Modified method for selective enrichment and isolation of Bacillus thuringiensis from soil

Many colonies of Bacillus thuringiensis (Bt) were found to be lost using the isolation technique described by Martin and Travers modified by Morris (Morris method). Hence, a modified method for isolation of B. thuringiensis is described and compared with the Morris method. Screening methodology adopted by this method delivers an immensely rich and potentially more useful library of Bt colonies with ‘presumptive-positive’ morphology and 10-fold higher per cent frequency of Bt colonies as compared to the Morris method.     

Multidimensional Dynamics of the Proteome in the Neurodegenerative and Aging Mammalian Brain

The amount of any given protein in the brain is determined by the rates of its synthesis and destruction, which are regulated by different cellular mechanisms. Here, we combine metabolic labeling in live mice with global proteomic profiling to simultaneously quantify both the flux and amount of proteins in mouse models of neurodegeneration. In multiple models, protein turnover increases were associated with increasing pathology. This method distinguishes changes in protein expression mediated by synthesis from those mediated by degradation. In the App^NL-F knockin mouse model of Alzheimer’s disease, increased turnover resulted from imbalances in both synthesis and degradation, converging on proteins associated with synaptic vesicle recycling (Dnm1, Cltc, Rims1) and mitochondria (Fis1, Ndufv1). In contrast to disease models, aging in wild-type mice caused a widespread decrease in protein recycling associated with a decrease in autophagic flux. Overall, this simple multidimensional approach enables a comprehensive mapping of proteome dynamics and identifies affected proteins in mouse models of disease and other live animal test settings.     

Artifact-inducing enrichment of ethylenediaminetetraacetic acid and ethyleneglycoltetraacetic acid on anion exchange resins

Multivalent metal chelators, ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA), are used extensively during protein purification. Both strong (Q) and weak (DEAE) anion exchange resins were found to adsorb surprisingly large quantities of EDTA and EGTA that elute from the resin at NaCl concentrations of approximately 240 mM (EDTA) and 140 mM (EGTA). The EDTA/EGTA elution and saturation parameters were determined for five commonly used anion exchange resins. The resulting concentration of eluted EDTA was 10- to 200-fold higher than that originally present in the sample or in the mobile phase. Samples from fractions containing such a high concentration of EDTA were found to inhibit Mg²⁺-dependent polymerase chain reaction (PCR). EDTA binding to the anion exchange resins could saturate the resin, decrease its binding capacity, and displace weakly bound proteins such as green fluorescent protein (GFP). Several steps are suggested to minimize on-column EDTA concentration, including column equilibration in the absence of any EDTA, lower concentrations (0.1–0.5 mM) of EDTA, monitoring eluate absorbance at 280 nm as well as at 215 nm, adding EDTA back into fractions eluting before the EDTA peak, and performing blank column runs to control for the effect of changes in EDTA concentration in downstream assays.     

An integrated exploratory approach to examining the relationships of environmental stressors and fish responses

The Chesapeake Bay is one of the mostproductive systems in the world. It is theNation's largest estuary (64,000 square miles)and is home to about 13 million people. Itsupports a variety of aquatic resources offlora and fauna. However, for the past 350years and especially in the last two to threedecades, there has been substantialdeterioration of the natural resources. Manyspecies of submerged aquatic vegetation andbenthic invertebrates have been diminished orbecome extinct. Commercial harvests of fish,crab and shell fish have also declined.In 1983, a Chesapeake Bay Agreement was signedby Pennsylvania, Maryland, the District ofColumbia, Virginia and the Bay Commission. Itwas subsequently amended in 1987 and 1992. TheAgreement identified the improvement andmaintenance of water quality as the mostcritical elements in the overall restorationand protection of the Chesapeake Bay. In orderto restore the Bay area and to conserve thefish resources, the causal relationshipsbetween the environmental stressors and thecomposition and health of the fish communitiesmust be understood.Multivariate ordination techniques are usefulexploratory tools to help elucidate latentenvironmental relationships, define specificbiocriteria and to generate hypotheses. Geographical information systems (GIS) is ananalytical technique for identifying spatialrelationships. In this project, an integratedmethodology involving the use of multivariateordination, statistical, and GIS techniques wasadopted. A non-metric multi-dimensionalscaling (NMDS) ordination technique wasemployed in conjunction with other statisticaltechniques (such as correlation analysis) andArcView GIS to analyze a huge data set from theMaryland Biological Stream Survey (MBSS). Theobjectives were to elucidate the intricaterelationships between a suite of environmentalfactors and fish conditions in the riverinesystem in the Chesapeake Bay and to evaluatethe effectiveness of this approach inexploratory analyses.The results showed that landuse issignificantly related to nutrient loading. Toa large extent, landuse and nitrates are alsoaffecting the composition and health of thefish communities in some subwatersheds in theChesapeake Bay. It was also found that theapproach adopted in this study is flexible,requiring few model assumptions. But it iscomprehensive and reliable, capable ofrevealing the impacts of environmentalstressors on the ecology, structure,composition and health of the fishcommunities.     

Automation of a fluorometric method for the determination of 2-deoxy-D-glucose

A continuous-flow fluorometric procedure for the determination of 2-deoxy-D-glucose (2DG) is described. The method utilizes Technicon Autoanalyzer equipment and modules, and is based on the acid-catalyzed condensation of 3,5-diaminobenzoic acid with 2DG. The procedure permits analysis of 20 samples/h, is sensitive to concentrations of 2DG as low as 0.2 mg/100 ml, and requires sample volumes of only 0.25 ml. 2DG can be quantitatively measured in serum samples or tissue extracts without requiring deproteinization. Glucose does not interfere with the assay while 2-deoxy-D-ribose develops a fluorescence which is about 15% of that produced by the same amount of 2DG and is additive when both deoxy sugars are present together. The procedure is accurate, reproducible, and fast, and can be run continuously.     

Effect of crude oil on denitrification and sulfate reduction in marine sediments

The denitrifying activity was measured in different types of sediment from the Mediterranean coast of France before, and after, a massive contamination (30–100 g kg^-1 sediment) of hydrocarbons. A closed system was used in order to maintain anoxic conditions and to control substrates and gaseous products concentrations. We have demonstrated that the respiratory metabolism was inhibited in all cases following an incubation time of 20 to 50 days. At this time, the addition of lactate restore the denitrifying activity. The inhibitory effect of crude oil was not related to an alteration of bacterial cells, but to changes in environmental conditions allowing denitrification. The presence of hydrocarbons in the sediments causes a decrease in the redox potential and a concomitant stimulation of the sulfate reduction.     

A Bayesian Decision Procedure for Selecting Prognostic Variables Associated with Survival for Data in which Censoring is Prevalent

A Bayesian procedure is developed for the selection of concomitant variables in survival models. The variables are selected in a step-up procedure according to the criterion of maximum expected likelihood, where the expectation is over the prior parameter space. Prior knowledge of the influence of these covariates on patient prognosis is incorporated into the analysis. The step-up procedure is stopped when the Bayes factor in favor of omitting the variable selected in a particular step exceeds a specified value. The resulting model with the selected variables is fitted using Bayes estimates of the coefficients. This technique is applied to Hodgkin's disease data from a large Cooperative Clinical Trial Group and the results are compared to the results from the classical likelihood selection procedure.     

Facility for studying the effects of elevated carbon dioxide concentration and increased temperature on crops

The requirements for the experimental study of the effects of global climate change conditions on plants are outlined. A semi-controlled plant growth facility is described which allows the study of elevated CO₂ and temperature, and their interaction on the growth of plants under radiation and temperature conditions similar to the field. During an experiment on winter wheat (cv. Mercia), which ran from December 1990 through to August 1991, the facility maintained mean daytime CO₂ concentrations of 363 and 692 cm³ m^?3 for targets of 350 and 700 cm³ m^?3 respectively. Temperatures were set to follow outside ambient or outside ambient +4°C, and hourly means were within 0.5°C of the target for 92% of the time for target temperatures greater than 6°C. Total photosynthetically active radiation incident on the crop (solar radiation supplemented by artifieal light with natural photoperiod) was 2% greater than the total measured outside over the same period.     

Solid substrate fermentation of leached beet pulp withTrichoderma aureoviride

Optimum production of cellulases on leached beet pulp by native and mutant strains ofTrichoderma aureoviride in column-type, solid-substrate fermenters, at controlled temperature and aeration rate, was with a mutant strain, that produced 11 and 29 filter paper units of activity/g solids on raw and acid pre-treated leached beet pulp, respectively. The former value was 40% higher than in the native strain and 70% higher than in the reference strain. protein enrichment of residual solid was significant in all cases, with values up to 40% of total dry weight. Results are suitable for scale-up to pilot level.