Presence Of Ribonuclease Inhibitor Research Articles

Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach

Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor β (hERβ). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERβ (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.

Extraction of total RNA in the developing chicken forebrain

Background:Gene expression of Gama-Aminobutyric acid (GABAA) receptor subunits may change during development. Procedures in molecular biology are required to understand the gene expression profile GABAA R in chicken. The outcome of the results depends on good-quality high-molecular-weight RNA. Several procedures can be used to isolate RNA from the brain of chicken; however, most of them are time-consuming and require disruption of cells or freeze and thaw in the presence of RNase inhibitors. The aim of this experiment was isolation of RNA from chicken embryonic brain tissues using appropriate RNA extraction kit.Materials and Methods:Fertilized eggs from Ross breed (Gallus gallus) were incubated at 38°C and 60% relative humidity in a forced-draft incubator and were turned every 3 h. After 3, 7, 14 and 20 days of incubation, eggs were cooled on ice to induce deep anesthesia. Then whole brains were dissected out. As brains could not be excised in a reproducible way from earlier embryos (embryonic days 4 and 6), whole heads were collected. Chicken embryos between day 7 to 20 and 1 day after birth were decapitated, and their brains removed. Samples were immediately inserted into lysis buffer and stored at −70°C. Total RNA was isolated and a contaminating genomic deoxyribonucleic acid (DNA) was digested. RNA quality was checked using gel electrophoresis.Results:We obtained 52 mg/ml to 745 mg/ml with A260/280 1.7-2.2. Only high-quality RNA, with no signs of degradation, was used for further experiments.Conclusion:In conclusion, protocol was found to be suitable for the isolation of total RNA from embryonic chicken cells.

Technical note: A rapid method for 5-bromo-2′-deoxyuridine (BrdU) immunostaining in bovine mammary cryosections that retains RNA quality

A rapid method of 5-bromo-2′-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue while preserving RNA quality of the stained section. A thymidine analog that is incorporated into DNA of proliferating cells, BrdU serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open double-stranded DNA, and exposure to the BrdU antigen. Although these established treatments permit staining, they preclude use of cells within the tissue section for further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. A protocol was developed for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserved RNA integrity and quantity. This protocol used an initial acetone:polyethylene glycol 300 [9:1 (vol/vol)] fixation (2min) followed by staining with methyl green (0.5% aqueous; 2min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline; 4min incubation), antibody incubation in the presence of RNase inhibitors (5min), and minimal washing to facilitate recovery of RNA from cells from the stained sections. Applicability of this protocol to other nuclear antigens was evaluated by testing its suitability for staining estrogen receptor α and Ki-67 antigen. In both cases, use of the protocol provided good immunostaining and tissue morphology. The RNA quality of estrogen receptor α- and Ki-67-stained sections was not evaluated. Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative reverse transcription-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA or cDNA amplification, this immunostaining protocol provided a means for future transcriptome analysis of BrdU-labeled cells within a complex tissue.

