Pscroph,a parasitic plant EST database enriched for parasite associated transcripts

Background  

Parasitic plants in the Orobanchaceae develop invasive root haustoria upon contact with host roots or root factors. The development of haustoria can be visually monitored and is rapid, highly synchronous, and strongly dependent on host factor exposure; therefore it provides a tractable system for studying chemical communications between roots of different plants.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

Nucleotide Binding to Na,K-ATPase: The Role of Electrostatic Interactions.

The contribution of electrostatic forces to the interaction of Na,K-ATPase with adenine nucleotides was investigated by studying the effect of ionic strength on nucleotide binding. At pH 7.0 and 20 degrees C, there was a qualitative correlation between the equilibrium dissociation constant (K(d)) values for ATP, ADP, and MgADP and their total charges. All K(d) values increased with increasing ionic strength. According to the Debye-Hückel theory, this suggests that the nucleotide binding site and its ligands have "effective" charges of opposite signs. However, quantitative analysis of the dependence on ionic strength shows that the product of the effective electrostatic charges on the ligand and the binding site is the same for all nucleotides, and is therefore independent of the total charge of the nucleotide. The data suggest that association of nucleotides with Na,K-ATPase is governed by a partial charge rather than the total charge of the nucleotide. This charge, interacting with positive charges on the protein, is probably the one corresponding to the alpha-phosphate of the nucleotide. Dissociation rate constants measured in complementary transient kinetic experiments were 13 s(-1) for ATP and 27 s(-1) for ADP, independent of the ionic strength in the range 0.1-0.5 M. This implies similar association rate constants for the two nucleotides (about 40 x 10(6) M(-1) s(-1) at I = 0.1 M). The results suggest that long-range Coulombic forces, affecting association rates, are not the main contributors to the observed differences in affinities, and that local interactions, affecting dissociation rates, may play an even greater role.     

Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.     

Glutathione and Its Metabolic Enzymes in Gliomal Tumor Tissue and the Peritumoral Zone at Different Degrees of Anaplasia

This research was aimed at investigating the features of free radical activity and the parameters of glutathione metabolism in tumor tissues and the peritumoral zone at different degrees of glial tumor anaplasia. We analyzed postoperative material from 20 patients with gliomas of different degrees of anaplasia. The greatest differences compared to adjacent noncancerous tissues were found in the tumor tissue: an increased amount of glutathione and glutathione-related enzymes at Grades I and II, and a decrease of these parameters at Grades III and IV. For the peritumoral zone of Grades I and II, the indices changed in different directions, while for Grades III and IV, they occurred synchronously with the tumor tissue changes. For Low Grade and High Grade gliomas, opposite trends were revealed regarding changes in the level of glutathione and the enzymes involved in its metabolism and in the free radical activity in the peritumoral zone. The content of glutathione and the enzymes involved in its metabolism decreased with the increasing degree of glioma anaplasia. In contrast, free radical activity increased. The glutathione system is an active participant in the antioxidant defense of the body and can be used to characterize the cell condition of gliomas at different stages of tumor development.     

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.     

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.     

Nucleotide-binding kinetics of Na,K-ATPase: cation dependence

Correlation between the Na,K-ATPase affinity to ADP and the cation (its nature and concentration) present in the medium was investigated. In buffer with low ionic strength (I approximately 1 mM) high-affinity ADP binding was not observed, while a stepwise increase in the concentrations of added cation (Na(+), Tris(+), imidazole(+), N-methylglucamine(+), choline(+)) induced an increase in the ADP affinity. The effect was fully saturated at 30-50 mM for all of the cations tested. The maximal affinity for ADP was slightly higher in the presence of Na(+), Tris(+), or imidazole(+) than in the presence of N-methylglucamine(+) or choline(+) (equilibrium dissociation constant K(d) 0.2-0.3 vs 0.7 microM). The ADP dissociation rates from its complex with enzyme in the presence of Na(+) or Tris(+) were similar, implying identity of the nucleotide-binding enzyme conformations, which therefore are assigned to E(1). The ability to compete with K(+) clearly distinguished Na(+) from other cations, which speaks against the sole involvement of the transport sites in the induction of the ADP-binding E(1) conformation. Since the cations are similar in their mode of induction of the high ADP affinity but they demonstrate a pronounced difference in ability to compete with K(+), their effects cannot be combined within any scheme with only one type of cation-binding sites. We suggest that the high affinity toward nucleotide is induced by cation interactions within the protein or lipid and that these nucleotide-domain-related sites coexist with the transport sites, which bind only Na(+) or K(+).     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 microM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03 microM (for arginine) and 3.7 microM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose.