Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography

The bacterial production of recombinant rat calpain II has beenimproved greatly by the use of two compatible plasmids for thetwo subunits. The calpain small subunit C-terminal fragment(21 kDa) was expressed from a new A15-based vector created bycloning T7 control elements into pACYC177. This vector is compatiblewith the ColElbased pET-24d(+) vector containing the calpainlarge subunit, and the yield of calpain activity was increasedat least 16-fold by co-expression from these two vectors. Ahigh level of activity was also obtained from a bicistronicconstruct containing both subunit cDNAs under the control ofone T7 promoter. The addition of a C-terminal His-tag to thelarge subunit simplified purification without affecting subunitassociation or enzyme activity. The active-site cysteine 105was mutated to alanine, causing complete loss of activity. Theyield of purified C105A-calpain II (80 + 21 kDa) dimer followingthree column chromatography steps was 10 mg/l of cell culture.This provides a purified calpain, stable to autolysis and oxidation,which is likely to facilitate crystallization in both the presenceand absence of calcium.     

Biotransformation of N-ethyl perfluorooctanesulfonamide by rainbow trout (Onchorhynchus mykiss) liver microsomes

Rainbow trout (Onchorhynchus mykiss) liver microsomes were incubated with N-ethyl perfluorooctanesulfonamide [N-EtPFOSA, C8F17SO2NH(C2H5)], to examine the possibility of in vitro biotransformation to perfluorooctane sulfonate (PFOS, C8F17SO3-) and perfluorooctanoate (PFOA, C7F15COO-). Incubations were performed by exposing trout liver microsomes to N-EtPFOSA at 8 degrees C in the dark. Reaction mixtures were analyzed after incubation periods of 0, 2, 4, 8, 16, and 30 h for N-EtPFOSA, PFOS, PFOA, and perfluorooctanesulfonamide (PFOSA, C8F17SO2NH2), a suspected intermediate. Amounts of PFOS and PFOSA were found to increase with incubation time, but only background levels of PFOA were detected. Three possible reaction pathways are proposed for the conversion of N-EtPFOSA to PFOS: (i) direct conversion of N-EtPFOSA to PFOS by deethylamination accompanied by conversion of the sulfone group to sulfonate, (ii) deethylation of N-EtPFOSA to PFOSA, followed by deamination to form PFOS, and (iii) direct hydrolysis of N-EtPFOSA. These findings represent the first report indicating a possible biotransformation of a perfluorosulfonamide to PFOS in fish and may help to explain the detection of PFOS, which is relatively involatile, and thus not likely to undergo atmospheric transport, in biota from remote regions.     

Phase Ambiguity Correction and Visualization Techniques for Complex-Valued ICA of Group fMRI Data

Analysis of functional magnetic resonance imaging (fMRI) data in its native, complex form has been shown to increase the sensitivity both for data-driven techniques, such as independent component analysis (ICA), and for model-driven techniques. The promise of an increase in sensitivity and specificity in clinical studies, provides a powerful motivation for utilizing both the phase and magnitude data; however, the unknown and noisy nature of the phase poses a challenge. In addition, many complex-valued analysis algorithms, such as ICA, suffer from an inherent phase ambiguity, which introduces additional difficulty for group analysis. We present solutions for these issues, which have been among the main reasons phase information has been traditionally discarded, and show their effectiveness when used as part of a complex-valued group ICA algorithm application. The methods we present thus allow the development of new fully complex data-driven and semi-blind methods to process, analyze, and visualize fMRI data.We first introduce a phase ambiguity correction scheme that can be either applied subsequent to ICA of fMRI data or can be incorporated into the ICA algorithm in the form of prior information to eliminate the need for further processing for phase correction. We also present a Mahalanobis distance-based thresholding method, which incorporates both magnitude and phase information into a single threshold, that can be used to increase the sensitivity in the identification of voxels of interest. This method shows particular promise for identifying voxels with significant susceptibility changes but that are located in low magnitude (i.e. activation) areas. We demonstrate the performance gain of the introduced methods on actual fMRI data.     

