Guanidine Thiocyanate Research Articles

Preservation of nucleic acid integrity in guanidine thiocyanate lysates of whole blood

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4 degrees C, and more than a year at -20 degrees C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.

Aggregation as the basis for complex behaviour of cutinase in different denaturants

We have previously described the complexity of the folding of the lipolytic enzyme cutinase from F. solani pisi in guanidinium chloride. Here we extend the refolding analysis by refolding from the pH-denatured state and analyze the folding behaviour in the presence of the weaker denaturant urea and the stronger denaturant guanidinium thiocyanate. In urea there is excellent consistency between equilibrium and kinetic data, and the intermediate accumulating at low denaturant concentrations is off-pathway. However, in GdmCl, refolding rates, and consequently the stability of the native state, vary significantly depending on whether refolding takes place from the pH- or GdmCl-denatured state, possibly due to transient formation of aggregates during folding from the GdmCl-denatured state. In GdmSCN, stability is reduced by several kcal/mol with significant aggregation in the unfolding transition region. The basis for the large variation in folding behaviour may be the denaturants' differential ability to support formation of exposed hydrophobic regions and consequent changes in aggregative properties during refolding.

SPR-based immunocapture approach to creating an interfacial sensing architecture: mapping of the MRS18-2 binding site on retinoblastoma protein

Biosensor technologies based on optical readout are widely used in protein-protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb-MRS18-2 protein complex was examined and the pRb binding site (A-box-spacer-B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein-Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.

Homemade viral RNA isolation protocol using silica columns: A comparison of four protocols

The Wrst technique for RNA isolation described in the literature was developed by Chirgwin and coworkers in 1979 [1]. They standardized a method for breaking cellular membranes without denaturing nucleic acids through homogenization of tissue in a mixture of guanidinium thiocyanate and -mercaptoethanol followed by an ethanol extraction or ultracentrifugation in a cesium chloride gradient. Although this was a signiWcant advance at that time, the technique consumed a lot of time and was ineYcient, hazardous, and inconsistent. With more recent isolation techniques, the quality, speed, and eYciency of this process have improved. In 1987, Chomczynski and Sacchi improved on the Chirgwin method, converting the guanidinium–hot phenol method to a single-step extraction called the AGPC (acid guanidinium–phenol–chloroform) method [2]. This modiWcation had at least two beneWts: the possibility of processing more samples simultaneously and the isolation of unbroken RNA with a higher purity and yield. Some years later, in 1993, Chomczynski published a reagent for the single-step simultaneous isolation of RNA, DNA, and proteins from cell and tissue samples [3]. Nowadays, there are several nucleic acid isolation kits on the market based on this reagent. More recently, some commercial alternatives have been developed, but it was the development of new techniques employing silica columns for the isolation of nucleic acids

Sup35NM-His6 aggregates: A prion-like protein useful in prion degradation studies

A prion-like protein, Sup35NM-His6, which is safe, easy to produce and able to aggregate into stable amyloid, was developed. This study was to test the feasibility of using Sup35NM-His6 amyloid as a marker for standard sterilization methods known to be effective in inactivating infectious prions. Also Sup35NM-His6 aggregates were spiked into cow brain tissue homogenates to mimic prions in tissues to determine if the aggregate protein could be specifically detected by Western blot at the nanogram level. Like mammalian prions, the proteinase K (PK) resistant fractions of Sup35NM-His6 remain intact after autoclaving. Treatments with 0.1N NaOH or 1.0% NaOCl also resulted in PK undigestible residues. Exposure to the strong denaturant guanidine thiocyanate (>3 M) destabilized and made the protein PK-digestible. Under strong alkaline conditions of 1.0N NaOH, 2.5% NaOCl, or a combination of NaOH (0.1 and 1.0N) with autoclaving, Sup35NM-His6 was completely hydrolyzed such that it was no longer detectable by Western blot. Overall, these tests suggest that Sup35NM-His6 could be a useful tool to assess the effectiveness of prion degradation for the prevention of TSE.

