Modulating signaling networks by CRISPR/Cas9-mediated transposable element insertion

In a recent past, transposable elements (TEs) were referred to as selfish genetic components only capable of copying themselves with the aim of increasing the odds of being inherited. Nonetheless, TEs have been initially proposed as positive control elements acting in synergy with the host. Nowadays, it is well known that TE movement into host genome comprises an important evolutionary mechanism capable of increasing the adaptive fitness. As insights into TE functioning are increasing day to day, the manipulation of transposition has raised an interesting possibility of setting the host functions, although the lack of appropriate genome engineering tools has unpaved it. Fortunately, the emergence of genome editing technologies based on programmable nucleases, and especially the arrival of a multipurpose RNA-guided Cas9 endonuclease system, has made it possible to reconsider this challenge. For such purpose, a particular type of transposons referred to as miniature inverted-repeat transposable elements (MITEs) has shown a series of interesting characteristics for designing functional drivers. Here, recent insights into MITE elements and versatile RNA-guided CRISPR/Cas9 genome engineering system are given to understand how to deploy the potential of TEs for control of the host transcriptional activity.Fil: Vaschetto, Luis Maria Benjamin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Cátedra de Diversidad Animal I; Argentin

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases

Changes in apyrase activity in uterus and mammary gland during the lactogenic cycle

1. 1. The purpose of this present research was to explore the possible roles of ATP-diphosphohydrolase (apyrase) in two tissues with high energetic demands during cell proliferation and differentiation. 2. 2. Changes in apyrase activities during the pregnancy lactation cycle were examined in the rat uterus and mammary gland. 3. 3. A significant decrease in apyrase activity (ATPase-ADPase) was observed in the pregnant uterus; this observation correlates with a minor inhibitory effect on platelet aggregation. 4. 4. In mammary gland, the enzyme activity increases during lactation in parallel with an increase in blood supply, synthesis of glycoproteins and cell proliferation. 5. 5. Apyrase activity did not change during the estrous cycle. Estradiol administration to rats slightly increased (20%) both ATPase-ADPase activities. 6. 6. The probable function of apyrase in finally discussed, based on its substrate specifity and subcellular localization. © 1992

Properties of two apyrases from Solanum tuberosum

Two homogeneous isoenzymes of apyrase from Pimpernel and Desirée varieties of Solanum tuberosum were obtained by affinity chromatography on agarose-Cibacron Blue or agarose-ATP-phosphonate columns. Both enzymes split POP bonds of organic and inorganic di- and triphosphates. The ratio of ATPase/ADPase is different for the two apyrases: 10 for Pimpernel and 1 for Desirée. All these activities require bivalent metals. Both isoapyrases have the same MW (49 000) but differ in their pI (8.74 for Pimpernel and 6.69 for Desirée). The optimum pH of hydrolysis of organic di- and triphosphates is 6 (except for Pimpernel ADPase) and 5 for inorganic substrates. Chemical modification of tryptophan, tyrosine, arginine and carboxylic residues decreased all enzymic activities of both enzymes. Protection by substrates and inactivation rates of the individual activities are different for each isoenzyme. © 1982

Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms.

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel

Gelatinase activitiy of matrix metalloproteinases in the cerebrospinal fluid of various patient populations

Artículo de publicación ISIWe have studied the enzymatic gelatinolytic activity of matrix metalloproteinases (MMPs) present in cerebrospinal fluid (CSF) of samples obtained from 67 individuals, twenty-one nonneurological patients (considered controls) and 46 subjects with various neurological disorders e.g., vascular lesions, demyelination, inflammatory, degenerative and prion diseases. Biochemical characterization of MMPs, a family of neutral proteolytic enzymes involved in extracellular matrix modeling, included determination of substrate specificity and Ca+2 dependency, as well as the effects of protease inactivators, carboxylic and His (histidine) residue modifiers, and antibiotics. Whereas all CSF samples expressed MMP-2 (gelatinase A) activity, it corresponded in most cases (normal and pathological samples) to its latent form (proenzyme; pMMP-2). In general, inflammatory neurological diseases (especially meningitis and neurocisticercosis) were associated with the presence of a second enzyme, MMP-9 (or gelatinase B). Whereas MMP-9 was found in the CSF of every tropical spastic paraparesis patient studied, its presence in samples from individuals with vascular lesions was uncommon. Patients blood-brain barrier damage was ascertained by determining total CSF protein content using both, the conventional polyacrylamide gel electrophoresis procedure under denaturing conditions and capillary zone electrophoresis

Inhibition of cyclin-dependent kinase 5 but not of glycogen synthase kinase 3-β prevents neurite retraction and tau hyperphosphorylation caused by secretable products of human T-cell leukemia virus type I-infected lymphocytes

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease characterized by selective loss of axons and myelin in the corticospinal tracts. This central axonopathy may originate from the impairment of anterograde axoplasmic transport. Previous work showed tau hyperphosphorylation at T 181 in cerebrospinal fluid of HAM/TSP patients. Similar hyperphosphorylation occurs in SH-SY5Y cells incubated with supernatant from MT-2 cells (HTLV-I-infected lymphocytes secreting viral proteins, including Tax) that produce neurite shortening. Tau phosphorylation at T 181 is attributable to glycogen synthase kinase 3-β (GSK3-β) and cyclin-dependent kinase 5 (CDK5) activation. Here we investigate whether neurite retraction in the SH-SY5Y model associates with concurrent changes in other tau hyperphosphorylable residues. Threonine 181 turned out to be the only tau hyperphosphorylated residue. We also evaluate the role of GSK3-

Stimulatory response of neutrophils from periodontitis patients with periodontal pathogens

Publicación ISIOBJECTIVE: Neutrophils play a crucial role in the defense of invading bacteria by releasing biologically active molecules. The response of peripheral blood neutrophils was studied in periodontitis-affected patients and in healthy controls towards stimulation to Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) extracts. MATERIALS AND METHODS: Peripheral venous blood was drawn from 23 adult patients with moderate to advanced chronic periodontitis (probing depth >= 5 mm, attachment loss >= 3 mm), and 30 healthy volunteers. Neutrophil response followed by metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) secretion was assayed by zymography and enzyme-linked immunosorbent assay, respectively, on both whole blood and purified neutrophils. In addition to periodontal pathogen extracts, known stimulating agents were tested, such as Escherichia coli-lipopolysaccharide (LPS), phytohemagglutinin, and zymosan A. RESULTS: Neutrophil response, expressed as a secretion ratio under stimulated and non-stimulated conditions, measured in whole blood, showed no differences between periodontitis and healthy controls. Instead, in purified neutrophils from patients, MMP-9 exhibited a significantly higher secretion ratio with LPS and Pg (1.5- to 2-fold), whereas IL-8 showed a larger increase in secretion ratio (3- to 7-fold) in the presence of Pg, Aa, LPS, and zymosan A. CONCLUSION: Peripheral neutrophils of periodontitis-affected patients are more reactive as suggested by their significantly higher response toward periodontal pathogen extracts and other stimulating agents