Addition Of Nucleic Acids Research Articles

Synthesis of the full-length hepatitis B virus core protein and its capsid formation.

Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer. Capsid assembly modulators can induce error-prone assembly of HBV core proteins to prevent the formation of infectious virions, representing promising candidates for treating chronic HBV infections. To explore novel capsid assembly modulators from unexplored mirror-image libraries of natural products, we have investigated the synthetic process of the HBV core protein for preparing the mirror-image target protein. In this report, the chemical synthesis of full-length HBV core protein (Cp183) containing an arginine-rich nucleic acid-binding domain at the C-terminus is presented. Sequential ligations using four peptide segments enabled the synthesis of Cp183 via convergent and C-to-N direction approaches. After refolding under appropriate conditions, followed by the addition of nucleic acid, the synthetic Cp183 assembled into capsid-like particles.

Influence of 5′‐inosine monophosphate and 5′‐guanosine monophosphate on growth, feed digestibility and activity of digestive enzymes in turbot Scophthalmus maximus

We evaluated the influence of different proportions of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on growth, feed digestibility and activity of digestive enzymes of turbot Scophthalmus maximus. Weight gain and daily feed intake were significantly higher in S. maximus fed with IMP or GMP, in comparison with fish fed with neither IMP nor GMP. The growth of 0.05% IMP + 0.05% GMP group was the best, and the intestinal digestive function was improved. The addition of IMP and GMP to fish diets significantly increased the apparent feed digestibility coefficient of dry matter and protein, as well as intestinal protease activity. The highest intestinal protease activity was observed in fish fed with 1 g/kg IMP. However, the lipase activity in hepatopancreas decreased significantly after addition of nucleic acid. According to our results, the optimal level of dietary IMP is 1 g/kg, which is in line with most of the growth performance and feed digestibility.

Lipids as Biomarkers of Cancer and Bacterial Infections.

Lipids are ubiquitous molecules, known to play important roles in various cellular processes. Alterations to the lipidome can therefore be used as a read-out of the signs of disease, highlighting the importance to consider lipids as biomarkers in addition of nucleic acid and proteins. Lipids are among the primary structural and functional constituents of biological tissues, especially cell membranes. Along with membrane formation, lipids play also a crucial role in cell signalling, inflammation and energy storage. It was shown recently that lipid metabolism disorders play an important role in carcinogenesis and development. As well, the role of lipids in disease is particularly relevant for bacterial infections, during which several lipid bacterial virulence factors are recognized by the human innate immune response, such as lipopolysaccharide in Gram-negative bacteria, lipoteichoic acid in Gram-positive bacteria, and lipoglycans in mycobacteria. Compared to nucleic acids and proteins, a complete analysis of the lipidome, which is the comprehensive characterization of different lipid families, is usually very challenging due to the heterogeneity of lipid classes and their intrinsic physicoproperties caused by variations in the constituents of each class. Understanding the chemical diversity of lipids is therefore crucial to understanding their biological relevance and, as a consequence, their use as potential biomarkers for non-infectious and infectious diseases. This mini-review exposes the current knowledge and limitations of the use of lipids as biomarkers of the top global killers which are cancer and bacterial infections.

Nucleic Acids Interfere with PF4 Tetramer Formation and Stabilise PF4 Monomers to Bind Non-Pathogenic HIT Antibodies: Subtypes of Non-Pathogenic Antibodies Emerge

