Increase In Ribonuclease Activity Research Articles

Changes in RNase activities and in expression of two RNase genes in powdery mildew infected barley, wheat and Brachypodium distachyon leaves

AbstractChanges in RNase activities were investigated in extracts from barley near isogenic lines without or with various powdery mildew resistance genes and were compared to changes in wheat and Brachypodium distachyon leaves after powdery mildew infections. In barley, the compatible interaction with powdery mildew induced the highest increase in RNase activity as measured spectrophotometrically. The incompatible interaction that accompanied with hypersensitive reaction in Mla leaves gave less increase, whereas incompatible interactions in Mlg and mlo barley leaves without visible symptoms gave the least increase of RNase activity. In wheat, the largest RNase activity was found in leaves infected with the compatible wheat powdery mildew or wheat stem and leaf rusts. RNase activity in B. distachyon was higher than that in healthy wheat and especially barley leaves. The electrophoretic RNase enzyme activity patterns were different in barley, wheat and B. distachyon plants, but showed similar activities as determined spectrophotometrically. Barley genes encoding endonuclease 2 and ribonuclease 3-like protein X3 showed the highest expression in the compatible barley - barley powdery mildew interaction as measured by RT-qPCR. This correlated with RNase activities in leaf extracts suggesting that RNases in barley and wheat may act as susceptibility factors of powdery mildew and rust diseases.

Relationship between formation of gametophore buds in the protonema of mosses and increase in ribonuclease activity

Changes in RNase activity similar to those accompanying cytokinin-induced formation of gametophore buds in mosses (a decrease in the early phase of bud formation and later an increase in enzyme activity) have also been found during spontaneous formation of gametophores in moss ontogenesis. Using various factors affecting the cytokinin-induced process of bud formation a correlation has been found between this process and the increase in RNase activity.

Influence of gibberellic acid on hydrolytic enzymes activity in germinating different-aged lupin seeds

The influence of GA<sub>3</sub> -on the germination of different-aged lupin seed and α-amylase, protease and ribonuclease activity in the embryonic axis and cotyledons of these seeds were investigated after 24, 48, 72 and 96 h of germination. GA<sub>3</sub> treatment of older seeds greatly reduced the per cent of abnormal seedlings and increased ribonuclease activity, and, at the beginning of germination, also of amylase, whereas protease activity remained unchanged. Young seeds did not show any definite reaction to GA<sub>3</sub> with the exception of a slight increase in ribonuclease activity.

Ribonuclease activity of buckwheat

Ribonucleases (RNases) are present in base-level amounts in intact plants, but this level is able to increase greatly under stress conditions. The possible cause for such an increase is protection against plant RNA-virus attack. Buckwheat burn virus (BBV) is a highly virulent pathogen that belongs to Rhabdoviridae family. In our study, we have analyzed the correlation between RNase activity and resistance of different buckwheat cultivars to BBV infection. Two cultivars, Kara-Dag and Roksolana, with different sensitivities to BBV have been used. Kara-Dag is a cultivar with medium sensitivity to virus and Roksolana is a tolerant cultivar. It has been shown that the base level of RNase activity in Roksolana cultivar was in most cases higher than the corresponding parameter in Kara-Dag cultivar. Both infected and uninfected plants of Roksolana cultivar demonstrated high RNase activity during two weeks. Whereas infected plants of Kara-Dag cultivar demonstrated unstable levels of RNase activity. Significant decline in RNase activity was detected on the 7th day post infection with subsequent gradual increase in RNase activity. Decline of the RNase activity during the first week could promote the virus replication and therefore more successful infection of upper leaves of plants. Unstable levels of RNase activity in infected buckwheat plants may be explained by insufficiency of virus-resistant mechanisms that determines the medium sensitivity of the cultivar to BBV. Thus, plants of buckwheat cultivar having less sensitivity to virus, displayed in general higher RNase activity.