Large parallel machines can be extremely slow for small problems

We consider concurrent-write PRAMs with a large number of processors of unlimited computational power and an infinite shared memory. Our adversary chooses a small number of our processors and gives them a 0–1 input sequence (each chosen processor gets a bit, and each bit is given to one processor). The chosen processors are required to compute thePARITY of their input, while the others do not take part in the computation. Ifat most q processors are chosen andq 1/2 log logn, then we show that computing PARITY needsq steps in the worst case. On the other hand, there exists an algorithm which computes PARITY inq steps (for anyq <n) in this model, thus our result is sharp. Surprisingly, if our adversary choosesexactly q of our processors, then they can compute PARITY in [q/2] + 2 steps, and in this case we show that it needs at least [q2] steps. Our result implies that large parallel machines which are efficient when only a small number of their processors are active cannot be constructed. On the other hand, a result of Ajtai and Ben-Or [1] shows that if we haven input bits which contain at most polylogn 1's and polynomially many processors which are all allowed to work, then thePARITY can be solved inconstant time.     

Bitumen Recovery with Oily Air Bubbles

Air‐kerosene bubbles were used in a novel laboratory scale pipeline loop to assess the extraction performance of poor processing oil sand ores. The addition of kerosene to air, whereby producing oily bubbles, substantially enhanced bitumen recovery from poor processing oil sand ores. The oily bubbles were added in a pipeline loop during bitumen liberation from the sand grains. The bitumen recovery from poor processing ores with the addition of the oily bubbles to the conditioning slurry becomes comparable to that of good processing ores. The present findings can be of substantial benefit to the oil sands industry.     

Genetic regulation of elevated stearic acid concentration in soybean oil

Soybean [Glycine max (L.) Merr.] oil from commercial cultivars typically contains ca. 3% stearic acid (18∶0). However, germplasm carrying different mutations at the locus governing stearic acid (Fas) may contain 3% to about 35% 18∶0. Among these germplasm, a newly developed line, FAM94-41 (9% 18∶0), carries a serendipitous natural mutation that is temporarily designated as the recessive fas _nc allele, and the germplasm A6 (26% 18∶0) carries the recessive fas ^a allele. Mendelian genetic analysis of progeny from FAM94-41×A6 revealed that fas _nc and fas ^a are allelic to each other and represent different mutations in the same structural gene. However, the gene products (enzymes) produced by these alleles are unknown. The observation that 18∶0 concentrations among progeny from FAM94-41×A6 increased primarily at the expense of unsaturated C₁₈ FA suggests that fas alleles may reduce either 18∶0-acyl carrier protein (AcP) desaturase or 18∶1-ACP thioesterase activity. However, it also is conceivable that elevated 18∶0 concentrations may result from increased 3-keto-acyl-ACP synthetase (KAS) II activity. To test the latter possibility, a population was created that segregated for the fas _nc and the fap ₂ alleles (the latter of which is associated with reduced KAS-II activity). Mendelian genetic analysis showed that these alleles represent independent genes at different gene loci and interact in an additive genetic manner to increase the total saturate concentration in this population. Based on this finding, we speculate that fas alleles probably encode 18∶0-ACP desaturase or 18∶1-ACP thioesterase in soybeans.     

Molecular mapping and identification of soybean fatty acid modifier quantitative trait loci

Altering FA content in soybean [Glycine max (L.) Merr.] oil for improved functionality is a research goal of many soybean breeders. Several of the genes that alter palmitic, stearic, oleic, linoleic, and linolenic acids are modifier genes with small effects, causing these FA traits to act as quantitative traits. The objective of this study was to identify modifier FA quantitative trait loci (QTL) in soybean. A recombinant inbred line population was created from two prominent ancestors of currently available U.S. cultivars (Essex and Williams) and grown in five environments. One hundred simple sequence repeat markers spaced throughout the genome were mapped in this population. QTL were found for all five FA traits on the soybean linkage groups C2, D2, D1b, F, K, and L. A single marker interval on linkage group L contained the largest QTL for palmitic (r ²=13.1%), oleic (r ²=35.3%), linoleic (r ²=50.5%), and linolenic acids (r ²=24.8%); however, this interval also contained the gene for growth habit (Dt1) and was significantly associated with maturity. Other modifier QTL found in this study may be of use in marker-assisted selection to enable breeders to increase genetic gains for desirable FA composition of soybean.     

A new approach to genetic alteration of soybean protein composition and quality

Although soybeans produce high-quality meal, modern animal and fish production systems often require synthetic essential amino acid supplements to fortify feed rations. However, biotechnology may enable development of soybeans with naturally adequate levels of certain essential amino acids for advanced feed formulations. One approach involves genetic manipulation of glycinin (11S) and β-conglycinin (7S) contents, the principal components of soybean storage proteins. Because 11S contains more cysteine and methionine than 7S protein, a higher 11S:7S ratio could lead to beneficial changes in the nutritional quality of soybean meal. Although genotypic variation for 11S:7S may be low among soybean [Glycine max (L.) Merr.] germplasm, ratios ranging from 1.7–4.9 were observed among accessions of the wild ancestor of cultivated soybean (Glycine soja Sieb, and Zucc.). Thus, wild soybean germplasm was evaluated as a potential source of genes that govern protein synthesis that may have been lost during the domestication of G. max. Change in the amount of 11S protein accounts for a significant portion of the genotypic variation in protein concentration and composition among wild soybeans. Strong positive correlation exists between the 11S:7S ratio and methionine or cysteine concentration of total protein. Moderate positive associations were found for threonine or tyrosine. A moderate negative correlation was found between lysine and 11S:7S. No association was found for leucine and phenylalanine or for total essential amino acid concentration. Based on these data, G. soja may contain a different complement of genes that influence expression of 11S and 7S proteins than G. max germplasm. Thus, through interspecific hybridization, wild soybeans may be a useful genetic resource for the further improvement of protein quality in cultivated soybeans.     

Fines/Water Interactions and Consequences of the Presence of Degraded Illite on Oil Sands Extractability

The chemical composition of the aqueous phase in oil sand slurries influences bitumen recovery from oil sands, especially those containing greater than 10% fines. The composition is controlled by a combination of mixing and dilution, ion exchange with clay surfaces and precipitation of divalent ions as carbonate minerals. Elevated levels of soluble potassium in the oil sand, which appear to be a marker for degraded illite or smectitic clays, are associated with depressed bitumen recovery. These clays have a swelling character and can contribute divalent ions to the slurry by ion exchange between the clay mineral surfaces and the process water.     

Genetic Gain for Soybean Seed Protein,Oil, and Yield in a Recombinant Inbred Line Population

The use of soybean [Glycine max (L.) Merrill] is widespread, with applications in food, fuel, livestock feed, and industrial products. We developed a soybean population of 239 F₄-derived recombinant inbred lines (RIL) from the cross “5601T” × U99-310255 to estimate the heritability and genetic gain for seed protein, oil, and yield, and to assess the utility of making selections for these important traits. Heritability estimates on an entry means basis for protein (0.78), oil (0.65), seed yield (0.38) varied by trait with regard to genetic influence. Genetic gain was calculated for each trait at 5%, 10%, 15%, and 20% selection intensities. With the genetic gain that was obtained through the selections for each trait, some lines excelled above that of the checks and parents in this population. The second and third highest yielding selections (56U99-081 and 56U99-244) are notable in producing 50.2% and 49.8% meal protein, respectively, while maintain high yields above the high-yielding parent, 5601T. Seeking balance for these important soybean traits is essential for breeders looking to make optimal improvements for the wide variety of soybean uses. Further research on this population could prove to be of value to breeding programs wishing to increase seed yield, oil, and protein.