IntroductionThe pathogenesis of heparin-induced thrombocytopenia (HIT) primarily involves anti-platelet factor(PF)4/heparin antibodies engaging FcγRIIa receptors on platelets to trigger a deleterious cascade of activation, thrombosis, and thrombocytopenia. Recent work with murine monoclonal antibodies has shed light on epitopic determinants that potentiate platelet activation (pathogenic KKO vs non-pathogenic RTO antibodies), but these mechanisms may not fully reflect the gamut of polyspecific antibodies seen in human clinical practice. We examined the development and characteristics of anti-PF4/heparin antibodies in patients undergoing cardiac surgery.MethodsAfter obtaining informed consent from patients undergoing cardiac bypass surgery, we performed ELISA using: (1) commercial Diagnostica Stago Asserachrom IgG kit (Cat.Nr 00624); or (2) in-house ELISA with platelet derived native(n)PF4. ELISA performed as per manufacturer instructions and published methods. Bacterial genomic nucleic acids (NA) were extracted from overnight culture of BL21(DE3)pLysS-wtPF4 using Promega kit Wizard® SV Genomic DNA Purification System (#A2361, Promega). Cross linking assays were performed as previously described using Bis(sulfosuccinimidyl)suberate (BS3) obtained commercially (#21585, Thermo Scientific Pierce). Optical density was normalised with controls to assist interassay comparability.All samples were screened with the commercial kit day 7 post-operatively, or the nearest available serum between day 4-10. Positive samples were then examined longitudinally to chart the onset of antibody formation and their duration.ResultsCommercial ELISA identified 30/123 (24%) patients positive for anti-PF4/heparin antibodies following cardiac surgery, but there was no HIT observed in the complete cohort of 123 patients and therefore all 30 were false positive.Surprisingly, in-house ELISA using nPF4/heparin was negative for 22/30, but the addition of NA to PF4 rescued the OD of these to become positive again.In contrast, OD fell for all true HIT controls with the addition of NA, as they did for a subset of the false-positives 7/30 [Figure A].Five patients had persistently raised OD despite the addition of NA, suggesting the presence of both NA/nPF4/heparin antibodies and nPF4/heparin antibodies.Overall, the incidence of false-positive anti-PF4/heparin antibodies was only 12/123 (10%) using the in-house ELISA method with nPF4. SRA was performed on all patients with nPF4/heparin antibodies, but they were all negative.Spiking nPF4 with increasing concentrations of NA caused reduction of tetramer formation and an increasing proportion of dimeric and monomeric forms seen on cross-linkage assays [Figures B and C].Longitudinal studies demonstrated that all 13 patients positive by commercial ELISA with only low range absolute results (OD <0.4), and without increase at later timepoints, possessed only NA/nPF4/heparin antibodies.By contrast, of the 14 patients whose ELISA OD increased over time, seven had evidence of nPF4/heparin antibodies. Three patients were ELISA positive pre-operatively, and only one of these had evidence of nPF4/heparin antibodies.ConclusionsIncreasing amounts of NA inhibit PF4 tetramer formation, and reduce the ability for HIT antibodies to bind. Thus, NA may participate in the preferential presentation of PF4 monomers that are recognised by non-pathogenic NA/PF4 monomer/heparin antibodies, similar to RTO. There is a smaller subset of “true” non-pathogenic antibodies that bind to PF4 tetramers, and an even smaller number of patients who may possess both classes of non-pathogenic antibodies. We speculate that pre-formed antibodies to NA/PF4 monomers are responsible for the majority of false positive results from commercial ELISA kits, and these are unrelated to the pathogenic anti-PF4/heparin antibodies of HIT.Figure Caption ARepresentative example of five patients with increasing ELISA OD in the presence of NA (NA1-5), two patients with OD that fell in the presence of NA (TFP1-2), and known HIT serum with reduced OD in the presence of NA. B: Reduction in PF4 tetramer formation and increasing PF4 monomers with increasing NA concentration. Lanes from left-right: control PF4 without BS3, and then increasing concentration of NA from 0, 5, 30 and 45ng/μL with BS3. C: Percentage of PF4 as tetramers or monomers with increasing concentration of NA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MP87-07 THE BIOLOGICAL RAMIFICATIONS OF PROSTATE CANCER ASSOCIATED CELL FREE DNA: EFFECT ON PLATELET FUNCTION AND DISEASE PROGRESSION

