MORPHOLOGICAL AND HISTOCHEMICAL CHANGES IN THE SPLEEN AND LYMPH NODE OF WHITE MICE IN RESPONSE TO RADIATION AND ANTIGENIC EFFECTS (in Russian)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akopova, V.A.

1961-01-01

Research was carried out on 90 white ntice subjected to x irradiation (Series I), vaccination with a brucellosis vaccine (Series II), and to a combined action of these factors (Series III and IV). The spleen and lymph nodes were studied both morphologically and histochemically. When exposing the mice to a total x-ray dose of 335 r, visible destructive changes and the lowering of desoxyribonucleic and ribonucleic acid level were found to occur in the spleen and lymph nodes. In the case of a single vaccination of the mice with the brucellosis vaccine, a certain vaccinal hyperplasia was observed in themore » spleen and lymph nodes, the level of desoxyribonucleic and ribonucleic acid being normal. In vaccinating the white mice, following total irradiation, destructive changes along with a gradual lowering of desoxyribonucleic and ribonucleic acid level are observed in the spleen and lymph nodes. The total x irradiation following a singie vaccination with the brucellosis vaccine, brings about slight destructive changes along with a simultaneous reticular hyperplasia in the spleen and lymph nodes. The levels of desoxyribonucleic and ribonucleic acids are high at all times. (auth)« less

Comparison of Two Serologically Distinct Ribonucleic Acid Bacteriophages I. Properties of the Viral Particles

PubMed Central

Overby, L. R.; Barlow, G. H.; Doi, R. H.; Jacob, Monique; Spiegelman, S.

1966-01-01

Overby, L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman. Comparison of two serologically distinct ribonucleic acid bacteriophages. I. Properties of the viral particle. J. Bacteriol. 91:442–448. 1966.—Two ribonucleic acid (RNA) coliphages, MS-2 and Qβ, have been characterized physically and serologically. MS-2 has an S20, w value of 79, a molecular weight of 3.6 × 106, a density of 1.422, and pH 3.9 as its isoelectric point. Qβ has an S20, w of 84, a molecular weight of 4.2 × 106, a density of 1.439, and an isoelectric point at pH 5.3. One host (Escherichia coli A-19) permits a distinction between the two on the basis of a marked difference in plaque size. They are distinct immunochemically, no serological cross-reaction being detectable. Images PMID:5903109

Future Technology Themes: 2030 to 2060

DTIC Science & Technology

2013-07-01

Rocket-Based Combined Cycle RF Radio Frequency RNA Ribonucleic Acid SA Situational Awareness SEAD Suppression of Enemy Air Defences SME...and re-routing light in information processing and optical communications ; or for processing radio signals in mobile phones [44]. UNCLASSIFIED DSTO...make use of network polymorphism technologies from 2020 onwards to create frequency -agile and adaptive14 communications links that would change network

A Contemporary, Laboratory-Intensive Course on Messenger RNA Transcription and Processing

ERIC Educational Resources Information Center

Carson, Sue; Miller, Heather

2012-01-01

Messenger ribonucleic acid (mRNA) plays a pivotal role in the central dogma of molecular biology. Importantly, molecular events occurring during and after mRNA synthesis have the potential to create multiple proteins from one gene, leading to some of the remarkable protein diversity that genomes hold. The North Carolina State University…

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Chromatographic Differences Between Transfer Ribonucleic Acids from Spores and Cells in Exponential Growth

PubMed Central

Vold, Barbara S.

1973-01-01

Differences between the transfer ribonucleic acid (tRNA) of spores and exponentially growing cells of Bacillus subtilis 168 were compared by co-chromatography on reversed-phase column RPC-5. This system gave excellent resolution of isoaccepting species in 1 to 2 hr using a 200-ml gradient. Two methods were used to extract spore tRNAs, a procedure using a Braun homogenizer and a pretreatment with dithiothreitol followed by lysis with lysozyme. Where changes were observed, column elution profiles of spore tRNAs were independent of the extraction method used. Three kinds of changes between the profiles of vegetative cell tRNA and spore tRNA were observed: (i) no change; phe-, val-, ala-, asp-, ileu-, pro-, met-, fmet-, and his-tRNAs, (ii) a change in the ratio of existing peaks; gly-, tyr-, leu-, ser-, thr-, aspn-, and arg-tRNAs, and (iii) the appearance or disappearance of unique peaks; lys-, glu-, and trp-tRNAs. PMID:4632322

Bringing RNA into View: RNA and Its Roles in Biology.

