An intracellular inhibitory activity of RNase secreted by Bacillus intermedius

Bacillus intermedius cells producing extracellular RNAse were found to contain its inhibitor and an RNAse-inhibitor complex. Bacillus subtilis and Escherichia coli cell lysates did not inhibit the activity of homogeneous extracellular RNAse produced by B. intermedius. The inhibitor was shown to be specific for this RNAse and did not interact with other RNAses. As was demonstrated by biochemical tests and electrophoretic analysis, the inhibitor is released when the protoplasts are disintegrated, i.e. it is located in the cytoplasm. A correlation has been established between the biosynthesis of extracellular RNAse and its intracellular inhibitor.     

Comparative pyrimidine- and purine-specific RNAse-gold labeling on pancreatic acinar cells and isolated hepatocytes.

We applied the enzyme-gold approach to investigate the potential of various ribonucleases displaying different affinities for ultrastructural localization of particular RNA molecules. Five specific ribonucleases were used: three from a pancreatic source, RNAses A, B, and S with affinities for pyrimidine bases; and two from Aspergillus oryzae, RNAses T1 and T2 specific for purine bases. Conditions required for preparing each RNAse-gold complex, as well as for obtaining specific labelings, were determined. Application of the probes on thin sections of pancreatic acinar cells yielded labeling patterns that differed according to the enzyme used. Pancreatic RNAses labeled mostly the rough endoplasmic reticulum and the nucleolus, whereas fungal RNAses labeled more intensely the interchromatin space and the nucleolus, the rough endoplasmic reticulum being labeled to a lesser extent. Areas rich in interchromatin granules were intensely labeled by the RNAses T1 and T2. This was confirmed on DRB-treated hepatocytes, which displayed large clusters of interchromatin granules. Perichromatin granules were labeled by the RNAse A- and T1-gold complexes. These results provide a strong indication for the presence of RNA molecules in both types of granules. Nuclear pores were labeled, particularly by the RNAses T1 and T2, thus supporting the hypothesis for the site of RNA transit between nucleus and cytoplasm. The differences in patterns of labeling among the various enzyme-gold complexes could be related to differences in affinities. The use of a panel of specific RNAses, displaying different affinities, could thus allow for the topographical distribution of particular RNA molecules according to their relative content of specific bases.     

RIBONUCLEASE ACTIVITIES IN VARIOUS CLASSES OF NUCLEI FROM BOVINE CEREBRUM

Abstract— Nuclei of adult bovine cerebrum were separated by dissection of grey matter from white matter and discontinuous sucrose gradient centrifugation, into neuron, astrocyte-enriched and oligodendrocyte fractions. The activities of alkaline and acidic RNAses were determined in the different nuclei types. The acidic and alkaline RNAse activities of the neuronal nuclei were much higher than those of the glial nuclei. In each type of nuclei the acidic RNAse activity was lower than the measured alkaline RNAse activity.     

Kinetic properties of Ca2+/calmodulin-dependent phosphodiesterase isoforms dictate intracellular cAMP dynamics in response to elevation of cytosolic Ca2+

