential chemomarkers is demonstrated.

Acquisition of chemoresistance following discontinuous exposures to cisplatin is associated in ovarian carcinoma cells with progressive alteration of FAK, ERK and p38 activation in response to treatment.

OBJECTIVE: Recurrence and cisplatin resistance that progressively develops in the course of treatments are major impediments in ovarian cancer therapy. We investigated the involvement of alterations of different signaling pathways in this acquired chemoresistance. METHODS: We studied the activation of these pathways in a model of progressive acquisition of resistance that we established, by discontinuously exposing a sensitive ovarian carcinoma cell line, OAW42, to increasing concentrations of cisplatin. RESULTS: OAW42-T1 and -T2 variants, which emerged after the first two treatments of OAW42 cells with 5 microg/ml cisplatin, showed enhanced but transient resistance. OAW42-R cells, obtained following successive reiterations of the treatment, displayed both a stronger resistance to cisplatin-induced apoptosis and an increased capacity to recover a normal proliferation after treatment. The measurement of DNA adducts demonstrated that the mechanisms leading to a decreased DNA platination could not explain the level of resistance of OAW42-R cells. The simultaneous study of activation pattern of key proteins of different signaling pathways revealed that cisplatin induced both activation of ERK and p38 and inhibition of P-FAK in the sensitive cells, whereas it progressively failed to elicit such a response in the resistant variants. In contrast, STAT3 and Akt did not seem to be involved in the acquired chemoresistance in our model. CONCLUSIONS: Our results suggested that emergence of chemoresistance was accompanied with the progressive loss of ERK and p38 activation and with the maintenance of FAK activation in response to cisplatin, and they demonstrated that these alterations were early events in the course of treatments.

Estimation of the sulfate content of hydrothermal vent bacterial polysaccharides by Fourier transform infrared spectroscopy.

Deep-sea hydrothermal vents are characterized by unusual chemical and physical parameters, including high pressure and temperature. In this extreme environment, unusual microorganisms of biotechnological importance survive. Polymer-producing bacteria have been specifically studied for several years with the aim of demonstrating their ability to produce unusual polysaccharides in terms of physical, chemical, and biological properties. Because sulfates play an important role in the biological properties of polysaccharides, it is very important to determine their content with accuracy. Fourier transform infrared spectroscopy (FT-IR) was used for sulfate analysis and the results were compared with those of other analytical techniques. We found a good correlation between FT-IR and other analytical techniques for sulfate concentrations ranging from 2.4 to 20%. The data indicated that this technique could be used to determine the chemical composition of the polymers along with a semiquantitative estimation of the sulfate content.

An HPLC method for the assay of UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in Solieria chordalis (Rhodophyceae).

An efficient method to assay both UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in a crude extract of the red seaweed, Solieria chordalis is described. The method is based on the direct quantification by reverse-phase high-performance liquid chromatography of the UDP-sugars generated in the reaction mixture. UDP-glucose, UDP-galactose and UTP were detected by spectrophotometry at 254 nm and their recoveries ranged from 97 to 100%. In the course of the reaction, a correlation was observed between the reduction in the area of the substrate peak and the occurrence of product peak(s). This highly reproducible method for enzyme assay is fast since no intermediate reaction mixture is required.

Acquisition of chemoresistance in a human ovarian carcinoma cell is linked to a defect in cell cycle control.

Chemoresistance is a major concern in cancer erradication; it involves various mechanisms, including defects in the apoptosis program induced by anticancer drugs. In order to further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin treatment, we established an in vitro model, mimicking a clinical protocol of administration of cisplatin. Therefore, IGROV1 ovarian carcinoma cells were exposed for 2 hr to the drug and allowed to recover for several weeks; this way of exposure was reiterated with escalating doses. We followed changes in cytotoxicity of the drug, cell cycle kinetics and long-term survival of cells after cisplatin treatment, and found that resistance to cisplatin was not associated with altered apoptosis pathway, since both cisplatin sensitive and resistant cells underwent apoptosis in a similar way. Acquisition of resistance to cisplatin was associated with the ability of the treated cells to progress through the cell cycle beyond the G1/S checkpoint; although most cells died by apoptosis, a few surviving cells proliferated and recolonized the cultures. Compared to sensitive cells, the chemoresistant variants were able to override the G1/S checkpoint whatever the dose, and the recurrent cells recolonized the cultures much faster. Analysis of alterations in gene expression suggests that the defect in cell cycle regulation could take place at the level of the cdk inhibitor p21(CIP1/WAF1).

Early detection of staurosporine-induced apoptosis by comet and annexin V assays.

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction.

Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis.

BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during apoptosis in comparison with standard DNA flow cytometry analysis. METHODS: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. RESULTS: Nuclear staining with DAPI confirmed the induction of apoptosis with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.

Physical, chemical, and microbiological characteristics of microbial mats (kopara) in the South Pacific atolls of French Polynesia.

Microbial mats that develop in shallow brackish and hyposaline ponds in the rims of two French polynesian atolls (Rangiroa and Tetiaroa) were intensively investigated during the past three years. Comparative assessment of these mats (called kopara in polynesian language) showed remarkable similarities in their composition and structure. Due to the lack of iron, the color of the cyanobacterial pigments produced remained visible through the entire depth of the mats (20-40 cm depth), with alternate green, purple, and pink layers. Profiles of oxygen, sulfide, pH, and redox showed the anoxia of all mats from a depth of 2-3 mm. Analyses of bacterial pigments and bacterial lipids showed that all mats consisted of stratified layers of cyanobacteria (mainly Phormidium, Schizothrix, Scytonema) and purple and green phototrophic bacteria. The purple and green phototrophic bacteria cohabit with sulfate reducers (Desulfovibrio and Desulfobacter) and other heterotrophic bacteria. The microscopic bacterial determination emphasized the influence of salinity on the bacterial diversity, with higher diversity at low salinity, mainly for purple nonsulfur bacteria. Analyses of organic material and of exopolymers were also undertaken. Difference and similarities between mats from geomorphological, microbiological, and chemical points of view are discussed to provide multicriteria of classification of mats.