RNAase Treatment Research Articles

Comparison Of The Gut Microbiota Composition Between Obese And Non-obese Young Children Using 16s Gene Sequening

PURPOSE: The gut microbiota regulates metabolic function and energy balance, and an altered microbial ecology contributes to the development of several metabolic diseases including obesity. To examine the association between obesity and the human gut microbiota composition in Japanese children, fecal concentrations of Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria and Firmicutes/Bacteroidetes ratio were analyzed in 42 young children. METHODS: The subjects were 42 young children (6 obese, 36 non-obese) aged 4.5-6.5 years. Obesity was determined upon an obesity index score ((real weight - standard weight) / standard weight×100) of more than +15%. To extract enterobacterial DNA, 0.2 g of feces was used to crush cells, centrifuge several times, and collect precipitates to prepare a DNA solution. Next, RNAase treatment was performed on the DNA solution to prepare a PCR solution. Submit the amplified DNA to the next-generation sequencer team of the Faculty of Applied Biological Sciences, Gifu University, and perform metagenomic analysis using the Illumina MiSeq(TM)II system by quantitative RT-PCR targeting bacterial 16S rRNA It was. The items to be detected were phylum classification, bifidobacteria, lactic acid bacteria, and F/B ratio. RESULTS: The fecal concentrations of Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria in 42 young children were 31.9±9.4%, 58.6±10.5%, 7.0±4.8% and 1.7±1.4%. The obtained data indicate that obese children have a significantly higher level of Firmicutes (Effects size=1.08) and lower level of Bacteroidetes (Effects size=1.13) compared to non-obese children (p<0.05). The Firmicutes to Bacteroidetes ratio was higher in obese children compared with non-obese subjects (p<0.05, Effects size=0.74). However, the gut microbiota diversity, bifidobacteria and lactic acid bacteria were not different between the obese and non-obese groups. CONCLUSIONS: Gut microbial properties differ between obese and non-obese subjects in Japan, suggesting that gut microbiota composition is related to obesity.

Antinucleolar antibodies in diagnostics of antiphospholipid syndrome

Anti "nucleolar" antibodies are estimated to be present in about 5-10% of anti-nuclear antibodies-positive (ANA), but only in about 15% of cases (sometimes in lower percentages) it is possible to assess precisely the specificity of antibodies responsible for this pattern of ANA. Nucleoli are composed of many proteins, e.g., nucleolin, fibrillarin, Pm-Scl (spliceosome component), RNA-polymerases and different sizes rRNA transcripts. Nucleoli also contain annexins (ANX) II and V. Many of these proteins are target molecules for autoantibodies generated in connective tissue diseases (CTD)--especially in scleroderma and overlap syndromes. The aim of this study was to work out methods for differentiation of antibody specificities in the case of sera ANA-positive with the nucleolar pattern in indirect immunofluorescence (IIF), with a particular concern directed to anti-Annexin V antibodies (belonging to anticofactor antibody system--characteristic of antiphospholipid syndrome [APS]). In the sera of the tested group of 12 (selected from 150 subjects suffering from different kinds of CTD) ANA-positive (Western-blott negative) and showing the nucleolar type of ANA pattern patients, the following autoantibodies were assessed: ANA-IIF, anti-Pm-Scl (overlap syndrome marker), anticardiolipin, antiannexin V, anti-RNA-ase--all using the ELISA method. With the use of the Western-blott method a basic set "marker" autoantibody in CTD was estimated, and the RNA-ase was used for elimination (by enzymatic digestion) of RNA-s and RNA-protein complexes--a possible target for autoantibodies giving the nucleolar pattern. In 5 out of 12 sera (41.7%) of ANA-positive/Western-blott-negative CTD patients antibodies against Annexin V, cardiolipin, Pm-Scl were detected. In 2 out of 12 patient sera antibodies to the RNA-ase were found. None of the tested sera showed the presence of autoantibodies against antigen Scl-70 (scleroderma marker), and SS-A (Sjögren syndrome marker), which can also give the atypical nucleolar ANA-pattern. Only in few sera "my ositis"--blotts showed antibodies to antigen Ku (nuclear protein), Jo-1 and PI-7 (anti-tRNA synthetases--polymyositis markers)--myositis and overlap syndrome markers. In 80% of tested sera the co-appearance of anti-ANX-V and anticardiolipin antibodies was observed. The presence of antibodies against ANX-V together with Pm-Scl was confirmed in 60% of sera The RNA-ase, used as a specific rRNA and RNP-protein blocker, resulted in a partial or total disappearance of the "nucleolar" pattern, but independently of the tested serum specificity (anti-ANX-V, anti-Pm-Scl or both together). The observed RNA-ase treatment of the Hep-2 cells effects are equivocal and they may result from either binding or digestion the antigen by the RNA-ase, but also from forming a sterical hindrance for the autoantigen binding to another "nucleolar" antigens. These preliminary results are interesting, especially as concerns anti-ANX-V autoantigens (associated with APS), but they require further research including bigger groups of CTD patients.

Using fluorescence for improvement of the quantitative analysis of micronucleus in cell culture

The micronucleus test is of unquestionable importance, being widely used in studies with several purposes. Standardization of the technique has been proposed by several groups mainly when in vivo assays are performed. In cell cultures the staining method that predominates is the Feulgen's reaction, since it is specific for DNA. In this work, we evaluated the use of two fluorescent stains, SYTOX green ® and propidium iodide, in substitution for the Feulgen's reaction. Based on the results we concluded that the fluorescence microscopy allows the reliable detection of micronuclei, presenting the following advantages: the technique is easy and fast to perform, it avoids acid treatments; the fluorescence is long lasting; micronucleus counting becomes easier and more reliable by the use of RNAase treatment; the technique is more sensitive to detect the smallest micronuclei.

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238.

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.

Formation of 5-aminolevulinate via glutamate-1-semialdehyde and 4,5-dioxovalerate with participation of an RNA component in Scenedesmus obliquus mutant C-2A′

In the yellow mutant C-2A′ of the unicellular green alga Scenedesmus obliquus the participation of an RNA species in the conversion of glutamate to 5-aminolevulinate is clearly demonstrated by the fact that RNAase treatment of a soluble enzyme preparation drastically decreases the formation of 5-aminolevulinate. The involvement of 4,5-dioxovalerate in the C 5 pathway is demonstrated by the decrease of label in enzymatically formed 5-aminolevulinate from [ 14C]glutamate by providing an increased unlabelled pool of 4,5-dioxovalerate. Evidence supporting the role of glutamate-1-semialdehyde as an additional intermediate in the reaction sequence is also presented. We propose a new reaction scheme, consistent with the results reported here, for the formation of 5-aminolevulinate via the C 5 pathway.

Purification of a novel 30,000 Da calcium-binding protein from bovine cerebellum.

A novel very acidic calcium-binding protein (CaBP) was purified from bovine cerebellum, using 45Ca autoradiography as a marker, through a preparative procedure involving salting out with a very high concentration of ammonium sulfate, DE52 column chromatography, RNAase treatment, and HPLC gel filtration. This protein showed a molecular weight of 30,0000 dalton (Da) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and of 120,000 on in gel filtration chromatography analysis under physiological ionic strength. The calcium binding activity of this 30,000 Da CaBP was monitored on the basis of calcium-dependent changes in tyrosine fluorescence (Kd = 3.0 microM).

Immunological difference between ribonuclease and temperature, time and salt-induced forms of the estrogen receptor detected by a monoclonal antibody

The effect of RNAase A on the activation of the estrogen receptor from fetal guinea pig uterus was studied by DNA-cellulose binding assay and immunorecognition of the estradiol-receptor complex by the monoclonal antibody D547 raised against the human estrogen receptor. After RNAase treatment at 4°C or 25°C the binding of the receptor to DNA-cellulose doubled. This stimulation was partially prevented by sodium molybdate. RNAase treatment did not modify the interaction of the receptor with the monoclonal antibody D547; this antibody, as was demonstrated previously, selectively recognizes the activated form of the receptor when activation has been induced by temperature, time or high salt concentrations. In addition, RNAase had little or no effect on the transformation of the 8–9 S receptor to more slowly sedimenting forms under low salt concentrations. These observations suggest that even if RNAase induces receptor activation, which can be inferred from the increase in its binding to DNA-cellulose, the conformational modifications of the receptor molecule involved in this process are apparently different from those induced by factors such as temperature, time or high-salt concentrations.