ERIC Educational Resources Information Center

Atkins, John F.; Ellington, Andrew; Friedman, B. Ellen; Gesteland, Raymond F.; Noller, Harry F.; Pasquale, Stephen M.; Storey, Richard D.; Uhlenbeck, Olke C.; Weiner, Alan M.

This guide presents a module for college students on ribonucleic acid (RNA) and its role in biology. The module aims to integrate the latest research and its findings into college-level biology and provide an opportunity for students to understand biological processes. Four activities are presented: (1) "RNA Structure: Tapes to Shapes"; (2) "RNA…

Clearance of HCV RNA following acute hepatitis A superinfection.

PubMed

Cacopardo, B; Nunnari, G; Nigro, L

2009-05-01

A transient reduction of hepatitis C virus replication during the course of acute hepatitis A virus infection has already been reported in the literature. The present study reports the case study of a subject with chronic hepatitis due to hepatitis C virus who went on to develop an acute hepatitis A. From the early onset of acute disease, hepatitis C virus ribonucleic acid became undetectable. Following recovery from acute hepatitis, alanine amino-transferase levels became persistently normal and liver biopsy revealed a reduction in the Knodell histological activity index score. Hepatitis C virus ribonucleic acid clearance was maintained up to 4 years after the onset of acute hepatitis A. During the course of the acute disease, a sharp increase in interferon gamma levels was detected in serum and in the supernatant of both unstimulated and phytoemagglutinin/lipopolysaccharide-stimulated peripheral blood mononuclear cells. Interferon gamma levels were still high 3 months later. We hypothesize that acute hepatitis A virus superinfection during the course of chronic hepatitis C may lead to hepatitis C virus ribonucleic acid clearance through an immunological mechanism related to interferon gamma production.

Exploring the trans-acting short interfering RNAs (ta-siRNAs) technology for virus control in plants

USDA-ARS?s Scientific Manuscript database

Small ribonucleic acid (RNAs) (~20-24nt) processed from double-stranded RNA in plants can trigger degradation of the target mRNAs in cytoplasm or de novo DNA methylation in nucleus leading to gene silencing. Trans-acting short-interfering RNAs (ta-siRNAs) have been shown to enhance the target mRNA d...

Ribonucleic Acid Synthesis and Glutamate Excretion in Escherichia coli

PubMed Central

Broda, Paul

1968-01-01

Cultures of Escherichia coli excreted glutamate into the medium when protein synthesis was blocked in RCrel strains or when it was blocked with chloramphenicol in either RCstr or RCrel strains. Both of these conditions resulted in continued ribonucleic acid (RNA) synthesis in the absence of protein synthesis. Glutamate was also excreted by both RCstr and RCrel strains when RNA synthesis was inhibited by uracil starvation or by treatment with actinomycin D. It is proposed that, in each of these cases, glutamate excretion resulted from an increase in the permeability of the cell membrane. PMID:4973126

Discrimination of Self and Non-Self Ribonucleic Acids

PubMed Central

Gebhardt, Anna; Laudenbach, Beatrice T.