Multiply regulated adenylyl cyclases (AC) and phosphodiesterases (PDE) can yield complex intracellular cAMP signals. Ca2+-sensitive ACs have received far greater attention than the Ca2+/calmodulin-dependent PDE (PDE1) family in governing intracellular cAMP dynamics in response to changes in the cytosolic Ca2+ concentration ([Ca2+]i). Here, we have stably expressed two isoforms of PDE1, PDE1A2 and PDE1C4, in HEK-293 cells to determine whether they exert different impacts on cellular cAMP. Fractionation and imaging showed that both PDEs occurred mainly in the cytosol. However, PDE1A2 and PDE1C4 differed considerably in their ability to hydrolyze cAMP and in their susceptibility to inhibition by the non-selective PDE inhibitor, IBMX and the PDE1-selective inhibitor, MMX. PDE1A2 had an approximately 30-fold greater Km for cAMP than PDE1C4 and yet was more susceptible to inhibition by IBMX and MMX than was PDE1C4. These differences were mirrored in intact cells when thapsigargin-induced capacitative Ca2+ entry (CCE) activated the PDEs. Mirroring their kinetic properties, PDE1C4 was active at near basal cAMP levels, whereas PDE1A2 required agonist-triggered levels of cAMP, produced in response to stimulation of ACs. The effectiveness of IBMX and MMX to inhibit PDE1A2 and PDE1C4 in functional studies was inversely related to their respective affinities for cAMP. To assess the impact of the two isoforms on cAMP dynamics, real-time cAMP measurements were performed in single cells expressing the two PDE isoforms and a fluorescent Epac-1 cAMP biosensor, in response to CCE. These measurements showed that prostaglandin E1-mediated cAMP production was markedly attenuated in PDE1C4-expressing cells upon induction of CCE and cAMP hydrolysis occurred at a faster rate than in cells expressing PDE1A2 under similar conditions. These results prove that the kinetic properties of PDE isoforms play a major role in determining intracellular cAMP signals in response to physiological elevation of [Ca2+]i and thereby provide a rationale for the utility of diverse PDE1 species.     

Extracellular guanyl-specific ribonuclease Sa from the actinomycete Streptomyces aureofaciens. Primary structure and homology with ribonucleases from bacteria and fungi

The complete amino acid sequence of a guanyl-specific RNAse from Streptomyces aureofaciens has been established using a rapid method of primary structure analysis which eliminates the peptide fractionation. The automated Edman degradation of the carboxymethylated RNAse Sa and of non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the modified protein were used. The RNAse contains 96 amino acid residues, Mr 10,566. The secondary structures of RNAse Sa and microbial RNAses have been calculated using a modified Chou--Fasman procedure. A comparison of the primary and secondary structures of the RNAses revealed different degrees of sequence homology and a similar distribution of predicted structural regions (alpha-helices, beta-structure and beta-turn). The predicted secondary structure patterns are discussed in the light of the RNAse X-ray analysis date.     

Extracellular ribonuclease from Bacillus pumilus]

The extracellular ribonuclease (RNAse Bp) was isolated from the cultural medium filtrate of Bacillus pumilus by ammonium sulfate precipitation and two stages of ion-exchange chromatography on carboxymethyl- and phospho-cellulose columns. The amino acid composition and N-terminal amino acid residue have been determined. The kinetic parameters of cleavage reaction of synthetic polynucleotides have been measured. According to their structural homology RNAse Bp has been shown to be similar to RNAses Ba and Bi. Catalytic properties of the enzyme are very close to RNAse Bi.     

The Aicardi-Goutières syndrome. Molecular and clinical features of RNAse deficiency and microRNA overload

Intracellular RNAses are involved in various functions, including microRNA maturation and turnover. Mutations occurring in genes encoding RNAses cause Aicardi-Goutiéres syndrome (AGS). AGS mutations silence RNAse activity, thus inducing accumulation of endogenous RNAs, mainly consisting of short RNAs and microRNAs. Overload of intracellular RNA triggers Toll like receptor-dependent interferon-alpha production in the brain, which in turn activates neurotoxic lymphocytes and inhibits angiogenesis thus inducing the typical clinical phenotype of AGS. However, these pathogenic mechanisms are attenuated after three years of age by the endogenous production of DNAJP58IPK and Cystatin F, which arrest AGS progression. Because RNAses are involved in microRNA turnover, we evaluated the expression of 957 microRNAs in lymphocytes from AGS patients and control patients. Our results indicate that microRNA overload occurs in AGS patients. This upregulation inhibits microRNA turnover impeding the synthesis of the novel microRNAs required for the differentiation and myelination of the brain during the initial period of postnatal life. These pathogenic mechanisms result in AGS, a neurological syndrome characterized by irritability, mild hyperpyrexia, pyramidal and extrapyramidal signs, and spastic-dystonic tetraplegia. Typical cerebrospinal fluid alterations include lymphocytosis and elevated interferon-alpha levels. Brain imaging demonstrates cerebral calcifications, white matter abnormalities, and progressive cerebral atrophy.Thus, evidence exists that mutations silencing intracellular RNases affect microRNA turnover resulting in the severe clinical consequences in the brain characterizing the clinical feature of AGS.     

