Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems

A 3.0-kb genomic fragment has been isolated from Rhodospirillum rubrum (ATCC 25903) that contains an open reading frame (ORF) with strong homology to other known polyhydroxyalkanoate (PHA) synthase genes. This ORF has lower homology to the R. rubrum strain Ha PHA synthase than would be expected within the same species. We have conducted a series of heterologous expression studies evaluating the in vivo substrate specificity of PHA synthase genes from Rhodobacter sphaeroides, Ralstonia eutropha (formerly Alcaligenes eutrophus), Thiocystis violacea, and Nocardia corrallina, within the PHA-synthase-negative hosts, Ralstonia eutropha DSM541 and Pseudomonas putida GpP104. The N. corrallina PHA synthase incorporated the highest percentage of C5 monomers in the polymer when fermented in medium supplemented with 0.1% heptanoate as the sole carbon source. When the T. violacea and R. sphaeroides were expressed in the PHA-negative host DSM541, a greater percentage of C5 monomer was observed in the polymer as compared to the expression of the PHA synthase of R. eutropha, when the transconjugants were fermented in medium supplemented with 0.4% propionate. Evaluation for preference of medium-chain-length monomers demonstrated the flexibility of the N. corrallina, T. violacea, and R. eutropha synthase genes to polymerize a copolyester composed of short- and medium-chain-length monomers when the respective transconjugants were fermented in medium supplemented with 0.5% octanoate. These studies demonstrate that the PHA synthase from N. corrallina, T. violacea, and R. eutropha are able to polymerize a copolyester composed of short- and medium-chain-length monomers, while the PHA synthase from R. sphaeroides lacks this ability and only produces a short-chain-length polymer. These observations suggest that the composition of the PHA from the PHA-producing organisms does not necessarily reflect the inherent specificity of the PHA synthase. Received: 16 March 1999 / Received revision: 24 August 1999 / Accepted: 24 September 1999     

A logistic regression of risk factors for disease occurrence on Asian shrimp farms

Serious shrimp-disease outbreaks have reduced shrimp production and slowed industry growth since 1991. This paper tests factors such as farm sitting and design, and farm-management practices for relationships with disease occurrence. Logistic regression is used to analyze farm-level data from 3951 shrimp farms in 13 Asian countries. Disease occurrence is modeled as a 0-1 variable where 1 = disease loss of > or = 20% to any 1 crop, and 0 = losses of < 20%. Logistic regression is performed for each of 3 levels of shrimp culture intensity, i.e. extensive, semi-intensive, and intensive. Attempts to apply logistic regression models to each country were not successful due to insufficient data for most countries. Factors affecting disease occurrences were quite different for different farming intensities. Farms that had larger pond production areas, with larger number of farms discharging effluent into their water supply canals, and removed silt had greater disease occurrence. On the other hand, farms that practiced polyculture and took water from the sea through a canal had lower disease occurrence.     

Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins

Endogenously produced asymmetrically methylated arginine residues are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) specifically hydrolyzes these asymmetrically methylated arginine residues to citrulline and methylamines. Previously we have proposed that regulation of asymmetric methylarginine concentration by DDAH may provide a novel mechanism for the regulation of NOS activity in vivo. Recently we reported the cloning of human DDAH and identified a novel human DDAH isoform (DDAH I and DDAH II, respectively). Here we report that the DDAH1 gene maps to chromosome 1p22 and confirm that DDAH2 maps to the MHC III region of chromosome 6p21.3. Extensive analysis of the distribution of DDAH1 and DDAH2 mRNA in 50 human tissues indicates differential expression of DDAH isoforms in brain regions, in immune cells, and during development. DDAH2 expression predominates in highly vascularized tissues that express the endothelial NOS isoform and in immune tissues that can express iNOS. Whereas DDAH2 is expressed at relatively high levels in all fetal tissues examined, DDAH1 expression varies little between fetal and adult tissues. The chromosomal localization of the DDAHs is consistent with gene duplication, and consistent with this, comparison of the gene structures indicates that the intron/exon organization is highly conserved. Phylogenetic analysis of DDAH sequences from diverse species suggests that DDAH gene duplication occurred prior to the emergence of bony fish some 400 million years ago. Overall the data suggest that DDAH2 may be the more ancient of the two genes.     