2017-01-01

Most virus infections are controlled through the innate and adaptive immune system. A surprisingly limited number of so-called pattern recognition receptors (PRRs) have the ability to sense a large variety of virus infections. The reason for the broad activity of PRRs lies in the ability to recognize viral nucleic acids. These nucleic acids lack signatures that are present in cytoplasmic cellular nucleic acids and thereby marking them as pathogen-derived. Accumulating evidence suggests that these signatures, which are predominantly sensed by a class of PRRs called retinoic acid-inducible gene I (RIG-I)-like receptors and other proteins, are not unique to viruses but rather resemble immature forms of cellular ribonucleic acids generated by cellular polymerases. RIG-I-like receptors, and other cellular antiviral proteins, may therefore have mainly evolved to sense nonprocessed nucleic acids typically generated by primitive organisms and pathogens. This capability has not only implications on induction of antiviral immunity but also on the function of cellular proteins to handle self-derived RNA with stimulatory potential. PMID:28475460

Rift Valley Fever Virus: Molecular Biologic Studies of the M Segment RNA(Ribonucleic Acids) for Application in Disease Prevention

DTIC Science & Technology

1988-12-01

correctly expresses, processes , and transports all M segment proteins (virus 7; r-eferences 2,3) were pulse -labeled with 35S- methionine and subsequently...c-ranslationii~y processed to yield the mature proteins. The first ATG codon of tP’o _iinle OrF is required for production of the 78kd protein...employed for the expression of the 78kd and l4kd proteins serves to control glycosylation arnd proteolytic. processing of the resultant poI~peptidles

A METABOLITE OF METHOXYCHLOR,2,2-BIS(P-HYDOXYPHENYL)-1,1,1- TRICHLOROETHANE REDUCES TESTOSTERONE BIOSYNTHESIS IN RAT LEYDIG CELLS THROUGH SUPPRESSION OF STEADY-STATE MESSENGER RIBONUCLEIC ACID LEVELS OF THE CHOLESTEROL SIDE-CHAIN CLEAVAGE ENZYME

EPA Science Inventory

Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the ma...

Human health and ecological risk assessments for SmartStax PRO (MON 89034 x TC1507 x MON 87411 x DAS-59122-7), a plant-incorporated protectant intended to control corn rootworm through ribonucleic acid (RNA) interference

EPA Science Inventory

The use of RNA interference (RNAi) gene silencing technology, particularly RNAi for pesticidal purposes to control macroorganism pests, is a relatively recent innovation. Post-transcriptional silencing of gene function is a very rapid process where double-stranded RNA (dsRNA) dir...

Phylogenetic and Biochemical Studies on Stomatococcus mucilaginosus.

PubMed

Stackebrandt, E; Scheuerlein, C; Schleifer, K H

1983-01-01

The degree of relatedness of nine strains of Stomatococcus mucilaginosus (formerly classified as 'Micrococcus mucilaginosus') was investigated by deoxyribonucleic acid hybridization. We confirm that all strains are highly related. Differences in the peptidoglycan type and the cytochrome pattern between S. mucilaginosus and members of Micrococcus, together with the results of 23 S ribosomal ribonucleic acid cistron similarity studies and the analysis of the 16S ribosomal ribonucleic acid support the exclusion of this species from the genus Micrococcus and justifies its reclassification as a member of a new genus, Stomatococcus (Bergan and Kocur, 1982). Phylogenetically, S. mucilaginosus represents an independent line of descent within a broad group of Gram-positive bacteria that contains arthrobacteria, micrococci, cellulomonads, brevibacteria and microbacteria. Copyright © 1983 Gustav Fischer Verlag, Stuttgart/New York. Published by Elsevier GmbH.. All rights reserved.

Structural and metabolic characterization of RNAs from rats with experimental Guerin tumor - I. Nucleotide composition of RNAs from the liver and tumor tissues of rats.

PubMed

Ratkiewicz, A; Galasinski, W

1976-01-01

The characteristics of the ribonucleic acids of Guerin tumor was the subject of this work. The effect of tumor development on the structure of the ribonucleic acids in the liver of tumor bearing rats was studied. Some differences of nucleotide compositions in RNAs isolated from subcellular fractions of liver of control and tumor bearing rats and of cancer tissue were observed. The nucleotide compositions of cancer nuclear RNA is distinctly different from liver RNA. The changes in primary structure of liver RNAs due by development of tumor in rats may be result of metabolic peculiarities of these RNAs.

Evaluation of the reversal of multidrug resistance by MDR1 ribonucleic acid interference in a human colon cancer model using a Renilla luciferase reporter gene and coelenterazine.

PubMed

Jeon, Yong Hyun; Bae, Seon-ae; Lee, Yong Jin; Lee, You La; Lee, Sang-Woo; Yoon, Ghil-Suk; Ahn, Byeong-Cheol; Ha, Jeoung-Hee; Lee, Jaetae

2010-12-01

The reversal effect of multidrug resistance (MDR1) gene expression by adenoviral vector-mediated MDR1 ribonucleic acid interference was assessed in a human colon cancer animal model using bioluminescent imaging with Renilla luciferase (Rluc) gene and coelenterazine, a substrate for Rluc or MDR1 gene expression. A fluorescent microscopic examination demonstrated an increased green fluorescent protein signal in Ad-shMDR1- (recombinant adenovirus that coexpressed MDR1 small hairpin ribonucleic acid [shRNA] and green fluorescent protein) infected HCT-15/Rluc cells in a virus dose-dependent manner. Concurrently, with an increasing administered virus dose (0, 15, 30, 60, and 120 multiplicity of infection), Rluc activity was significantly increased in Ad-shMDR1-infected HCT-15/Rluc cells in a virus dose-dependent manner. In vivo bioluminescent imaging showed about 7.5-fold higher signal intensity in Ad-shMDR1-infected tumors than in control tumors (p < .05). Immunohistologic analysis demonstrated marked reduction of P-glycoprotein expression in infected tumor but not in control tumor. In conclusion, the reversal of MDR1 gene expression by MDR1 shRNA was successfully evaluated by bioluminescence imaging with Rluc activity using an in vivo animal model with a multidrug resistance cancer xenograft.

Evolution of proteins.

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.

1971-01-01

The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

PubMed Central

Mellert, Hestia S.; Alexander, Kristin E.; Jackson, Leisa P.; Pestano, Gary A.

2018-01-01

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt. PMID:29683453

Glyceraldehyde and glycolaldehyde in interstellar ice analogues and the role of aldehydes in cosmochemical evolution

NASA Astrophysics Data System (ADS)

Meierhenrich, U.; de Marcellus, P.; Meinert, C.; Myrgorodska, I.; Nahon, L.; Buhse, T.; d'Hendecourt, L.

2015-10-01

Our understanding of the molecular origin of life is based on amino acids, ribose, and nucleobases that - after their selection by prebiotic processes - initiated the evolutionary assembly of catalytic and informational polymers, being proteins and ribonucleic acids. Following previous amino acid identifications in the room-temperature residues of simulated circumstellar/interstellar ices [1,2] we have searched for a different family of molecules of potential prebiotic interest. Using multidimensional gas chromatography coupled to time-of-flight mass spectrometry, we have detected ten aldehydes, including the sugar-related glycolaldehyde and glyceraldehyde - two species considered as key prebiotic intermediates in the first steps toward the synthesis of ribonucleotides in a planetary environment.

THE CONTENT AND RELATIVE BASE RATIOS OF RIBONUCLEIC ACID IN AMOEBA

PubMed Central

Iverson, Ray M.

1964-01-01

The amount and relative base ratios of ribonucleic acid (RNA) in the nucleus and cytoplasm of Amoeba proteus and A. dubia, and of homospecies cells obtained by nuclear transfer with A. proteus, have been determined by microelectrophoresis. In A. proteus the average amounts of RNA in the nucleus and the cytoplasm were 134. micromicrograms and 2520. micromicrograms; in A. dubia the averages for the nucleus and cytoplasm were 67. micromicrograms and 1427. micromicrograms. The relative base ratio of RNA of the nucleus is similar to that of the RNA of the cytoplasm within a species, but the two species differed in this respect. Homospecies nuclear transfer did not affect the relative base ratio or amount of RNA. PMID:14105213

Reovirus-induced Ribonucleic Acid Polymerase

PubMed Central

Watanabe, Y.; Gauntt, C. J.; Graham, A. F.