124 EXPRESSION OF PLURIPOTENCY MARKERS IN GENITAL RIDGES OF OVINE FOETUSES

Fundamental problems of primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Historically, these cells can be identified using criteria as alkaline phosphatase, but also by the expression of other pluripotency markers suchas OCT4, Nanog, cKIT, SSEA1, SSEA4. Much less is known about PGC inother mammals. The objective ofthis study wasto describe, for the first time, the expression of multiple pluripotency markers in the genital ridges of ovine fetuses evaluating the binding of antibodies for stem cell surface markers (SSEA-1 and EMA-1) and the expression of Oct4, Nanog, and Sox2 genes. Ovine fetuses (24 and 27 days) were collected from slaughtered ewes and transported in a PBS solution at 4°C, to be processed as follows: Immunohistochemistry: 4 foetuses for each age were frozen in OCT TissueTek freezing compound and stored at -80°C. Tissue blocks were cut in 6-μm sections, placed on slides, and immediately prepared for indirect immunofluorescent staining. Section were fixed in 4% paraformaldehyde (10 min) and incubated overnight with SSEA-1 (1:100) and EMA-1 (1:50) antibodies (Hybridoma bank). Antibodies were detected with fluorescently-labelled goat anti-mouse secondary antibodies (1:100 dilution; Sigma) in 15% goat serum in DPBS for 1 h at room temperature. After washing, slides were mounted using ProLong antifade mounting medium (Molecular Probes). Gene expression: total RNA was extracted from fetal genital ridge samples with Trizol™ (Invitrogen). Any contaminating DNA was removed with RNase-free DNase I (Invitrogen). First strand cDNA synthesis was performed with SuperScript™ III RNase H-Reverse Transcriptase (Invitrogen) in the presence of RNase inhibitor (Promega). Real Time PCR amplification was performed in a BioRad iCycler™, with Platinum SYBR Green qPCR Super Mix UDG (Invitrogen). The relative quantification of the target genes was calculated with the 2-ddCt method (Livak and Schmittgen 2001 Methods 25, 402-408), after normalization against β-actin expression levels. Fragment identity was confirmed by sequencing. Our results indicate that the cortical region of the genital ridges is positive to stem cell surface markers. A larger number of positive cells was recorded in Day 24 fetuses with respect to Day 27 ones. The SSEA-1 and EMA-1 showed identical expression patterns. Similar levels of Oct4 and Nanog mRNA were detected in the genital ridges at both stages, whereas Sox2 displayed a lower abundance at Day 27. In conclusion, ovine PGC can be recognized by stem cell surface antigens and by the expression of Oct4, Nanog, and Sox2 pluripotency markers, as in mouse. The combined biomolecular and immunohistochemical approach provides a useful tool to follow the ovine PGC during in vitro culture to derive pluripotent stem cells in this species.

Selective Targeting of Leukemic Cell Growth in Vivo and in Vitro Using a Gene Silencing Approach to Diminish S-Adenosylmethionine Synthesis

We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIbeta subunit in Jurkat cells and accordingly shifted the K(m L-Met) of the enzyme 10-15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 microm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II K(m L-Met) due to MAT-IIbeta ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rgamma(null) humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIbeta failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIbeta ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

Characterization of mRNA species that are associated with postsynaptic density fraction by gene chip microarray analysis

We previously reported the partial identification by random sequencing of mRNA species that are associated with the postsynaptic density (PSD) fraction prepared from the rat forebrain [Tian et al., 1999. Mol. Brain Res. 72, 147–157]. We report here further characterization by gene chip analysis of the PSD fraction-associated mRNAs, which were prepared in the presence of RNase inhibitor. We found that mRNAs encoding various postsynaptic proteins, such as channels, receptors for neurotransmitters and neuromodulators, proteins involved in signaling, scaffold and adaptor proteins and cytoskeletal proteins, were highly concentrated in the PSD fraction, whereas those encoding housekeeping proteins, such as enzymes in the glycolytic pathway, were not. We extracted ∼1900 mRNA species that were highly concentrated in the PSD fraction. mRNAs related to certain neuronal diseases were also enriched in the PSD fraction. We also constructed a cDNA library using the PSD fraction-associated mRNAs as templates, and identified 1152 randomly selected clones by sequencing. Our data suggested that the PSD fraction-associated mRNAs are a very useful resource, in which a number of as yet uncharacterized mRNAs are concentrated. Identification and functional characterization of them are essential for complete understanding of synaptic function.

Modulation of the two-pore domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume.

The molecular identity of K(+) channels involved in Ehrlich cell volume regulation is unknown. A background K(+) conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K(+) channels)-type of two pore-domain K(+) channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K(+) currents in the absence intracellular Ca(2+). Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK(a) in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K(+) > Rb(+) Cs(+) > NH(4)(+) > Na(+) congruent with Li(+), similar to that for the native conductance (I(K, vol)). Clofilium, a quaternary ammonium blocker of I(K, vol), blocked the mTASK-2-mediated K(+) current with an IC(50) of 25 microm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I(K, vol), and its modulation by changes in cell volume suggest that this two-pore domain K(+) channel participates in the regulatory volume decrease phenomenon.

Экспрессия гена тиреоглобулина при врожденном зобе

A polyA-containing thyroglobulin mRNA (mRNAptf) was isolated from human thyroid in health and congenital goiter. A singlestranded complementary DNA (cDNA) was synthesized on a polyA-containing mRNA Tl [ matrix by reverse transcription in the presence of RNAse inhibitor from human placenta; this cDNA was used in hybridization experiments for measurements of mRNA. The amount of mRNApH in the presence of TH deficiency was normal in the examined goiter case, indicating an impaired TH gene expression at the translation level.

Ribonuclease A variants with potent cytotoxic activity.

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.