Glucocorticoids Modulate MicroRNA Expression and Processing during Lymphocyte Apoptosis

Glucocorticoids modulate immune development and function through the induction of lymphocyte apoptosis via mechanisms requiring alterations in gene expression. Recently, short, noncoding, microRNAs have been identified as key regulators of lymphocyte function; however, it is unknown whether glucocorticoids regulate noncoding microRNAs and whether this regulation contributes to lymphocyte apoptosis. We now show by both microarray and deep sequencing analysis that microRNAs are substantially repressed during glucocorticoid-induced apoptosis of primary rat thymocytes. Mechanistic studies revealed that primary microRNA transcripts were not repressed, whereas the expression of the key microRNA processing enzymes: Dicer, Drosha, and DGCR8/Pasha, were significantly reduced at both the mRNA and protein levels during glucocorticoid-induced apoptosis. To delineate the role of Dicer depletion and microRNA repression in apoptosis, we silenced Dicer expression in two human leukemic cell lines and demonstrated that Dicer depletion significantly enhanced glucocorticoid-induced apoptosis in both model systems. Finally, in vitro and in vivo overexpression of the conserved miR-17-92 polycistron, which was repressed significantly by dexamethasone treatment in both our microarray and deep sequencing studies, blunted glucocorticoid-induced apoptosis. These studies provide evidence of altered post-transcriptional microRNA expression and the repression of the microRNA bioprocessing pathway during glucocorticoid-induced apoptosis of lymphocytes, suggesting a role for microRNA processors and specific microRNAs in cell life/death decisions.

RNA polymerase activities in isolated nuclei of guinea pig seminal vesicle epithelium: influence of castration and androgen administration.

Nuclei from seminal vesicle epithelium of adult guinea pigs were isolated in hypertonic sucrose solution. The incorporation of [3H]UTP by the isolated nuclei into acid-precipitable products was studied. Incorporation required ATP, GTP, CTP, UTP, and Mg+2. It was inhibited by addition of actinomycin D, deoxyribonuclease, or pyrophosphate to the reaction mixture. Thus, incorporation of [3H]UTP by isolated nuclei had the same characteristics that have been demonstrated for the reactions catalyzed by nuclear RNA polymerases. Using alpha-amanitin as a metabolic tool, we established concentrations of (NH4)2SO4. Mg+2, and nucleotides that give maximum assayable activities of nuclear RNA polymerases I and II. When the activities of polymerases I and II were measured in isolated seminal vesicle nuclei of guinea pigs that had been castrated 4 days earlier, a marked decrease in activities was found relative to control values (nuclei from intact animals). No further decrease was found 8 days after castration. Diminished accessibility to the nuclear DNA template and a decrease in the concentration of RNA polymerase molecules seemed to be responsible for the observed effects of castration on activities of RNA polymerases. An increase in ribonuclease activity did not seem to be responsible for the effects of castration. Activities of the enzymes did not change 2, 3, or 4 hours after intraperitoneal injection (2 mg/kg body weight) of each of five different androgens. Similarly, a single intraperitoneal injection of testosterone did not restore enzyme activity of polymerade I or II at any time during the first 24-hour period after hormone administration.

Ribonuclease activity in roots of soybean seedlings subjected to chilling stress and recovery

Many developmental and physiological changes, including alterations of enzyme activities, occur in plants under low temperature stress. In this study the total ribonuclease activity was determined in crude extracts from root tips of soybean seedlings germinated at 25 °C, subjected to chilling conditions (10°C) and recovered at optimal temperature (25°C). Measurements of RNase activity were performed every 24 hours starting from the third to the 10-th day of growth. We found that chilling caused a considerable increase in ribonuclease activity (in comparison with the control), with an activity peak on the fourth day of the cold treatment. The enzyme activity in root extracts of the plants recovered after cold stress decreased along with the time of recovery.

Induction of RNase and nuclease activity in cultured maize endosperm cells following sucrose starvation

The activity of RNases and nucleases in plants often increases following exposure to many types of stress, including prolonged exposure to dark or phosphate starvation. In cereals, the activity of RNases and nucleases is also regulated developmentally during late seed development. In this study, we investigated the effect that the absence of sugar or phosphate in culture medium has on the activity of RNases and nucleases expressed in maize endosperm suspension cells. Withdrawal of sugar from the culture medium resulted in a substantial increase in RNase and nuclease activities, whereas deprivation of phosphate during the same period of growth had no detectable effect on either of these activities. The increase in RNase activity was limited to the neutral RNases, demonstrating that the effect of sugar starvation is specific to one class of RNase. Elimination of asparagine from the medium resulted in a transient reduction in nuclease but not in RNase activity. These observations suggest that sugar starvation constitutes a stress to which maize endosperm responds, in part, by increasing neutral RNase and nuclease activity.

Parallel acceleration of phosphoenolpyruvate carboxykinase mRNA degradation and increase in ribonuclease activity induced by insulin in cultured rat hepatocytes.