1968-01-01

A virus-induced ribonucleic acid (RNA) polymerase activity was found in L cells infected with type 3 reovirus. Most of the enzyme is associated with the “large particle” fraction of the infected cells. The enzyme first appeared at 3 to 5 hr after infection and increased in amount until 7 to 9 hr. All four ribonucleoside triphosphates are incorporated in vitro into an acid-insoluble form by the enzyme. The major part of the product formed in vitro is a double-stranded RNA indistinguishable from viral RNA by electrophoresis on polyacrylamide gel. Approximately 40% of the product is a single-stranded RNA of relatively small molecular weight. More than 95% of the nucleotides incorporated into double-stranded RNA by the enzyme are bound in internal 3′-5′-phosphodiester linkages extending back from both 3′- and 5′-termini of the RNA strands. PMID:5725319

Informational biopolymer structure in early living forms.

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.; Mclaughlin, P. J.; Barker, W. C.; Hunt, L. T.

1972-01-01

Some studies devoted to the search in various organisms for 'relics' of the biochemical nature of ancient organisms, preserved by the conservative nature of the evolutionary process in all living species, are reviewed. Investigations of five families of informational molecules constituting such 'relics' in very diverse organisms are reported. They include: cytochrome c, ferredoxin, trypsin, transfer ribonucleic acid (RNA), and 5S ribosomal RNA. It is shown that, even from these few informational molecules, some interesting inferences about early living organisms can be drawn.

Fundamental Approaches in Molecular Biology for Communication Sciences and Disorders

ERIC Educational Resources Information Center

Bartlett, Rebecca S.; Jette, Marie E.; King, Suzanne N.; Schaser, Allison; Thibeault, Susan L.

2012-01-01

Purpose: This contemporary tutorial will introduce general principles of molecular biology, common deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein assays and their relevance in the field of communication sciences and disorders. Method: Over the past 2 decades, knowledge of the molecular pathophysiology of human disease has…

Genetics Home Reference: Pol III-related leukodystrophy

MedlinePlus

... two largest parts (subunits) of an enzyme called RNA polymerase III. This enzyme is involved in the production (synthesis) of ribonucleic acid (RNA), a chemical cousin of DNA. The RNA polymerase ...

Micro-ribonucleic acid-binding site variants of type 2 diabetes candidate loci predispose to gestational diabetes mellitus in Chinese Han women.

PubMed

Wang, Xiaojing; Li, Wei; Ma, Liangkun; Ping, Fan; Liu, Juntao; Wu, Xueyan; Mao, Jiangfeng; Wang, Xi; Nie, Min

2018-01-20

Emerging evidence has suggested that the genetic background of gestational diabetes mellitus (GDM) was analogous to type 2 diabetes mellitus. In contrast to type 2 diabetes mellitus, the genetic studies for GDM were limited. Accordingly, the aim of the present study was to extensively explore the influence of micro-ribonucleic acid-binding single-nucleotide polymorphisms (SNPs) in type 2 diabetes mellitus candidate loci on GDM susceptibility in Chinese. A total of 839 GDM patients and 900 controls were enrolled. Six micro-ribonucleic acid-binding SNPs were selected from 30 type 2 diabetes mellitus susceptibility loci and genotyped using TaqMan allelic discrimination assays. The minor allele of three SNPs, PAX4 rs712699 (OR 1.366, 95% confidence interval 1.021-1.828, P = 0.036), KCNB1 rs1051295 (OR 1.579, 95% confidence interval 1.172-2.128, P = 0.003) and MFN2 rs1042842 (OR 1.398, 95% confidence interval 1.050-1.862, P = 0.022) were identified to significantly confer higher a risk of GDM in the additive model. The association between rs1051295 and increased fasting plasma glucose (b = 0.006, P = 0.008), 3-h oral glucose tolerance test plasma glucose (b = 0.058, P = 0.025) and homeostatic model assessment of insulin resistance (b = 0.065, P = 0.017) was also shown. Rs1042842 was correlated with higher 3-h oral glucose tolerance test plasma glucose (b = 0.056, P = 0.028). However, no significant correlation between the other included SNPs (LPIN1 rs1050800, VPS26A rs1802295 and NLRP3 rs10802502) and GDM susceptibility were observed. The present findings showed that micro-ribonucleic acid-binding SNPs in type 2 diabetes mellitus candidate loci were also associated with GDM susceptibility, which further highlighted the similar genetic basis underlying GDM and type 2 diabetes mellitus. © 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Selective Methylation: an Incorrect Hypothesis

