The distribution of acid and alkaline ribonuclease activities in preneoplastic and neoplastic rat livers.

Ribonuclease (RNase) activities revealed by the substrate film method were compared with reactions for acid and alkaline RNase obtained by lead precipitation technique in serial sections of preneoplastic livers and hepatomas. The preneoplastic parenchymal tissue giving positive reactions with ribonucleic acid films showed both acid and alkaline RNase activities by lead precipitation technique, and the area of hyperplastic nodules nonreactive against substrate films were deficient in acid and alkaline RNase activities. Preneoplastic hyperbasophilic foci and hepatoma gave weak or negative reactions by either method, but necrotic areas and stromal tissue showed appreciable RNase activities. Thus a good correlation was observed in these tissues between the RNase activities revealed by the film method and those demonstrated by lead precipitation.     

Site-Specific RNase A Activity Was Dramatically Reduced in Serum from Multiple Types of Cancer Patients

Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10⁻⁵ mg/ml- 10⁻¹ mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types.     

Messenger and ribosomal RNA hydrolysis by ribonucleases I. The action of two barley leaf ribonucleases on the messenger and ribosomal RNA of isolated polysomes

When barley (Hordeum vulgare L.) leaf polysomes are incubatedwith two RNase fractions (the pH 5 insoluble and soluble RNases)under limit digestion conditions, the two enzymes exhibit characteristicpreference for messenger and ribosomal RNA (mRNA and rRNA) hydrolysis.The pH 5 insoluble RNase from a cultivar of barley, Prior, andthe corresponding enzyme from two near-isogenic lines (M1622and M1623) cleave polysomal mRNA at specific sites and generatepolysome profiles that are unique to the cultivar. By contrast,the soluble RNase from barley leaves, although a typical endoribonuclease,catalyzes no detectable hydrolysis of polysomal mRNA. Both of these barley leaf RNases hydrolyze rRNA when eitherpolysomes or monosomes are treated with these enzymes. Withpolysomes as substrate, the pH 5 insoluble RNase hydrolyzesthe high molecular weight RNA component of both large and smallsubunits of chloroplast and cytoplasmic ribosomes. The solubleRNase preferentially hydrolyzes the high molecular weight RNAcomponent of the small subunit of chloroplast and cytoplasmicribosomes. Analytical gel electrophoresis of the RNA of theRNase-treated monosomes has revealed that both enzymes hydrolyzerRNA into very small fragments. However, despite scission inrRNA at multiple sites, the RNase-treated monosomes remain activein polyuridylate-directed polyphenylalanine synthesis. (Received January 31, 1980; )     

RNase MC2: a new <Emphasis Type="Italic">Momordica charantia</Emphasis> ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways

Ribonucleases (RNases) are ubiquitously distributed nucleases that cleave RNA into smaller pieces. They are promising drugs for different cancers based on their concrete antitumor activities in vitro and in vivo. Here we report for the first time purification and characterization of a 14-kDa RNase, designated as RNase MC2, in the seeds of bitter gourd (Momordica charantia). RNase MC2 manifested potent RNA-cleavage activity toward baker’s yeast tRNA, tumor cell rRNA, and an absolute specificity for uridine. RNase MC2 demonstrated both cytostatic and cytotoxic activities against MCF-7 breast cancer cells. Treatment of MCF-7 cells with RNase MC2 caused nuclear damage (karyorrhexis, chromatin condensation, and DNA fragmentation), ultimately resulting in early/late apoptosis. Further molecular studies unveiled that RNase MC2 induced differential activation of MAPKs (p38, JNK and ERK) and Akt. On the other hand, RNase MC2 exposure activated caspase-8, caspase-9, caspase-7, increased the production of Bak and cleaved PARP, which in turn contributed to the apoptotic response. In conclusion, RNase MC2 is a potential agent which can be exploited in the worldwide fight against breast cancer.     

Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

RNA processing enzymes RNase III, E and P in Escherichia coli are not ribosomal enzymes.

We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures. Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+. It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells. However, the possibility of membrane preparation contamination with ribosomes could not be ruled out. Here we show that RNase III, E and P are not associated with ribosomes. E. coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions. The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme. Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity. RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane. Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane. Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out. RNase E activity was also found to be present mostly in the post-ribosomal supernatant. These findings show that RNase III, E and P are not ribosomal enzymes.     

RNase 3 (ECP) is an extraordinarily stable protein among human pancreatic-type RNases

There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

Messenger and ribosomal RNA hydrolysis by ribonucleases II. Changes in ribonuclease activities and ribosomes of barley leaves during the early stages of powdery mildew infection

We have monitored changes in the properties of two barley (Hordeumvulgare L.) leaf RNases with respect to their action on polysomalmessenger RNA (mRNA) and the RNA of isolated ribosomes duringthe early stages of infection by the powdery mildew fungus (Erysiphegraminis f. sp. hordei). The results presented support the followingconclusions. (i) At 48 hr after inoculation, the pH 5 insolubleRNase undergoes significant changes in its catalytic properties.This is evident from the finding that under limit digestionconditions, the enzyme from inoculated leaves hydrolyzes chloroplastpolysomal mRNA and produces far greater quantities of chloroplastmonosomes than does the corresponding enzyme from healthy leaves.(ii) The acid soluble oligonucleotide fragments produced bythe soluble RNase from healthy and inoculated leaves (at 48hr after inoculation) in the RNA of isolated ribosomes are quantitativelysignificantly different. This suggests a change in the propertiesof the soluble RNase during the initial stages of host-parasiteinteractions. (iii) As early as 24 hr after inoculation, thereis a dramatic change in the distribution of the pH 5 insolubleand soluble RNase cleavage sites in the RNA of ribosomes indicatinga readily detectable conformational change in the ribonucleoproteinparticles. (iv) These changes in the RNases and ribosomes areonly detectable in the susceptible cultivars of barley and notin a cultivar which is genetically resistant to race 3 of thepowdery mildew fungus. (Received January 31, 1980; )     

Purification and properties of two ribonucleases in different intracellular compartments in pea root tissue.

Two RNases in bound forms associated with the microsomal membrane and with the ribosomes or unknown particles in pea root tissue were solubilized by subjecting the membrane to sonic oscillation in the presence of EDTA and KC1 and by treating the particles with EDTA, respectively. The RNases were than purified by DEAE-cellulose and Sephadex G-75 column chromatographies. The elution profiles of RNases from the columns were very similar. No significant differences were observed in their electrophoretic mobilities in polyacrylamide gels, in molecular weight, in activation by inorganic ions, urea or phospholipid micelles or in the dependence of their activities upon pH. The purified RNASES were not different from the bound enzymes as regards activation by inorganic ions and urea and the dependence of the activity upon pH. Triton X-100 stimulated the activity only if RNase was in a bound form associated with the microsomal membrane. We propose that the two RNases may be the same molecular species and differ only in the form of association with intracellular structures.     

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(2)S(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.     

Partial Purification and Properties of Tea Leaf Ribonuclease

Four fractions with ribonuclease activity have been isolated from tea leaves by DEAE-cellulose column chromatography and designated as RNase Tf-1, RNase Tf-2, RNase Tf-3 and RNase Tf-4. The bigger fractions of both RNase Tf-3 and RNase Tf-4 have been partially purified by Sephadex G-100 column chromatography.

RNase Tf-3 and RNase Tf-4 were respectively found to have their optimum pH at 4.75 and 4.9 and molecular weights of approximately 13,000 and 16,000, as determined by gel filtration. Both enzymes were inhibited by Cu²⁺ and Hg²⁺, and inactivated by heating at over 50°C. By addition of yeast RNA to the two enzymes, however, their thermostabilities increased. The activities of the enzymes were stable in a pH range of 4.5 to 6.5. Like other plant RNases, RNase Tf-3 and RNase Tf-4 appeared to have no preference for base in RNA.     

On the track of antitumour ribonucleases

Ribonucleases (RNases) are potential alternatives to non-mutagenic antitumour drugs. Among these enzymes, onconase, bovine-seminal ribonuclease and the Rana catesbeiana and Rana japonica lectins exert a cytotoxic activity that is selective for tumour cells. A model for the mechanism of cytotoxicity of these RNases which involves different steps is generally accepted. The model predicts that cytotoxicity requires interaction of the RNases with the cell membrane and internalisation to occur by endocytosis. Then, at a precise point, the RNases are translocated to the cytosol where they cleave cellular RNA if they have been able to preserve their ribonucleolytic activity. The cleavage of cellular RNA induces apoptosis but there is evidence suggesting that RNase-triggered apoptosis does not entirely result from the inhibition of protein synthesis. How efficiently a particular RNase carries out each of the steps determines its potency as a cytotoxin.     

Why ribonucleases cause death of cancer cells

Molecular properties and possible mechanisms of action of cytotoxic ribonucleases (RNases), potential antitumor therapeutics, are characterized. The analysis of recent publications and own experimental results have allowed the authors, on the one hand, to distinguish cellular components that are responsible for selective activity of exogenous RNases towards malignant cells, and on the other--to identify the contribution of definite molecular determinants to the enzyme cytotoxicity. The predominant effect of the RNase molecule charge on the cell death induction is shown. The RNase cytotoxic effects are caused by catalytic cleavage of available RNA, by products of its hydrolysis, as well as by non-catalytic electrostatic interaction of exogenous enzyme with cell components. Potential targets for RNase action in a cancer cell have been revealed. The role of modulation of the membrane calcium-dependent potassium channels and ras-oncogene functions in the RNase-induced cell damage is defined. The effect of cytotoxic RNases on gene expression via influencing the RNA interference is discussed.     