In cultured rat hepatocytes, glucagon increased phosphoenolpyruvate carboxykinase mRNA transiently. Insulin, given at the maximal increase, enhanced the degradation by 3-fold. The levels of beta-actin mRNA and ribosomal RNA, which served as a control, remained unchanged. The transcriptional inhibitor, actinomycin D, or the serine/threonine phosphatase IIA inhibitor, okadaic acid, prevented the degradation of phosphoenolpyruvate carboxykinase mRNA. This indicated that the degradation of phosphoenolpyruvate carboxykinase mRNA requires the de novo synthesis of a bona fide destabilizing factor and/or active protein phosphatase. In vitro RNA degradation assays were developed in order to investigate whether insulin-treated cells contained enhanced ribonuclease activity. Fractionated cytosolic extracts were prepared by removing cell organelles by differential centrifugation and thereafter part of the cytosolic proteins by heat treatment. These extracts were incubated with exogenously added total RNA and the degradation of phosphoenolpyruvate carboxykinase mRNA, beta-actin mRNA and 28S ribosomal RNA was studied. In this assay, phosphoenolpyruvate carboxykinase mRNA and the otherwise stable beta-actin mRNA and ribosomal RNA were degraded 3-fold faster by extracts from insulin-treated, than from untreated, cells. The increase in RNase activity induced by insulin could be prevented by treatment of cultured rat hepatocytes with actinomycin D, indicating that ongoing gene transcription was required. The 'in vivo' specificity of the insulin effect on PCK mRNA degradation in cultured hepatocytes seemed to be lost in the in vitro assay in cytosolic extracts due to the disruption of the intracellular environment. Also in whole cell lysates, which were obtained by hypo-osmotic shock of the cells, and which contained the disrupted particulate and all soluble cellular components, PCK mRNA as well as beta-actin mRNA and ribosomal RNA, was degraded. The increase in ribonuclease activity due to insulin paralleled the insulin-induced acceleration of phosphoenolpyruvate carboxykinase mRNA degradation in cultured hepatocytes, which might indicate a functional correlation.

Temperature-sensitive mRNA degradation is an early event in hepatocyte de-differentiation.

The isolation and culture of metabolically active hepatocytes by proteolytic digestion of the extracellular matrix of the liver results in the transcriptional silencing of liver-specific genes encoding cytochromes P-450 (CYP) and albumin together with an induction of cellular RNase activity. The levels of albumin mRNA are maintained in cultured hepatocytes at similar levels to that present in the intact liver for at least 24 h, whereas the major constitutively expressed CYP2C11 mRNA is rapidly degraded. Hepatocytes heat-shocked at 40 degrees C during the isolation procedure (which results in an induction of heat-shock protein mRNA species) blocks the increase in RNase activity and abrogates the loss of CYP2C11 mRNA for at least 4 h. Cycloheximide-dependent inhibition of protein synthesis blocks the temperature-dependent induction of heat-shock proteins without affecting the protection afforded to CYP2C11 mRNA, indicating that CYP2C11 mRNA levels are not directly dependent on heat-shock protein induction and suggesting that the induction of RNase activity might be responsible for the specific loss of CYP2C11 mRNA in hepatocytes isolated at 37 degrees C. Differential rates of degradation of CYP2C11 transcribed in vitro and of albumin mRNA are observed in the presence of cellular extracts from cultured hepatocytes isolated at 37 degrees C (which have maximally induced levels of cellular RNase activity) but not in comparable extracts from cultured hepatocytes isolated at 40 degrees C, supporting the hypothesis that an RNase activity is induced in culture that specifically degrades CYP2C11 mRNA but not albumin mRNA. These results suggest that an early event in hepatocyte de-differentiation involves the induction of RNase activity in addition to transcriptional silencing of liver-specific genes and that the induced RNase activity demonstrates specificity within liver-specific gene products.

Improvement ofescherichia coli cellfree system by utilization of cell extract having additional property

In the Escherichia coli cell-free system, the modification of cell extract can be achieved by preparation of the strains carrying additional property or those being induced with a certain gene expression prior to harvesting. In this study, we analyzed the cell-free system with S30 extract containing T7 RNA polymerases (S30 extract-T7pol) prepared from E. coli BL21(DE3) strain, which includes T7 RNA polymerase from extrinsic genes by IPTG induction, as a model for the improvement of the cell-free system. The fact that a significant degree of mRNA degradation was observed in the cell-free system with S30 extract-T7pol indicates the increase of ribonuclease activity was an unfavorable influence derived from the cell-extract modification process. We also showed that this influence was settled by the addition of an effective ribonuclease inhibitor, such as copper (II) ion, to the reaction mixture.

Myocardial mRNA content and stability, and enzyme activities of Ca-cycling and aerobic metabolism in canine dilated cardiomyopathies.

Idiopathic dilated cardiomyopathy is associated with derangement of myocardial sarcoplasmic Ca-homeostasis and energy production. The molecular mechanism for these changes is unknown. Accordingly, we used genetic and experimentally-induced models of canine dilated cardiomyopathy and tested the hypothesis that these metabolic changes resulted from altered gene expression, as indicated by mRNA content. We studied dilated cardiomyopathy occurring naturally (n = 9) in Doberman pinschers, and in dogs subjected to rapid ventricular pacing (n = 5), in comparison with normal dogs (n = 9). We determined content and integrity of mRNA's using Northern and slot blotting, and measured activities of their translated product for the Ca-release channel and Ca-ATPase of sarcoplasmic reticulum, lactate dehydrogenase of glycolysis, citrate synthase of the tricarboxylic acid cycle, and for myoglobin, ATP-synthetase and the adenine nucleotide transporter, which are integral in oxidative phosphorylation. We found that, whereas both mRNA content and enzyme activity for markers of Ca-cycling, glycolysis, and oxidative phosphorylation were downregulated (20-80%) in dilated cardiomyopathy, they were upregulated (10-15%) for tricarboxylic acid cycling and for ribosomal RNA. RNA from cardiomyopathic tissue was up to 50% more degraded than for normal hearts in association with a 150% increase in ribonuclease activity. Downregulation of the Ca-cycle was asymmetric, with the Ca-channel being 65% more affected than the Ca-ATPase. This work supports the general paradigm that transcriptional and translational responses to pathophysiology are major determinants of the metabolic response seen in cardiac failure.

A protein RNase inhibitor (RNasin) expresses anti-angiogenic properties in mice.

Angiogenin is a protein which in addition to its ability to induce angiogenesis can function as a ribonuclease [1].1 First described and isolated by Vallee and his collaborators, it has since been fully sequenced and subjected to extensive biochemical analysis. Angiogenin is a potent angiogenesis-inducing molecule. Angiogenin shows selective ribonuclease activity, readily breaking down tRNA but yielding larger intermediate fragments than pancreatic ribonulease A when acting on 18S or 28S RNA. Angiogenin has been synthesized by recombinant technology and site-specific mutagenesis studies have been carried out to help identify those moieties significant for ribonuclease and angiogenic activity of the molecule. Interestingly, while in most instances the efficacy of angiogenin as an angiogenesis inducer and as a ribonuclease were correlated, mutants have now been identified which show an increase in ribonuclease activity with a concomitant decrease in angiogenic activity. Recently, Vallee and his colleagues have suggested that the Rnase active site is required but not sufficient for angiogenic activity and that a second site on the molecule is required in addition [2]. What mechanism allows angiogenin to act as an inducer of new blood vessel formation is not known, although binding studies suggest that angiogenin can act directly on endothelial cell targets.

Glucocorticoid-mediated muscle atrophy: alterations in transcriptional activity of skeletal muscle nuclei

Glucocorticoid-induced musle atrophy is associated with a decrease in the level of protein synthesis and a loss of RNA. This paper reports the behaviour of RNA polymerase I- and RNA polymerase II-directed transcription (EC 2.7.7.6) in nuclei isolated from skeletal muscles of rats given a catabolic dose of dexamethasone acetate (5 mg per Kg body weight) over a period of 4 days. Both activities were altered by teh dexamethasone treatment. In the case of RNA polymerase I-mediated transcription there was a loss of template-engaged indicating the existence of an inhbition of initiation of transcription while the rate of elongation of bound enzymes was unaltered. The number of RNA polymerase II-chromatin bound enzymes was increased, but the mean polynucleotide elongation rate was reduced. The possiblity that glucocorticoids may impair the elongation stage of transcription in skeletal muscle by increasing the frequency of premature termination of transcripts is discussed. No evidence was obtained for any increase in ribonuclease activity in muscle nuclei of dexamethasone-treated animals.

Formation of enzyme—substrate disulfide linkage during catalysis by protein disulfide isomerase

During the regeneration of native ribonuclease A (RNase) from the disulfide scrambled molecule by protein disulfide isomerase (PDI), the substrate forms a covalent intermediate with the enzyme through disulfide linkage(s). This has been shown by the appearance of a band at the molecular weight position expected in SDS-PAGE at the same time as the increase in RNase activity. The new band decreased when the regeneration of RNase activity approached completion and disappeared by treatment of the reaction mixture with excess dithiothreitol.

Extracellular RNase activity in healthy and rust infected wheat leaves

Intercellular washing fluids (IWFs) from six spring wheat cultivars were tested for RNase activities. IWFs from all of the cultivars showed relatively high RNA-splitting activity. The activities of the IWFs were raised by 25–55 % after infection with stem or stripe rust in compatible combinations. The incompatible combinations resulted only in a 10–17 % increase in RNase activity. Polyacrylamide gradient gel electrophoresis of the IWFs revealed up to six bands with RNase activity. In IWFs from infected leaves in susceptible combination the activity of bands of about 12 and 16 kD was raised. There were only slight changes in the activity of the higher molecular weight bands, except that the band of about 100 kD in the IWFs from the cultivar Arkas showed a very characteristic increase of activity after infection of the leaves. Moreover, rust infection increased not only the activity but also the pH optimum of the same RNase enzymes. Comparing the RNA-splitting pattern of the IWFs with that of the leaf homogenates in gel, the differences were more pronounced in the case of the infected plants. In homogenates of susceptible leaves after infection not only was the increase in total RNase activity much higher than in the IWFs, but besides the low molecular weight bands, the activities of bands of about 75 kD and of 20–30 kD were strengthened. On the other hand, the much higher specific RNase activity (calculated on protein basis) of the IWFs, as compared to the homogenates, suggests that RNases found in the IWFs are located in the cell wall and in the outer cell membranes. The significance of the extra- and intracellular RNases in rust infection is discussed.

Effect of Salinity Stress on Ribonucleolytic Activities in Glycophytic and Halophytic Plant Species

Summary The ribonuclease activities (RNase, EC 2.7.7.17) of the halophyte, Suaeda maritima (L.) DUM var. macrocarpa MOQ, and the glycophytes, Phaseolus vulgaris (L.) and Vicia faba (L.), were studied in relation to the salinity (NaCl) of the nutrient medium. A reduction of growth was observed for the three species subjected to «salt stresso (i.e. hypersalinity for glycophytes or absence of salt for the halophyte). Both short-term (salt shock) and long-term treatments with NaCl induced a marked increase in RNase activity, and a close relationship was established between «salt stress» and a high RNase activity. The stimulation of RNase activity was independent of the species (halophyte or glycophyte), the analyzed organ (root or leaf) and the nature of the salt treatment (saline shock or steady state culture). Ribonuclease activities of S. maritima leaves and V. faba roots were studied in detail by ion-exchange chromatography. Under the influence of salt treatment it appears that both anionic and cationic RNase activities were increased. It has been demonstrated for the first time that an anionic RNase is stimulated to a greater extent than any other ribonucleolytic enzyme in both the halophyte and the glycophyte. The RNases, particularly the anionic enzymes, can thus be considered as good markers for conditions of saline contraint.

Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

The nature of the increase in ribonuclease activity in germinating seeds of cowpea treated with gibberellic acid or cyclic-amp

There was a two- to three-fold stimulation of RNAase activity by the application of GA 3 and cAMP to cowpea seedlings. The increase in activity was inhibited by the administration of actinomycin D, cycloheximide, cordycepin, 5-fluorouracil and amino acid analogues. Purification of labelled RNAase revealed that GA 3 and cAMP enhanced the RNAase activity predominantly by its fresh synthesis.

Changes in brain protein synthesis during the life span of male Fischer rats.

The cell-free protein synthetic activity of the postmitochondrial supernatant isolated from whole brain of 6- to 32-month-old male Fischer F344 rats was compared. Protein synthesis decreased 56% from 6 to 32 months of age. The decrease in cell-free protein synthesis was not due to an age-related increase in RNase activity. Although monomeric ribosomes (ribosomes stripped of mRNA) isolated from the brains of older rats were less active in polyuridylic acid directed polyphenylalanine synthesis, the fidelity of polyuridylic acid translation by monomeric ribosomes did not decrease with increasing age.