Polymerase chain reaction in comparison with serological tests for early diagnosis of human leptospirosis

The aim of this study was to compare the sensitivity and specificity of polymerase chain reaction (PCR) using two primer pairs and combined with blood culture, immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA), microscopic agglutination test (MAT) and slide agglutination test (SAT) in the diagnosis of human leptospirosis. We analysed 124 serum samples: 60 from patients with confirmed leptospirosis, 20 from patients with other diseases and 44 from healthy individuals. Analysing the first serum sample collected during the first 3-8 days of disease, the sensitivities of the four tests MAT, IgM ELISA, SAT and PCR were, respectively, 69.0%, 79.3%, 72.4% and 62%. In subsequent samples, those same sensitivities were, respectively, 95.4%, 100%, 100% and 72.7% in samples collected from days 9 to 14 and 88.9%, 88.9%, 77.8% and 44.4% in those collected from days 15 to 42. The most specific method (at 100%) was PCR and the least specific (at 89.1%) was IgM ELISA. Although we found PCR to be less sensitive than the serological tests over the course of the disease, our data indicate that PCR was the most sensitive in those initial serum samples presenting no specific antibodies detectable by any of the serological methods tested. We also recommend that PCR can be used in combination with serological tests as we found that this improves the sensitivity of the diagnosis of leptospirosis in the first phase of the disease (93.1-96.5%).

Human astroviruses in raw sewage samples in Hungary

Routine procedures for monitoring viruses in water samples have not been drawn up for the water-microbiology screening panel. Enteric viruses, including astroviruses, are able to persist under environmental conditions and may cause public health problems by contaminating natural and drinking water resources. The aim of this study was to detect human astroviruses (HAstVs) from raw wastewater samples. To obtain data on whether human astroviruses are shed in the environment, 35 raw sewage samples from 22 sewage plants in different regions of Baranya County, Hungary were tested for astrovirus using a polyethylene glycol method for concentration and a guanidinium thiocyanate-silica procedure for extraction of viral RNA. Reverse transcription-polymerase chain reaction (RT-PCR) with HAstV-specific primer pairs was used for amplification and the specificity of amplicons was confirmed by nucleotide sequencing and phylogenetic analysis. Among the 35 raw sewage samples, 15 (43%) contained HAstV and by sequence analysis, 10 genotype HAstV-1 and one genotype HAstV-2 were identified. The high detection rate of astroviruses we encountered in this study provide convincing evidence that HAstVs circulate at a relatively high frequency in the Hungarian population. No correlation between the standard indicators of faecal pollution and the presence of HAstVs was found. Our study is the first report on detection of HAstV in sewage in Hungary and suggests that HAstV might be potent indicators of viral pollution in environmental specimens.

Hypothyroidism Reduces Tricarboxylate Carrier Activity and Expression in Rat Liver Mitochondria by Reducing Nuclear Transcription Rate and Splicing Efficiency

The tricarboxylate carrier (TCC), also known as citrate carrier, is an integral protein of the mitochondrial inner membrane. It is an essential component of the shuttle system by which mitochondrial acetyl-CoA, primer for both fatty acid and cholesterol synthesis, is transported into the cytosol, where lipogenesis occurs. The effect of hypothyroidism on the activity and expression of the hepatic mitochondrial TCC was investigated in this study. TCC activity was significantly decreased in hypothyroid rats as compared with euthyroid animals. This hormone deficiency effect was due to a reduction in the amount of carrier protein, which resulted from a proportionate decrease of the specific mRNA. Hypothyroidism did not influence TCC mRNA stability. On the other hand, nuclear run-on assay revealed that the transcriptional rate of TCC mRNA decreased by approximately 40% in the nuclei from hypothyroid versus euthyroid rats. In addition, the ribonuclease protection assay showed that, in the nuclei of hypothyroid rats, the ratio of mature to precursor RNA decreased, indicating that the splicing of TCC RNA is affected. Furthermore, we found that the ratio of polyadenylated/unpolyadenylated TCC RNA as well as the length of the TCC RNA poly(A) tail were similar in both euthyroid and hypothyroid rats. Thus, the rate of formation of the TCC 3'-end is not altered in hypothyroidism. These results suggest that hypothyroidism affects TCC expression at both the transcriptional and post-transcriptional levels.

The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on

Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years.

