RNAase Activity Research Articles

BCL-2 Modulates IRE1α Activation to Attenuate Endoplasmic Reticulum Stress and Pulmonary Fibrosis.

BCL-2 family members are known to be implicated in survival in numerous biological settings. Here, we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix accumulation. Days after an intratracheal bleomycin challenge, mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1α. This fraction is dramatically increased by BCL-2 inhibition, because IRE1α activation is dependent on its physical association with the BCL-2-proapoptotic family member BAX, and we found BCL-2 to disrupt this association invitro. In vivo, navitoclax (a BCL-2/BCL-xL inhibitor) given 15-21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress, but not apoptosis. Remarkably, after BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at Day 42, compared with resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1α downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in precision-cut lung slice cultures of Day-42 lungs from bleomycin-exposed wild-type and MRC2 null mice. Soluble and tissue collagen accumulated in precision-cut lung slice cultures from navitoclax-treated, bleomycin-challenged mice compared with controls, in a manner nearly identical to that of challenged but untreated MRC2 null mice. Thus, apart from mitochondrial-based antiapoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair.

The evolution and immunomodulatory role of Zc3h12 proteins in zebrafish (Danio rerio)

Zc3h12 family is an important RNA-binding protein family regulating mRNA of inflammatory cytokines in mammals. However, there are few studies on their post-transcriptional level regulation of inflammatory cytokines in fish. Here, we investigated the evolution of zebrafish Zc3h12 family and explored their immunomodulatory role. Phylogenetic and syntenic analysis indicated the number of zc3h12 family members had increased ranging from a single member in invertebrates to a single copy of four members in mammals. As the most evolutionarily diverse group of vertebrates, the number of zc3h12 family members was more complex and diverse in the teleost, each member experienced different fates and followed different rules in multiple rounds of whole-genome duplication events. Thereinto, zebrafish contained three zc3h12 genes, among which zc3h12aa and zc3h12ab were duplicated from the same gene. Zebrafish Zc3h12 family could recognize the 3′-UTR regions of inflammatory cytokines through binding to the specific RNA secondary structure and negatively regulate their expression. Deletion of either Zc3h12 domains or mutation of the key amino acid in RNAase domain attenuated their modulatory effect, suggesting both domain and RNAase activity are important to the immunomodulatory role. These results elucidated the evolution of Zc3h12 family and uncovered Zc3h12-mediated post-transcriptional regulation of cytokines in zebrafish.

Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro

To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms. The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells. The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1. rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.

Evaluation of circulatory microRNA-196 levels during the progression of chronic renal (kidney) disease of unknown etiology

Chronic renal disease of unknown etiology is prevalent &amp; clinically silent until its late stages at which point patients may suffer significant irreversible damage or mortality in the lack of early screening &amp; intervention. If diagnosed early stage 1-3 the progression of chronic renal disease can be altered and complications reduced. Current diagnosing markers of renal function, serum creatinine, blood urea and uric acid have been shown to be lacking high predictive value. So, there is a need for next generation biomarkers suitable for Chronic renal disease of unknown etiology and should not be influenced by age, sex, nutritional status, current health concern. microRNAs appears to be very stable in tissues &amp; biological fluids even under adverse conditions like extreme pH, long term room temperature storage, multiple freeze thaw cycles &amp; RNAase activity. microRNAs elaborate in renal development, maintenance of renal function &amp; progression of renal disease. miRNA-196 is a renal derived biomarker, extensively distributed in renal, shows an important role in renal diseases &amp; superior to other renal biomarkers. The aim of present study is to evaluate circulatory miRNA-196 during progression of chronic renal disease of unknown etiology.

Effect of pollutants on biochemical properties of microorganisms

Introduction. The development of different ways of identifying hazard of substances, polluting the environment, that are alternative to routine methods for assessing health of different professional groups of people, as well as to tests on laboratory animals, has been assessed and summarized in a number of papers, and there is a growing volume of actual data on this issue. The analyzing pathogenic properties of microflora, isolated from polluted habitats or changed under the effect of pollutants, allows both assessing the risk of diseases with microbial etiology and identifying the influence of modifying factors on this process. Materials and methods. The research was performed on 150 cultures of Klebsiella pneumoniae, exposed to diethanolamine, phenol, sulfuric and nitric acids, taken at the level of their threshold limit value. The same cultures incubated without pollutants were used as a control group. Pathogenic factors were determined with the use of traditional methods, our modified methods (RNAase activity) and standard agents, kits and media. Results. The contact with all the pollutants resulted in a fair increase in adhesive, anti-Ig, lysozyme, anti-lysozyme and RNAase activities of microorganisms, their resistance to antibacterial action of blood serum, as well as to damaging action of lactoferrin. The bacteria increased their ability to synthesize a substance, immunologically similar to human lactoferrin. At the same time, there was a decrease in lipase activity, primarily referred to factors determining the bacterial ability to survive in the environment. Discussion. Therefore, the paper results can be presented as follows: being an independent and holistic section of modern microbiology, bacterial biochemistry unites the issues appeared to be far from each other - changes in factors of pathogenicity and persistence of microorganisms and technogenic changeability of the environment. Being quite urgent today, this problem may become aggravated in the course of further technological progress.

Effect of pollutants on biochemical properties of microorganisms

Introduction. The development of different ways of identifying hazard of substances, polluting the environment, that are alternative to routine methods for assessing health of different professional groups of people, as well as to tests on laboratory animals, has been assessed and summarized in a number of papers, and there is a growing volume of actual data on this issue. The analyzing pathogenic properties of microflora, isolated from polluted habitats or changed under the effect of pollutants, allows both assessing the risk of diseases with microbial etiology and identifying the influence of modifying factors on this process. Materials and methods. The research was performed on 150 cultures of Klebsiella pneumoniae, exposed to diethanolamine, phenol, sulfuric and nitric acids, taken at the level of their threshold limit value. The same cultures incubated without pollutants were used as a control group. Pathogenic factors were determined with the use of traditional methods, our modified methods (RNAase activity) and standard agents, kits and media. Results. The contact with all the pollutants resulted in a fair increase in adhesive, anti-Ig, lysozyme, anti-lysozyme and RNAase activities of microorganisms, their resistance to antibacterial action of blood serum, as well as to damaging action of lactoferrin. The bacteria increased their ability to synthesize a substance, immunologically similar to human lactoferrin. At the same time, there was a decrease in lipase activity, primarily referred to factors determining the bacterial ability to survive in the environment. Discussion. Therefore, the paper results can be presented as follows: being an independent and holistic section of modern microbiology, bacterial biochemistry unites the issues appeared to be far from each other - changes in factors of pathogenicity and persistence of microorganisms and technogenic changeability of the environment. Being quite urgent today, this problem may become aggravated in the course of further technological progress.

Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2)

Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5'-phosphomonoesterase activities at optimum temperature 37 degrees C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process.

Retention of enzyme activity following freeze-drying the mycelium of ectomycorrhizal isolates

Qualitative enzyme assays were used as a tool to investigate the stability of freeze-dried mycorrhizal fungi. Both lyophilized (L) and non-lyophilized (NL) mycelia of individual isolates showed identical response for all the enzymes tested (nitrate reductase, protease, pectinase, and nuclease). All the isolates showed positive nitrate reductase activity, except two isolates of Thelephora terrestris (both L and NL). Both L and NL cultures of individual isolates showed substrate specificity (between gelatin and casein) for protease activity. Though both L and NL mycelia of all the culture isolates grew upon pectin substrate, there was no pectinase activity expressed. RNAase activity was variously exhibited (little activity, little growth–no activity, and no growth–no activity) by individual test cultures. The consistencies in growth and enzyme activity of the cultures before and after lyophilization imply the stability of the freeze-dried vegetative mycelium.