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2017MP87-07 THE BIOLOGICAL RAMIFICATIONS OF PROSTATE CANCER ASSOCIATED CELL FREE DNA: EFFECT ON PLATELET FUNCTION AND DISEASE PROGRESSION Mackenzie Adams, James Henderson, Mathew Lee, Todd Morgan, Ganesh Palapattu, and Alexander Zaslavsky Mackenzie AdamsMackenzie Adams More articles by this author , James HendersonJames Henderson More articles by this author , Mathew LeeMathew Lee More articles by this author , Todd MorganTodd Morgan More articles by this author , Ganesh PalapattuGanesh Palapattu More articles by this author , and Alexander ZaslavskyAlexander Zaslavsky More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2711AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The risk of developing venous thromboembolic events (VTE) is increased up to 4-fold in in men diagnosed with metastatic prostate cancer (PCa). While the precise reasons for this are unknown previous work has established the capacity of platelets (clot initiators) to absorb factors from the bloodstream that may alter platelet functions. Notably, one such factor, cell free DNA (cfDNA) has been shown to strongly correlate with disease severity and outcome. In this study, we sought to examine the effects of cfDNA from men with aggressive PCa on platelets in the context of clot formation and disease progression. METHODS Platelets were isolated from venous blood of 10 men with metastatic castration resistant prostate cancer (mCRPC). Platelet aggregation in response to agonists (i.e. thrombin and collagen) was established using platelet aggregometer. Platelet ability to bind to cancer cells (in vitro) was determined using flow cytometry. Platelet effect on prostate tumor growth in vivo was assessed using human platelet transfusions into 3 different PCa mouse models (intracardiac, subcutaneous, and orthotopic) in combination with standard bioluminescence techniques. RESULTS We assessed the effects of tumor associated nucleic acids on circulating platelets. We found that PCa derived cfDNA - loaded platelets were more reactive to agonists (thrombin and collagen), vs. unloaded platelets, making them more susceptible to spontaneous clot formation. We established that platelets from men with mCRPC (10 men) were significantly (p<0.001) more adherent to cancer cells in vitro as compared to platelets from healthy controls. Addition of nucleic acids (i.e. DNA) to healthy donor platelets in vitro converted them to be more thrombogenic and similar to platelets from men with mCRPC. Finally, we observed that platelets from men with mCRPC strongly induced cell proliferation and tumor growth in vivo (p<0.005), as compared to platelets from healthy controls. CONCLUSIONS High levels of cancer associated cfDNA have been previously demonstrated to correlate with poor prognosis and increased risk of VTE in men with PCa. Intriguingly, we found that PCa associated cfDNA modulates platelet behavior, revealing, for the first time, a potential molecular basis for Trousseau syndrome in PCa. Further, our findings also shed new light on how cfDNA may alter cancer growth via modulating platelets in the tumor microenvironment. Taken together, our study provides novel insights into the possible biological consequences of cfDNA seen in the advanced cancer state. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1168 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Mackenzie Adams More articles by this author James Henderson More articles by this author Mathew Lee More articles by this author Todd Morgan More articles by this author Ganesh Palapattu More articles by this author Alexander Zaslavsky More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.

miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-016-4043-6) contains supplementary material, which is available to authorized users.

Yeast-Expressed Bacteriophage-Like Particles for the Packaging of Nanomaterials

Virus-like particles (VLPs) generated by heterologous expression of viral structural genes have become powerful tools in vaccine development. Recently, we and others have reported on the assembly of VLPs of the RNA bacteriophages MS2, Qβ, and GA in yeast. Here, we investigate the formation of VLPs of five additional phages in the yeasts Saccharomyces cerevisiae and Pichia pastoris, namely, the coliphages SP and fr, Acinetobacter phage AP205, Pseudomonas phage PP7, and Caulobacter phage φCb5. In all cases except SP, particle formation was detected, although VLP outcome varied from 0.2 to 8mg from 1g of wet cells. We have found that phage φCb5 VLPs easily dissociate into coat protein dimers when applied to strong anion exchangers. Upon salt removal and the addition of nucleic acid or its mimics and calcium ions, the dimers re-assemble into VLPs with high efficiency. A variety of compounds, including RNA, DNA, and gold nanoparticles can be packaged inside φCb5 VLPs. The ease with which phage φCb5 coat protein dimers can be purified in high quantities and re-assembled into VLPs makes them attractive for downstream applications including the internal packaging of nanomaterials and the chemical coupling of peptides of interest on the surface.

Functional Redundancy in HIV-1 Viral Particle Assembly

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Application of linear-sweep voltammetry to the determination of nucleic acids using crystal violet as an electrochemical probe

A new linear-sweep voltammetric assay of nucleic acids (NAs) based on their interaction with crystal violet (CV) is proposed. In a pH 3.5 Britton—Robinson (B-R) buffer solution, CV had an irreversible voltammetric reductive peak at −0.77 V and the peak current greatly decreased by the addition of NAs. Under the experimental conditions, the decrease in the peak current was used for the NAs assay 0.5–18.0 μg/mL of fish sperm DNA, 0.6–15.0 μg/mL of calf thymus DNA, and 0.8–12.0 μg/mL of yeast RNA. The detection limits (3σ) were 0.32, 0.47, and 0.61 μg/mL for fsDNA, ctDNA, and yRNA, respectively. The binding reaction can be completed after mixing DNA with CV within 10 min and the electrochemical response is stable for 2 h. There are seldom interferences in this method and three synthetic samples were analyzed with satisfactory results. The stoichiometry of the supramolecular complex with the binding number 3 and the binding constant 2.78 × 1014 is calculated using electrochemical data.

Determination of nucleic acids at nanogram level using resonance light scattering technique with Congo Red

Based on the enhancement of the resonance light scattering (RLS) of Congo Red (CR) by nucleic acid, a new quantitative method for nucleic acid is developed. In the Tris–HCl buffer (pH 10.5), the weak light scattering of CR is greatly enhanced by addition of nucleic acid and CTMAB, the maximum peak is at 560 nm and the enhanced intensity of RLS is in proportion to the concentration of nucleic acid. The linear range is 1.0×10 −9 to 1.0×10 −6 g ml −1, 7.5×10 −8 to 1.0×10 −6 g ml −1 and 7.5×10 −8 to 2.5×10 −6 g ml −1 for herring sperm DNA, calf thymus DNA and yeast RNA, and the detection limits are 0.019, 0.89 and 1.2 ng ml −1 ( S/ N = 3), respectively. Actual biological samples were satisfactorily determined.

A kinetic fluorometric method for the determination of nucleic acids using a ternary equilibrium system of nucleic acids?iron (III) tetracarboxy phthalocyanine?poly-lysine coupled with the oxidation reaction between hydrogen peroxide and -tyrosine

Using the oxidation reaction between hydrogen peroxide and dl -tyrosine as fluorescence indication, the evident tuning effect of nucleic acids on catalytic activity of mimetic enzyme iron (III) tetracarboxy phthalocyanine (FeC 4 Pc) in the presence of poly-lysine was observed and studied. The oxidation reaction between hydrogen peroxide and dl -tyrosine with FeC 4 Pc as catalyst gave an intensively fluorescent compound, which has an excitation wavelength of 325 nm and an emission wavelength of 418 nm. The fluorescence was quenched by a proper concentration of poly-lysine due to its association with FeC 4 Pc and consequently the descent of the catalytic activity of FeC 4 Pc, but recovered by addition of nucleic acids. Under optimal conditions, the recovered fluorescence is proportional to the concentration of nucleic acids. Based on the fact, a kinetic fluorescent method was developed for the determination of nucleic acids. The calibration graphs are linear over the range 10–2000 ng/mL both for fish sperm DNA (FS DNA) and calf thymus DNA (CT DNA). The corresponding detection limits are 1.04 ng/mL for FS DNA and 1.18 ng/mL for CT DNA, respectively. Four synthetic and three real nucleic acid samples were determined with satisfactory results.

A kinetic fluorometric method for the determination of nucleic acids using a ternary equilibrium system of nucleic acids—iron (III) tetracarboxy phthalocyanine–poly-lysine coupled with the oxidation reaction between hydrogen peroxide and dl-tyrosine

Using the oxidation reaction between hydrogen peroxide and dl-tyrosine as fluorescence indication, the evident tuning effect of nucleic acids on catalytic activity of mimetic enzyme iron (III) tetracarboxy phthalocyanine (FeC 4Pc) in the presence of poly-lysine was observed and studied. The oxidation reaction between hydrogen peroxide and dl-tyrosine with FeC 4Pc as catalyst gave an intensively fluorescent compound, which has an excitation wavelength of 325 nm and an emission wavelength of 418 nm. The fluorescence was quenched by a proper concentration of poly-lysine due to its association with FeC 4Pc and consequently the descent of the catalytic activity of FeC 4Pc, but recovered by addition of nucleic acids. Under optimal conditions, the recovered fluorescence is proportional to the concentration of nucleic acids. Based on the fact, a kinetic fluorescent method was developed for the determination of nucleic acids. The calibration graphs are linear over the range 10–2000 ng/mL both for fish sperm DNA (FS DNA) and calf thymus DNA (CT DNA). The corresponding detection limits are 1.04 ng/mL for FS DNA and 1.18 ng/mL for CT DNA, respectively. Four synthetic and three real nucleic acid samples were determined with satisfactory results.

Interaction of morin-cetyltrimethylammonium bromide with nucleic acids and determination of nucleic acids at nanograms per milliliter levels based on the enhancement of preresonance light scattering.

A new preresonance light scattering (PRLS) assay of nucleic acids is presented. At pH 7.30, the weak PRLS of morin-cetyltrimethylammonium bromide (CTMAB) can be greatly enhanced by the addition of nucleic acids, owing to the interaction between the nucleic acid and morin-CTMAB. After the addition of morin and CTMAB to DNA, the zeta potential of DNA decreases and changes from negative to positive, which is due to the formation of an associate, the aggregation of morin on nucleic acids and the electric neutralization between DNA and the cationic surfactant CTMAB. Mechanism studies showed that the enhanced PRLS comes from the aggregation of morin in the presence of nucleic acids and CTMAB. The enhanced intensity of PRLS is in proportion to the concentration of nucleic acids in the range 7.5 x 10(-9)-1.0 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-1.0 x 10(-6) g ml(-1) for salmon sperm DNA and 1.0 x 10(-8)-1.0 x 10(-6) g ml(-1) for yeast RNA. The detection limits are 3.4, 6.2 and 4.1 ng ml(-1) for calf thymus DNA, salmon sperm DNA and yeast RNA, respectively. Synthetic samples were analyzed satisfactorily.

Molecular spectroscopy study of the reaction of nucleic acids with brilliant cresol blue

The interaction of brilliant cresol blue (BCB) with nucleic acids in aqueous solution has been studied by spectrophotometry and Rayleigh light scattering (RLS) spectroscopy. Under suitable conditions, the RLS spectra of BCB changed significantly due to the presence of nucleic acids. RLS intensity of BCB at 364 nm is greatly enhanced with the addition of nucleic acids, and a new RLS peak is observed at 552 nm. This peak is about half the intensity of that at 364 nm. The results of this study show that BCB interacts with DNA possibly due to the cooperative effect of electrostatic attraction, intercalation, coordination and hydrophobic effect. Under optimum conditions, the increase of RLS at 364 nm of a BCB solution is proportional to the concentration of nucleic acids added. This result is the basis for a new RLS method for determination of nucleic acids. The linear range of ctDNA, fsDNA and yRNA is 0.12–4.70, 0.11–4.64 and 0.43–7.07 μg ml −1, respectively.

Determination of Nucleic Acids Using Rivanol as the Fluorescent Probe in the Presence of SDS

ABSTRACT The quantitative method for nucleic acids using rivanol as the fluorescent probe in the presence of SDS was proposed. Under proper conditions, addition of nucleic acids to a mixture of rivanol and SDS resulted in enhanced fluorescence and spectral shifts of rivanol-SDS system. The calibration graph was linear in the range of 0–10.0 μg/ml for CT DNA and 0–9.0 μg/ml for yeast RNA, the limit of detection was 62 ng/ml for CT DNA and 156 ng/ml for yeast RNA. CT DNA could be determined in the presence of 20%(w/w) yeast RNA.

Signal transduction in the carnivorous plant Sarracenia purpurea. Regulation of secretory hydrolase expression during development and in response to resources.

