The pharmacological role of nucleotidases in snake venoms

Several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as nucleotidases (5′nucleotidase, ATPase, and ADPase) are less studied and their pharmacological role in venoms is not clearly defined. Very few studies have shown the pharmacological importance of these endogenous purine release related enzymes in venoms. The near‐ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It is suggested that their major function is in the generation of purines (mainly adenosine)—a multitoxin. Therefore, it appears that these enzymes play a central role in liberating adenosine and through the action of adenosine help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification and biochemical characterization of unique secretory nucleases of the human enteric pathogen, Entamoeba histolytica

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.     

Genome modifications in plant cells by custom-made restriction enzymes

Genome editing, i.e. the ability to mutagenize, insert, delete and replace sequences, in living cells is a powerful and highly desirable method that could potentially revolutionize plant basic research and applied biotechnology. Indeed, various research groups from academia and industry are in a race to devise methods and develop tools that will enable not only site-specific mutagenesis but also controlled foreign DNA integration and replacement of native and transgene sequences by foreign DNA, in living plant cells. In recent years, much of the progress seen in gene targeting in plant cells has been attributed to the development of zinc finger nucleases and other novel restriction enzymes for use as molecular DNA scissors. The induction of double-strand breaks at specific genomic locations by zinc finger nucleases and other novel restriction enzymes results in a wide variety of genetic changes, which range from gene addition to the replacement, deletion and site-specific mutagenesis of endogenous and heterologous genes in living plant cells. In this review, we discuss the principles and tools for restriction enzyme-mediated gene targeting in plant cells, as well as their current and prospective use for gene targeting in model and crop plants.     

Variations in the enzymatic activity of S1-type nucleases results from differences in their active site structures

BackgroundS1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn²⁺-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‑zinc-dependent S1-like nucleases and what is the molecular basis of their activities.MethodsS1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay.ResultsWe discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues.ConclusionsOur findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases.General significanceOur findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‑zinc-dependent members of this family.     

Modulatory role of purines in neuromuscular transmission

The data on purine modulation of myoneural transmission are reviewed. A particular attention is paid to adenosine-5′-triphosphoric acid (ATP), the co-transmitter of the principal mediator (acetylcholine), and adenosine, the final ATP metabolite in the synaptic cleft. The effects of these endogenous modulators on pre- and postsynaptic current are discussed. The contributions of purines to the process of quantal and non-quantal acetylcholine release into the synaptic cleft and the effects of ATP and adenosine on cholinoceptor function have been assessed. It is concluded that the role of endogenous purines is mainly confined to enhancement of the efficiency of neuromuscular transmission and synaptic adjustment of a motor unit at different modes of function.     

Metabolism of DNA secondary structures at the eukaryotic replication fork

DNA secondary structures are largely advantageous for numerous cellular processes but can pose specific threats to the progression of the replication machinery and therefore genome duplication and cell division. A number of specialized enzymes dismantle these structures to allow replication fork progression to proceed faithfully. In this review, we discuss the in vitro and in vivo data that has lead to the identification of these enzymes in eukaryotes, and the evidence that suggests that they act specifically at replication forks to resolve secondary structures. We focus on the role of helicases, which catalyze the dissociation of nucleotide complexes, and on the role of nucleases, which cleave secondary structures to allow replication fork progression at the expense of local rearrangements. Finally, we discuss outstanding questions in terms of dismantling DNA secondary structures, as well as the interplay between diverse enzymes that act upon specific types of structures.     

Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.

     

Propentofylline and Other Adenosine Transport Inhibitors Increase the Efflux of Adenosine Following Electrical or Metabolic Stimulation of Rat Hippocampal Slices

Abstract: Propentofylline is a novel neuroprotective agent that has been shown to act as an adenosine transport inhibitor as well as an adenosine receptor antagonist. In the present series of experiments we have compared the effects of propentofylline with those of known adenosine transport inhibitors and receptor antagonists on the formation of adenosine in rat hippocampal slices. The ATP stores were labeled by incubating the slices with [³H]-adenine. The total ³H overflow and the overflow of endogenous and ³H-labeled adenosine, inosine, and hypoxanthine were measured. Adenosine release, secondary to ATP breakdown, was induced both by hypoxia/hypoglycemia and by electrical field stimulation. Propentofylline (20–500 µM) increased the release of endogenous and radiolabeled adenosine, without increasing the total release of purines. Thus, the drug altered the pattern of released purines, i.e., increasing adenosine and decreasing inosine and hypoxanthine. This pattern, which was observed when purine release was induced both by electrical field stimulation and by hypoxia/hypoglycemia, was shared by the nucleoside transport inhibitor dipyridamole (1 µM) and by mioflazine (1 µM) and nitrobenzylthioinosine (1 µM). By contrast, other xanthines, including theophylline (100 µM) and 8-cyclopentyltheophylline (10 µM), enprofylline (100 µM), or torbafylline (300 µM), if anything, increased the total release of purines without alterations of the pattern of release. These results indicate that nucleoside transport inhibitors can decrease the release of purines from cells and at the same time increase the concentration of extracellular adenosine, possibly by preventing its uptake and subsequent metabolism. This change in purine metabolism may be beneficial with regard to cell damage after ischemia. The results also indicate that propentofylline behaves in such a potentially beneficial manner.     