Characterization of prokaryotic recombinant Aspergillus ribotoxin α-sarcin

The Aspergillus ribonuclease α-sarcin is toxic to intact mammalian cells but the mechanism by which it enters the cells to reach its ribosomal RNA substrate is unclear. Here we have compared the cytotoxicity of α-sarcin to that of ricin, another catalytic toxin that targets the same rRNA sequence but whose mechanism of cell entry is better understood. Intact ricin binds to cell surface components and enters the cells by receptor-mediated endocytosis, whereas the catalytic polypeptide of ricin (the A chain or RTA) which, like α-sarcin, is unable to bind to surface components directly and enters cells by fluid phase uptake. Recombinant α-sarcin was produced in Escherichia coli and purified to homogeneity. The protein was soluble, stable and its ability to inhibit in vitro protein synthesis was indistinguishable from that of native α-sarcin. Further, recombinant α-sarcin had the same in vitro protein synthesis inhibition activity as ricin A chain. The cytotoxicity of α-sarcin and ricin A chain to HeLa cells was also the same. The cytotoxicity of α-sarcin was due to its RNAase activity rather than to specific membrane effects at the cell surface, since a mutant containing a single substitution at a putative key catalytic residue had reduced ribonuclease activity and an equivalent reduction in cytotoxicity. One interpretation of the data is that α-sarcin enters mammalian cells in the same way as free ricin A chain.

A protein disulfide-thiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY181984)

Plasma membrane vesicles isolated from HeLa cells grown in suspension culture contain a protein disulfide-thiol interchange (protein disulfide-like) activity. The activity was estimated from the restoration of activity to inactive (scrambled) pancreatic RNAase. RNAase activity was measured either by hydrolysis of cCMP or by a decrease in acid precipitable yeast RNA. The ability of plasma membrane vesicles to restore activity to inactive (scrambled) pancreatic ribonuclease was inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY181984). The activity correlated with that of a cyanide-resistant NADH oxidase also associated with the plasma membrane vesicles that exhibited a similar pattern of drug response. The activity was stimulated by reduced glutathione and inhibited by oxidized glutathione but did not depend on either for activity. The antitumor sulfonylurea-inhibited activity was greatest in the presence of reduced glutathione and least in the presence of oxidized glutathione. The antitumor sulfonylurea-inhibited activity was unaffected by a monoclonal antibody to protein disulfide isomerase. Also the antitumor sulfonylurea-inhibited activity was unaffected by peptide antisera to the consensus active site sequence of protein disulfide isomerase. Thus the antitumor sulfonylurea-inhibited activity appeared to reside with a novel cell surface protein capable of oxidation of both NADH and protein thiols and of carrying out a protein disulfide isomerase-like protein disulfide-thiol interchange activity in the absence of NADH or other external reductants.

Spectrophotometric Assay of Endo-β-N-acetylglucosaminidase H Activity

An endoglycosidase H (Endo H) assay based on the use of a glycoprotein enzyme as substrate, in conjunction with immobilised concanavalin A (Con A), was devised and investigated. In this system, ribonuclease B (RNAase B) functions as both glycosidase substrate and reporter enzyme. Separation of glycosylated from deglycosylated RNAase B is effected by mixing with colloidal Con A-agarose, followed by centrifugation. The activity of carbohydrate-denuded RNAase B in the supernate is used to calculate endoglycosidase activity. Results obtained with this assay correlated closely with analysis of digest products by denaturing electrophoresis and lectin reactivity on western blots. Effective removal of non-hydrolysed RNAase B from assay digests was dependent on both lectin-agarose bead concentration and time of exposure. The assay was linear over the range 10–100 fmol of Endo H protein; a Km value of 0.48 mmol/l and turnover number of 7200 mol RNAase B/mol enzyme/min was determined for this endoglycosidase. The suitability of the assay for measuring the activity of other endoglycosidases was also investigated, and the usefulness of the assay is discussed.