Carnivory in plants has developed as an evolutionary adaptation to nutrient-poor environments. A significant investment of the resources of a carnivorous plant is committed to producing the traps, attractants, and digestive enzymes needed for the carnivory. The cost:benefit ratio of carnivory can be improved by either maximizing the prey capture rate or by reducing the metabolic commitment toward carnivory. Using the pitcher plant Sarracenia purpurea, we have investigated whether the expression of the hydrolytic enzymes needed for digestion is regulated in response to the presence of prey. Expression of protease, RNase, nuclease, and phosphatase activities could be induced in the fluid of nonactive traps by the addition of nucleic acids, protein, or reduced nitrogen, suggesting that hydrolase expression is induced upon perception of the appropriate chemical signal. Hydrolase expression was also developmentally controlled since expression commenced upon opening of a trap, increased for several days, and in the absence of prey largely ceased within 2 weeks. Nevertheless, the traps remained competent to induce expression in response to the appropriate signals. These data suggest that in young traps hydrolase expression is developmentally regulated, which is later replaced by a signal transduction mechanism, and they demonstrate the ability of a carnivorous species to respond to the availability of resources.

Effects of DNA and synthetic oligodeoxyribonucleotides on the binding properties of a cGMP-binding protein from Dictyostelium discoideum

Previously, we identified a cGMP-binding protein (cGBP) in Dictyostelium discoideum that can exist in two forms: a fast-dissociating (F-type) activity and a slow-dissociating (S-type) activity. Moreover, the F-type activity was converted effectively to S-type by the addition of nucleic acids, especially DNA (Parissenti, A.M. and Coukell, M.B. (1989) J. Cell Sci. 92, 291-301). In this study, we examined the effects of heterologous DNA and various synthetic homooligodeoxyribonucleotides on the cGMP-binding properties of partially purified F-type activity. Equilibrium and kinetic binding experiments revealed that DNA increased the affinity of the protein for cGMP without altering the number of binding sites. However, the presence of DNA decreased only slightly the apparent K d of the protein for cGMP becouse the nucleic acid also reduced the rate of cGMP association. Addition of oligo(dGMP) 8 or oligo(dCMP) 8 to the protein increased both total cGMP binding and the conversion of F-type activity to S-type; in contrast, oligo(dAMP) 8 or oligo(dTMP) 8, at the same concentration, had no effect. Oligodeoxycytidylic acids with chain lengths less than about eight nucleotides were also ineffective or inhibitory. Analysis of cGMP binding to intact, filipin-permeabilized cells revealed a binding activity with association and dissociation rates comparable to isolated S-type activity. This observation suggests that in vivo the cGBP might exist in its S-form.

Inhibition of DNA synthesis in BHK cells infected with western equine encephalitis virus 2. Properties of the inhibitory factor of DNA polymerase induced in infected cells

Infection of baby hamster kidney cells with western equine encephalitis virus results in rapid inhibition of cellular DNA synthesis. We previously reported that an inhibitory factor of cellular DNA polymerase activity was found in infected cell extracts. The mechanism of this inhibitory action was studied by varying the amount of each component involved in the DNA polymerase reaction mixture. Increase in DNA polymerase or template DNA showed no effect on the inhibitory activity, whereas that in substrate deoxynucleotides markedly suppressed the inhibition. Accordingly, the target of the inhibitory factor may be deoxynucleotides, and the factor was expected to be a phosphohydrolytic enzyme. In fact, deoxynucleotides were markedly hydrolyzed in the reaction mixture incubated with the inhibitory factor. This phosphohydrolytic enzyme activity was identified as a nucleoside triphosphate phosphohydrolase, which had little specificity to the base or the sugar of nucleotides, but high specificity to the triphosphate moiety. This enzyme showed the maximal activity at pH 7.5, and required a low concentration of Mg 2+, which could not be replaced by Mn 2+ or Ca 2+. Monovalent cations were not required. Furthermore, the enzyme activity was strikingly enhanced by addition of nucleic acids. The significance of this unique enzyme induced in infected cells is discussed in relation to inhibition of cellular DNA synthesis and viral replication.