Hyaluronic acid production in Bacillus subtilis

The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.     

Mechanical properties of laser cut poly(L-lactide) micro-specimens: implications for stent design, manufacture, and sterilization

BACKGROUND: The development of endoluminal stents from polymeric materials requires an understanding of the basic mechanical properties of the polymer and the effects of manufacturing and sterilization on those properties. METHODS: Pure poly(L-lactide) (PLLA) and PLLA containing varying amounts of triethylcitrate (TEC) as a plasticizer (5-10-15%) were studied. The specimens were solution-cast and CO2 laser-cut. Specimen dimensions were adapted to the strut size of polymeric vascular stents. The properties of the PLLA micro-specimens were assessed before and after sterilization (EtO cold gas, H2O2-plasma, beta- and gamma-irradiation). Tensile tests, and creep and recovery tests were carried out at 37 degrees C. Additionally the thermal and thermo-mechanical characteristics were investigated using dynamic-mechanical analysis (DMA) and differential scanning calorimetry (DSC). RESULTS: The results showed the dramatic influence of the plasticizer content and sterilization procedure on the mechanical properties of the material. Laser cutting had a lesser effect. Hence the effects of processing and sterilization must not be overlooked in the material selection and design phases of the development process leading to clinical use. Altogether, the results of these studies provide a clearer understanding of the complex interaction between the laser machining process and terminal sterilization on the primary mechanical properties of PLLA and PLLA plasticized with TEC.     

Characterization of starvation-induced dispersion in Pseudomonas putida biofilms

The biofilm lifestyle, where microbial cells are aggregated because of expression of cell-to-cell interconnecting compounds, is believed to be of paramount importance to microbes in the environment. Because microbes must be able to alternate between sessile and planktonic states, it is anticipated that they must be able to regulate their ability to form biofilm and to dissolve biofilm. We present an investigation of a biofilm dissolution process occurring in flow-chamber-grown Pseudomonas putida biofilms. Local starvation-induced biofilm dissolution appears to be an integrated part of P. putida biofilm development that causes characteristic structural rearrangements. Rapid global dissolution of entire P. putida biofilms was shown to occur in response to carbon starvation. Genetic analysis suggested that the adjacent P. putida genes PP0164 and PP0165 play a role in P. putida biofilm formation and dissolution. PP0164 encodes a putative periplasmic protein of previously unknown function, and PP0164 mutant bacteria are sticky, and unable to reduce their adhesiveness and dissolve their biofilm in response to carbon starvation. PP0165 encodes a putative transmembrane protein containing GGDEF and EAL domains, and PP0165 mutant bacteria are unable to increase their adhesiveness and form biofilm. We suggest that the PP0164 and PP0165 proteins are involved in the regulation of the adhesiveness of the bacteria; the PP0165 protein through c-di-GMP signalling, and the PP0164 protein as a transducer of the signal.     

Testosterone-Fatty Acid esterification: a unique target for the endocrine toxicity of tributyltin to gastropods

Over the past thirty years, a global occurrence of sexual aberrationhas occurred whereby females among populations of prosobranchsnails exhibit male sex characteristics. This condition, calledimposex, has been causally associated with exposure to the biocidetributyltin. Tributyltin-exposed, imposex snails typically haveelevated levels of testosterone which have led to the postulatethat this endocrine dysfunction is responsible for imposex.This overview describes recent evidence that supports this postulate.Gastropods maintain circulating testosterone levels and administrationof testosterone to females or castrates stimulates male sexdifferentiation in several snail species. Studies in the mudsnail (Ilyanassa obsoleta) have shown that gastropods utilizea unique strategy for regulating free testosterone levels. Excesstestosterone is converted to fatty acid esters by the actionof a testosterone-inducible, high capacity/low affinity enzyme,acyl-CoA:testosterone acyl transferase, and stored within theorganisms. Free testosterone levels are regulated during thereproductive cycle apparently due to changes in esterification/desterificationsuggesting that testosterone functions in the reproductive cycleof the organisms. Testosterone esterification provides a uniquetarget in the testosterone regulatory machinery of snails thatis altered by tributyltin. Indeed, imposex and free testosteronelevels were elevated in field collected snails containing hightin levels, while testosterone-fatty acid ester pools were reducedin these organisms. These observations indicate that tributyltinelevates free testosterone by reducing the retention of testosteroneas fatty acid-esters. This endocrine effect of tributyltin maybe responsible for imposex.     