Measurement of purine release with microelectrode biosensors

Purinergic signalling departs from traditional paradigms of neurotransmission in the variety of release mechanisms and routes of production of extracellular ATP and adenosine. Direct real-time measurements of these purinergic agents have been of great value in understanding the functional roles of this signalling system in a number of diverse contexts. Here, we review the methods for measuring purine release, introduce the concept of microelectrode biosensors for ATP and adenosine and explain how these have been used to provide new mechanistic insight in respiratory chemoreception, synaptic physiology, eye development and purine salvage. We finish by considering the association of purine release with pathological conditions and examine the possibilities that biosensors for purines may one day be a standard part of the clinical diagnostic tool chest.     

Nucleases in homologous recombination as targets for cancer therapy

Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.     

Patatin-related phospholipase A: nomenclature, subfamilies and functions in plants

The release of fatty acids from membrane glycerolipids has been implicated in a variety of cellular processes, but the enzymes involved and their regulation are poorly understood in plants. One large group of acyl-hydrolyzing enzymes is structurally related to patatins. Patatins are potato tuber proteins with acyl-hydrolyzing activity, and the patatin catalytic domain is widely spread in bacterial, yeast, plant and animal enzymes. Recent results have indicated that patatin-related enzymes are involved in different cellular functions, including plant responses to auxin, elicitors or pathogens, and abiotic stresses and lipid mobilization during seed germination. In this review, we highlight recent developments regarding these enzymes and propose the nomenclature pPLA for the patatin-related phospholipase A enzyme.     

Characterization of orthophosphate release from dissolved organic phosphorus by gel filtration and several hydrolytic enzymes

The molecular weight distribution of dissolved organic phosphorus (DOP) and the possible mechanisms of orthophosphate (Pi) release were examined by gel filtration and incubation with some hydrolytic enzymes. Sixty five percent of the DOP appeared to have apparent molecular weights between 300 to 10000 daltons. Less than 10% of the DOP estimated higher molecules greater than 10000 daltons. Alkaline phosphatase released Pi more easily from low molecular weight (< 1500 daltons) DOP than from high molecular weight fractions. While, addition of nucleases or phosphodiesterase alone did not appear Pi release from high molecular weight DOP compounds. Pi release from those DOP compounds increased markedly (more than 30%) when alkaline phosphatase was incubated with nucleases or phosphodiesterase. However, 60% of DOP did not release Pi when alkaline phosphatase was incubated with either enzymes.     

Retake the center stage--new development of rat genetics

The rat is a powerful model for the study of human physiology and diseases,and is preferred by physiologists,neuroscientists and toxicologists.However,the lack of robust genetic modification tools has severely limited the generation of rat genetic models over the last two decades.In the last few years,several gene-targeting strategies have been developed in rats using N-ethyl-N-nitrosourea(ENU), transposons,zinc-finger nucleases(ZFNs),bacterial artificial chromosome(BAC) mediated transgenesis,and recently established rat embryonic stem(ES) cells.The development and improvement of these approaches to genetic manipulation have created a bright future for the use of genetic rat models in investigations of gene function and human diseases.Here,we summarize the strategies used for rat genetic manipulation in current research.We also discuss BAC transgenesis as a potential tool in rat transgenic models.     

Extracellular purines from cells of seminiferous tubules

It has been long postulated that extracellular purines can modulate the function of the male reproductive system by interacting with different purinergic receptors of Sertoli and germinative cells. Many authors have described the biological changes induced by extracellular ATP and/or adenosine in these cells, and some hypothetical models for paracrine communication mediated by purines were proposed; however, the cellular source(s) of these molecules in seminiferous tubules remains unknown. In this study, we demonstrated for the first time that Sertoli cells are able to release ATP (0.3 nmol/mg protein) and adenosine (0.1 nmol/mg protein) in the extracellular medium, while germinative and myoid peritubular cells are able to secrete adenosine (0.02 and 0.37 nmol/mg protein, respectively). Indeed, all the three types of cells were able to release inosine at significant concentrations (about 0.4 nmol/mg protein). This differential secretion depending on the cellular type suggests that these molecules may be involved in the paracrine regulation and/or control of the maturation processes of these cells.