A new method for rapid extraction of high quality RNA from recalcitrant tissues of grapevine

A quick, inexpensive, and reliable protocol for the extraction of RNA from grapevine berry skins containing large quantities of polyphenols, procyanidins, and polysaccharides is described. The method involves an extraction step in the presence of ribonuclease inhibitors and compounds that compete with vacuolar contaminants for binding to RNA. After extraction with organic solvents, RNA is bound to a fibrous cellulose matrix and processed to eliminate the remaining contaminants and ribonucleases. Following this method, highly stable RNA, sufficiently pure for northern hybridizations and enzymatic processing, may be obtained from as little as 200 mg of starting amounts of fresh material and without multiple, time consuming precipitations or ultracentrifugation steps. This procedure may also prove useful for extracting RNA from recalcitrant tissues of other plant species. Abbreviations: ATA, aurintricarboxylic acid; CF11, cellulose fibrous medium (type 11); PVPP, polyvinylpolypyrrolidone; RT room temperature; VRC, vanadyl ribonucleoside complex.

Eosinophils inhibit retroviral transduction of human target cells by a ribonuclease-dependent mechanism.

Human eosinophils contain a number of granule proteins for which specific physiological roles remain unclear. The combined ribonucleolytic and membrane disruptive properties of the eosinophil-derived neurotoxin and eosinophil cationic protein, respectively, suggest the possibility that eosinophils might participate in host defense against enveloped single-stranded RNA viruses. To test this hypothesis, stocks of a replication-defective retrovirus encoding the reporter gene beta-galactosidase were pretreated with isolated human eosinophils, then used to transduce human erythroleukemia (K-562) target cells. Histochemical staining for beta-galactosidase activity was used to detect and quantitate the transduced cells. Co-incubation of retrovirus with eosinophils (0.4 x 10[6]/mL) before target cell transduction resulted in a marked decrease in transduction efficiency corresponding to an approximately 20-fold dilution of viral stock (P < 0.01), an effect that was directly proportional to the concentration of eosinophils, and that was reversed in the presence of ribonuclease inhibitor. Reverse transcriptase-polymerase chain reaction analysis demonstrated loss of the retroviral RNA genome as a result of eosinophil pretreatment, indicating that eosinophils are capable of mediating direct ribonucleolytic destruction of the isolated retroviral particles. Our results demonstrate that eosinophils function as effective anti-retroviral agents in vitro via the actions of their secreted ribonucleases, and suggest that eosinophils may represent an unrecognized arm of host defense against enveloped single-stranded RNA viral pathogens.

Rapid isolation of total RNA from mammalian tissues

A rapid procedure for the isolation of total RNA from small amounts of mammalian tissue (35 to 150 mg) is described. Tissues were homogenized in the presence of RNase inhibitors but in the absence of strong detergents. Contaminants were removed by phenol/chloroform extraction and Sephadex column chromatography. Total RNAs were precipitated with ethanol and sodium acetate. The RNAs isolated were intact and suitable for mRNA quantitation via Northern blot or slot-blot analyses. This procedure isolates total RNAs in high yield and purity, without CsCl ultracentrifugation, and is especially useful when mRNAs must be quantitated from many samples.

A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.

Isolation and characterization of rat intestinal polyribosomes and RNA during absorption of fat. Increased translation in vitro of apo-AIV

Polyribosomes were isolated from the intestinal mucosa of fasted and fat-fed rats in the presence of ribonuclease inhibitors. Polyribosomes from fat-fed rats were larger and more efficient in incorporating radioactive aminoacids into proteins than those from fasted rats. Total RNA prepared by guanidine-HCl extraction, from the intestine of fasting and fat-fed rats was translated in vitro in a mRNA-dependent rabbit reticulocyte lysate system in the presence of 35S-methionine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of the synthesized peptides showed a relative increase in the radioactivity of some peptides of RNA from fat-fed animals and particularly a two fold increase in preapo-AIV indicating that the intestinal apo-AIV synthesis is under transcriptional regulation by the metabolic processes involved in fat transport, that is, triglyceride-rich lipoprotein production.

Ribonuclease and ribonuclease inhibitor in the human pancreas.