Stromal cells and extracellular matrix components in spontaneous canine transmissible venereal tumour at different stages of growth

Stromal cells and extracellular matrix (ECM) components are important for tumour cell behaviour. Little is known about the role of stromal cells and ECM components in the progression and regression of spontaneous canine transmissible venereal tumour (CTVT). In this study, the stromal cell type was determined by immunohistochemical labelling with antibodies to desmin, vimentin and alpha-smooth muscle actin (alpha-SMA) during the progressive and regressive stages of spontaneous CTVT. The distribution of ECM components tenascin-C, chondroitin sulphate and versican were determined immunohistochemically, and hyaluronan distribution was determined using a biotinylated protein complex with specific affinity for hyaluronan. Stromal cells of tumours in both the progressive and regressive stage were positive for vimentin and negative for desmin. The number of stromal cells expressing alpha-SMA was significantly higher (P=0.001) in regressing tumours, than progressing tumours. These results suggest that the modulation of stromal cells that occurs during the regression of CTVT is similar to that occurring during wound healing. Tenascin-C was weakly expressed in the stroma of tumours in the progressive stage and in regions of the regressing tumours with tumour infiltrating lymphocytes (TILs), but intensely expressed in the stroma of tumours in late regressive stage. In addition, tenascin-C was also expressed in the cytoplasm of some tumour cells in the late regressive stage. A strong stromal tenascin-C intensity was significantly associated with regressing tumours (P=0.001). Strong stromal hyaluronan intensity and a high proportion of hyaluronan-positive tumour cells were significantly associated with progressing tumours (P=0.001). This suggests that hyaluronan is involved in the growth of the tumour. There was no significant difference in the expression of chondroitin sulphate and versican in progressing and regressing tumours.     

Activities of RNases in different cell compartments in different regions of pea roots

RNase activity in three different regions of pea roots—thetip, middle and basal regions—was analyzed. There werethree types of RNases differing from each other in their intracellularlocalization; the enzymes in a soluble form and two bound formsassociated with unknown, small particles or ribosomes and withthe microsomal membrane. The top region showed a high activityper DNA content of RNase in the microsomal membrane and lowactivities for the other two RNases, as compared with the otherregions. The middle region contained a low amount of RNA perDNA and showed a higher activity per DNA content of RNase inthe unknown particles or ribosomes than in the basal region.The activity of RNase in the unknown particles or ribosomesvaried greatly among the regions, but that in microsomal membranevaried only slightly. ¹ Present address: Okitsu Branch, Fruit Tree Research Station,Okitsu, Shizuoka, Japan. ² Present address: Asahi Denka Co. Ltd., 7–1 Higashiogu,Arakawa, Tokyo, Japan. (Received July 25, 1974; )     

Junction ribonuclease activity specified in RNases HII/2

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA-DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA-DNA/RNA heteroduplex in a manner similar to the RNA-DNA/DNA substrate. The cleavage site of the RNA-DNA/RNA substrate corresponds to the RNA/RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA-DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single-stranded RNA-DNA, it can recognize a single-stranded RNA-DNA junction if a double-stranded region is located adjacent to the junction.     

Human ribonucleases. Quantitation of pancreatic-like enzymes in serum, urine, and organ preparations

Antibodies against pure human pancreatic ribonuclease (RNase) were used to study ribonuclease levels in human tissues and body fluids. The antibodies completely inhibit the activity of purified RNase as well as ribonuclease activity in crude pancreatic extracts. RNase activity is inhibited by 70-80% in serum and urine, indicating that a significant proportion of the RNases in these preparations are structurally like the pancreatic enzyme. In contrast, inhibition of RNase activities from spleen (8%) and liver (30%) was inefficient suggesting that most of the RNases in these tissues are structurally unlike the pancreatic enzyme. A competitive binding radioimmunoassay (RIA), sensitive in the range of 1-100 ng of RNase, was developed to quantitate the pancreatic like enzymes. The RIA of crude tissue preparations and samples fractionated by gel filtration was compatible with inhibition results. Enzymes structurally like pancreatic RNase could be quantitated despite the presence of other RNase activities. Immunological quantitation of pancreatic like RNases was also found to be much more simple and precise than enzymatic assays comparing RNA and polycytidylate substrates. We suggest the immunological assays will be useful in the quantitation and definition of tissue of origin of RNases in serum of patients with pancreatic carcinoma.     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.