Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Do Rates and Splitting of Phosphogypsum Applications Influence the Soil and Annual Crops in a No-Tillage System?

ABSTRACT Applications of phosphogypsum (PG) provide nutrients to the soil and reduce Al3+ activity, favoring soil fertility and root growth, but allow Mg2+ mobilization through the soil profile, resulting in variations in the PG rate required to achieve the optimum crop yield. This study evaluated the effect of application rates and splitting of PG on soil fertility of a Typic Hapludox, as well as the influence on annual crops under no-tillage. Using a (4 × 3) + 1 factorial structure, the treatments consisted of four PG rates (3, 6, 9, and 12 Mg ha-1) and three split applications (P1 = 100 % in 2009; P2 = 50+50 % in 2009 and 2010; P3 = 33+33+33 % in 2009, 2010 and 2011), plus a control without PG. The soil was sampled six months after the last PG application, in stratified layers to a depth of 0.8 m. Corn, wheat and soybean were sown between November 2011 and December 2012, and leaf samples were collected for analysis when at least 50 % of the plants showed reproductive structures. The application of PG increased Ca2+ concentrations in all sampled soil layers and the soil pH between 0.2 and 0.8 m, and reduced the concentrations of Al3+ in all layers and of Mg2+ to a depth of 0.6 m, without any effect of splitting the applications. The soil Ca/Mg ratio increased linearly to a depth of 0.6 m with the rates and were found to be higher in the 0.0-0.1 m layer of the P2 and P3 treatments than without splitting (P1). Sulfur concentrations increased linearly by application rates to a depth of 0.8 m, decreasing in the order P3>P2>P1 to a depth of 0.4 m and were higher in the treatments P3 and P2 than P1 between 0.4-0.6 m, whereas no differences were observed in the 0.6-0.8 m layer. No effect was recorded for K, P and potential acidity (H+Al). The leaf Ca and S concentration increased, while Mg decreased for all crops treated with PG, and there was no effect of splitting the application. The yield response of corn to PG rates was quadratic, with the maximum technical efficiency achieved at 6.38 Mg ha-1 of PG, while wheat yield increased linearly in a growing season with a drought period. Soybean yield was not affected by the PG rate, and splitting had no effect on the yield of any of the crops. Phosphogypsum improved soil fertility in the profile, however, Mg2+ migrated downwards, regardless of application splitting. Splitting the PG application induced a higher Ca/Mg ratio in the 0.0-0.1 m layer and less S leaching, but did not affect the crop yield. The application rates had no effect on soybean yield, but were beneficial for corn and, especially, for wheat, which was affected by a drought period during growth.

Behavior of Eucalyptus urophylla and Eucalyptus citriodora Seedlings Grown in Soil Contaminated by Arsenate

ABSTRACT Persistent areas of tailings and deposits from coal and gold mining may present high levels of arsenic (As), mainly in the arsenate form, endangering the environment and human health. The establishment of vegetation cover is a key step to reclaiming these environments. Thus, this study aimed to evaluate the potential of Eucalyptus urophylla and E. citriodora seedlings for use in phytoremediation programs of arsenate-contaminated areas. Soil samples were incubated at increasing rates (0, 50, 100, 200 and 400 mg dm-3) of arsenic (arsenate form, using Na2HAsO4) for 15 days. The seedlings were produced in a substrate (vermiculite + sawdust) and were transplanted to the pots with soil three months after seed germination. The values of plant height and diameter were taken during transplanting and 30, 60 and 90 days after transplanting. In the last evaluation, the total leaf area and biomass of shoots and roots were also determined. The values of available As in soil which caused a 50 % dry matter reduction (TS50%), the As translocation index (TI) from the roots to the shoot of the plants, and its bioconcentration factor (BF) were also calculated. Higher levels of arsenate in the soil significantly reduced the dry matter production of roots and shoots and the height of both species, most notably in E. urophylla plants. The highest levels of As were found in the root, with higher values for E. citriodora (ranging from 253.86 to 400 mg dm-3). The TI and BF were also reduced with As doses, but the values found in E. citriodora were significantly higher than in E. urophylla. E. citriodora plants presented a higher capacity to tolerate As and translocate it to the shoot than E. urophylla. Although these species cannot be considered as hyperaccumulators of As, E. citriodora presented the potential to be used in phytoremediation programs in arsenate-contaminated areas due to the long-term growth period of this species.

Optimization of Soil Stabilization with Class C Fly Ash, January 1987

Previous Iowa DOT sponsored research has shown that some Class C fly ashes are ementitious (because calcium is combined as calcium aluminates) while other Class C ashes containing similar amounts of elemental calcium are not (1). Fly ashes from modern power plants in Iowa contain significant amounts of calcium in their glassy phases, regardless of their cementitious properties. The present research was based on these findings and on the hyphothesis that: attack of the amorphous phase of high calcium fly ash could be initiated with trace additives, thus making calcium available for formation of useful calcium-silicate cements. Phase I research was devoted to finding potential additives through a screening process; the likely chemicals were tested with fly ashes representative of the cementitious and non-cementitious ashes available in the state. Ammonium phosphate, a fertilizer, was found to produce 3,600 psi cement with cementitious Neal #4 fly ash; this strength is roughly equivalent to that of portland cement, but at about one-third the cost. Neal #2 fly ash, a slightly cementitious Class C, was found to respond best with ammonium nitrate; through the additive, a near-zero strength material was transformed into a 1,200 psi cement. The second research phase was directed to optimimizing trace additive concentrations, defining the behavior of the resulting cements, evaluating more comprehensively the fly ashes available in Iowa, and explaining the cement formation mechanisms of the most promising trace additives. X-ray diffraction data demonstrate that both amorphous and crystalline hydrates of chemically enhanced fly ash differ from those of unaltered fly ash hydrates. Calciumaluminum- silicate hydrates were formed, rather than the expected (and hypothesized) calcium-silicate hydrates. These new reaction products explain the observed strength enhancement. The final phase concentrated on laboratory application of the chemically-enhanced fly ash cements to road base stabilization. Emphasis was placed on use of marginal aggregates, such as limestone crusher fines and unprocessed blow sand. The nature of the chemically modified fly ash cements led to an evaluation of fine grained soil stabilization where a wide range of materials, defined by plasticity index, could be stabilized. Parameters used for evaluation included strength, compaction requirements, set time, and frost resistance.

Morphology and segregation in two-component diffusion-limited aggregation

A diffusion-limited-aggregation (DLA) model with two components (A and B species) is presented to investigate the structure of the composite deposits. The sticking probability PAB (=PBA) between the different species is introduced into the original DLA model. By using computer simulation it is shown that various patterns are produced with varying the sticking probabilities PAB (=PBA) and PAA (= PBB), where PAA (=PBB) is the sticking probability between the same species. Segregated patterns can be analyzed under the condition PAB < PAA, assumed throughout the paper. With decreasing sticking probability PAB, a clustering of the same species occurs. With sufficiently small values of both sticking probabilities PAB and PAA, the deposit becomes dense and the segregated patterns of the composite deposit show a striped structure. The effect of the concentration on the pattern morphology is also shown.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Development of an aggregation assay to screen FimH antagonists.

Alpha-D-mannopyranosides are potent FimH antagonists, which inhibit the adhesion of Escherichia coli to highly mannosylated uroplakin Ia on the urothelium and therefore offer an efficient therapeutic opportunity for the treatment and prevention of urinary tract infection. For the evaluation of the therapeutic potential of FimH antagonists, their effect on the disaggregation of E. coli from Candida albicans and guinea pig erythrocytes (GPE) was studied. The mannose-specific binding of E. coli to yeast cells and erythrocytes is mediated by type 1 pili and can be monitored by aggregometry. Maximal aggregation of C. albicans or GPE to E. coli is reached after 600 s. Then the FimH antagonist was added and disaggregation determined by light transmission over a period of 1400 s. A FimH-deleted mutant of E. coli, which does not induce any aggregation, was used in a control experiment. The activities of FimH antagonists are expressed as IC(50)s, the half maximal inhibitory concentration of the disaggregation potential. n-Heptyl alpha-D-mannopyranoside (1) was used as a reference compound and exhibits an IC(50) of 77.14 microM , whereas methyl alpha-D-mannopyranoside (2) does not lead to any disaggregation at concentrations up to 800 microM. o-Chloro-p-[N-(2-ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl alpha-D-mannopyranoside (3) shows a 90-fold and 2-chloro-4-nitrophenyl alpha-D-mannopyranoside (4) a 6-fold increased affinity compared to 1. Finally, 4-nitrophenyl alpha-D-mannopyranoside (5) exhibits an activity similar to 1. As negative control, D-galactose (6) was used. The standardized aggregation assay generates concentration-dependent, reproducible data allowing the evaluation of FimH antagonists according to their potency to inhibit E. coli adherence and can therefore be employed to select candidates for experimental and clinical studies for treatment and prevention of urinary tract infections.