Glycolytic enzyme Pgk1 is strongly expressed in the developing tooth germ of the mouse lower first molar

This study examined detailed in situ expression patterns and possible functional roles of phosphoglycerate kinase 1 (Pgk1) gene in the developing tooth germ of the mouse lower first molar. The strong expression of Pgk1 mRNA was seen in the odontogenic epithelial cells and surrounding mesenchymal cells of the tooth germ from embryonic day 10.5 (E10.5) to E18.0. Western blotting analysis demonstrated that Pgk1 protein formed 84-kDa protein complex in these embryonic organs. The results of immunoprecipitation-western blotting also suggested this complex to be formed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, the immunofluorescence expression of those proteins was shown to overlap each other in the tooth germ at E15.0. A strong immunofluorescence expression of both Pgk1 and GAPDH also corresponded to the in situ expression of those mRNAs. These results suggested that Pgk1 plays some functional roles in the development of tooth germ and other embryonic organs by forming protein complex with GAPDH.     

Chloroxylenol- and triclosan-tolerant bacteria from industrial sources

Potential development of bacterial tolerance to biocides in the industrial environment is examined in this study. Bacteria tolerant to the phenolic-type agent para-chloro-meta-xylenol (PCMX) and the bis-phenol 2,4,4′-trichloro-2′-hydroxydiphenylether (triclosan) were isolated from industrial sources and identified. Minimum inhibitory concentrations (MICs) were determined and compared with those of culture collection (standard) strains. Of around 100 isolates originally obtained, most were naturally tolerant species such as Pseudomonas spp., or showed low tolerance levels. PCMX-tolerant isolates of Pseudomonas stutzeri and triclosan-tolerant isolates of Citrobacter freundii and Acinetobacter johnsonii were retained for further study. Of these, only P. stutzeri and A. johnsonii showed elevated tolerance compared with the standard strains. There was no evidence of tolerance to the other biocide except for Pseudomonas aeruginosa (an intrinsically tolerant microorganism), and tolerances were stable in the absence of selective pressure except for A. johnsonii. Attempts to select or generate increased tolerance in the standard strains were unsuccessful. High tolerances in terms of MIC were not reflected in terms of lethal effects. This study did not produce any evidence suggesting that the presence of residual biocide concentrations in the industrial environment promotes the emergence of bacterial tolerance for them. Received 01 February 2002/ Accepted in revised form 09 August 2002     

Microbiological monitoring in the biodegradation of sewage sludge and food waste

AIM: To study the microbiology of intensive, in-vessel biodegradation of a mixture of sewage sludge and vegetable food waste. METHODS AND RESULTS: The biodegradation was performed in a closed reactor with the addition of a starter culture of Bacillus thermoamylovorans SW25 under conditions of controlled aeration, stirring, pH and temperature (60 degrees C). The content of viable bacterial cells, determined by flow cytometry, increased from 5 x 108 g-1 of dry matter to 61 x 108 g-1 for 6 days of the process and then dropped to the initial value at the end of the process. The reductions of organic matter, 16S rRNA of methanogens and coenzyme F420 fluorescence during 10 days of the treatment were 67, 54 and 87% of the initial values, respectively. The biodegradability of the organic matter decreased during the 10 days of the treatment from 3.8 to 1.3 mg CO2 g-1 of organic matter per day. The treatment of sewage sludge and food waste at 60 degrees C did not remove enterobacteria, which are the agents of intestinal infections, from the material. The percentage of viable enterobacterial cells, determined by fluorescent in situ hybridization (FISH) with Enterobacteriaceae-specific oligonucleotide probe and flow cytometry, varied from 1 to 14% of the viable bacterial cells. CONCLUSIONS: The mixture of sewage sludge and food waste can be degraded by the aerobic thermophilic bacteria; the starter culture of Bacillus thermoamylovorans SW25 can be used to perform this process; and enterobacteria can survive under treatment of sewage sludge and food waste at 60 degrees C for 13 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that FISH with an oligonucleotide probe can be used to study not only the growth but also the degradation of biomass. Obtained results could be used to design the bioconversion of sewage sludge and food waste into organic fertilizer.     

Use of the 'ex vivo' test to study long-term bacterial survival on human skin and their sensitivity to antisepsis

AIMS: To determine bacterial survival on human skin and their sensitivity to antisepsis. METHODS AND RESULTS: An 'ex vivo' protocol which uses human skin samples placed into diffusion cells, and electron microscopy (EM), were used to study the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa inoculated onto skin samples over a 46-h incubation period at 32 degrees C. Concurrently variation in skin pH was evaluated at different time intervals during this period. In addition the antimicrobial activity of three antiseptics against the incubated micro-organisms was assessed quantitatively with the 'ex vivo' test, while their detrimental effects against bacteria were observed by EM. All three bacteria were still present in high number after 46 h inoculation on skin, although the concentration of E. coli and S. aureus were reduced by 2.74 and 1.58 log(10) reduction, respectively, over this period of time. Electron micrographs showed clear evidence of cell division and some bacteria appeared to be embedded into the skin layers. The antiseptics tested had some antibacterial activity against bacteria incubated on skin for 3 and 10 h, and EM evidence showed some morphological damages including cellular blebbing and the presence of fibrillar material around the cells. All micro-organisms had an acidifying effect on skin samples. CONCLUSIONS: Here, it was shown that bacterial pathogens can survive and grow when incubated on human skin. In addition, it is possible that they can penetrate the stratum corneum, which can provide some protection against antisepsis. SIGNIFICANCE AND IMPACT OF THE STUDY: The apparent low bactericidal activity of biocides attributed in part to bacterial protection from skin layers is particularly important to assess in order to ensure antisepsis efficacy.     

Explaining diversity of livestock-farming management strategies of multiple-job holders: importance of level of production objectives and role of farming in the household

We characterised the livestock-farming management strategies of multiple-job holders and identified which variables contributed most to the differentiation of these strategies. We hypothesised that they would mainly be differentiated by the contribution of the farming income to the total household income and the availability of the household members for farming. The multiple-job holding livestock-farmer's motivations, decisions and actions about both multiple-job holding and livestock farming were obtained in semi-directed interviews of 35 sheep farmers who held multiple jobs, on farm and off farm. They were synthesised into six variables characterising the diversity of the livestock-farming objectives and management guidelines. Thanks to a multiple factorial analysis, we showed that the diversity of the sheep-farming management strategies of multiple-job holders was better explained by two factors 'level of motivation of the farmer to get high technical results' and 'more personal fulfilling v. the family business conception of farming', than the factors we hypothesised. Within our sample, the performances ranged from 0.7 to 1.4 weaned lambs per ewe per year. Six sheep-farming management strategies were identified. They illustrated the importance of the level of production objectives and of farming income expectation, which were found to be independent, in explaining diversity. No direct relationship between farm work organisation and sheep-farming management strategy was identified. Explaining the diversity of the livestock-farming management strategies of multiple-job holders appears to require that all the benefits expected from farming and their hierarchy be identified before analysing how they are translated into production objectives and management guidelines.     

Quantification of zidovudine and its monophosphate in cell extracts by on-line solid-phase extraction coupled to liquid chromatography

A simple and rapid analytical method for the simultaneous quantification of zidovudine (AZT) and its monophosphate (AZTMP) in cell extracts has been developed using high-performance liquid chromatography (HPLC) with on-line solid-phase extraction and 2-aminoethyl-3'-azido-2',3'-dideoxythymidin-5'-yl phosphodiester sodium salt as internal standard (IS). The cell extract samples were directly injected on a short reversed-phase precolumn using an aqueous buffer containing an ion-pairing reagent as a mobile phase. Under these conditions, the analytes were retained on the precolumn whereas the proteins were discarded. The analytes were then transferred onto the analytical column by increasing the strength of the eluent. The calibration curve was linear over a concentration range of 0.5-100 microg/ml. Inter- and intra-day accuracy and precision results satisfied the accepted criteria for bioanalytical validation. This method was used to study the decomposition pathway of a model pronucleotide in an in vitro approach.     

Enameloid/enamel transition through successive tooth replacements in <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> (Lissamphibia,Caudata)

Study of the evolutionary enameloid/enamel transition suffers from discontinuous data in the fossil record, although a developmental enameloid/enamel transition exists in living caudates, salamanders and newts. The timing and manner in which the enameloid/enamel transition is achieved during caudate ontogeny is of great interest, because the caudate situation could reflect events that have occurred during evolution. Using light and transmission electron microscopy, we have monitored the formation of the upper tooth region in six successive teeth of a tooth family (position I) in Pleurodeles waltl from late embryos to young adult. Enameloid has only been identified in embryonic tooth I₁ and in larval teeth I₂ and I₃. A thin layer of enamel is deposited later by ameloblasts on the enameloid surface of these teeth. From post-metamorphic juvenile onwards, teeth are covered with enamel only. The collagen-rich enameloid matrix is deposited by odontoblasts, which subsequently form dentin. Enameloid, like enamel, mineralizes and then matures but ameloblast participation in enameloid matrix deposition has not been established. From tooth I₁ to tooth I₃, the enameloid matrix becomes ever more dense and increasingly comes to resemble the dentin matrix, although it is still subjected to maturation. Our data suggest the absence of an enameloid/enamel transition and, instead, the occurrence of an enameloid/dentin transition, which seems to result from a progressive slowing down of odontoblast activity. As a consequence, the ameloblasts in post-metamorphic teeth appear to synthesize the enamel matrix earlier than in larval teeth.