Inactivation during denaturation of ribonuclease A by guanidinium chloride is accompanied by unfolding at the active site

Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

Lactoferrin contains structural motifs of ribonuclease

A high molecular weight ribonuclease isolated from human milk (hmRNAase) shares identical immunological, physical, structural features and considerable sequence homology with human lactoferrin; and it has been demonstrated to be an isoform of lactoferrin. We have analyzed the sequence data of lactoferrin looking for the existence of specific features corresponding to the consensus sequence of pyrimidine-specific ribonucleases. The analysis was done by comparing sequence features with respect to elements which are, in principle, responsible for RNAase activity. This revealed the existence of a ribonuclease-signature pattern in lactoferrin. Further analysis of X-ray data together with molecular modeling studies have revealed close similarities between the spatial geometry of the constituent groups of the active site of pyrimidine-specific ribonucleases and the corresponding groups comprising the potential active site of lactoferrin. Our results provide the strong structural basis for the existence of ribonuclease activity in lactoferrin.

Possible role for mRNA stability in the ammonium-controlled regulation of nitrate reductase expression.

Ammonium, or a metabolite of ammonium, represses the expression of nitrate reductase (NR) in Chlorella vulgaris. The removal of ammonium and addition of nitrate (induction) resulted in a rapid (20 min) peaked synthesis of NR mRNA. Nitrate reductase protein and activity increased at a much lower rate, reaching their maxima by 8 h. Ammonium added to nitrate-grown cells resulted in a dramatic decrease in NR mRNA from a steady-state level to undetectable levels within 15 min of ammonium addition. Nitrate reductase activity and protein levels decreased to 20% and 40% of initial levels respectively over 8 h. The half-life for NR mRNA under these conditions was estimated to be less than 5 min, compared with 120 min for NR protein. Such rapid decreases in NR mRNA suggested a degradation and/or cessation in NR mRNA transcription. No apparent difference in NR mRNA-specific RNAase activity of crude cell extracts (NR-induced or repressed) was observed. However, a significant difference in the susceptibility to degradation of NR mRNA from long-term nitrate-grown cells compared with the NR mRNA isolated from short-term induced cells (20 min in nitrate) was observed. NR mRNA isolated from long-term-nitrate-grown cells was completely degraded by RNAases in cell extracts under conditions in which the NR mRNA isolated from short-term induced cells was resistant to degradation. These results suggest that mRNA stability may be an important factor in the metabolic regulation of assimilatory nitrate reductase in Chlorella.

Expression cloning of 2-5A-dependent RNAase: A uniquely regulated mediator of interferon action

2-5A-dependent RNAase, an interferon-induced enzyme that is activated by 5′-phosphorylated, 2′,5′-linked oligoadenylates (2-5A), is implicated in both the molecular mechanisms of interferon action and the fundamental control of RNA stability in mammalian cells. Here we report the expression cloning and analysis of murine and human 2-5A-dependent RNAases. The 2-5A binding properties and RNAase activities of recombinant and naturally occurring forms of 2-5A-dependent RNAase were identical. Interferon induction of 2-5A-dependent RNAase expression was demonstrated by measuring the mRNA levels in cells treated with interferon and cycloheximide. Analysis of aligned murine and human 2-5A-dependent RNAase sequences revealed several intriguing features, including similarity to RNAase E, which is implicated in the control of mRNA stability in E. coli. Interestingly, a duplicated phosphate-binding loop motif was determined by deletion analysis and site-directed mutagenesis to function in the binding of 2-5A.

Catabolism in mollicutes.