NUCLEAR RIBONUCLEASE ACTIVITIES OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— The activities of alkaline and acidic RNAses were determined in soluble and insoluble fractions from nuclei of brain hemispheres of rats, aged from 1 day to adult. The activities increased rapidly and reached a maximum, at 30 days, of about 10 times (alkaline RNAsel or 5 times (acidic RNAse) that seen at day 1.     

Multiple activities contribute to Pc2 E3 function

Pc2 is a polycomb protein, which has SUMO E3 activity for the corepressors CtBP and CtBP2. Here we demonstrate that, in vivo, Pc2 adapter function contributes to enhancement of CtBP sumoylation. Mutation of the CtBP binding site on Pc2 abolishes E3 activity toward CtBP. However, a carboxyl-terminal fragment of Pc2 that recruits both Ubc9 and CtBP lacks E3 activity. We identify a second domain, which, when coexpressed with the carboxyl-terminal adapter region, restores E3 function. In vitro, this domain has E3 activity in isolation, suggesting that it is a functional domain, and that adapter function is required to selectively corecruit E2 and substrate in vivo. These results demonstrate the presence of two domains in Pc2 that contribute to full in vivo E3 activity, and suggest that SUMO E3s are more than simple platforms to which E2 and substrate bind.     

High-resolution mapping adjacent to the Pc71 crown-rust resistance locus in hexaploid oat

The D526-derived BC₁F₂ population of hexaploid oat segregates for resistance to crown rust isolate 345. A mapping population consisting of 440 F2 individuals was used to develop a high-resolution RFLP map of the area of the genome where Pc71, the locus conferring this resistance, is located. Twelve RFLP markers have been identified within ca. 6 cM of Pc71, with cosegregating loci Xcdo1502 and Xcdo783 positioned 0.2 cM from the locus. All of the RFLP markers map to the same side of the locus, suggesting either that the Pc71 resistance locus resides at the end of a linkage group, or that there is no detectable heterozygosity on the opposite side. Some degree of microcollinearity between rice and oat is present in this area, as the two markers, Xcdo783 and Xrz69, linked to Pc71 are linked also in rice; however the genetic distance in oat is much less than the genetic distance in rice (6.2 cM and 20 cM, respectively).     

Immunological studies on the interaction of polyadenylic acid and human liver ribonuclease

The effect of polyadenylic acid, a potent inhibitor of mammalian and bacterial RNAses, on the binding of human liver RNAse to its antibody was studied. To do this, a human liver RNAse antibody was immobilized on Sepharose 4B. Examination of the ability of the enzyme to bind to the immobilized anti-RNAse in the presence or absence of polyadenylic acid indicated that enzyme-antibody binding was more sensitive to the presence of polyadenylic acid than was enzyme activity. Furthermore, the effect of polyadenylic acid on enzyme-antibody binding was specific since neither polycytidylic acid nor polyuridylic acid had much effect on the antigenicity of the enzyme. The metal cation, Mg2+, and the polyamine, spermidine, but not putrescine, readily reversed the effects of polyadenylic acid on enzyme-antibody binding.     

Intratumor Administration of the Photosensitizer Pc 4 Affords Photodynamic Therapy Efficacy and Selectivity at Short Drug-Light Intervals

We evaluated intratumor (IT) versus intravenous (IV) administration of the photosensitizer Pc 4 with respect to tumor photosensitizer concentration, specificity, and responses to irradiation. BALB/c mice bearing intradermal EMT6 tumors were given 0.3 mg/kg Pc 4 injected IT or IV through the tail vein. Photosensitizer concentration was evaluated by chloroform extraction and localization assessed by fluorescence imaging and spectroscopy in vivo. Tumors were irradiated at 667 nm, 50 mW/cm², and 100 J/cm². Cures were defined as no palpable tumor 90 days after irradiation. Tumor Pc 4 concentrations 1 hour after IT administration were 35,000-fold higher than measured 24 hours after IV administration (0.112 vs 0.317 x 10^-5 µg Pc 4/mg tumor). Exquisite tumor selectivity was observed 1 hour after IT injection. Fluorescence imaging of freshly sectioned tumors revealed no regions devoid of sensitizer at this time point, with pixel intensities in a midline section within a factor of 3 of the peak intensity. For identical photosensitizer doses, IT administration significantly improved tumor responses to irradiation, with more than 70% of tumors cured with IT-Pc 4-PDT. In this model, IT-Pc 4 administration provides improved tumor control, greater selectivity, and opportunity for a short drug-light interval.     

Description of a genetic polymorphism of a human proline-rich salivary protein,Pc, and its relationship to other proteins in the salivary protein complex (SPC)

A new polymorphism, Pc, has been identified in human saliva. Two proteins, Pc 1 and Pc 2, are determined by alleles Pc1 and Pc2, respectively, which show autosomal codominant inheritance. No null phenotype has been encountered in 225 randomly collected salivas. The frequencies of the two alleles differ in the Black and White American populations, with Pc1 and Pc2 being 0.670 and 0.330 in the Black (N = 47) and 0.461 and 0.539 in the White (N = 178) populations, respectively. The alleles are in equilibrium in the two populations and segregation analyses (30 families) do not suggest the existence of a null allele in either population. Of seven polymorphic human salivary proteins determined by genes in the salivary protein complex (SPC), Pc phenotypes show association only with Ps phenotypes. Based on that association, our linkage studies, and the biochemical similarities with other SPC proteins, we tentatively conclude that Pc is a member of the SPC, bringing the total number of genes in that group to 13.     

A Role for Non-Covalent SUMO Interaction Motifs in Pc2/CBX4 E3 Activity

Background

Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified.

Methodology/Principal Findings

Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci.

Conclusions/Significance

This work provides the first clear evidence for a role for SIMs in SUMO E3 activity.     

Study of splitting dinucleoside monophosphates by Penicillium brevicompactum RNAse]

In studies of splitting of transferase substrates cytidylyl-(3' leeds to 5')-adenosine and adenylyl-(3'leds to to 5')-cytidine by Penicillium brevicompactum RNAase the pH-optimum activity of enzyme has been found to fall within the range of 4.7 +/- 0;1; temperature optimum--within 41 degrees--43 degrees C; adenine-nucleotides, their constituent components and polyphosphates display the properties of competitive inhibitors on splitting substrates and that amino-acid residues (presumably weakly- and strongly-protonated imidasole groups) function in the active site of this enzyme (pK 5.88 +/- 0,1 AND 6.6 +/- 0.1). A comparison of some physico-chemical and kinetic parameters of Penicillium breviocompactum RNAse to those of other nonspecific RNAses of fungi is made.     

RNAse substrate specificity in Acholeplasma laidlawii PG-8]

A substrate specificity of RNAses of A. laidlawii PG-8 to polynucleotides - poly (C), poly (U), poly (A) has been studied. Due to the data obtained both intracellular and extracellular RNAses of A. laidlawii possess similar specificity to different polynucleotides. Both RNAses preferentially break cytidine-bonds. Specificity of the studied enzymes in respect to polyuridylic and polyadenylic acids was less expressed.     

The yeast cyclins Pc16p and Pc17p are involved in the control of glycogen storage by the cyclin-dependent protein kinase Pho85p

Pho85p is a yeast cyclin-dependent protein kinase (Cdk) that can interact with 10 cyclins (Pcls) to form multiple protein kinases. The functions of most of the Pcls, including Pc16p and Pc17p, are poorly defined. We report here that Pc16p and Pc17p are involved in the metabolism of the branched storage polysaccharide glycogen under certain conditions and deletion of PCL6 and PCL7 restores glycogen accumulation to a snf1 pcl8 pcl10 triple mutant, paradoxically activating both glycogen synthase and phosphorylase. Pho85p thus affects glycogen accumulation through multiple Cdks composed of different cyclin partners.