Kinetic mechanism of human dUTPase, an essential nucleotide pyrophosphatase enzyme

Human dUTPase is essential in controlling relative cellular levels of dTTP/ dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize gamma-(32) P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway

Immunoglobulin A Deposition in Jejunal Mucosa of Children with Dermatitis Herpetiformis

Previously we have shown by indirect immunofluorescence (IF) technique that a special IgA antibody in the sera of patients with dermatitis herpetiformis (DH) binds to the structures of the normal jejunum [1]. Now we show by direct IF that specific IgA deposits are present in the proximal jejunum of 11/12 DH and 2/2 celiac patients before a gluten-free diet (GFD). The IgA deposition was in a tubular pattern underlying the villous and crypt epithelial basement membranes and in the lamina propria. This IGA deposition diminished or was not detectable in DH patients under a GFD for a year, and became detectable under gluten challenge in three DH patients. One patient with celiac disease and IGA deficiency, four with other intestinal diseases, and four without jejunal damage had neither jejunal IgA deposition nor circulating IgA anti-jejunal antibody. The deposition of IgA in the jejunum seemed to be correlated with the presence of IgA anti-jejunal antibody in the serum and with the presence of jejunal damage, but the degree of jejunal atrophy, the titer of the anti-jejunal antibody, and the intensity of jejunal IgA deposition in DH patients were not clearly related. Deposition of IgA in the jejunum in DH did not clearly correlate with the activity of the skin symptoms and thus may not be directly related to the pathogenesis of the skin disease of DH

Új és hagyományos irányok a gyermekkori akut lymphoblastos leukaemia biológiájában és ellátásában

Owing to clinical trials and improvement over the past few decades, the majority of children with acute lymphoblastic leukemia (ALL) survive by first-line chemotherapy and combat with the problems of returning to community. However, many patients may have severe acute or late therapeutic side effects, and the survival rate in some groups (e.g., patients with MLL rearrangements, hypodiploidy, IKZF1 mutation or early precursor T cell phenotype) is far behind the average. Innovative strategies in medical attendance provide better clinical outcomes for them: complete gene diagnostics, molecularly targeted anticancer treatment, immuno-oncology and immune cell therapy. The number of genes with identified alterations in leukemic lymphoblasts is over thirty and their pathobiologic role is only partly clear. There are known patient groups where the use of specific drugs is based on gene expression profiling (e.g., tyrosine kinase inhibitors in Philadelphia-like B-cell ALL). The continuous assessment of minimal residual disease became a routine due to the determination of a leukemia-associated immunophenotype by flow cytometry or a sensitive molecular marker by molecular genetics at diagnosis. Epitopes of cluster differentiation antigens on blast surface (primarily CD19, CD20 and CD22 on malignant B cells) can be attacked by monoclonal antibodies. Moreover, antitumor immunity can be strengthened utilizing either cell surface markers (bispecific T cell engagers, chimeric antigen receptor T cell therapy) or tumor-specific immune cells (immune checkpoint inhibitors). This review gives an insight into current knowledge in these innovative therapeutic directions. Orv Hetil. 2018; 159(20): 786-797

Oscillations in Quantum Entanglement During Rescattering

We study the time evolution of quantum entanglement between an electron and its parent ion during the rescattering due to a strong few-cycle laser pulse. Based on a simple one-dimensional model, we compute the Neumann entropy during the process for several values of the carrier-envelope phase. The local maxima of the oscillations in the Neumann entropy coincide with the zero crossings of the electric field of the laser pulse. We employ the Wigner function to qualitatively explain the quantum dynamics of rescattering in the phase space.Comment: 2 page

Disturbance and stress - different meanings in ecological dynamics?

There is an increasing frequency of papers addressing disturbance and stress in ecology without clear delimitation of their meaning. Some authors use the terms disturbance and stress exclusively as impacts, while others use them for the entire process, including both causes and effects. In some studies, the disturbance is considered as a result of a temporary impact, which is positive for the ecosystem, while stress is a negative, debilitating impact. By developing and testing simple theoretical models, the authors propose to differentiate disturbance and stress by frequency. If the frequency of the event enables the variable to reach a dynamic equilibrium which might be exhibited without this event, then the event (plus its responses) is a disturbance for the system. If frequency prevents the variable’s return to similar pre-event dynamics and drives or shifts it to a new trajectory, then we are facing stress. The authors propose that changes triggered by the given stimuli can be evaluated on an absolute scale, therefore, direction of change of the variable must not be used to choose one term or the other, i.e. to choose between stress and disturbance