Effect of modifications of the P-Bray no. 1 method on soil test results

The importance of typical modifications of the Bray no. 1 procedure for the determination of “available” soil phosphorus was assessed using soil samples from Rwanda. Variations in shaking time (1 or 5 min.), soil to extraction solution ratio (1:7 or 1:10), and soil sieve size (2 mm vs. 0.5 mm) were found to significantly affect available P values. The modifications gave values which were up to 75 % higher than those of the original Bray no. 1 procedure.     

Sodium dodecyl sulfate capillary electrophoresis of wheat proteins. 1. Uncoated capillaries.

Four different polymer/buffer systems (a commercial polymer from Bio-Rad, dextran, poly(ethylene oxide) (PEO), and non-crosslinked poly(acrylamide)) were evaluated for use in sodium dodecyl sulfate capillary electrophoresis (SDS-CE) separations of wheat proteins. These polymers were chosen on the basis of published reports of their use in uncoated or dynamically coated capillaries. Each polymer was optimized (where possible) by manipulating the polymer concentration and buffer concentration, and through the use of organic modifiers such as methanol and ethylene glycol. The addition of ethylene glycol to the separation buffer was found to improve the resolution of the separations, despite dilution of the sieving polymers. When PEO was used as the sieving polymer, however, no improvement was seen when ethylene glycol was added. Despite producing similar separations of molecular mass markers, the polymers did not all produce similar wheat protein separations. The commercial reagent and dextran produced similar separations, while the poly(acrylamide) produced faster separations than either. The poly(acrylamide) displayed much lower resolution in the 40-60 kDa range than the other polymers, though this polymer was able to separate the high molecular mass glutenin subunits (HMM-GS) without the use of added organic solvent. PEO produced much different wheat protein separations than the other polymers, despite similar separations of the molecular weight markers. This may have been due to interaction between the wheat proteins and PEO. Each polymer system also predicted different molecular masses of the various wheat protein fractions separated, with the PEO and poly(acrylamide) grossly overestimating the masses for all protein classes. This could have been due to protein-polymer interactions. Further work was done with the Bio-Rad buffer modified by the addition of ethylene glycol. Several different wheat protein fractions as well as proteins extracted from several different cultivars were separated with this buffer and compared. SDS-CE separations were also compared to SDS-poly(acrylamide) gel electrophoresis (PAGE) and several differences in the migration pattern of HMM-GS were noted.     

Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.     

Ovalbumin, ovotransferrin, lysozyme: three model proteins for structural modifications at the air-water interface

Structural modifications of ovalbumin, ovotransferrin, and lysozyme at the air-water interface have been investigated using SDS-PAGE, both intrinsic and ANS fluorometry, and circular dichroism experiments. Ovalbumin contact with an interface induced an exposure of aromatic residues, a slight decrease in alpha-helix structures (-1.7%), and an increase in both beta-sheet (+3.4%) and beta-turn (+7.9%) structures. Moreover, these conformational changes led to the formation of insoluble polymers of ovalbumin through intermolecular disulfide bonds. Ovotransferrin contact with an interface led to an increase in its surface hydrophobicity (+30%) and modifications of its secondary structure (-33% of alpha-helices, +96.4% of beta-sheets, +13.2% of beta-turns, and +21.2% of random coils), characteristic of major conformational changes. On the other hand, lysozyme did not undergo any structural modification. These results clearly underscore that at the air-water interface proteins are susceptible to denaturation.     

Isolation and characterization of glyoxal-arginine modifications

5-(4,5-Dihydroxy-2-imino-1-imidazolidinyl)norvaline (1) was identified as the only product of the early reaction of arginine with glyoxal, which was slowly degraded to N(5)-[[(carboxymethyl)amino](imino)methyl]ornithine (3, N(7)-carboxymethylarginine). No other structures could be detected within a range of pH 4-8 and 20-50 degrees C in reaction conditions. The rates of formation for both products increased with pH and temperature. In equilibrium, the vincinal diol groups of 1 were 86% trans configured. The formation of 1 was reversible, as could be shown by cis-trans isomerization of the separated isomers and by regeneration of arginine in the presence of the alpha-dicarbonyl trapping reagents, o-phenylenediamine and aminoguanidine. Both 1 and 3 were converted to 5-(2-imino-5-oxo-1-imidazolidinyl)norvaline (2) only under strong acidic conditions.     