Feed-borne Outbreak of Salmonella Cubana in Swedish Pig Farms: Risk Factors and Factors Affecting the Restriction Period in Infected Farms

   

In 2003, a feed-borne outbreak of Salmonella Cubana occurred in Sweden as a result of contamination in a feed plant. Salmonella Cubana was detected in 49 out of 77 pig farms having received possibly contaminated feed. In this study, potential risk factors for farms being salmonella positive were examined, and a survival analysis was performed to investigate risk factors affecting the restriction period for salmonella positive farms.     

GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space analysis

The target of the in-situ research of optical activity in extraterrestrial samples stimulated an extended investigation of a GC-MS method based on the derivatization of amino acids by using a mixture of perfluorinated alcohols and perfluorinated anhydrides. Amino acids are converted to their N(O,S)-perfluoroacyl perfluoroalkyl esters in a single-step procedure, using different combinations of the derivatization reagents trifluoroacetic anhydride (TFAA)-2,2,2-trifluoro-1-ethanol (TFE), TFAA-2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB), and heptafluorobutyric anhydride (HFBA)-HFB. The derivatives obtained are analyzed using two different chiral columns: Chirasil-L-Val and gamma-cyclodextrin (Rt-gamma-DEXsa) stationary phases which show different and complementary enantiomeric selectivity. The mass spectra of the derivatives are studied, and mass fragmentation patterns are proposed: significant fragment ions can be identified to detect amino acid derivatives. The obtained results are compared in terms of the enantiomeric separation achieved and mass spectrometric response. Linearity studies and the measurement of the limit of detection (LOD) show that the proposed method is suitable for a quantitative determination of enantiomers of several amino acids. The use of the programmed temperature vaporiser (PTV) technique for the injection of the untreated reaction mixture is a promising method for avoiding manual treatment of the sample and decreasing the LOD.     

sli-3 negatively regulates the LET-23/epidermal growth factor receptor-mediated vulval induction pathway in Caenorhabditis elegans

The LIN-3-LET-23-mediated inductive signaling pathway plays a major role during vulval development in C. elegans. Studies on the components of this pathway have revealed positive as well as negative regulators that function to modulate the strength and specificity of the signal transduction cascade. We have carried out genetic screens to identify new regulators of this pathway by screening for suppressors of lin-3 vulvaless phenotype. The screens recovered three loci including alleles of gap-1 and a new gene represented by sli-3. Our genetic epistasis experiments suggest that sli-3 functions either downstream or in parallel to nuclear factors lin-1 and sur-2. sli-3 synergistically interacts with the previously identified negative regulators of the let-23 signaling pathway and causes excessive cell proliferation. However, in the absence of any other mutation sli-3 mutant animals display wild-type vulval induction and morphology. We propose that sli-3 functions as a negative regulator of vulval induction and defines a branch of the inductive signaling pathway. We provide evidence that sli-3 interacts with the EGF signaling pathway components during vulval induction but not during viability and ovulation processes. Thus, sli-3 helps define specificity of the EGF signaling to induce the vulva.     

Incorporating Genomics into the Toolkit of Nematology

The study of nematode genomes over the last three decades has relied heavily on the model organism Caenorhabditis elegans, which remains the best-assembled and annotated metazoan genome. This is now changing as a rapidly expanding number of nematodes of medical and economic importance have been sequenced in recent years. The advent of sequencing technologies to achieve the equivalent of the $1000 human genome promises that every nematode genome of interest will eventually be sequenced at a reasonable cost. As the sequencing of species spanning the nematode phylum becomes a routine part of characterizing nematodes, the comparative approach and the increasing use of ecological context will help us to further understand the evolution and functional specializations of any given species by comparing its genome to that of other closely and more distantly related nematodes. We review the current state of nematode genomics and discuss some of the highlights that these genomes have revealed and the trend and benefits of ecological genomics, emphasizing the potential for new genomes and the exciting opportunities this provides for nematological studies.