Characterization of Na/K-ATPase in Macrobrachium rosenbergii and the effects of changing salinity on enzymatic activity

A ouabain-sensitive Na/K-ATPase kinetic assay system based on the hydrolysis of ATP and the oxidation of NADH was adapted in order to characterize enzymatic activity in gills and examine the effects of changing salinity in Macrobrachium rosenbergii. Maximum inhibition by ouabain occurred at a concentration of 1.4 mM, and the K(m) of the reaction was 0.2 mM. In a first experiment, animals were acclimated to freshwater, 1/3 seawater, 2/3 seawater and full seawater for up to 1 week. Na/K-ATPase activity in front gills was 1. 62+/-0.19 micromol ADP/mg protein per h in freshwater, and was seen to increase slightly in 1/3 seawater (1.88+/-0.19 micromol ADP/mg protein per h) and 2/3 seawater (2.09+/-0.24 micromol ADP/mg protein per h), decreasing slightly in full seawater (1.92+/-0.43 micromol ADP/mg protein per h); however, differences were not significant. Back gills showed slightly higher levels, and a similar pattern of Na/K-ATPase activity. In a second experiment, animals were acclimated to 1/3 seawater and 2/3 seawater, and then transferred to freshwater. However, no changes in activity were seen, indicating that exposure to dilute media did not effect enzymatic activity. Whereas Na/K-ATPase is important in osmoregulatory function in marine euryhaline crustaceans, it may not play a significant role in adaptation in freshwater crustaceans that inhabit a more narrow range of salinities.     

Enthalpy measurement using calorimetry shows a significant difference in potential energy between the active and latent conformations of PAI-1

A central feature of the serpin inhibition mechanism is insertion of the reactive center loop into the central beta-sheet (beta-sheet A). This insertion also occurs when the reactive center loop is cleaved without protease inhibition. Using this effect, we have measured the enthalpy (DeltaH) of loop cleavage and insertion for plasminogen activator inhibitor 1 (PAI-1) as -38 kcal/mol. Because loop insertion can be blocked by incorporating a peptide into the central beta-sheet, it was possible to assign -7 kcal/mol to loop cleavage and -31 kcal/mol to loop insertion. These values are lower than values reported for the serpins alpha 1 -proteinase inhibitor and antithrombin of -53 to -63 kcal/mol, respectively, for loop insertion with negligible enthalpy for loop cleavage. A free energy difference of -9 kcal/mol has been reported between the active and spontaneously loop inserted "latent forms" of PAI-1, which is significantly smaller in magnitude than the -31 kcal/mol of enthalpy we measured for loop insertion. Because the enthalpy should relate closely to those regions of PAI-1 that have moved to lower potential energy, a difference distance matrix is presented that identifies regions of PAI-1 that move during loop insertion.     

Synthetic multifunctional pores that open and close in response to chemical stimulation

Studies on synthetic multifunctional pores with external and internal active sites for ligand gating and noncompetitive blockage are presented, with emphasis on the contribution of external ligands to the characteristics of pore. A comparison between different synthetic multifunctional pores reveals that the location of functional groups in rigid-rod beta-barrel pores is precisely reflected in the function: molecular recognition at the outer barrel surface results in pore opening, while molecular recognition at the inner barrel surface results in pore closing. Negligible nonspecific leakage, disappearance of pH gating, inhibition of intervesicular pore transfer, and maybe also the flickering of currents of single open pores characterize external ligands as adhesive cushions that liberate the pore from lateral pressure exerted by the surrounding membrane. Refined molecular models show good agreement with pore design and experimental facts with regard to function.     

Cell migration and invasion assays

The processes of cell migration and invasion are integral to embryonic development and the functioning of adult organisms. Deregulation of these processes contributes to numerous diseases. Ras GTPases and in particular members of the Rho subfamily of GTPases play critical roles in cell migration and invasion. Here, we provide a collection of protocols to assay these functions. We describe two cell migration assays. The monolayer wound healing assay is very easy to implement, whereas the microliter-scale migration assay allows examination of cell behavior on defined extracellular matrices. We also describe two methods that allow the quantification of tumor cell invasion, a versatile transwell Matrigel invasion assay and an organotypic assay that examines the invasion of glioma cells through a rat brain slice.     

TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium

Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G protein-coupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.