Effects of aluminum stress on alfalfa root proteins 1

An estimated 40% of arable soils worldwide contain phytotoxic levels of aluminum (Al). Recent evidence indicates that Al‐stress‐induced low molecular weight proteins may bind Al in Al‐tolerant plants. The objective of this study was to investigate protein patterns in young roots of two Al‐sensitive and two Al‐tolerant alfalfa (Medicago sativa L.) clones grown at 0 and 111 μmol Al in pH 4.5 nutrient solution. Based on SDS‐PAGE of supernatant, Al stress resulted in an increase in detectable root proteins in all clones and results are consistent with results reported for other plant species. A proliferation of new low‐molecular‐weight proteins in the tolerant clones could be related to Al tolerance. One protein (18.7 kD) was produced in both tolerant clones yet not detected in the sensitive clones. Protein levels were more often reduced than enhanced under Al stress and reduction was more prevalent in sensitive than in tolerant clones. Aluminum stress may initiate the production of some of the same proteins in alfalfa and wheat (Triticum aestivum L. Thell.). General stress proteins could be produced in reaction to a variety of chemical, environmental, and pathological stresses.     

Methylene group modifications of the N-(Isothiazol-5-yl)phenylacetamides. Synthesis and insecticidal activity.

It has been shown that oxidation at the alpha-carbon of N-(4-chloro-3-methyl-5-isothiazolyl)-2-[p-[(alpha,alpha, alpha-trifluoro-p-tolyl)oxy]phenyl]acetamide (1) is conveniently brought about using dimethylformamide dimethylacetal to give N-(4-chloro-3-methyl-5-isothiazolyl)-beta-(dimethylamino)-p-[(alpha, alpha,alpha-trifluoro-p-tolyl)oxy]atropamide (2), which has served as a common starting point for a variety of functional group transformations. These transformations were found to proceed in moderate to good yields to give derivatives of 1 that retained much of the efficacy associated with the parent amide and have allowed for an expansion of the SAR to be developed. Examples of enamines, enols, enol (thio)ethers, oximes, and hydrazones were prepared. In particular, the enamines derived from low molecular weight amines and amino acids were most active as broad-spectrum insecticides and were found to be even more active than 1 on root-knot nematode.     

Physicochemical modifications of high-pressure-treated soybean protein isolates

Changes induced by high pressure (HP) treatment (200-600 MPa) on soybean protein isolates (SPI) at pH 3 (SPI3) and pH 8 (SPI8) were analyzed. Changes in protein solubility, surface hydrophobicity (Ho), and free sulfhydryl content (SH(F)) were determined. Protein aggregation and denaturation and changes in secondary structure were also studied. An increase in protein Ho and aggregation, a reduction of free SH, and a partial unfolding of 7S and 11S fractions were observed in HP-treated SPI8. Changes in secondary structure were also detected, which led to a more disordered structure. HP-treated SPI3 was partially denatured and presented insoluble aggregates. A major molecular unfolding, a decrease of thermal stability, and an increase of protein solubility and Ho were also detected. At 400 and 600 MPa, a decrease of the SH(F) and a total denaturation were observed.     

Engineering nutritious proteins: improvement of stability in the designer protein MB-1 via introduction of disulfide bridges.

Protein design is currently used for the creation of new proteins with desirable traits. In this laboratory the focus has been on the synthesis of proteins with high essential amino acid content having potential applications in animal nutrition. One of the limitations faced in this endeavor is achieving stable proteins despite a highly biased amino acid content. Reported here are the synthesis and characterization of two disulfide-bridged mutants derived from the MB-1 designer protein. Both mutants outperformed their parent protein MB-1 with their bridge formed, as shown by circular dichroism, size exclusion chromatography, thermal denaturation, and proteolytic degradation experiments. When the disulfide bridges were cleaved, the mutants' behavior changed: the mutants significantly unfolded, suggesting that the introduction of Cys residues was deleterious to MB-1-folding. In an attempt to compensate for the mutations used, a Tyr62-Trp mutation was performed, leading to an increase in bulk and hydrophobicity in the core. The Trp-containing disulfide-bridged mutants did not behave as well as the original MB-1Trp, suggesting that position 62 might not be adequate for a compensatory mutation.     

Inhibition of proteolysis in oxidized lipid-damaged proteins.

The proteolysis of bovine serum albumin (BSA) modified by reaction with the lipid peroxidation product 4,5(E)-epoxy-2(E)-heptenal was studied to better understand the loss of digestibility observed in oxidized lipid-damaged proteins. BSA was incubated for different periods of time with eight concentrations of the epoxyalkenal and, then, treated for 24 h with chymotrypsin, pancreatin, Pronase, or trypsin. The treatment of BSA with the aldehyde always decreased its proteolysis in relation to that of native BSA, and this inhibition of the proteolysis was related to the concentration of the epoxyalkenal and the reaction time. In fact, this inhibition was correlated with the damage suffered by the protein as a consequence of its reaction with the aldehyde: mainly the development of browning, the denaturation of the protein, and the formation of the oxidized lipid/amino acid reaction product epsilon-N-pyrrolylnorleucine (p < or = 0.0011, 0.0045, and 0.0031, respectively). In addition, epsilon-N-pyrrolylnorleucine added at 0.1 or 1 mM inhibited the proteases assayed and suggested that the inhibition of the proteolysis observed in oxidized lipid-damaged proteins may be related to the formation and accumulation of pyrrolized amino acid residues.     

Microclimate modifications,growth and yield of intercrops under Hardwickia binata Roxb. based agroforestry system

An experiment was conducted by planting Hardwickia binata Roxb. at 200, 400 and 800 trees ha^???1, intercrops viz., Brassica campestris (mustard) and Glycine max (soybean) were sown from the second year onwards in winter and summer respectively at the National Research Centre for Agroforestry, Jhansi, U.P., India with the objectives of evaluating agrosilvicultural potential of the system and to study the effect of tree densities on ecophysiology of understorey crops. Tree growth was affected by both density and intercrop in the initial years of growth. Photosynthetic photon flux density (PPFD) available to the intercrops reduced with increasing densities. Transpiration rate and stomatal conductance in intercrops decreased due to the presence of trees. No significant changes in leaf temperature were observed till the fifth year of the growing season. Yield was significantly higher in pure crop in comparison with all the densities in mustard. Soybean yield under 200 trees ha^???1 was comparable to that of the pure crop. Trees at the density of 200 trees ha^???1 provided a conducive microenvironment to the intercrops.     

Earthworm populations in a northern U.S. Cornbelt soil are not affected by long-term cultivation of Bt maize expressing Cry1Ab and Cry3Bb1 proteins

Earthworms, which play a key role in biogeochemical processes in soil ecosystems, could be negatively affected by the cultivation of transgenic Bt crops. Studies to date have found few effects of Bt maize on earthworm species. If adverse effects occur, they are likely to be chronic or sub-lethal and expressed over large spatial and temporal scales. Our objective in the present study was to investigate potential effects on earthworm populations in soil cultivated with Bt maize in a large multiple-year field study. We surveyed the earthworm populations in 0.16-ha experimental field plots of two varieties of Cry1Ab Bt maize, one variety of Cry3Bb1 Bt maize, and three non-transgenic control varieties cultivated for four years. Four earthworm species were found in our sample: Aporrectodea caliginosa, Aporrectodea trapezoides, Aporrectodea tuberculata (collectively, the A. caliginosa species complex), and Lumbricus terrestris. We found no significant differences in the biomass of juveniles and adults for all four species between Bt and non-Bt maize varieties. From this and previous studies, we conclude that the effects of Cry1Ab and Cry3Bb1 Bt maize on the A. caliginosa species complex and L. terrestris are small. Nonetheless, general conclusions about the effects of Bt maize on earthworm populations are not warranted due to the small number of species tested. In future laboratory studies, earthworm species should be selected according to their association with a Bt crop and the impact of that species to valued soil ecosystem processes.     

Kinetics of thermal modifications in a grape seed extract

The thermal modification kinetics of a commercial grape seed extract (GSE) was investigated. A GSE was exposed to 60, 90, and 120 °C for 5, 10, 15, 30, 45, and 60 min. The antioxidant activity (AA) and the absorbance at 420 nm (A(420)) were measured. (+)-Catechin, (-)-epicatechin, procyanidins B1 and B2, and gallic acid were identified and measured. After the thermal treatments, the AA did not show a significant difference (p > 0.05) and both procyanidins and gallic acid increased as well as A(420). (+)-Catechin and (-)-epicatechin decreased. To obtain the activation energy (E(a)) of the changes, a modified Weibull and a combined zero- and first-order model were compared, both followed by the Arrhenius equation. The Weibull model was more accurate. The E(a) values for browning and (+)-catechin, (-)-epicatechin, gallic acid, and procyanidins B1 and B2 were 170, 286, 42, 102, 249, and 95 kJ/mol, respectively. The results were valid at a confident level of 95%.     

Macronutrient deficiency and anatomic modifications in crambe leaves

Abstract

This study had the objective of assessing growth, deficiency symptoms and leaf anatomy of crambe plants submitted to macronutrient availability. The experimental design was the complete randomized with four replications. The first treatment consisted of cultivating crambe plants in a nutrient solution completed with N, P, K, Ca, Mg, and S. Using the diagnosis by subtraction, the other treatments consisted of the same solution with individual omission of each nutrient, totaling seven treatments. Supplement of different solutions took place two weeks after emergence. One week forward, visual symptoms of deficiency started to be evaluated. By the end of the experiment, the number of leaves, number of branches, shoot dry matter and leaf anatomic parameters were evaluated. Nutrient deficiency limited shoot dry matter in the following order: N?>?Ca?>?P>Mg?>?S?>?K. Subtracting Ca from the solution was most limiting to crambe growth once plants did not even reach reproductive stages. Individual subtractions of each macronutrient anatomically altered crambe leaves, especially omitting Ca, K, and S, which reduced tissue thickness.     

Amaranth globulin structure modifications induced by enzymatic proteolysis

Globulin-P was partially hydrolyzed with papain under specific conditions to study the resulting structural modifications. Under mild hydrolytic conditions, globulin-P polymers were cleaved to render their unitary constituents (280 kDa molecules). Under stronger hydrolytic conditions these unitary molecules were 13% smaller than those from nonhydrolyzed globulin. Moreover, these molecules remained assembled even though they contained degraded polypeptides. The monomeric (M) subunit and the A chains were preferentially cleaved under mild and intermediate hydrolytic conditions, whereas B chains remained with the same size. These results suggest that the M and A polypeptides might be located at an exposed site of the molecules resembling the structure of the legumins. The M subunit may be participating in the stabilization of globulin-P polymers, on the basis that these two species disappeared under the same hydrolytic conditions. Similar events such as those described in this paper might be taking place on globulin-P during germination of amaranth grain.     

Calcium- and magnesium-dependent aggregation of legume seed storage proteins.

The solubility characteristics and sedimentation behavior of total or individual globulins from legume seeds [Lupinus albus L., Pisum sativum L., and Glycine max (L.) Merr.] were investigated. The typical insolubility of globulins detected during their extraction seems to be due to the presence of a low molecular weight factor(s) in the seed extract. The solubility of the purified globulins decreases with increasing concentrations of calcium and/or magnesium, but not of other cations, showing minimum values at concentrations that vary with the particular globulin considered. Ultracentrifugation analyses revealed that the Ca(2+)- and/or Mg(2+)-induced insolubilization of the globulins involves the formation of high-order aggregates of molecules of the same or of different globulins. These macromolecular structures are dissociated under conditions of high ionic strength, suggesting the involvement of electrostatic interactions in the aggregation process. The degree of association relies heavily on the amount of Ca(2+) and/or Mg(2+) available, on the presence of chelating agents for these divalent cations, and on the ionic strength of the surrounding medium. The possible physiological significance of the findings is discussed.