High sensitivity of human epidermal keratinocytes (HaCaT) to topoisomerase inhibitors

In the panorama of the numerous established cell lines, the human keratinocyte line HaCaT has a very interesting feature, having a close similarity in functional competence to normal keratinocytes. This cell line has been used in many studies as a paradigm for epidermal cells and therefore we selected HaCaT as a cell model for investigating the activity of three antitopoisomerase drugs (Camptothecin, Doxorubicin, Ciprofloxacin) on in vitro cell growth. The effect was evaluated both by a 24-h cytotoxicity test and by a 7-day antiproliferation assay, in which the cell viability was assessed by an MTT (3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenil-2-H-tetrazolium bromide) test. DNA topoisomerase I was also partially purified from a nuclear extract of HaCaT cells, the level of topo I catalytic activity was measured by a pBR322 DNA relaxation assay and then the in vitro effect of antitopoisomerase drugs on the target enzyme was also assessed. The results indicated that the in vitro sensitivity of human epidermal HaCaT cells to antitopoisomerase drugs is comparable to that of many human tumour cell lines. HaCaT cells express a high level of topoisomerase I activity that is significantly inhibited by both Camptothecin and Doxorubicin and to a minor degree by Ciprofloxacin. A high correlation between the cell sensitivity to the antitopoisomerase I drug measured by the MTT test and the in vitro direct inhibition of HaCaT topoisomerase I was observed, suggesting that HaCaT cells can represent a very interesting model both for studying cellular pharmacokinetics of antineoplastic drugs on keratinocytes and for predicting possible secondary effects, exerted by these drugs on cutaneous cells, during treatment with chemotherapy.     

Interaction between Wheat-Germ Agglutinin and acid phosphatase present in dry de-embryonated wheat grains

Abstract

Different hydrolases were found to be present in the crude extract of dry de-embryonated wheat grains, including acid phosphatase. When the crude extract was chromatographed on a Wheat-Germ Agglutinin-Sepharose affinity column, an aliquot of acid phosphatase bound to the column and could be eluted specifically by N-acetyl-D-glucosamine solution. Some properties of the WGA-binding acid phosphatase (pH optimum, heat stability, Triton X-100 sensitivity, inhibition by some ions, molecular weight and K_m) were studied. WGA appeared to have a stabilizing effect on the enzyme, while it was ineffective on the K_m.     

Semi-Supervised Multi-View Learning for Gene Network Reconstruction

The task of gene regulatory network reconstruction from high-throughput data is receiving increasing attention in recent years. As a consequence, many inference methods for solving this task have been proposed in the literature. It has been recently observed, however, that no single inference method performs optimally across all datasets. It has also been shown that the integration of predictions from multiple inference methods is more robust and shows high performance across diverse datasets. Inspired by this research, in this paper, we propose a machine learning solution which learns to combine predictions from multiple inference methods. While this approach adds additional complexity to the inference process, we expect it would also carry substantial benefits. These would come from the automatic adaptation to patterns on the outputs of individual inference methods, so that it is possible to identify regulatory interactions more reliably when these patterns occur. This article demonstrates the benefits (in terms of accuracy of the reconstructed networks) of the proposed method, which exploits an iterative, semi-supervised ensemble-based algorithm. The algorithm learns to combine the interactions predicted by many different inference methods in the multi-view learning setting. The empirical evaluation of the proposed algorithm on a prokaryotic model organism (E. coli) and on a eukaryotic model organism (S. cerevisiae) clearly shows improved performance over the state of the art methods. The results indicate that gene regulatory network reconstruction for the real datasets is more difficult for S. cerevisiae than for E. coli. The software, all the datasets used in the experiments and all the results are available for download at the following link: http://figshare.com/articles/Semi_supervised_Multi_View_Learning_for_Gene_Network_Reconstruction/1604827.     

Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells

Objective

Computational models of calcium (Ca²⁺) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca²⁺ signaling model for RPE based on our experimental data of mechanically induced Ca²⁺ wave in the in vitro model of RPE, the ARPE-19 monolayer.

Methods

We combined six essential Ca²⁺ signaling components into a model: stretch-sensitive Ca²⁺ channels (SSCCs), P₂Y₂ receptors, IP₃ receptors, ryanodine receptors, Ca²⁺ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca²⁺ waves reproduced our control experimental data and data where gap junctions were blocked.

Results

Our model was able to explain Ca²⁺ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca²⁺ through plasma membrane SSCCs and gap junctions conduct the Ca²⁺ and IP³ between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca²⁺ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP₃ receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca²⁺ signal in the distance.

Conclusions

The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P₂Y₂ receptors or accelerate P₂Y₂ receptor phosphorylation, which may partially be the reason for Ca²⁺ wave attenuation by suramin. Being the first RPE Ca²⁺ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.     

Una Iscrizione Commeaiorativa Già Esistente All'Orto Botanico di Padova

Abstract

The use of the mini-plug system for the production of container seedlings is relatively new, so there is little information on the potential impact of method on the quality of planting stock. The objective in this study was to evaluate the impact of mini-plug growing method on quality of Pinus brutia seedlings, and compare the performance of this stock type with that of standard container nursery stock. Seedling survival, growth and physiological status (root growth potential, shoot electrolyte leakage) were measured after pre-cultivation in mini-plugs, at the end of the first growing season in standard containers and after field transplanting. Our results showed that mini-plug transplants of P. brutia seedlings performed as well as the standard planting stock currently used in nursery operation in Greece. For the pre-cultivation of P. brutia seedlings in mini-plugs, the use of peat and a density of 2000 mini-plugs m^?2 are recommended.     

Preparation of a glycosynthase from the β-glycosidase of the Archaeon Pyrococcus horikoshii

The β-glycosidase from the hyperthermophilic Archaeon Pyrococcus horikoshii (Phoβ-gly) is a monomeric enzyme with wide substrate specificity belonging to family 1 of glycoside hydrolases classification. Inspection of the three-dimensional structure of the enzyme, recently resolved, showed that Phoβ-gly is membrane bound and that the residues putatively involved in the catalytic activity are Glu155 and Glu324 working as the general acid/base and the nucleophile of the reaction, respectively. We show here that mutation of the latter completely eliminated the activity of the enzyme and that it could be reactivated in the presence of sodium formate. Analysis of the products obtained in the presence of sodium formate buffer pH 4.0 at 75°C showed that the Glu324Gly mutant acts as a hyperthermophilic glycosynthase.     

Physics and the canalization of morphogenesis: a grand challenge in organismal biology

Morphogenesis takes place against a background of organism-to-organism and environmental variation. Therefore, fundamental questions in the study of morphogenesis include: How are the mechanical processes of tissue movement and deformation affected by that variability, and in turn, how do the mechanic of the system modulate phenotypic variation? We highlight a few key factors, including environmental temperature, embryo size and environmental chemistry that might perturb the mechanics of morphogenesis in natural populations. Then we discuss several ways in which mechanics-including feedback from mechanical cues-might influence intra-specific variation in morphogenesis. To understand morphogenesis it will be necessary to consider whole-organism, environment and evolutionary scales because these larger scales present the challenges that developmental mechanisms have evolved to cope with. Studying the variation organisms express and the variation organisms experience will aid in deciphering the causes of birth defects.     

Efficient extension of slipped DNA intermediates by DinB is required to escape primer template realignment by DnaQ

We show that Escherichia coli DinB polymerase, which creates single-base deletions, prefers to extend slipped DNA substrates with the skipped base at the -4 position. A DinB(Y79L) variant, which extends these substrates less efficiently in vitro, allows the proofreading function of polymerase III to reverse their formation in vivo.