Detection of colorectal neoplasia associated K-Ras mutations in human urine

1005 Background: Since colorectal neoplasia-associated genes have been detected in human blood, we hypothesized that small DNA fragments containing genetic mutations associated with colorectal neoplasias are filtered and excreted in the urine. If so, genes associated with colorectal cancer will be detected in the urine. K-ras mutations are commonly associated with colorectal neoplasia and do not occur in the urinary tract. Methods: K-ras mutation detection: 200 microl of urine was extracted with guanidine thiocyanate and purifed using a Wizard DNA isolation kit. Codon 12 K-ras mutation detection methods–1: restriction enriched PCR, 20 cycles, with primers that amplify both wild type and mutated DNA but with an artificial BstNI site at the 5’ end of the amplified product (>2 K-ras copies per assay); 2: Peptide nucleic acid clamping real time PCR (>15 K-ras copies per assay). Human subjects: Training set = 20 patients with known K-ras mutations in colorectal cancer tissue. Test set = blinded urine samples from colorectal adenocarcinoma (N=48), adenoma (N=31), hyperplastic polyp (N=12) and endoscopically normal colon and rectum (N=60). Results: 1. Human urine contains 150–250 base pair DNA fragments derived from the circulation. These fragments can be readily distinguished from high molecular weight DNA from sloughed urinary tract cells. 2. Training set for K-ras detection (tissue confirmed K-ras mutations): Serum 6/20 (30%), Plasma (11/20 (55%), Urine: 18/20 (90%), (p<0.15 for plasma, p<0.001 for serum). 3. Test set (blinded): a. >2 copies of mutated K-ras genes were detected in: 16/48 (33%) adenocarcinomas; 23/31 (74%) adenomas; 5/12 (42%) hyperplastic polyps, and 19/60 (31%) non-neoplasia controls. b. >15 copies of mutated K-ras genes were detected in: 12/48 (25%) adenocarinomas; 15/31 (48%) adenomas; 3/12 (25%) hyperplastic polyps, and 11/60 (18%) non-neoplasia controls. Conclusions: Small DNA fragments in human urine contain K-Ras mutations identical to those found in colorectal cancer DNA. The sensitivity for detection of K-ras mutations in urine appears equivalent or superior to K-ras mutation detection in feces or serum. DNA mutations from systemic epithelial neoplasias may be detected in filtered urinary DNA fragments and may be useful for early detection of neoplasia. No significant financial relationships to disclose.

Guanidinium thiocyanate

Guanidinium thiocyanate

Guanidine thiocyanate

Guanidine thiocyanate

Distinct modulation of angiotensin II-induced early left ventricular hypertrophic gene programming by dietary fat type

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.

Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

Stimulation of beta-adrenergic receptors (betaARs) leads to sequential recruitment of beta-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for betaAR endocytosis is not known. In this study, we have used beta(1)- and beta(2) ARs, two ubiquitously expressed members of the betaAR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of beta(2)ARs, beta(1)ARs, and mutants of beta(1)ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit beta-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for beta(2)AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of beta(2)AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated beta(2)AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of beta(2)AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal betaAR internalization.

ALTERATIONS IN ANDROGEN RECEPTORS AND ANDROGEN RECEPTOR mRNA LEVELS IN PROSTATE GLANDS OF VITAMIN E DIFICIENT RATS WITH AND WITHOUT CASTRATION AND ANDROGEN ADMINISTRATION

Vitamin E has been reported to be essential for germ cell development and maturation, gonadotropin secretion, steroid hormone synthesis and metabolism, and reproduction and fertility. The present study was designed to study if vitamin E deficiency changes prostate androgen receptor (AR) and AR mRNA levels. Male King-Holtzman rats were fed with vitamin E deficient diet and control rats were fed with Purina rat chow for 3 months. Two weeks prior to autopsy, other groups of rats were castrated and administered with testosterone. Prostate tissues were obtained for determination of nuclear AR levels and quantitation of mRNA encoding androgen receptors. Total cellular RNA was prepared by guanidine thiocyanate method and determined by Northern and slot blot analyses. Prostate androgen receptor levels in vitamin E deficient rats were significantly lower than in control animals. Androgen receptor mRNA levels were decreased in vitamin E deficient rats when compared to those of normal controls. However, prostate AR and AR mRNA levels were increased following castration, whereas AR and AR mRNA levels were decreased in castrated rats with androgen administration. These findings indicate that vitamin E deficiency causes a reduction of AR levels in rats with the decline of circulating androgen levels and a decreased transcription of AR mRNA which leads to a defective translation of AR protein, suggesting an important role of dietary vitamin E on prostate AR levels, AR mRNA expression, and reproduction and fertility.

