Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction

A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 μg soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time. Received: 22 October 1996 / Received revision: 24 January 1997 / Accepted: 10 February 1997     

Distal Val346Ile mutation in inducible NO synthase promotes substrate-dependent NO confinement

The function of inducible NO synthase (WT iNOS) depends on the release of NO from the ferric heme before the enzyme is reduced. Key parameters controlling ligand dynamics include the distal and proximal heme pocket amino acids, as well as the inner solvent molecules. In this work, we tested how a point mutation in the distal heme side of WT iNOS affected the geminate rebinding of NO by ultrafast kinetics and molecular dynamics simulations. The mutation sequestered much of the photodissociated NO close to the heme compared to WT iNOS, with a main picosecond phase accounting for 78% of the rebinding to the arginine-bound Val346Ile protein. Consequently, the probability of NO release from Val346Ile decreased as compared to that from WT iNOS, provided the substrate binding site is filled. These data are rationalized by a steric effect of the Ile methyl group inducing events mediated by the substrate, transmitted via the propionates to the NO and the protein. This model is consistent with the role of the H-bonding network involving the heme, the substrate, and the BH4 cofactor in controlling NO release, with a key role of the heme propionates [Gautier et al. (2006) Nitric Oxide 15, 312]. These data support the effect of Val346Ile mutation in decreasing NO release and slowing down NO synthesis compared to WT iNOS determined by single turnover catalysis [Wang et al. (2004) J. Biol. Chem. 279, 19018].     

Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells

Cell-based therapies are used to treat bone defects. We recently described that human multipotent adipose-derived stem (hMADS) cells, which exhibit a normal karyotype, self renewal, and the maintenance of their differentiation properties, are able to differentiate into different lineages. Herein, we show that hMADS cells can differentiate into osteocyte-like cells. In the presence of a low amount of serum and EGF, hMADS cells express specific molecular markers, among which alkaline phosphatase, CBFA-1, osteocalcin, DMP1, PHEX, and podoplanin and develop functional gap-junctions. When loaded on a hardening injectable bone substitute (HIBS) biomaterial and injected subcutaneously into nude mice, hMADS cells develop mineralized woven bone 4 weeks after implantation. Thus hMADS cells represent a valuable tool for pharmacological and biological studies of osteoblast differentiation in vitro and bone development in vivo.     

Effect of ions on the organization of phosphatidylcholine/phosphatidic acid bilayers

Lipid bilayers are two-dimensional fluids. Here, the effect of monovalent ion concentration on the mixing, and consequently the organization, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) bilayers has been examined. Epifluorescence microscopy was used to visualize the organization. Fluorescence recovery after photobleaching and attenuated total reflection-Fourier transform infrared spectroscopy were used to assess the fluidity of the lipids. At high ionic strength the DOPC and DOPA lipids appear uniformly mixed. Upon lowering the ionic strength, rapid separation is observed. The DOPA-rich regions appear fractal-like and exhibit hysteresis in their properties. The lipids freely exchange between the two regions. These experiments clearly demonstrate the significant effect that electrostatics can have on membrane organization.     

Response to De Silva et al. (2007): Endemic freshwater finfish of Asia: distribution and conservation status

De Silva et al . (2007) present an overview of the distribution and conservation status of the endemic freshwater fish of Asia. Within that review they use data from the IUCN Red List of Threatened Species™ (2006) to conduct an analysis of the conservation status of those endemic fish species. Their analysis is incorrect and provides a very misleading impression of the level of threat to Asian freshwater fish and to freshwater fish at the global scale. The errors stem from a misinterpretation of the data presented on the IUCN Red List. The sources of errors are discussed below and the opportunity is taken to clarify what the information on the IUCN Red List represents.     

Fingerprinting the genetic diversity of the biotin carboxylase gene (accC) in aquatic ecosystems as a potential marker for studies of carbon dioxide assimilation in the dark

We designed and tested a set of specific primers for specific PCR amplification of the biotin carboxylase subunit gene (accC) of the Acetyl CoA carboxylase (ACCase) enzyme. The primer set yielded a PCR product of c. 460 bp that was suitable for denaturing gradient gel electrophoresis (DGGE) fingerprinting followed by direct sequencing of excised DGGE bands and sequence analysis. Optimization of PCR conditions for selective amplification was carried out with pure cultures of different bacteria and archaea, and laboratory enrichments. Next, fingerprinting comparisons were done in several aerobic and anaerobic freshwater planktonic samples. The DGGE fingerprints showed between 2 and 19 bands in the different samples, and the primer set provided specific amplification in both pure cultures and natural samples. Most of the samples had sequences grouped with bacterial accC, hypothetically related to the anaplerotic fixation of inorganic carbon. Some other samples, however, yielded accC gene sequences that clustered with Crenarchaeota and were related to the 3-hydroxypropionate/4-hydroxybutyrate cycle of autotrophic crenarchaeota. Such samples came from oligotrophic high mountain lakes and the hypolimnia of a sulfide-rich lake, where crenarchaeotal populations had been previously reported by 16S rRNA surveys. This study provided a fast tool to look for presence of accC genes in natural environments as potential marker for studies of carbon dioxide assimilation in the dark. After further refinement for better specificity against archaea, the new and novel primers could be very helpful to establish a target for crenarchaeota with implications for our understanding of archaeal carbon biogeochemistry.     

Survival of Bifidobacterium animalis DN-173 010 in the faecal microbiota after administration in lyophilised form or in fermented product - a randomised study in healthy adults

The survival of Bifidobacterium animalis strain DN-173 010 was assessed after its ingestion in a fermented product or in a lyophilised form. Twelve healthy subjects were included in a randomised, open study with 2 parallel groups. The composition and activities of the faecal microbiota were monitored before (10-day baseline step), during (1-week product administration step) and after (10-day follow-up step) the ingestion of 1 of the 2 products. A colony immunoblotting method, fluorescent in situ hybridisation with group-specific DNA probes, and temporal temperature gradient gel electrophoresis using group-specific primers were carried out to compare survival of B. animalis strain DN-173 010 after ingestion of the 2 products, together with analyses of enzyme activities and faecal metabolites. At the end of the supplementation step, the mean number of B. animalis DN-173 010 quantified by immunodetection in the faeces of 5 of 6 subjects in each treatment group was >/=10(8) colony-forming units/g faeces. These numbers corresponded to an average survival of 22% for the lyophilised form and 20% for the fermented product. At the same step, the PCR temporal temperature gradient gel electrophoresis profiles showed a double band corresponding to the B. animalis DN-173 010 pattern for 11 subjects. No major modification was observed during the trial in either the dominant members of the faecal microbiota assessed by fluorescent in situ hybridisation or their activities. In conclusion, we show that the lyophilised form of B. animalis DN-173 010 survives transit and could represent a more convenient form to administer for long-term clinical trials.     

Ability of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> to overcome host α-galactosidase deficiency,as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides

Background  

Soya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharides in situ.     

Dissecting the genetic components of adaptation of Escherichia coli to the mouse gut

While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments.