Introduction of sulfhydryl groups and disulfide linkage to mungbean 8Sα globulin and effects on physicochemical and functional properties

Sulfhydryl groups and disulfide bond were introduced in mungbean's major storage protein, 8Sα globulin, by protein engineering to improve structural stability and functional properties. Five modified proteins or mutants (F59C, I99C, A213C, one free sulfhydryl group; I99C/A213, one disulfide bridge; F59C/I99C/A213C, one free sulfhydryl group and one disulfide linkage) were expressed in Escherichia coli at a yield similar to that of the unmodified protein or wild type (WT) in soluble form (38%). The number of introduced groups in the mutants was confirmed by Ellman analysis. Mutant and WT proteins exhibited similar elution patterns on gel filtration indicating their trimeric native conformation. Mutants had 2 to 3.8 °C higher T_m values than WT and were digested by chymotrypsin at 52–58% in 60 min but exhibited different digestion patterns. All mutants showed greater hardness of heat-induced gels than WT, especially I99C/A213C and F59C/I99C/A213C. Results indicate the improved structural stability of the modified 8Sα globulin.     

Spatial and temporal distribution of singlet oxygen in Lake Superior

A multiyear field study was undertaken on Lake Superior to investigate singlet oxygen ((1)O(2)) photoproduction. Specifically, trends within the lake were examined, along with an assessment of whether correlations existed between chromophoric dissolved organic matter (CDOM) characteristics and (1)O(2) production rates and quantum yields. Quantum yield values were determined and used to estimate noontime surface (1)O(2) steady-state concentrations ([(1)O(2)](ss)). Samples were subdivided into three categories based on their absorbance properties (a300): riverine, river-impacted, or open lake sites. Using calculated surface [(1)O(2)](ss), photochemical half-lives under continuous summer sunlight were calculated for cimetidine, a pharmaceutical whose reaction with (1)O(2) has been established, to be on the order of hours, days, and a week for the riverine, river-impacted, and open lake waters, respectively. Of the CDOM properties investigated, it was found that dissolved organic carbon (DOC) and a300 were the best parameters for predicting production rates of [(1)O(2)](ss). For example, given the correlations found, one could predict [(1)O(2)](ss) within a factor of 4 using a300 alone. Changes in the quantum efficiency of (1)O(2) production upon dilution of river water samples with lake water samples demonstrated that the CDOM found in the open lake is not simply diluted riverine organic matter. The open lake pool was characterized by low absorption coefficient, low fluorescence, and low DOC, but more highly efficient (1)O(2) production and predominates the Lake Superior system spatially. This study establishes that parameters that reflect the quantity of CDOM (e.g., a300 and DOC) correlate with (1)O(2) production rates, while parameters that characterize the absorbance spectrum (e.g., spectral slope coefficient and E2:E3) correlate with (1)O(2) production quantum yields.     

Extended study on the influence of z-value(s) of single and multicomponent time-temperature integrators on the accuracy of quantitative thermal process assessment

The possibilities and limitations of single- and multicomponent time-temperature integrators (TTIs) for evaluating the impact of thermal processes on a target food attribute with a Ztarget value different from the zTTI value(s) of the TTI is far from sufficiently documented. In this study, several thousand time-temperature profiles were generated by heat transfer simulations based on a wide range of product and process thermal parameters and considering a Ztarget value of 10 degrees C and a reference temperature of 121.1 degrees C, both currently used to assess the safety of food sterilization processes. These simulations included 15 different Ztarget=10 degrees CF121.1 degrees C values in the range 3 to 60 min. The integration of the time-temperature profiles with ZTTI values of 5.5 to 20.5 degrees C in steps of 1 degrees C allowed generation of a large database containing for each combination of product and process parameters the correction factor to apply to the process value FmultiTTI, which was derived from a single- or multicomponent TTI, to obtain the target process value 10 degrees CF121.1 degrees C. The table and the graph results clearly demonstrated that multicomponent TTIs with z-values close to 10 degrees C can be used as an extremely efficient approach when a single-component TTI with a z-value of 10 degrees C is not available. In particular, a two-component TTI with z1 and z2 values respectively above and below the Ztarget value (10 degrees C in this study) would be the best option for the development of a TTI to assess the safety of sterilized foods. Whatever process and product parameters are used, such a TTI allows proper evaluation of the process value 10 degrees CF121.1 degrees C.     

The esterified plasma fatty acid profile is altered in early HIV-1 infection

The online version of the original article can be found at     

Efficient Total Chemical Synthesis of 13C=18O Isotopomers of Human Insulin for Isotope‐Edited FTIR

Isotope‐edited two‐dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site‐specific incorporation of stable ¹³C=¹⁸O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis—via a key ester insulin intermediate—of 97 % enriched [(1‐¹³C=¹⁸O)Phe^B24] human insulin: stable‐isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X‐ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1‐¹³C=¹⁸O)Phe^B24] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red‐shifted amide I carbonyl band peak at 1595 cm^?1 resulting from the (1‐¹³C=¹⁸O)Phe^B24 backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.     

Solvent and Extraction Conditions Control the Assayable Phenolic Content and Antioxidant Activities of Seeds of Black Beans,Canola and Millet

The effects of extraction solvent and conditions on the total phenolic content (TPC) and antioxidant activity of black beans, canola and foxtail millet were investigated. The antioxidant activity was assayed using 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity (DRSA) and oxygen radical absorbance capacity (ORAC). Four solvent systems, namely 70 % acetone, 80 % ethanol, 80 % methanol and a mixture of acetone/methanol/water (7:7:6, v/v/v) were used. The extraction methods adopted in this study included refluxing, homogenization, cold extraction and sonication. The TPC as measured using the Folin Ciocalteu's method were 12.35–28.39, 2.43–16.73, and 1.78–5.06 µmol catechin equivalents/g dry matter (dm) for canola, black beans and foxtail millet, respectively. Aqueous acetone afforded the highest TPC for black beans and canola. Within the same solvent system used, the TPC, DRSA and ORAC obtained from different extraction techniques differed for black beans, canola and foxtail millet. The results demonstrated that the solvent system as well as method influenced the extraction of phenolic compounds and their antioxidant activities, depending on the type of matrix in which phenolics were embedded.     

Response of winter wheat to N-fertiliser: Quantitative relations for components of growth

Measurements were made of yield of dry matter, plant-N content, and the distribution of mineral-N down the soil profile in 10 fertiliser-N experiments. In one of them detailed measurements were made throughout growth. Rate of N-uptake by the crop was unaffected by the amount of mineral-N in the upper 90 cm of soil when it was above about 30 kg N ha^–1. The %N in plants that received ample N-fertiliser declined with increase in plant mass according to a previously derived equation. During senescence there was an apparent loss of N from the crop.N-nutrition in the different experiments had little effect on the partition of assimilate between grain and straw. At harvest grain and straw weights were well related by a linear model which had the same gradient but different intercepts for each experiment. Grain %N was about four times greater than straw %N. Regression analysis supported the view that high evaporative conditions or temperatures during the growing period induced earlier harvest dates, less grain relative to straw, and a higher %N in the plant when ample N-fertiliser was applied but not when N-fertiliser was withheld.Other analyses indicated that cereal roots were generally unable to extract mineral nitrogen from the soil when the concentration was less than about 0.18 kg N ha^–1 cm^–1, that at low levels of N-nutrition the recovery of available inorganic-N from soil by the grain and straw was about 80%, and that the average mineralisation rates from early spring to shortly after harvest date varied between 0.22 and 0.88 kg N ha^–1 d^–1 from site to site.     

The simultaneous determination of both the quantity and the fatty acid composition of the triglycerides in three to ten microliters of plasma

An integrated system using chromatography on silica gel-impregnated glass paper, an internal standard and gas-liquid chromatography (GLC) makes possible the isolation of triglycerides, the determination of triglyceride, fatty acid composition, and the quantitative determination of triglyceride. Recoveries are good and values for total triglyceride obtained by this system compare favorably with values obtained by application of the hydroxamate colorimetric method to the triglyceride eluate from silicic acid columns. The fatty acid composition of the cholesteryl ester (CE) fraction is obtainable at the same time by GLC of methyl esters prepared from the CE fractions which also appears as a distinct spot on these chromatograms. Also, a densitometric method is described for triglyceride determination by photometry of the charred spots obtained after chromatography on the gel-paper. Presented at the AOCS Meeting, Houston, April, 1965     

Influence of medium-chain triglycerides on lipid metabolism in the rat

Lipid metabolism was studied in rats fed diets containing corn oil, coconut oil, or medium-chain triglyceride (MCT), a glyceride mixture containing fatty acids of 8 and 10 carbons in length. The ingestion of MCT-supplemented, cholesterolfree diets depressed plasma and liver total lipids and cholesterol as compared with corn oil-supplemented diets. In rats fed cholesterol-containing diets, plasma cholesterol levels were not influenced by dietary MCT, but liver cholesterol levels were significantly lower than in animals fed corn oil. In vitro cholesterol synthesis from acetate-1-¹⁴C was lower in liver slices of rats that consumed MCT than in similar preparations from corn oil-fed rats. Studies of fatty acid carboxyl labeling from acetate-1-¹⁴C and the conversion of palmitate-1-¹⁴C to C₁₈ acids by liver slices showed that chain-lengthening activity is greater in the liver tissue of rats fed MCT than in the liver of animals fed corn oil. The hepatic fatty acid desaturation mechanisms, evaluated by measuring the conversion of stearate-2-¹⁴C to oleate, was also enhanced by feeding MCT. Adipose tissue of rats fed MCT converts acetate-1-¹⁴C to fatty acids at a much faster rate than does tissue from animals fed corn oil. Evidence is presented to show that the enhanced incorporation of acetate into fatty acids by the adipose tissue of rats fed MCT represents de novo synthesis of fatty acids and not chain-lengthening activity. Data are also presented on the fatty acid composition of plasma, liver, and adipose tissue lipids of rats fed the different fats under study.     

A reproducible and high-throughput HPLC/MS method to separate sarcosine from α- and β-alanine and to quantify sarcosine in human serum and urine

While sarcosine was recently identified as a potential urine biomarker for prostate cancer, further studies have cast doubt on its utility to diagnose this condition. The inconsistent results may be due to the fact that alanine and sarcosine coelute on an HPLC reversed-phase column and the mass spectrometer cannot differentiate between the two isomers, since the same parent/product ions are generally used to measure them. In this study, we developed a high-throughput liquid chromatography-mass spectrometry (LC-MS) method that resolves sarcosine from alanine isomers, allowing its accurate quantification in human serum and urine. Assay reproducibility was determined using the coefficient of variation (CV) and intraclass correlation coefficient (ICC) in serum aliquots from 10 subjects and urine aliquots from 20 subjects across multiple analytic runs. Paired serum/urine samples from 42 subjects were used to evaluate sarcosine serum/urine correlation. Both urine and serum assays gave high sensitivity (limit of quantitation of 5 ng/mL) and reproducibility (serum assay, intra- and interassay CVs < 3% and ICCs > 99%; urine assay, intra-assay CV = 7.7% and ICC = 98.2% and interassay CV = 12.3% and ICC = 94.2%). In conclusion, this high-throughput LC-MS method is able to resolve sarcosine from α- and β-alanine and is useful for quantifying sarcosine in serum and urine samples.