Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase.

Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and T75S, under a wide range of collision energies (40-160 eV). The results established the order of dimer stability as TWT > T75S > Q64E âˆ¼ Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies.

"Clickable", trifunctional magnetite nanoparticles and their chemoselective biofunctionalization.

A multifunctional iron oxide based nanoformulation for combined cancer-targeted therapy and multimodal imaging has been meticulously designed and synthesized using a chemoselective ligation approach. Novel superparamagnetic magnetite nanoparticles simultaneously functionalized with amine, carboxyl, and azide groups were fabricated through a sequence of stoichiometrically controllable partial succinylation and Cu (II) catalyzed diazo transfer on the reactive amine termini of 2-aminoethylphosphonate grafted magnetite nanoparticles (MNPs). Functional moieties associated with MNP surface were chemoselectively conjugated with rhodamine B isothiocyanate (RITC), propargyl folate (FA), and paclitaxel (PTX) via tandem nucleophic addition of amine to isothithiocyanates, Cu (I) catalyzed azide--alkyne click chemistry and carbodiimide-promoted esterification. An extensive in vitro study established that the bioactives chemoselectively appended to the magnetite core bequeathed multifunctionality to the nanoparticles without any loss of activity of the functional molecules. Multifunctional nanoparticles, developed in the course of the study, could selectively target and induce apoptosis to folate-receptor (FR) overexpressing cancer cells with enhanced efficacy as compared to the free drug. In addition, the dual optical and magnetic properties of the synthesized nanoparticles aided in the real-time tracking of their intracellular pathways also as apoptotic events through dual fluorescence and MR-based imaging.

Glycation of albumin and its implication in Diabetes: A comprehensive analysis using mass spectrometry.

AIM: To understand the mechanism of glycation of albumin and effects on cysteinylation and methionine oxidation. METHODS: The in vitro glycation of HSA and BSA was studied with varying concentrations of glucose. Clinical blood samples of diabetic subjects with varying HbA1c values, were analyzed to assess in vivo glycation. All samples and their tryptic digests were analyzed using liquid chromatography/mass spectrometry. Glycation sites were mapped on to the three-dimensional structure of the HSA and BSA. RESULTS: A total thirty-one sites for glycation and eight sites of Nε-carboxymethyl-lysine (CML) modification were identified on albumin. The site selectivity of glycation was correlated with the environment of the reactive residue in the three-dimensional structure. CONCLUSIONS: The maximum percentage glycation under extreme conditions was in the range of ~55 to 88% in four weeks. Two major glycation sites K-233 and K-525 were identified, which together accounted for 40-50% of total glycation. A correlation was observed between glycation and oxidation of methionine residues in samples glycated in vitro. The role of spatially proximate residues in facilitating the glycation process is evident. The tri- and tetra-glycated isoforms of albumin can serve as biomarkers for the severe uncontrolled diabetic state.

Incidental positive emotion modulates neural response to outcome valence in a monetarily rewarded gambling task.

Choices are influenced by incidental emotions. To understand the neural mechanisms underlying the potential effects of incidental emotions on outcome processing, we conducted two experiments measuring feedback-related negativity (FRN) as a function of outcome (gain and loss) and emotional context. Experiment 1 used happy, neutral, and sad faces. Experiment 2 used pleasant, neutral, and unpleasant emotional scenes. We expected that incidental emotions would influence outcome processing at the behavioral level in line with the cognitive themes associated with each emotion. At a neural level, the effect of emotion based on outcome was expected in FRN. Participants chose one of two gambles presented on either side of an emotional face (Experiment 1) or on the scene (Experiment 2), and were later shown the outcome. Behaviorally, both the experiments showed emotion specific carryover effects on outcome experience in line with the cognitive appraisal tendencies associated with specific emotions. In both experiments, mean amplitude of FRN measured related to the outcome at Fz and FCz showed a significant effect of outcome with larger amplitude for loss compared to gain. The interaction between emotion and outcome was significant at FCz in Experiment 1 and at FPz in Experiment 2. The amplitude difference between loss and gain was larger for positive emotional context compared to neutral and negative emotional contexts, indicating a dopaminergic basis moderating the emotion-outcome processing interaction.

Probing the role of highly conserved residues in triosephosphate isomerase--analysis of site specific mutants at positions 64 and 75 in the Plasmodial enzyme.

UNLABELLED: Highly conserved residues in enzymes are often found to be clustered close to active sites, suggesting that functional constraints dictate the nature of amino acid residues accommodated at these sites. Using the Plasmodium falciparum triosephosphate isomerase (PfTIM) enzyme (EC 5.3.1.1) as a template, we have examined the effects of mutations at positions 64 and 75, which are not directly involved in the proton transfer cycle. Thr (T) occurring at position 75 is completely conserved, whereas only Gln (Q) and Glu (E) are accommodated at position 64. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The dimeric structure is weakened in the Q64E and Q64N mutants, whereas dimer integrity is unimpaired in all four T75 mutants. Measurement of the concentration dependence of enzyme activity permits an estimate of Kd values for dimer dissociation (Q64N = 73.7 ± 9.2 nm and Q64E = 44.6 ± 8.4 nm). The T75S/V/C mutants have activities comparable to the wild-type enzyme, whereas a fourfold drop is observed for T75N. All four T75 mutants show a dramatic fall in activity between 35 °C and 45 °C. Crystal structure determination of the T75S/V/N mutants provides insights into the variations in local interactions, with the T75N mutant showing the largest changes. Hydrogen-bond interactions determine dimer stability restricting the choice of residues at position 64 to Gln (Q) and Glu (E). At position 75, the overwhelming preference for Thr (T) may be dictated by the imperative of maintaining temperature stability of enzyme activity. DATABASE: Structural data have been deposited in the Protein Data Bank under accession numbers 4ZZ9, 5BMW, 5BMX, 5BNK and 5BRB.

Role of affect in decision making.

Emotion plays a major role in influencing our everyday cognitive and behavioral functions, including decision making. We introduce different ways in which emotions are characterized in terms of the way they influence or elicited by decision making. This chapter discusses different theories that have been proposed to explain the role of emotions in judgment and decision making. We also discuss incidental emotional influences, both long-duration influences like mood and short-duration influences by emotional context present prior to or during decision making. We present and discuss results from a study with emotional pictures presented prior to decision making and how that influences both decision processes and postdecision experience as a function of uncertainty. We conclude with a summary of the work on emotions and decision making in the context of decision-making theories and our work on incidental emotions.