Saccharopine Dehydrogenase Activity in the High-Lysine Opaque and Floury Maize Mutants

Lysine is an essential amino acid normally present in very low concentration in cereal seeds. In previous reports we have studied the metabolism of lysine in several distinct high-lysine maize mutants and observed drastic variations in the activity of saccharopine dehydrogenase (SDH), a key enzyme involved in lysine degradation. We have now analyzed the activity of SDH using non-denaturing polyacrylamide gel electrophoresis (PAGE) to identify possible isoenzymes that could explain the patterns of activity previously observed. The results indicated the presence of at least two SDH isoenzymes, one contributing to approximately 90% of the total enzyme activity and a minor form only present in the wild type lines and the opaque-1 mutant. The results suggest that the differences in total SDH activity among the genotypes tested are due to alterations in the predominant SDH isoenzymic form, which is likely to be the bifunctional polypeptide containing lysine 2-oxoglutarate reductase.     

Comparisons of α-amylase inhibitors from seeds of common bean mutants extracted through three phase partitioning

This study compared the inhibitory activity of α-amylase inhibitor (αAI) extracted from common bean (Phaseolus vulgaris L.) variety Hwachia, its nine mutants and two introduced varieties by using three-phase partitioning (TPP). A commercially prepared Phase 2 was also used to serve as a comparative reference. The optimal purification parameters for TPP were 30% saturation ammonium sulphate and pH 5.25. Considerable variations were detected in αAI content, total inhibitory activity and specific inhibitory activity of αAI purified from different common beans. Mutant SA-05 had the αAI inhibitory activity of 6267 units g⁻¹ dry seed weight, which was higher than Hwachia (5062 units g⁻¹ dry seed weight) and Phase 2 (3200 units g⁻¹ dry weight). Moreover, it had an extremely lower IC₅₀ (0.40 μg) than Phase 2 (10.22 μg). Thus, the mutant SA-05 may be used as raw material in commercial preparation of αAI extracts for controlling appetite and energy intake.     

Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane

Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.     

On the relation between the MXL family of algorithms and Gröbner basis algorithms

The computation of Gröbner bases remains one of the most powerful methods for tackling the Polynomial System Solving (PoSSo) problem. The most efficient known algorithms reduce the Gröbner basis computation to Gaussian eliminations on several matrices. However, several degrees of freedom are available to generate these matrices. It is well known that the particular strategies used can drastically affect the efficiency of the computations. In this work, we investigate a recently-proposed strategy, the so-called “Mutant strategy”, on which a new family of algorithms is based (MXL, MXL₂ and MXL₃). By studying and describing the algorithms based on Gröbner basis concepts, we demonstrate that the Mutant strategy can be understood to be equivalent to the classical Normal Selection Strategy currently used in Gröbner basis algorithms. Furthermore, we show that the “partial enlargement” technique can be understood as a strategy for restricting the number of S-polynomials considered in an iteration of the _F4$F_4$ Gröbner basis algorithm, while the new termination criterion used in MXL₃ does not lead to termination at a lower degree than the classical Gebauer–Möller installation of Buchberger’s criteria. We claim that our results map all novel concepts from the MXL family of algorithms to their well-known Gröbner basis equivalents. Using previous results that had shown the relation between the original XL algorithm and _F4$F_4$, we conclude that the MXL family of algorithms can be fundamentally reduced to redundant variants of _F4$F_4$.     

Determination of Oil Content and Fatty Acid Composition of Sesame Mutants Suited for Intensive Management Conditions

Sesame mutants with closed capsules, determinate growth habit and wilt resistance, have been proposed to be suitable for intensive management conditions facilitating mechanized harvesting. The objective of our experiment was to determine the oil content and fatty acid composition of these mutants before they are placed on the market. Oil content and fatty acids were studied in 19 mutants, 6 breeding lines and 4 control source genotypes. The oil contents of the seeds ranged from 46.4 to 62.7%. The mutants had generally a lower oil content than the control genotypes except the wilting tolerant group. For unsaturated fatty acids, oleic acid was higher in the mutants and breeding lines while linoleic acid was lower in the seed oil. However, no mutants or breeding lines were found with radically different composition or with any undesirable lipid component. The closed capsule and determinate growth habit mutants need to be improved for oil content while their fatty acid composition is fine.     

Saccharopine Dehydrogenase Activity in the High-Lysine Opaque and Floury Maize Mutants

Lysine is an essential amino acid normally present in very low concentration in cereal seeds. In previous reports we have studied the metabolism of lysine in several distinct high-lysine maize mutants and observed drastic variations in the activity of saccharopine dehydrogenase (SDH), a key enzyme involved in lysine degradation. We have now analyzed the activity of SDH using non-denaturing polyacrylamide gel electrophoresis (PAGE) to identify possible isoenzymes that could explain the patterns of activity previously observed. The results indicated the presence of at least two SDH isoenzymes, one contributing to approximately 90% of the total enzyme activity and a minor form only present in the wild type lines and the opaque-1 mutant. The results suggest that the differences in total SDH activity among the genotypes tested are due to alterations in the predominant SDH isoenzymic form, which is likely to be the bifunctional polypeptide containing lysine 2-oxoglutarate reductase.     

A Genetic Approach to Studying the Morphology,Structure and Function of Starch Granules using Pea as a Model

A series of near‐isogenic lines have been developed for mutations induced and identified at six independent loci controlling steps in starch biosynthesis of pea (Pisum sativum) seeds. The review describes the methodology associated with the development of these lines and with the characterisation of the starches produced, in particular the effects that the mutations have on the morphology, structure and properties of the starch granules. The shape of starch granules from lines containing mutations at either the r or rug5 loci, encoding a starch branching enzyme and a soluble starch synthase, differ significantly from granules found in the non‐mutant parental line. Mutations at each of the six loci result in characteristic changes in granular structure, which has been studied using polarised light microscopy, wide angle X‐ray diffraction, solid state NMR and differential scanning calorimetry. Relatively small but significant changes were found in the proportion of crystalline material within the granules and much greater differences in the spatial distribution of the crystals within the granules. In particular, granules from r and rug5 mutants do not show the characteristic ‘Maltese cross’ pattern when viewed under polarised light. Large differences were found between the mutants in the proportion of A‐ and B‐type crystallites within the granules, ranging from about 60% A‐type in rug3 mutants to 100% B‐type in r mutant lines. It is suggested that the pea lines are an invaluable resource for answering fundamental questions on the genetic control of starch granule structure and function.     

Conformational flexibility decreased due to Y67F and F82H mutations in cytochrome c: Molecular dynamics simulation studies

Cytochrome c (cyt c), a mitochondrial protein, has dual functions in controlling both cellular energetic metabolism and apoptosis (programmed cell death). During apoptosis, cyt c (Fe³⁺) released into the cytosol initiates caspase activation leading to apoptosis. Since, X-ray crystallography gives only the static structure, we report here the dynamic behavior of holo and apo wild type (WT), Y67F and F82H mutant cyt c's (Fe³⁺) in their apoptotic states. Four nanosecond MD simulations were run for holo WT, Y67F and F82H cyt c's with and without FeS (Met-80) bond and also for apo WT and mutated cyt c's (Y67F and F82H) in water using GROMOS96 force field. Mutations of Y67F and F82H resulted in the decrease of backbone and Cα RMSDs, and radii of gyration (backbone and protein) in both the holo and apo forms. MD and ED results revealed that the flexibility of mutated holo cyt c's decreased perhaps affecting their ability to take part in mitochondrial electron/proton transfer process. Without FeS bond, the backbone and Cα RMSD increased in holo cyt c's perhaps resulting in enhanced peroxidase activity. ED revealed that four to six eigenvectors involved in over all motions of holo cyt c's without FeS bond, and six to eight eigenvectors in apo cyt c's in comparison to three to four eigenvectors for holo cyt c's with FeS bond.     

Insights into RpoB clinical mutants in mediating rifampicin resistance in Mycobacterium tuberculosis

Rifampicin (RIF) an essential first-line anti-tuberculosis (TB) drug, resistance to RIF is a potential threat to TB control program and widely considered as surrogate marker for detection of multi-drug resistant-TB (MDR-TB), molecular understanding of which is the utmost need of the hour. Mutations at RIF resistance-determining region (RRDR) of 81-bp in the rpoB gene coding for β subunit or RpoB protein is the major cause of RIF resistance in Mycobacterium tuberculosis (MTB). Mutation at positions 526 and 531 are generally associated with high-level RIF resistance and at codons 516, 521 and 533 with low-level resistance. Thus, in order to understand the interactions between the clinical mutants (MTs) of RpoB and RIF which are responsible for mediating both levels of RIF resistance from MTB. In the present study, models of wild type (WT) and seven MTs (D516V, L521M, H526D, H526R, H526Y, S531L and L533P) of RpoB from MTB were generated using crystal structure of 2A68 and 4KBM as templates, for deducing 3 domains structure. Molecular docking between RpoB proteins and RIF was carried out, which showed higher values for WT compared to MTs. The high score in WT may be due to the presence of favorable interactions with RIF and MT-L521M which lacks in other MTs. Molecular dynamics (MD) simulation was performed for over 10 nanoseconds, which suggest the root mean square deviation (RMSD) was more and root mean square fluctuation (RMSF) was less in WT compared to MTs. The ligand RMSD exhibited very unique deviation with the MT-D516V compared to other MTs and WT. The RMSF for MTs such as H526R–H526D, L521M and D516V were higher for residues such as 152, 265, 352, 402, 513, 552, and 577 compared to WT. Hydrogen bond interactions at RIF binding site after MD simulations were found comparatively lower in WT than MTs. Similarly, the binding energy of WT was observed to be lesser in comparison to MTs. All MTs demonstrated certain (2 Å) degree of structural deviation from the WT. Overall, these results suggest that RIF binding ability shows differences between WT and MTs, which could be because of different substitutions affecting the conformation of the MT proteins, leading to changes in binding interactions with RIF, eventually to the cause of RIF resistance.