The small genome size of mollicutes, and particularly mycoplasmas and ureaplasmas, precludes their possession of the extensive range of metabolic activities present in most other bacterial groups. Demonstrated catabolic activities appear primarily to be associated with energy generation, rather than the provision of substrates for synthetic pathways, and anabolism is largely dependent upon extracellular sources of amino acids, nucleic acid precursors and lipids. However, the pathways of energy generation in mollicutes are diverse and specialized, and may in vivo be dependent upon the presence of a single amino acid (arginine) or urea. Even in those species that utilize carbohydrates the range of substrates is restricted, and while Ac. laidlawii has both EMP and PP pathways and is able to oxidize pyruvate to acetate plus CO2, many mycoplasmas possess only a part of these activities. Such specialization and the infrequent demonstration of inducible enzyme activity in mollicutes implies adaptation to specific habitats in host species, and suggests that differences in the catabolic activities of mollicute strains may be significant in terms of their ecology and pathogenicity. The demonstrated energy-generating pathways of mollicutes produce low ATP yields. Thus, mollicute growth will generate relatively large quantities of metabolic end-products and may deplete host tissues of substrates. Arginine depletion may be of particular importance in pathogenesis and the close physical association between mollicutes and host cells will enhance the potential significance of NH4+ production from the hydrolysis of arginine and urea, and of H2O2 and superoxide formation during carbohydrate metabolism. In addition, lipid and protein catabolism may be associated with virulence where extracellular or membrane-bound enzyme activities exist. Membrane-bound DNAase and RNAase activities have also been demonstrated in mycoplasmas and Ac. laidlawii (Pollack et al., 1965) and U. urealyticum (Romano & La Licata, 1978). Many aspects of mollicute catabolism, including energy conservation in some groups, is poorly understood. Also, while substantial catabolic diversity has been demonstrated within mollicutes and new species are continually being isolated, metabolism has been studied in relatively few species, and even in these only single strains or small groups of strains have been used. In this review, therefore, an attempt to avoid generalizations concerning mollicute behaviour has been made. The lack of much basic knowledge concerning mollicute metabolism has also necessitated the widespread use of 'may be' and other equally vague terms.(ABSTRACT TRUNCATED AT 400 WORDS)

Secretion of mammalian ribonucleases from Escherichia coli using the signal sequence of murine spleen ribonuclease.

A nucleotide sequence identical with that of the recently identified murine pancreatic ribonuclease (RNAase) was isolated from a murine spleen cDNA library. Active RNAase was expressed and secreted from Escherichia coli lon-htpr- transformed with a plasmid containing the E. coli trp promoter followed by the murine RNAase gene sequence, including the original eukaryotic 26-amino-acid signal sequence. Approx. 1 mg of properly matured RNAase protein/litre was secreted into the medium of a fermentor culture after the promotor was induced by tryptophan starvation. When the signal sequence was deleted from the plasmid, intracellular RNAase activity was very low and there was no significant supernatant RNAase activity. Even higher RNAase yields were obtained with a synthetic gene for bovine pancreatic ribonuclease cloned after the signal sequence of the murine gene. About 2 mg of correctly processed RNAase A/litre was isolated from the growth medium, and a further 8-10 mg of correctly processed RNAase/litre could be isolated from the soluble fraction of the cells. Thus this eukaryotic signal sequence is both recognized by the E. coli transport and processing apparatus and gives efficient secretion, as well as export, of active, mature mammalian RNAases.

Inhibition of protein synthesis in liver and kidney of mice by bolesatine: mechanistic approaches to the mode of action at the molecular level

Protein synthesis was assayed in liver and kidney of mice treated with bolesatine, a toxic glycoprotein from the mushroom Boletus satanas (Lenz) which was previously shown to be an inhibitor of protein synthesis by cell-free systems in vitro and by cultured cell-lines. Protein synthesis in vivo (Swiss mice) is inhibited in a dose-dependent manner in liver and kidney. The mechanism of action does not appear to be due to RNA- N-glycosidase activity of bolesatine or a RNAase activity of this toxin on the ribosomal RNAs. Ribosomes do not appear to be damaged by pretreatment with bolesatine as judged by a poly(U) translation system. Thus bolesatine cannot be included in the group of protein synthesis inhibitors of plant origin known as ribosome-inactivating proteins (RIPs).

Effects of nutritional status of Aspergillus nidulans on its nuclease activity

Before nutrient limitation set in, mycelium of Aspergillus nidulans contained little free nuclease activity but treatment of extracts with trypsin led to the appearance of substantial DNAase and RNAase activity as a result of activation of a cryptic nuclease. Onset of nitrogen or sulphur limitation led to the appearance of DNAase activity but not necessarily RNAase activity. Carbon or phosphate limitation did not lead to a similar increase in free DNAase activity.