Carbonic anhydrase activators: the first activation study of the human secretory isoform VI with amino acids and amines

The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO(2) hydration reaction proved hCA VI to possess a k(cat) of 3.4 x 10(5)s(-1) and k(cat)/K(M) of 4.9 x 10(7)M(-1)s(-1) (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction, of the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of amino acids and amines were shown to act as CA VI activators, with variable efficacies. l-His, l-Trp, and dopamine showed weak CA VI activating effects (K(A)s in the range of 21-42 microM), whereas d-His, d-Phe, l-DOPA, l-Trp, serotonin, and some pyridyl-alkylamines were better activators, with K(A)s in the range of 13-19 microM. The best CA VI activators were l-Phe, d-DOPA, l-Tyr, 4-amino-l-Phe, and histamine, with K(A)s in the range of 1.23-9.31 microM. All these activators enhance k(cat), having no effect on K(M), participating thus in the rate determining step in the catalytic cycle, the proton transfer reactions between the enzyme active site and the environment.     

Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state

We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.     

Classification of small molecules by two- and three-dimensional decomposition kernels

MOTIVATION: Several kernel-based methods have been recently introduced for the classification of small molecules. Most available kernels on molecules are based on 2D representations obtained from chemical structures, but far less work has focused so far on the definition of effective kernels that can also exploit 3D information. RESULTS: We introduce new ideas for building kernels on small molecules that can effectively use and combine 2D and 3D information. We tested these kernels in conjunction with support vector machines for binary classification on the 60 NCI cancer screening datasets as well as on the NCI HIV data set. Our results show that 3D information leveraged by these kernels can consistently improve prediction accuracy in all datasets. AVAILABILITY: An implementation of the small molecule classifier is available from http://www.dsi.unifi.it/neural/src/3DDK.     

Caloric restrictions affect some factors involved in age-related hypercholesterolemia

Ageing has been defined as a progressive decrease in physiological capacity and a reduced ability to respond to environmental stresses. It has been observed that diet-restricted animals show a minor morbidity in age-related disease. Among these age-related diseases, hypercholesterolemia is the most recurring one and it is often associated with cardiac failure. Several studies have been published indicating age-dependent changes in circulating levels of cholesterol in both humans and in rodents; recently changes have also been reported in the proteins involved in cholesterol homeostasis, that is, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR), Insig-induced gene (Insig) protein, SREBP cleavage activating protein (SCAP), sterol regulatory element binding protein (SREBP), and low density lipoprotein receptor (LDLr). Most age-related modifications of biochemical parameters are normalized or very improved in food-restricted animals, so the aim of this work is to examine whether or not alterations of the factors involved in cholesterol homeostasis which occur during ageing could be counteracted by caloric restriction (CR). The data show that the diet restrictions used attenuate the age-related effects on the factors involved in the synthesis and the degradation rate of HMG-CoAR; in spite of this, CRs have a good effect on the age-related hypercholesterolemia whose reduction seems to depend both on the correct membrane LDLr localization and on the proper restored HMG-CoAR activity.     

Two different pathways are involved in peroxynitrite-induced senescence and apoptosis of human erythrocytes

CO(2) changes the biochemistry of peroxynitrite basically in two ways: (i) nitrating species is the CO(3)(-) / ()NO(2) radical pair, and (ii) peroxynitrite diffusion distance is significantly reduced. For peroxynitrite generated extracellularly this last effect is particularly dramatic at low cell density because CO(3)(-) and ()NO(2) are short-lived and decay mostly in the extracellular space or at the cell surface/membrane. This study was aimed to distinguish between peroxynitrite-induced extra- and intracellular modifications of red blood cells (RBC). Our results show that at low cell density and in the presence of CO(2) peroxynitrite induced the oxidation of surface thiols, the formation of 3-nitrotyrosine and DMPO-RBC adducts, and the down-regulation of glycophorins A and C (biomarkers of senescence). Reactivation of glycolysis reversed only the oxidation of surface thiols. Without CO(2) peroxynitrite also induced the oxidation of hemoglobin and glutathione, the accumulation of lactate, a decrease in ATP, the clustering of band 3, the externalization of phosphatidylserine, and the activation of caspases 8 and 3 (biomarkers of apoptosis). The latter biomarkers were all reversed by reactivation of glycolysis. We hypothesize that cell senescence could (generally) be derived by irreversible radical-mediated oxidation of membrane targets, while the appearance of apoptotic biomarkers could be bolstered by oxidation of intracellular targets. These results suggest that, depending on extracellular homolysis or diffusion to the intracellular space, peroxynitrite prompts RBCs toward either senescence or apoptosis through different oxidation mechanisms.     

Identification of Cdk targets that control cytokinesis

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast. We use phosphoproteome analysis of mitotic exit to identify Cdk targets that are dephosphorylated at the time of cytokinesis. We then apply a new and widely applicable tool to generate conditionally phosphorylated proteins to identify those whose dephosphorylation is required for cytokinesis. This approach identifies Aip1, Ede1 and Inn1 as cytokinetic regulators. Our results suggest that cytokinesis is coordinately controlled by the master cell cycle regulator Cdk together with its counteracting phosphatase and that it is executed by concerted dephosphorylation of Cdk targets involved in several cell biological processes.     

Comparative neurochemical features of the innervation patterns of the gut of the basal actinopterygian,Lepisosteus oculatus,and the euteleost,Clarias batrachus

The structure and physiology of enteric system are very similar in all classes of vertebrates, although they have been investigated only occasionally in non‐mammalian vertebrates. Very little is known about the distribution of the neurotransmitters in the gut of actinopterygian fishes. Anatomical and physiological studies of enteric nervous systems in the spotted gar (Lepisosteus oculatus) and airbreathing catfish (Clarias batrachus), a non‐teleost and teleost actinopterygian, respectively, have not been undertaken. This study provides the first comprehensive characterization of the range of neurochemical coding in the enteric nervous system of these two species, including the chemical diversity of the mucosal endocrine cells in the pyloric stomach of Clarias. Autonomic innervation of the secretory glands is also described and reported herein for the first time for fishes. We also report splanchnic (spinal) innervation of the stomach, submucosal ganglia (that also colocalize with nNOS) and caudal intestine of Clarias. In both fish species, numerous 5HT, ChAT, nNOS and TH‐positive nerve fibres have been observed. These discoveries demonstrate that much more physiological and pharmacological data are needed before a comprehensive model of enteric nervous system control in vertebrates can be developed.