Purification of RNA from cells and tissues by acid phenol–guanidinium thiocyanate–chloroform extraction

Purification of RNA from cells and tissues by acid phenol–guanidinium thiocyanate–chloroform extraction

Technical Note: Improved Method for Rapid DNA Extraction of Mastitis Pathogens Directly from Milk

Efficient control against bovine mastitis requires sensitive, rapid, and specific tests to detect and identify the main bacteria that cause heavy losses to the dairy industry. Molecular detection of pathogenic microorganisms is based on DNA amplification of the target pathogen. Therefore, efficient extraction of DNA from pathogenic bacteria is a major step. In this study, we aimed to develop a specific, sensitive, and rapid method to extract DNA directly from the main gram-positive bacteria known to cause bovine mastitis (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis) found in milk samples. The DNA extraction method is based on the lysing and nuclease-inactivating properties of the chaotropic agent, guanidinium thiocyanate, together with the nucleic acid-binding properties of the silica particles. An efficient protocol consisting of 6 basic steps (3 of which were done twice) was developed and applied directly to milk samples. Absence of PCR inhibitors and DNA quality were evaluated by PCR amplification of the species-specific DNA sequences of the target bacteria. The level of sensitivity achieved in our experiments is applicable to milk sample analysis without sample enrichment.

Highly Efficient Solar Cells Using Organic Dyes with Amorphous Moiety

ABSTRACTNovel organic sensitizers comprising donor; electron-conducting, and anchoring groups were engineered at molecular level and synthesized. The functionalized unsymmetrical organic sensitizers 3-{5-[N,N-Bis(9,9-dimethylfluorene-2-yl)phenyl]-thiophene-2-yl}-2-cyano-acrylic acid (JK-1) and 3-{5'-[N,N-Bis(9,9-dimethylfluorene-2-yl)phenyl]-2,2'-bithiophene-5-yl}-2-cyano-acrylic acid (JK-2), upon anchoring onto TiO2 film exhibit unprecedented incident photon to current conversion efficiency 91 %. The photovoltaic data using an electrolyte having composition of 0.6M M-methyl-N-butyl imidiazolium iodide, 0.04 M iodine, 0.025 M LiI, 0.05M guanidinium thiocyanate and 0.28 M tert.butylpyridine in 15/85 (v/v) mixture of valeronitrile and acetonitrile revealed a short circuit photocurrent density of 14.0 mA/cm2, an open circuit voltage of 753 mV and a fill factor of 0.76, corresponding to an overall conversion efficiency of 8.01 % under standard AM 1.5

A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration

We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.

Combined spectroscopic and calorimetric characterisation of rubredoxin reversible thermal transition

Rubredoxins are small iron proteins containing the simplest type of iron-sulphur centre, consisting of an iron atom coordinated by the thiol groups of four cysteines. Here we report studies on the conformational stability of a new type of rubredoxin from the hyperthermophile Methanocaldococcus jannaschii, having an atypical metal site geometry resulting from a modified iron-binding motif. Absorption and fluorescence spectroscopies were used in combination with differential scanning calorimetry to probe different aspects of the thermal unfolding transition: iron site degradation (absorption at 380 nm), tertiary structure unfolding (Trp emission), exposure of hydrophobic regions (1-anilinonaphalene-8-sulphonate fluorescence enhancement) and iron release. Thermal denaturation was found to be irreversible and caused by decomposition of the metal centre. The protein is hyperstable and between pH 4 and 10 it is only thermally denatured in the presence of a strong chemical denaturant. The study of the heating rate dependence of the melting temperature allowed us to determine the reaction equilibrium thermodynamic parameters. At pH 2 the protein is destabilised owing to the absence of salt bridges and it has a T(m) of 65 degrees C. In these conditions, there is excellent agreement between the parameters determined by the different spectroscopic methods and calorimetry. The highest stability was found to be at pH 8, and a detailed study of the heating rate dependence in the presence of guanidine thiocyanate in this condition allowed the determination of a reversible T(m) of 118 degrees C.