A proteomics view on the role of drought-induced senescence and oxidative stress defense in enhanced stem reserves remobilization in wheat

Drought is one of the major factors limiting the yield of wheat (Triticum aestivum L.) particularly during grain filling. Under terminal drought condition, remobilization of pre-stored carbohydrates in wheat stem to grain has a major contribution in yield. To determine the molecular mechanism of stem reserve utilization under drought condition, we compared stem proteome patterns of two contrasting wheat landraces (N49 and N14) under a progressive post-anthesis drought stress, during which period N49 peduncle showed remarkably higher stem reserves remobilization efficiency compared to N14. Out of 830 protein spots reproducibly detected and analyzed on two-dimensional electrophoresis gels, 135 spots showed significant changes in at least one landrace. The highest number of differentially expressed proteins was observed in landrace N49 at 20days after anthesis when active remobilization of dry matter was observed, suggesting a possible involvement of these proteins in effective stem reserve remobilization of N49. The identification of 82 of differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in leaf senescence, oxidative stress defense, signal transduction, metabolisms and photosynthesis which might enable N49 to efficiently remobilized its stem reserves compared to N14. The up-regulation of several senescence-associated proteins and breakdown of photosynthetic proteins in N49 might reflect the fact that N49 increased carbon remobilization from the stem to the grains by enhancing senescence. Furthermore, the up-regulation of several oxidative stress defense proteins in N49 might suggest a more effective protection against oxidative stress during senescence in order to protect stem cells from premature cell death. Our results suggest that wheat plant might response to soil drying by efficiently remobilize assimilates from stem to grain through coordinated gene expression.     

Mechanistically significant diastereoselection in the sulfoximine inhibition of carboxypeptidase A

The diastereomers of L-alpha-[ [S-(2-phenylethyl)sulfonimidoyl]methyl]benzenepropanoic acid bind differentially to carboxypeptidase A. These putative transition state-analogue inhibitors show unique and interpretationally significant pH dependences for Ki, as well as for the visible absorption spectra of their E.I complexes in the case of the cobalt-substituted enzyme. From the geometry of the enzymically preferred isomer, it may be concluded that the mechanism of peptide scission by the enzyme entails addition of a nucleophile to the si face of the bound-substrate prochiral carboxamide linkage. New interpretational constraints on the mode of action of the enzyme are thereby imposed.     

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

Torsional-strain considerations in enzymology : Some applications to proteases and ensuing mechanistic consequences

A stereoxplicit analysis of amide torsional strain reveals two distinct rehybridization modes, each of which correlates with a unique hydrolysis mechanism. These may be characterized as anti distortion of cis (Z) substitutents (yielding trans addition of HOR) and syn distortion of trans (E) substituents (correlating with cis addition). The latter mechanism is known to apply in the cases of the serine proteases, and supporting evidence for torsional activation is sought in crystallographic structures of trypsin with its inhibitors as well as from kinetic data. A contrast is made with the mechanism of carboxypeptidase A, which operates on the former principle. For this enzyme it is demonstrated that an unusual minor-protonation state is most probably the catalytically active species, a conclusion with some important ramifications. A general comparison of the torsional strain hypothesis is made with a recently propounded theory concerning stereoelectronic control of cleavage of hemiorthoamide tetrahedral intermediates. When these approaches to enzyme mechanism are used in tandem, a unique comprehension is achievable.     

The effect of diethyl- -fluoroglutarate on phosphates and glutamate in slices of rabbit cerebral cortex

Abstract— The diethyl ester of α-fluoroglutarate (DEFG), an inhibitor of glutamate dehydrogenase, was prepared, and its effect on glutamate and phosphates in slices of rabbit cerebral cortex was examined. The primary effect of the drug on cortical slices incubating in a Krebs-Ringer glucose medium was to decrease the tissue levels of glutamate in association with decreased levels and turnover of high-energy phosphates. Assimilation of exogenous glutamate by the slices was partially blocked in the presence of the drug and severely depressed oxidative phosphorylation resulted when glutamate and DEFG were both present in the incubation mixture. The results suggested a significant relationship between the activity of cerebral glutamate dehydrogenase and oxidative phosphorylation. During incubation in a Krebs-Ringer glucose medium the endogenous pool of free amino acids in the cortical slice partitioned with the medium. Little or no glutamate, aspartate or GABA was present in the medium after incubation, but glycine, alanine, threonine, serine and glutamine did partition to varying degrees, with over one-half of the glutamine present in the incubation medium. With the exception of ‘leakage’ of aspartate, the partitioning patterns were relatively unaffected by the presence of added glutamate or DEFG.     

Production of interleukin-6 in Arxula adeninivorans, Hansenula polymorpha and Saccharomyces cerevisiae by applying the wide-range yeast vector (CoMed) system to simultaneous comparative assessment

A wide-range yeast vector (CoMed) system has been applied to the comparative assessment of three different yeast platforms for the production of human interleukin-6. A vector equipped with an rRNA gene targeting sequence and an Arxula adeninivorans-derived LEU2 gene was used for simultaneous transformation of auxotrophic A. adeninivorans, Hansenula polymorpha and Saccharomyces cerevisiae strains. IL6 was expressed under control of the strong constitutive A. adeninivorans-derived TEF1 promoter, which is functional in all yeast species analyzed so far. Secreted IL-6 was found to be correctly processed from an MFalpha1-IL6 precursor in A. adeninivorans only, whereas N-terminally truncated proteins were observed in H. polymorpha and S. cerevisiae.     

Murine splenocytes produce inflammatory cytokines in a MyD88-dependent response to Bacillus anthracis spores

Bacillus anthracis is a sporulating Gram-positive bacterium that causes the disease anthrax. The highly stable spore is the infectious form of the bacterium that first interacts with the prospective host, and thus the interaction between the host and spore is vital to the development of disease. We focused our study on the response of murine splenocytes to the B. anthracis spore by using paraformaldehyde-inactivated spores (FIS), a treatment that prevents germination and production of products associated with vegetative bacilli. We found that murine splenocytes produce IL-12 and IFN-gamma in response to FIS. The IL-12 was secreted by CD11b cells, which functioned to induce the production of IFN-gamma by CD49b (DX5) NK cells. The production of these cytokines by splenocytes was not dependent on TLR2, TLR4, TLR9, Nod1, or Nod2; however, it was dependent on the signalling adapter protein MyD88. Unlike splenocytes, Nod1- and Nod2-transfected HEK cells were activated by FIS. Both IL-12 and IFN-gamma secretion were inhibited by treatment with B. anthracis lethal toxin. These observations suggest that the innate immune system recognizes spores with a MyD88-dependent receptor (or receptors) and responds by secreting inflammatory cytokines, which may ultimately aid in resisting infection.