Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes

Advances in genetic engineering methods have allowed the development of an increasing number of practical and scientific applications for bioluminescence with lux genes cloned from a variety of organisms. Bioluminescence derived from the shortened lux operon (luxAB genes) is a complex process, and applications seem to be proliferating in advance of an understanding of the underlying biochemical processes. In this report, we describe a two-phase kinetic behavior of the light emission which must be properly taken into account in any quantitative measurements of the bioluminescence signal. By using strains of Escherichia coli and Caulobacter crescentus, this behavior was characterized and interpreted in terms of the biochemistry underlying the bacterial luciferase mechanism. We show that the intensity profile of each of the two phases of the luminescence signal is responsive (and exhibits different sensitivities) to the concentration of added decanal and other components of the assay mix, as well as to the order of mixing and incubation times. This study illustrates the importance of appropriate protocol design, and specific recommendations for using the luxAB system as a molecular reporter are presented, along with versatile assay protocols that yield meaningful and reproducible signals.     

Evaluation of Wetland Gain and Loss of Abandoned Agricultural Impoundments in South Louisiana, 1978–1988

At least 43 agriculture fields were built in south Louisiana wetlands between 1900 and 1920 using pumps and levees. All but one project is now abandoned as a result of soil subsidence, financial difficulties, or multiple levee failures. Some are now in flood-protected urban zones, and the remainder are mostly open water. The total area of abandoned agricultural fields in 1990 is large (> 80,000 ha), and their levees continue to deteriorate naturally. Ten failed or abandoned coastal agricultural impoundments (22,680 ha) were examined to determine recent wetland restoration or regression rates from 1978 to 1988. Wetland area and levee length were determined from aerial photography for 1978,1983,1985, and 1988. Average wetland change rates for all areas ranged from ?4.28 to +2.54% per year from 1978 to 1988. One site gained wetland area between 1978 and 1988 (77 ha/yr), and four sites gained wetland area between 1985 and 1988 (range 14–439 ha/yr). Wetland area in the other sites either remained stable or declined during the study period. The results from a multiple regression model indicate that restoration is inversely related to impoundment size and directly related to levee reduction. Results from a multiple regression model indicate that active levee removal will probably enhance wetland restoration rates at a very favorable cost (< $1/ha) and will be sustainable with little additional management.     

Dry Matter Assimilation and Partitioning in Pepper Cultivars Differing in Susceptibility to Stress-induced Bud and Flower Abscission

Stress-induced abscission of buds and flowers is a constraintto the production of peppers (Capsicum annuum L.). Susceptibilityof pepper cultivars to stress-induced abscission may be relatedto assimilate levels and partitioning. Growth analysis experimentswere conducted in the field to determine whether cultivars differingin susceptibility to stress-induced abscission showed correspondingdifferences in growth rates and dry matter partitioning whensubjected to low light stress. With the stress susceptible cultivar'Shamrock', reduction in net assimilation rate (NAR) and relativegrowth rate (RGR) was greater under low light stress than withthe more tolerant cultivar 'Ace'. Compared to 'Ace', 'Shamrock'partitioned a smaller proportion of dry matter into reproductiveorgans and a larger proportion into expanded leaves. Cultivarsdid not differ in the amount of dry matter partitioned to youngleaves or in the extent to which accumulation of dry matterby young leaves was reduced by low light stress. Other experimentsconducted in growth chambers and greenhouse examined the effectof removal of leaves less than 3 cm long on high temperatureand low light stress-induced bud and flower abscission. Forboth 'Ace' and 'Shamrock', abscission was not altered by removalof these leaves. Preferential partitioning of assimilate toyoung leaves does not appear to be involved in stress-inducedabscission in these pepper cultivars.Copyright 1994, 1999 AcademicPress Pepper (Capsicum annuum L.), abscission, low light stress, high temperature stress, assimilate partitioning, assimilation/growth rate, growth analysis, defoliation     

Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.     

Effects of fire on landscape heterogeneity in Yellowstone National Park,Wyoming

Abstract. A map of burn severity resulting from the 1988 fires that occurred in Yellowstone National Park (YNP) was derived from Landsat Thematic Mapper (TM) imagery and used to assess the isolation of burned areas, the heterogeneity that resulted from fires burning under moderate and severe burning conditions, and the relationship between heterogeneity and fire size. The majority of severely burned areas were within close proximity (50 to 200 m) to unburned or lightly burned areas, suggesting that few burned sites are very far from potential sources of propagules for plant reestablishment. Fires that occurred under moderate burning conditions early during the 1988 fire season resulted in a lower proportion of crown fire than fires that occurred under severe burning conditions later in the season. Increased dominance and contagion of burn severity classes and a decrease in the edge: area ratio for later fires indicated a slightly more aggregated burn pattern compared to early fires. The proportion of burned area in different burn severity classes varied as a function of daily fire size. When daily area burned was relatively low, the proportion of burned area in each burn severity class varied widely. When daily burned area exceeded 1250 ha, the burned area contained about 50 % crown fire, 30 % severe surface burn, and 20 % light surface burn. Understanding the effect of fire on landscape heterogeneity is important because the kinds, amounts, and spatial distribution of burned and unburned areas may influence the reestablishment of plant species on burned sites.     

Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa.

The process of extracellular secretion in Pseudomonas aeruginosa requires specialized machinery which is widely distributed among bacteria that actively secrete proteins to the extracellular medium. One of the components of this machinery is the product of the xcpR gene, which is homologous to pilB, a gene encoding a protein essential for the biogenesis of type IV pili. Both XcpR and PilB are characterized by the presence of a conserved ATP-binding motif (Walker sequence). The codons of highly conserved glycine residues within the Walker sequences of xcpR and pilB were altered to encode a serine, and the effects of these substitutions were examined. Bacteria expressing mutant XcpR or PilB were unable to secrete exotoxin A or assemble pili, respectively. In addition, high-level expression of mutant XcpR in wild-type P. aeruginosa led to a pleiotropic extracellular secretion defect, resulting in the periplasmic accumulation of enzymes that are normally secreted from the cell. These studies show that the putative ATP-binding sites of XcpR and PilB are essential for their functions in protein secretion and assembly of pili, respectively. Moreover, the observed dominant negative phenotype of mutant XcpR suggests that this protein functions as a multimer or, alternatively, interacts with another essential component of the extracellular protein secretion machinery.     

Protein stabilization by compatible solutes. Effect of diglycerol phosphate on the dynamics of Desulfovibrio gigas rubredoxin studied by NMR.

Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of stabilizing solutes from hyperthermophiles. Rubredoxin from Desulfovibrio gigas was selected as a model protein and the effect of diglycerol phosphate on its dynamic behaviour was studied. The presence of 100 mM diglycerol phosphate induces a fourfold increase in the half-life for thermal denaturation of D. gigas rubredoxin. A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of fast-scale motions. Hydrogen exchange data acquired over a temperature span of 20 degrees C yielded thermodynamic parameters for the structural opening reactions that allow for the exchange. This shows that the closed form of the protein is stabilized by an additional 1.6 kJ x mol(-1) in the presence of the solute. The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions.