Acid Ribonuclease Research Articles

Effect of sucrose in a holding solution on some processes associated with ageing of cut carnations

Vase-life of cut carnations and their response to a preservative (8-HQS+S) depended on the sucrose and reducing sugar content in the petals at the time of harvest. The reducing sugar level decreased in the wilted flowers kept in water and increased above the initial level in the carnations given sucrose in a holding solution. This increase in reducing sugars was small or 2-3 fold in the flowers with high and low initial sugar levels, respectively. Changes in the endogenous sucrose levels in the petals were less pronounced but a trend (i.e. increase-decrease) was also dependent on the initial sucrose level. Considerable sucrose accumulation was found in the leaves of plants placed in the solution of 5% sucrose plus 200 ppm 8-HQS. Use of an exogenous sugar in a holding solution prevented an increase in free amino acids in carnation petals, a symptom for flower ageing. Carnations placed in a preservative solution had a had anthocyanin content then the control flowers. Lower activities of acid phosphatases and RNases were found in the flowers placed in the solutions tested as compared to those kept in water.

Effect of excretion-secretion products of some fouling species on the biochemical parameters of blue mussel Mytilus edulis L. (Mollusca: Bivalvia) in the White Sea

The effect of excretion-secretion products (ESP) of five abundant fouling invertebrate species (bivalve mollusks Hiatella arctica and Mytilus edulis, solitary ascidia Styela rustica, sponge Halichondria panicea, and sea starAsterias rubens, inhabiting the White Sea) on the biochemical status of blue mussel M. edulis was assessed by the dynamics of lysosomal enzymes activity (nucleases, glycoside hydrolases, and cathepsins). ESP of conspecific species had no effect on the metabolism of the mollusks of this species. ESP of A. rubens, S. rustica, and H. panicea activated the same enzymes. First, acid RNase and glycoside hydrolases activity increased, but in different ways. The metabolites of H. arctica affected the activity of proteometabolism enzymes.

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN CATHARANTHUS ROSEUS L. IN RESPONSE TO BORON NUTRITION

Catharanthus roseus L., a medicinally important plant was grown till maturity at varying levels of boron (0.033, 0.066, 0.33 and 3.3 mg B L−1) supply. Optimum yield was observed in plants receiving 0.33 mg B L−1. Plants receiving deficient boron showed growth reduction and visual symptoms such as chlorosis and cupping of young emerging leaves and apical tip necrosis. The number and size of the flowers, pods and seeds formed and the pollen viability of the boron deficient plants was markedly reduced. The threshold values for deficiency and toxicity were 57 and 79 μg B g−1 dry weight for vegetative growth and 60.4 and 68.9 μg B g−1 dry weight for reproductive growth, respectively, and is reported for the first time in periwinkle. An increase was observed in reducing and non-reducing sugars and in activity of acid phosphatase and ribonuclease in boron stressed plants.

Study of some Hydrolytic Enzymes in Sera and Saliva of Patients with Oral Epithelial Tumors

The present study was designed to investigate the changes in the activity and specific activity of some hydrolytic enzymes like - amylase, alkaline and acid RNase in sera and saliva of patients with oral epithelial tumors (benign and malignant). Then follow up the variation of these enzymes using polyacrylamide gel electrophoresis technique to evaluate the changes of these isoenzymes due to the presence of the tumors. Thirty three patients with oral tumors (benign and malignant) aged from 15-75 years attending the specialized surgery hospital in Baghdad medical city, were included in the present study. Thirty one healthy individuals of matched age and gender were utilized as control. The results indicated the presence of highly significant increase (P> 0.001)of sera alk. RNase activity (U/L) in both benign and malignant groups in comparison to that of control. The specific activity results (U/mg) showed also a significant increase (P> 0.05) in both benign and malignant groups. The results of acid RNase activity and specific activity reflected a non significant increase in both benign and malignant groups. The salivary amylase activity (U/L) and specific activity (U/mg) showed significant decrease (P> 0.001) in malignant group, whereas the sera amylase activity and specific activity revealed a non significant increase in both benign and malignant groups.

Correlation between influence of polysaccharides on hydrolase activity and their antiviral effect in tobacco leaves

The activities of hydrolases (acid phosphatase, RNase, and proteases) in healthy and tobacco mosaic virus-infected leaves of Nicotiana tabacum L. var. Samsun, both untreated and treated with polysaccharides (PS) (1,3;1,6-β-D-glucan, fucoidan, and κ/β-carrageenan), were determined. The PS lead to substantial increase in the hydrolase level. The percentage of viral particles undergoing destructive change also increases in leaves treated with PS 24 h before infection. We suppose that the PS-mediated hydrolase activation promotes intracellular destruction of the viral particles and, thus, comprises one of the PS-induced protective mechanisms limiting intracellular viral accumulation.

Comparison of the baculovirus‐insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2

We report the expression of recombinant RNASET2, the only human member of the Rh/T2/S family of acid ribonucleases, in the yeast Pichia pastoris and the baculovirus-insect cell heterologous systems. In both models, the yield of recombinant protein was comparable and ranged between 5 mg/L (for a catalytically impaired mutant version of RNASET2) and 30 mg/L for the wild-type protein. Thus, the produced protein version rather than the expression system used appears to influence protein yield after optimization of culture conditions. The recombinant protein was found to undergo heterogeneous glycosylation in both systems, particularly in P. pastoris. Most importantly, the wild-type protein purified from both systems was found to be catalytically competent. The expression of recombinant RNASET2 in both systems will allow the implementation of functional assays in vivo and in vitro to better define the antioncogenic properties of this member of the Rh/T2/S ribonuclease family.

The Structure of the Mammalian RNase H2 Complex Provides Insight into RNA·DNA Hybrid Processing to Prevent Immune Dysfunction

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

Impact of Boron Deficiency on Changes in Biochemical Attributes, Yield, and Seed Reserves in Chickpea

To determine the effect of boron (B) deficiency on biomass, reproductive yield, metabolism, and alterations in seed reserves of chickpea (Cicer arietinum L.) cv. ‘13.G‐256,’ plants were grown in refined sand until maturity at deficient (0.033 mg L−1) and adequate (0.33 mg L−1) B, supplied as boric acid (H3BO3). Boron‐deficient plants exhibited visible deficiency symptoms in addition to reduced number of pods and seeds, resulting in lowered biomass and economic yield. Boron deficiency lowered the concentration of B in leaves and seeds, photosynthetic pigments (leaves), Hill reaction activity, starch (in leaves and seeds), and proteins and protein N (in seeds), whereas phenols, sugars (in leaves and seeds), and nonprotein N (in seeds) were elevated. Specific activity of peroxidase (POX) increased in leaves and pod wall and decreased in seeds, while activity of acid phosphate and ribonuclease were stimulated in leaves, seeds, and pod wall in B‐deficient chickpea.

Paraquat and roundup effects on nucleases activities of alfalfa seedlings and alfalfa nucleases activities on paraquat-treated and roundup-treated nucleic acids

The specific activities of acid (pH 5.5) and neutral (pH 7) DNases and RNases were determined in alfalfa (Medicago sativa L.) seedlings grown in the dark in the presence of 3.7 mM paraquat (PQ) or 1 mM roundup (RD). Seedlings were taken at 0, 1, 3, and 5 days. Plant growth parameters (plant height and fresh weight) were dramatically reduced under these conditions of growth comparing to the control (grown in water). The DNase and RNase specific activities of herbicide-treated seedlings were reduced. The reduction of activities ranged by about 50–90 and 15–70% in PQ- and RD-treated seedlings, respectively. In vitro, PQ- and RD-treated nucleic acids [single-stranded DNA (ssDNA), RNA, and plasmid DNA (pl-DNA)] were incubated with acid and neutral nucleases. Both enzymes were isolated and purified from alfalfa seedlings. Electrophoretic analysis on agarose gel of the above incubated mixtures revealed the following: (a) neutral nuclease (pH 7) was capable of hydrolyzing PQ-treated ssDNA while acid nuclease (pH 5.5) was incapable. This could be due to the fact that acid and neutral nucleases displayed different base linkage specificity toward ssDNA; (b) RD formed strong complexes with ssDNA that were unable to be hydrolyzed by both nucleases; (c) in contrast, both enzymes were capable of hydrolyzing PQ- or RD-treated RNA; (d) neutral nuclease was capable of nicking and linearizing both PQ- and RD-treated pl-DNA while acid nuclease had the same activity only toward the PQ-treated pl-DNA; (e) the enzyme activities were not inhibited in the presence of both herbicides. The data suggest that the complexes of PQ or RD with DNA should not be functional substrates of nucleases, and consequently cell processes (e.g., metabolism of nucleic acids, gene expression, replication), in which DNA and nucleases are involved, could be disturbed.

Zebrafish RNase T2 genes and the evolution of secretory ribonucleases in animals

BackgroundMembers of the Ribonuclease (RNase) T2 family are common models for enzymological studies, and their evolution has been well characterized in plants. This family of acidic RNases is widespread, with members in almost all organisms including plants, animals, fungi, bacteria and even some viruses. While several biological functions have been proposed for these enzymes in plants, their role in animals is unknown. Interestingly, in vertebrates most of the biological roles of plant RNase T2 proteins are carried out by members of a different family, RNase A. Still, RNase T2 proteins are conserved in these animalsResultsAs a first step to shed light on the role of animal RNase T2 enzymes, and to understand the evolution of these proteins while co-existing with the RNase A family, we characterized RNase Dre1 and RNase Dre2, the two RNase T2 genes present in the zebrafish (Danio rerio) genome. These genes are expressed in most tissues examined, including high expression in all stages of embryonic development, and their expression corresponds well with the presence of acidic RNase activities in every tissue analyzed. Embryo expression seems to be a conserved characteristic of members of this family, as other plant and animal RNase T2 genes show similar high expression during embryo development. While plant RNase T2 proteins and the vertebrate RNase A family show evidences of radiation and gene sorting, vertebrate RNase T2 proteins form a monophyletic group, but there is also another monophyletic group defining a fish-specific RNase T2 clade.ConclusionBased on gene expression and phylogenetic analyses we propose that RNase T2 enzymes carry out a housekeeping function. This conserved biological role probably kept RNase T2 enzymes in animal genomes in spite of the presence of RNases A. A hypothetical role during embryo development is also discussed.

Ribonuclease from the hepatopancreas of the red king crab Paralithodes camtschatica

Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg2+ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT50 = 53-55 degrees C) and were strongly inhibited by NaCl (IC50, 0.1-0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates.

Effect of Hemodialysis on Acid Leukocyte-Type Ribonuclease, Alkaline Ribonuclease and Polymorphonuclear Elastase Serum Levels in Patients with End-Stage Renal Disease