Previous investigations have failed to confirm any specificity of elevated serum ribonuclease (RNase) in the diagnosis of pancreatic cancer. Although RNase had been known to be present in two forms (free and inhibitor-bound) in various rat tissues, little was known about its presence in the human pancreas. This report investigates the presence of RNase inhibitor in the human pancreas through the assay of both active (=free) and total (=sum of free and inhibitor-bound) RNases. Inhibitor-bound RNase was also named as latent RNase. RNase was classified into three types according to pH (acid, neutral, and alkaline RNases) in the pancreatic supernatant fraction. An inhibitor was separated from latent RNase by p-chloromercuribenzoic acid (PCMB), and the latent RNase was changed to an active form. Latent RNase was more active on the alkaline side with a maximum at pH 7.5. Hence, the presence of RNase inhibitor was highly suggested in the pancreatic supernatant fraction. RNase inhibitor is most likely a protein, which binds with both neutral and alkaline RNases. The presence of RNase inhibitor has not yet been demonstrated in the normal human serum.

A procedure for the rapid preparation of 14C-aminoacyl-transfer RNA and its use in the assay of class I ribonucleases and ribonuclease inhibitor.

The presence of ribonuclease inhibitor and/or the activity of class I ribonucleases can conveniently be measured, at all stages of purification, by a highly sensitive assay based on the loss of radioactivity during the concomitant hydrolysis of tRNA and small amounts of 14C-labeled aminoacyl-tRNA. The rapid, economical assay, which is readily adaptable to homologous tRNA substrates, eliminates the necessity of filtration, centrifugation and ultraviolet spectroscopy measurements required by most other assays and is particularly suitable for multiple samples and kinetic measurements.

Protein synthesizing activity of chicken liver and muscle

1. 1. Radioactivity incorporated in vivo into chicken liver proteins of the various cellular fractions was 4 to 5 times higher than that found in chicken muscle proteins of the corresponding cellular fractions when maximum radioactivity incorporated (per minute per mg. protein) into their respective proteins was compared. The specific radioactivity dropped by 52 to 75 % in the liver proteins and 22 to 30 % in the muscle proteins 96 hours after[ 3H]leucine administration. 2. 2. Amino acid incorporating activity of the post-mitochondrial (PM) fraction from chicken liver was 36 % of that a similar preparation from rat liver when chicken liver was homogenized in Tris-potassium-magnesium (TKM)-sucrose buffer and could be raised to 80 % by homogenizing in the presence of RNase inhibitor. PM fractions from the thigh muscles of the two animals showed similar activity although the activity was only 22 % of that of the rat liver preparation. 3. 3. Amino acid incorporating activity of the cell-free muscle preparation was 18% higher when the muscle was homogenized in the presence of 100 mM KCI and 16% lower when homogenized in the presence of 200 mM KCl compared to the amino acid incorporating activity of the preparation obtained by homogenizing the muscle in the presence of 25 mM KCl. 4. 4. The RNA concentration in chicken liver and thigh muscle was 6.92 and 2.05 mg. per g. of tissue and the lower amino acid incorporating activity of the muscle could be due to a lower RNA content in the tissue.

Determining the affinity in vitro of hepatic ribosomal subunits for derivatives of the rough endoplasmic reticulum

By a method employing centripetal floatation of membranes away from sedimenting particles, the binding capacity of pyrophosphate/citrate-treated rough membranes at 3 o for large ribosomal subunits has been found to be greater than for small subunits. Accurate comparison of the membrane affinity for the 2 subunits is apparently precluded however by greater instability of the small subunit, even in the presence of ribonuclease inhibitor.

The electrophoretic distribution of RNA synthesized in vitro by isolated rat liver nuclei

Both nuclear RNA newly synthesized in vivo and RNA synthesized by isolated rat liver nuclei in vitro are rapidly degraded in an RNA synthesizing media of low ionic strength. The presence of either a crude preparation of rat liver cytoplasmic ribonuclease inhibitor or 0.64 M (NH 4) 2SO 4 in the incubation media markedly retards this degradation. The RNA synthesized by isolated nuclei in media of low ionic strength in the presence of ribonuclease inhibitor is largely of high molecular weight with sedimentation values of greater than 18 S; whereas the RNA synthesized in media of high ionic strength is of a wider range of molecular weights with s values between 8 and 45 S. This difference may be due to the existence of two nuclear RNA polymerase activities that differ in high and low ionic strength: one polymerase may synthesize ribosomal precursor RNA and show activity at low (and possibly also high) ionic strength, and a second polymerase may transcribe nonribosomal portions of the genome and be active only at high ionic strength.