Carbon Metabolism Enzymes of Rhizobium meliloti Cultures and Bacteroids and Their Distribution within Alfalfa Nodules

Several carbon metabolism enzymes were measured in cultured cells and bacteroids of Rhizobium meliloti 102F51 and in alfalfa root nodule cytosol. The enzyme activity levels of the pentose phosphate pathway were much higher than those of the Embden-Meyerhof-Parnas or Entner-Doudoroff pathways in extracts of cultured cells. The pattern of enzyme activities in the bacteroids was different from that of cultured cells.     

Proteomic analysis of soybean nodule cytosol

An isolation procedure for soybean (Glycine max L. cv Williams 82) nodule cytosol proteins was developed which greatly improved protein resolution by two-dimensional polyacrylamide gel electrophoresis. The most abundant proteins were selected and analyzed by mass spectrometry. The identified proteins were categorized by function (% of total proteins analyzed): carbon metabolism (28%), nitrogen metabolism (12%), reactive oxygen metabolism (12%) and vesicular trafficking (11%). The first three categories were expected based on the known physiological functions of the symbiotic nitrogen fixation process. The number of proteins involved in vesicular trafficking suggests a very active exchange of macromolecules and membrane components. Among the 69 identified proteins were the enzymes of the three carbon portion of glycolysis, which were further characterized to support their roles in the sucrose synthase pathway to provide malate for the bacteroids. Proteomic analysis provides a functional tool by which to understand and further investigate nodule function.     

Pharmacophore-based discovery of 2-(phenylamino)aceto-hydrazides as potent eosinophil peroxidase (EPO) inhibitors

There is an increasing interest in developing novel eosinophil peroxidase (EPO) inhibitors, in order to provide new treatment strategies against chronic inflammatory and neurodegenerative diseases caused by eosinophilic disorder. Within this study, a ligand-based pharmacophore model for EPO inhibitors was generated and used for in silico screening of large 3?D molecular structure databases, containing more than 4 million compounds. Hits obtained were clustered and a total of 277 compounds were selected for biological assessment. A class of 2-(phenyl)amino-aceto-hydrazides with different substitution pattern on the aromatic ring was found to contain the most potent EPO inhibitors, exhibiting IC₅₀ values down to 10?nM. The generated pharmacophore model therefore, represents a valuable tool for the selection of compounds for biological testing. The compounds identified as potent EPO inhibitors will serve to initiate a hit to lead and lead optimisation program for the development of new therapeutics against eosinophilic disorders.     

Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones

Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements of this class with the bisindolylmethanone-structure led to a series of novel compounds. These compounds inhibited EGFR in the nanomolar range. Moreover, inhibition of EGFR autophosphorylation in intact A431 cells was shown, with IC₅₀ values ranging form 0.3–1 μM for compound 42, and 0.1–0.3 μM for 45. In a panel of 42 human tumor cell lines the sensitivity profile of the novel compounds was shown to be similar to that of the quinazoline class of tyrosine kinase inhibitors lapatinib and erlotinib (Tarceva®).     

Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D-PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight at -20 °C, it was carried out for 2 to 3 h at -80 °C. Ethanol was used for the final wash of the protein precipitate instead of routinely used acetone. Dithiothreitol (DTT) was used in all solutions from the beginning, considerably improving the solubilization of precipitated proteins. Solubilization was further improved by using a mixture of detergents and denaturants at high concentrations along with large amounts of DTT. Both in-gel rehydration and cup-loading methods were used for isoelectric focusing (IEF). For in-gel rehydration, samples reduced with DTT were diluted with sample buffer containing 2-hydroxyethyl disulfide (2-HED) (1:3) or were cup-loaded on a strip rehydrated with sample buffer containing 2-HED. Glycerol (5%) was used in the sample buffer, and the focusing was performed at 15 °C. The applicability of the method was demonstrated using several soybean tissues.     

A comparative proteomic evaluation of culture grown vs nodule isolated Bradyrhizobium japonicum

Total protein extract of Bradyrhizobium japonicum cultivated in HM media were resolved by 2-D PAGE using narrow range IPG strips. More than 1200 proteins were detected, of which nearly 500 proteins were analysed by MALDI-TOF and 310 spots were tentatively identified. The present study describes at the proteome level a significant number of metabolic pathways related to important cellular events in free-living B. japonicum. A comparative analysis of proteomes of free-living and nodule residing bacteria revealed major differences and similarities between the two states. Proteins related to fatty acid, nucleic acid and cell surface synthesis were significantly higher in cultured cells. Nitrogen metabolism was more pronounced in bacteroids whereas carbon metabolism was similar in both states. Relative percentage of proteins related to global functions like protein synthesis, maturation & degradation and membrane transporters were similar in both forms, however, different proteins provided these functions in the two states.     

Bradyrhizobium japonicum isocitrate dehydrogenase exhibits calcium-dependent hysteresis

Bradyrhizobium japonicum NADP(+)-dependent isocitrate dehydrogenase was purified both from cultured cells and from the symbiotic form of the bacteria and was found to be identical in terms of N-terminal amino acid sequence, kinetics, and physicochemical properties. Magnesium and glycerol were absolute requirements for maintaining enzyme activity. The N-terminal amino acid sequence of the enzyme was more similar to the sequences from soybean and yeast than to other bacterial sequences. There was no immunological cross-reaction of antibodies from B. japonicum isocitrate dehydrogenase to extracts of soybean, pea, or Escherichia coli, but there was detectable, although weak, cross-reaction of antibodies from E. coli with the B. japonicum enzyme. B. japonicum isocitrate dehydrogenase displayed strong inhibition by NADH, indicating that during symbiotic nitrogen fixation the enzyme activity would be markedly reduced in planta. The enzyme displayed a calcium-dependent hysteresis, with a pronounced lag lasting as long as 2 min. Hysteresis was evident at concentrations of magnesium less than 0.5 mM and calcium greater than 1 microM. The hysteresis could be alleviated by excess magnesium or by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The results suggest two roles for magnesium during catalysis; one magnesium may be needed to convert the enzyme into the steady-state form and the second needed for chelation of isocitrate for catalysis. The calcium-dependent hysteretic behavior of B. japonicum NADP(+)-isocitrate dehydrogenase suggested that this metal could serve as an intracellular regulator during symbiosis.     

Disruption of the aspartate to heme ester linkage in human myeloperoxidase: impact on ligand binding, redox chemistry, and interconversion of redox intermediates

In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here, monomeric recombinant myeloperoxidase (MPO) and the variants D94V and D94N were produced in Chinese hamster ovary cell lines. Disruption of the Asp(94) to heme ester bond decreased the one-electron reduction potential E'(0) [Fe(III)/Fe(II)] from 1 to -55 mV at pH 7.0 and 25 degrees C, whereas the kinetics of binding of low spin ligands and of compound I formation was unaffected. By contrast, in both variants rates of compound I reduction by chloride and bromide (but not iodide and thiocyanate) were substantially decreased compared with the wild-type protein. Bimolecular rates of compound II (but not compound I) reduction by ascorbate and tyrosine were slightly diminished in D94V and D94N. The presented biochemical and biophysical data suggest that the Asp(94) to heme linkage is no precondition for the autocatalytic formation of the other two covalent links found in MPO. The findings are discussed with respect to the known active site structure of MPO and its complexes with ligands.