Background: Low-molecular-weight proteins (LMWPs) are substances of molecular weights 10–35 kDa, which accumulate in plasma of patients with end-stage renal disease (ESRD) due to the abolishment of plasma renal filtration. LMWPs are considered as a separate group of uremic toxins. Aim: The influence of hemodialysis (HD) on the release of some LMWPs from leukocytes was assessed by comparing levels of serum pancreatic-type alkaline RNase and leukocyte-type acid RNase as well aspolymorphonuclear (PMN) elastase. Methods: The mentioned proteins were assayed in 58 ESRD patients on HD prior and after the dialysis session and compared with the results obtained from 36 healthy subjects. The levels of elastase and acid and alkaline RNase were correlated with HD parameters, residual diuresis, predialysis concentrations of serum creatinine, urea and albumin as well as pre- and postdialysis granulocyte count. Results: Changes in PMN elastase produced by the dialysis session positively correlate with changes in acid RNase levels (r = 0.3650; p = 0.0061), while there is no such correlation for alkaline RNase. There is a negative correlation between pre- and postdialysis differences in levels of acid and alkaline RNases (r = –0.3542; p = 0.008), indicating that HD induces liberation of a factor suppressing alkaline RNase. Levels of acid and alkaline RNase negatively correlate with residual diuresis, indicating its significance in control of LMWP accumulation (r = –0.3970; p = 0.0025; r = –0.2596; p = 0.0533, respectively). Conclusions: Dialysis treatment causes an increase in both acid leukocyte-type and alkaline pancreatic-type RNase activity in plasma. Dialysis-related increases in acid RNase activity correlate with the respective changes in PMN elastase, which suggests that leukocyte activation during dialysis contributes to an increase in plasma LMWPs.

Copper Stress Affects Metabolism and Reproductive Yield of Chickpea

ABSTRACT To observe the effects of copper (Cu) deficiency on growth, metabolism, and reproductive yield of chickpea (Cicer arietinum L.) cv. ‘13.G-256’, plants were grown in refined sand at deficient (0.1 μM) and adequate Cu (1 μM), supplied as copper sulfate (CuSO4·5H2O). At d 35–40, at deficient Cu, the growth of plants were depressed and the young leaflets appeared reduced in size, chlorotic, with narrow pointed tips. The primary branches collapsed later, and secondary branches were stunted with reduced number of leaves. The flowering was disturbed, less flowers matured, as a consequence pods and seeds were reduced, malformed and low in productivity, which lowered biomass and economic yield and is accompanied by decrease in Cu concentration in leaves and seeds. The quality of seeds deteriorated at deficient Cu as the concentration of proteins, carbohydrates (sugars and starch), protein nitrogen were lowered, and phenols, non-protein nitrogen, increased. Whereas in leaves, the concentration of carbohydrates (sugars and starch), phenols, and non-protein nitrogen were elevated and protein nitrogen was reduced. Copper deficiency also alleviated the concentration of chlorophyll (a and b) in leaves. At deficient Cu, the activity of antioxidative enzyme viz. peroxidase along with that of acid phosphatase and ribonuclease increased in leaves, seeds and pod wall of chickpea.

Dormancy and germination in wheat embryos: ribonucleases and hormonal control

Acidic and neutral ribonucleases (RNases) were studied in embryos of Triticum durum cv. Cappelli and the effects of abscisic acid (ABA) and gibberellic acid (GA3) were analysed. RNases activities increased during germination and were comparable in dormant and non-dormant embryos imbibed for 24 h. ABA generally inhibited ribonucleolytic activities, while GA3 only affected dormant embryos. To assess whether changes in RNase activities during germination or following hormonal treatment required new transcriptional or translational action, cycloheximide or cordycepin were used. The action of inhibitors of acidic RNase activity was found only in non-dormant-embryos. Findings obtained in the present work concur with a change of the ribonucleolytic pattern in the shift from dormant to non dormant metabolism.

BIOCHEMICAL STUDIES ON ACID AND ALKALINE RIBONUCLEASE IN SERA OF WOMEN WITH DIFFERENT OVARIAN TUMORS

Acid and alkaline ribonuclease (RNase) activities have been assayed in (14) serum samples of women with histological confirmed ovarian carcinoma, (26) samples of women with benign ovarian tumors, and (27) serum samples of healthy women without any clinically detectable diseases. The mean values of acid and alkaline RNase specific activities were statistically increased in the cancer group (p<0.001) in comparison with those of other groups. The ratio of acid to alkaline RNase (Ac/Al) activities was statistically decreased in the cancer group as compared with those of other groups. Serum alkaline RNase levels were estimated in sera of the above mentioned groups using bovine pancreatic RNase (A), as a standard. A markedly increase in this level in sera of the cancer group was observed when matched with those of other groups. Different alkaline RNase forms were detected in sera of control, benign and malignant tumors groups using gel filtration chromatography (1.6  60 cm. Sephadex G75 column). The results indicate the presence of five different forms in each case, which were different in their molecular weights.

Purification and characterization of a non-S-RNase and S-RNases from styles of Japanese pear ( Pyrus pyrifolia)

In this study we biochemically characterized stylar ribonucleases (RNases) of Japanese pear ( Pyrus pyrifolia), which exhibits S-RNase-based gametophytic self-incompatibility. We separated the RNase fractions NS-1, NS-2, and NS-3 from stylar extracts of the cultivar Nijisseiki (S 2S 4). The RNase in each fraction was purified to homogeneity through a series of chromatographic steps. Chemical analysis of the proteins revealed that the basic RNases in the NS-2 and NS-3 fractions were the S 4- and S 2-RNases, respectively. Five additional S-RNases were purified from other cultivars. An acidic RNase in the NS-1 fraction was also purified from other cultivars, and identified as a non-S-allele-associated RNase (non-S-RNase). The non-S-RNase is composed of 203 amino acids, is non-glycosylated and is a N-terminal-pyroglutamylated enzyme of the RNase T 2 family. The substrate specificities and optimum pH levels of the non-S-RNase and S-RNases were similar. Interestingly, the specific activity of the non-S-RNase was 7.5–221-fold higher than those of the S-RNases when tolura yeast RNA was used as the substrate. The specific activity of the S 2-RNase was 8.8–28.6-fold lower than those of the other S-RNases. These differences in specific activities among the stylar RNases are discussed.

Growth hormone modulates the degradative capacity of muscle nucleases but not of cathepsin D in post-weaning mice

We determined whether recombinant human growth hormone (rhGH) administration might modulate the enzyme degradative capacity of the muscle lysosomal system and influence muscle growth. Muscle cathepsin D, acid RNase and DNase II activities are determined in the gastrocnemius muscle of rhGH-treated post-weaning female BALB/c mice. Linear regressions were used to analyze the relationships of each enzyme with their respective substrate. GH induced a depletion-recovery response of muscle growth through a mechanism which is similar to catch-up growth. In these conditions, cathepsin D activity decreased with age in all animals (GH: 40%; saline: 79%), showing a substantial developmental decline that could reflect changes in the rate of protein breakdown. However, the degradative capacity of cathepsin D was paradoxically unmodified in rhGH-mice compared with saline mice (according to the enzyme vs. substrate linear regression slope), in spite of the increase in enzyme activity elicited by GH. This suggests that the muscle protein breakdown is not increased by GH-treatment in post-weaning mice. The enhancement of muscle protein deposition as indicated by the augmented muscle cell size (protein:DNA ratio) of rhGH-mice (increased 178% from 25 to 50 days) vs. saline, can be attributed to a higher muscle K(RNA). In contrast, acid RNase and DNase II activities directly participate in muscle RNA and DNA degradation. Both nucleases were inhibited by GH treatment (a decrease of 48% and 63%, respectively, vs. saline at 50 days). The decrease in RNase activity suggests an inverse relation between the rate of protein synthesis (high) and acid RNase activity (low), leading to spare muscle RNA for synthesizing protein during catch-up growth. Also, low DNase II activity could contribute to inhibiting of muscle DNA degradation, facilitating muscle growth. Thus, GH seems to act as a direct modulator of the degradative capacity of skeletal muscle nucleases but not of cathepsin D, influencing DNA and RNA degradation during the depletion-recovery response to GH of gastrocnemius muscle in female post-weaning mice.

Application of different test systems and biochemical indicators for environmental monitoring of the Troitsa Bay, Sea of Japan

The DNase express test system and the SF-bioassay (sea urchin sperm fertilization bioassay) were used to assess the quality of sea and bottom sediment water sampled at 8 stations of the Troitsa Bay (Possyet Bay, Sea of Japan). The specific activities of 8 enzymes (alkaline and acid DNases, RNases, phosphatases and phosphodiesterases) were determined for the liver and gill tissues of the mussel Crenomytilus grayanus sampled at the same stations. The activities of all enzymes were higher in the liver than in the gills, and the activity of acid nucleases in both tissues was significantly higher than that of alkaline nucleases. The specific activities of some nucleases in the mussel tissues correlated to the level of seawater pollution. The activities of acid RNases and phosphodiesterases decreased in both tissues upon an increase in water pollution, up to 1.5–3 times in the gills, while the activities of alkaline and acid phosphatases and phosphodiesterases increased 1.5–4 times. Thus, the specific activities of acid RNases, phosphatases and phosphodiesterases from mussel liver can be a useful indicator of marine pollution. This approach can be used for biotesting of ocean water and for assessment of the biological condition of invertebrates adapting to various ecological and anthropogenic effects.

Biochemical activity of selenium and glutathione on country made liquor (CML) induced hepatic damage in rats

The serum and hepatic enzymes of rats were studied after exposed to country made liquor (CML) along with two chelating agents (glutathione and Selenium). There was a significant increase in several serum enzyme levels (viz., aspartate transaminase, alanine transaminase, alkaline phosphatase, sorbitol dehydrogenase, glutamate dehydrogenase, bilirubin) and decrease in various hepatic enzymes (Succinic dehydrogenase, Glucose 6-phosphatase, 5'Nucleotiease, Acid phosphatase, Acid ribonuclease, Cytochrome P-450) due to repeated administration of CML (2ml/100g of body weight). Results of this study revealed that the GSH and Se could give a significant protective action in serum and hepatic enzymes of CML exposed rats.

The modulator effect of GH on skeletal muscle lysosomal enzymes is dietary protein dependent

Objective The purpose of this work is to determine whether changes in dietary protein level could alter the modulator effect that GH has on the muscle lysosomal system by influencing the hydrolytic activities of cathepsin D, acid RNase and DNase II and the participation of these enzymes in muscle growth. Design BALB/c female mice were fed a diet containing 20% (HP) or 12% (MP) protein ad libitum and were treated with either saline (s) or rhGH (GH) (74 ng/g) for 29 days. Body weight and feed intake were recorded daily. At 25, 30, 35, 40, 45 and 50 days of age, five mice from each group were slaughtered and nucleic acids and protein concentrations and cathepsin D, acid RNase and DNase II activities in gastrocnemius muscle were analysed. Correlation coefficients were used to analyse the links between the activity of each enzyme with its substrate. Results GH-treatment induced a depletion–recovery response in muscle growth through a compensatory mechanism. Changes in protein content, DNA and RNA concentrations were related to changes in lysosomal enzyme activities. Muscle cathepsin D activity in saline mice fell as the dietary protein concentration increased. GH-treatment reversed this effect by enhancing the proteolytic activity in muscle of well-fed mice and inhibiting it in mice fed a 12% protein diet. This inversion appears to be related to the different mechanism elicited by GH-treatment on skeletal muscle protein growth in each dietary group. An opposite trend was observed in muscle acid nuclease activities. Acid RNase and DNase II increased according to the dietary protein concentration, since a 12% protein diet induced a lower catabolism, especially on muscle DNA of saline mice. In contrast, GH-treatment decreased acid RNase and DNase II activities, but only in mice fed a 20% protein diet, perhaps leading to spare muscle RNA for protein synthesis, as well as to the inhibition of DNA degradation during catch-up growth. A lower dietary protein concentration appeared to reverse the GH protective effect on nucleic acids. Conclusions GH seems to act as a dietary protein-dependent modulator of the skeletal muscle lysosomal enzyme activity. These lysosomal enzymes play a role during muscle growth in GH-treated post-weaning mice by modifying muscle protein and DNA and RNA degradation.