Studies with chymotrypsin and RNAase showing a heterooligomeric structure of the glucocorticoid receptor complex from rat liver which is stabilized by a low molecular weight factor

The glucocorticoid receptor from rat liver displays a differential sensitivity toward digestion by chymotrypsin and RNAase A that is dependent on its activation state. Unactivated (9–10 S) receptor is not digested by these enzymes, while activated 7–8 S receptor is. Chymotrypsin treatment yields an approx. 3 S form, while RNAase treatment yields a 4.9 S form that is distinct from the high-salt 4 S form. To firmly establish that the results are due to specific hydrolytic activities of the particular enzymes, we show that the chymotrypsin effect is inhibited by diisopropylfluorophosphate and not RNAasin, while the reverse is true for RNAase A. We further show that the differential sensitivity toward chymotrypsin is due to the association of a proteinase-resistant, heat-stable low molecular weight factor with the unactivated glucocorticoid receptor. When this factor is removed by warming, dialysis or molecular sieving of the receptor complex, the complex becomes sensitive to chymotrypsin. We also show that moderate chymotrypsin treatment yields a 6–7 S form of the receptor which is composed of, at least, RNA and the 4 S receptor. On the basis of these results, we propose that the 9–10 S receptor is composed of a low molecular weight stabilizing factor whose presence apparently alters the conformation of the complex such that the RNA and the RNA-binding site of the receptor are protected, a chymotrypsin-sensitive factor, RNA and the 4 S receptor itself.

Low ionic strength extraction of nuclease-treated nuclei destroys the attachment of transcriptionally active DNA to the nuclear skeleton

We have studied how the conditions in which the nuclear matrix is isolated influence the association of transcribing DNA with the nuclear matrix. Extraction of nuclease-treated nuclei with a low ionic strength solution before a high salt nuclei with a low ionic strength solution before a high salt extraction completely abolishes this association. However, RNA removal by RNAase treatment does not affect the binding of transcribing DNA to the nuclear matrix. The nature of the association of active genes with the nuclear matrix is discussed.

Molecular properties of the Ro/SSA antigen and enzyme-linked immunosorbent assay for quantitation of antibody.

Antibodies to the Ro/SSA antigen occur in patients with systemic lupus erythematosus and Sjögren's syndrome. An immunoaffinity method for the preparation of electrophoretically homogeneous Ro/SSA antigen is described. Several molecular properties of the antigen have been determined. The native RNA protein particle has a molecular weight of approximately 100,000 D determined by gel filtration. Sodium dodecyl sulfate-analysis of the purified Ro/SSA antigen and analysis by staining of bands with silver and Coomassie Blue, Western blotting, and RNAase treatment leads to a hypothesis for the structure of the particle in which an antigenic 60,000 protein is bound to 24,000-27,000 RNA molecules which are not antigenic. An enzyme-linked immunoabsorbent assay method for assay of anti-Ro/SSA is also described which sensitively measures antigen binding at dilutions of sera containing anti-Ro/SSA precipitins up to 10(7) fold. Normal sera on average have 10(3) less binding activity.

Microfluorometric Assessment of Sperm Maturation in Testicular Biopsies From Men With Histologically Normal or Reduced Spermatogenesis

Spermatogenesis has been studied in testicular biopsies by means of microfluorometric assessment of the DNA-DNP complex using ethidium bromide in 10 men with histologically normal spermatogenesis and in 10 men with histologically reduced spermatogenesis. The expected haploid DNA value was found in the round spermatids, whereas only 70% of the haploid fluorescence value was found in the elongated spermatids and around 60% in the testicular spermatozoa. No difference was found in the mean fluorescence values between the pathological group and the controls. it is suggested that structural changes or an increase in basic nuclear proteins gradually exclude the ethidium bromide from the binding to the DNA molecule and that this phenomenon occurs concomitantly with a decrease in double-stranded RNA. The difference in fluorescence intensity before and after RNAase treatment was regarded as an approximate estimate of the RNA content. There was a significant decrease of these values during spermatogenesis.

Adenovirus DNA replication in vivo. Properties of short DNA molecules extracted from infected cells

Adenovirus type 5 (Ad5) DNA replicating in intact HeLa cells was pulse-labeled with [ 3H]thymidine extracted by the procedure of Hirt and analyzed on neutral sucrose gradients. In addition to viral replicative forms (over 33 kb), a slowly sedimenting species of DNA (0.05–3 kb) was observed. Hybridization analysis showed that this DNA contained about 30% Ad5 DNA sequences. Analysis of the sensitivity of this DNA to the 5′ OH-specific spleen exonuclease after alkali or RNAase treatment revealed that about 80% of these molecules contained ribonucleotides. DNA in this fraction was labeled at the 5′ end with 32P and hybridized together with control 3H-labeled Ad5 DNA to HpaI restriction fragments immobilized on nitrocellulose paper. Many of these small molecules were located near the termini. Alkali treatment prior to hybridization decreased the 32 P 3 H ratio throughout the genome. This suggests that some of these Ad5 molecules possess ribonuncleotides and therefore may be intermediates in discontinuous DNA replication. Longer molecules (over 0.5 kb) were found sedimenting with viral replicative forms and mature DNA. The Ad5 molecules in this fraction showed no evidence of alkali-labile termini.

Microfluorometric assessment of sperm maturation in testicular biopsies from men with histologically normal or reduced spermatogenesis.

Spermatogenesis has been studied in testicular biopsies by means of microfluorometric assessment of the DNA-DNP complex using ethidium bromide in 10 men with histologically normal spermatogenesis and in 10 men with histologically reduced spermatogenesis. The expected haploid DNA value was found in the round spermatids, whereas only 70% of the haploid fluorescence value was found in the elongated spermatids and around 60% in the testicular spermatozoa. No difference was found in the mean fluorescence values between the pathological group and the controls. it is suggested that structural changes or an increase in basic nuclear proteins gradually exclude the ethidium bromide from the binding to the DNA molecule and that this phenomenon occurs concomitantly with a decrease in double-stranded RNA. The difference in fluorescence intensity before and after RNAase treatment was regarded as an approximate estimate of the RNA content. There was a significant decrease of these values during spermatogenesis.

A small molecular weight peptide from P815 mastocytoma cells induces macrophage cytotoxicity.

In a previous article we have shown the presence of a "factor" in the cell free exudates of intraperitoneally growing P815 mastocytoma cells as well as in cell-free tumor cell extracts. This "factor" is able to induce nonspecific macrophage cytotoxicity against tumor target cells. The "factor" was sensitive to heating at 100 degree C for 1 min, destroyed by pronase treatment, and dialyzable. On the other hand, the "factor" was not removed by centrifugation (25,000 x g), and was resistant to chloroform extraction, ultraviolet irradiation, heating at 56 degree C (for 20 min), or RNAase treatment. Sephadex gel fractionation of the mastocytoma exudate showed that the "factor" has an approximate molecular weight of 650-700 daltons. Normal peritoneal macrophages incubated with an extract of murine mast cells, purified histamine or serotonin did not show detectable cytotoxicity against tumor cells. The results of our experiments suggest that the effect of the P815 cell-free exudate and tumor cell extract is due to a dialyzable small molecular weight peptide.

Interaction of AMP deaminase with RNA

tRNA, 18 S and 28 S ribosomal RNAs were found to activate muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) but inhibit liver and heart AMP deaminases. The macromolecular structures are essential for modulation of enzyme activity, since the effects of RNA disappeared after RNAase treatment. Sucrose density centrifugation experiments clearly demonstrated the binding of purified muscle AMP deaminase to tRNA, 18 S and 28 S RNAs. The binding is reversible and responsive to alterations of pH and KCl concentration. The binding was stable at pH 5.1–7.0 in 0.1 M KCl, but most of the enzyme dissociated at pH 7.5. KCl below 0.1 M concentration had no effect on dissociation of enzyme-RNA complex, but in 0.15 M KCl the complex was partially dissociated and in 0.2 M KCl most of the enzyme was released. Various nucleotides were also effective in dissociation of the enzyme from complex. The binding is saturable and the maximum number of muscle AMP deaminase molecules bound per mol 28 S RNA was calculated to be approx. 30. Liver and heart AMP deaminases were also found to interact with RNA.

Nucleoprotein cytochemistry during oogenesis in a unisexual fish, Poecilia formosa.

Cytochemical methods and electron microscopy were used to study changes in the chemical composition of nuclear, nucleolar and perinuclear bodies during the early stages of oocyte development in Poecilia formosa, an apomictic species of fish that produces only female offspring. Prominent accumulations of ribonucleoprotein (RNP) occur in nucleoli and appear on either side of the nuclear envelope during diplotene. In certain planes of section, RNP material seems to be in transit across this interface. En bloc acid extractions or RNAase treatment abolished basophilia and markedly reduced the electron density of both nucleoli and cytoplasmic nucleolar-like bodies. DNA-specific fluorescent probes such as mithramycin failed to reveal nucleolar cores in poeciliid oocytes, although the same procedures showed unequivocal localization of GC-rich DNA cores within multiple nucleoli of diplotene oocytes from Xenopus laevis or the rainbow trout, Salmo gairdneri. Also, cytological hybridization studies, utilizing [3H]rRNA as a probe for nucleolar DNA, failed to show localizations of silver grains above nucleoli in autoradiograms of poeciliid oocytes. Feulgen-stained pachytene oocytes of P. formosa have twice the number of chromosome strands seen in similar stages of oocytes from two, related bisexual species, P. mexicana and P. latipinna. Although the bivalent nature of these chromosomes could not be resolved with the light microscope, configurations resembling, but not identical to, synaptonemal complexes were identified by electron microscopy.

Functional Modification of the Plasmid RP1-specified Pilus by Caulobacter vibrioides

Summary: Caulobacter strains which contain plasmid RP1 are resistant to carbenicillin, kanamycin and tetracycline and can serve as donors of the plasmid, but such R+ strains are not susceptible to the RP1-specific phage PRR1. Since both conjugation and phage PRR1 infection are mediated by the plasmid-specified pilus, the lack of phage binding suggests some functional alteration of the pilus. In this report we demonstrate that RP1 pili are produced by Caulobacter R+ cells, but unlike the RP1 pili produced by PRR1-susceptible strains, the Caulobacter R+ pili do not inactivate phage PRR1. Antibody produced against Caulobacter R+ pili prevents phage PRR1 binding to RP1 pili from PRR1-susceptible strains of Pseudomonas aeruginosa and Escherichia coli, and also inhibits plasmid transfer by those strains. Antibody produced against RP1 pili from P. aeruginosa and E. coli inhibits plasmid transfer both by those strains and by Caulobacter R+ donors. Phage PRR1 rendered non-infectious by RNAase treatment prevents plasmid transfer by P. aeruginosa and E. coli but not by Caulobacter.

Characterization of a poly(A) +RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts

Poly(A) +RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A) +RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs 2SO 4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17–26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A) +RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A) +RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs 2SO 4-resistant structures, independent of ribosomes, but intimately associated with membrane components.

Physical properties of the complementary T 4 RNA

The complementary transcribed T 4 RNA after self-annealing and RNAase treatment was isolated by gel chromatography and then used for further studies. From salt-dependent RNAase resistance and melting studies it is evident that this RNA represents a genuine double-stranded structure. The base content of the isolated double-stranded RNA was found to be the same as total T 4 mRNA. Sucrose gradient analysis and hydroxyapatite chromatography of T 4 RNA, annealed early and late RNA, and of the isolated double-stranded RNA, gave results indicating that the complementary RNA is part of a RNA molecule and further that the size of the complementary regions are independent of the RNA molecules. Partial digestion of pulse-labelled late RNA with phosphodiesterase I prior to annealing with unlabelled early RNA, showed that the complementary regions on the mRNA are not located to the 5′- or 3′-end but randomly distributed along the T 4 RNA molecules.

A heat-stable protein inhibitor of cyclic 3′,5′-nucleotide phosphodiesterase

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodiesterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca 2+-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.