PubMed Central

Shugart, Lee

1976-01-01

“Selective methylation,” a hypothesis proposed to explain the discrepancy found in the degree of methyl deficiency of transfer ribonucleic acid, cannot be explained on the basis of some biological phenomenon. PMID:770445

The development of fixed-ratio performance under the influence of ribonucleic acid12

PubMed Central

Gott, C. Thomas; Weiss, Bernard

1972-01-01

The transition from fixed-ratio 1 performance (every response reinforced) to fixed-ratio 30 performance (every thirtieth response reinforced) was studied in nine pigeons. These were divided into three treatment groups given daily oral doses of saline, or 250 mg/kg/day or 500 mg/kg/day of yeast ribonucleic acid. Detailed computer-assisted analyses of how fixed-ratio behavior develops revealed the following typical sequence. After the transition, the first few ratios typically were emitted without long interresponse times within the ratio. Steady responding then ceased, and numerous long interresponse times occurred, with no systematic relationship to ordinal position within the ratio. Gradually, a new pattern evolved, characterized by a consistently long post-reinforcement time, a border region of the next few interresponse times within which the mean interresponse time monotonically decreased, and short interresponse times within the last 80% of the ratio. Long interresponse times were eliminated from this last section of the ratio without regard to proximity to reinforcement. Various analytical procedures suggested that the final pattern can be conceived, in part, as the shaping of a reliable response topography. The group of three pigeons given 250 mg/kg/day of yeast ribonucleic acid responded at higher rates than the saline and 500 mg/kg/day groups. The latter group, in contrast to the saline and lower dose groups, which continued to increase their rates, reached a rate asymptote very early. PMID:4574711

An ultrasensitive universal detector based on neutralizer displacement

NASA Astrophysics Data System (ADS)

Das, Jagotamoy; Cederquist, Kristin B.; Zaragoza, Alexandre A.; Lee, Paul E.; Sargent, Edward H.; Kelley, Shana O.

2012-08-01

Diagnostic technologies that can provide the simultaneous detection of nucleic acids for gene expression, proteins for host response and small molecules for profiling the human metabolome will have a significant advantage in providing comprehensive patient monitoring. Molecular sensors that report changes in the electrostatics of a sensor's surface on analyte binding have shown unprecedented sensitivity in the detection of charged biomolecules, but do not lend themselves to the detection of small molecules, which do not carry significant charge. Here, we introduce the neutralizer displacement assay that allows charge-based sensing to be applied to any class of molecule irrespective of the analyte charge. The neutralizer displacement assay starts with an aptamer probe bound to a neutralizer. When analyte binding occurs the neutralizer is displaced, which results in a dramatic change in the surface charge for all types of analytes. We have tested the sensitivity, speed and specificity of this system in the detection of a panel of molecules: (deoxy)ribonucleic acid, ribonucleic acid, cocaine, adenosine triphosphate and thrombin.

Ribonucleic Acid and Ribosomes of Bacillus stearothermophilus1

PubMed Central

Saunders, Grady F.; Campbell, L. Leon

1966-01-01

Saunders, Grady F. (University of Illinois, Urbana), and L. Leon Campbell. Ribonucleic acid and ribosomes of Bacillus stearothermophilus. J. Bacteriol. 91:332–339. 1966.—The ability of some thermophilic bacteria to grow at temperatures as high as 76 C emphasizes the remarkable thermal stability of their crucial macromolecules. An investigation of the ribonucleic acid (RNA) and ribosomes of Bacillus stearothermophilus was conducted. Washed log-phase cells were disrupted either by sonic treatment or by alumina grinding in 10−2m MgCl2–10−2m tris-(hydroxymethyl)aminomethane buffer, pH 7.4 (TM buffer). Ultracentrifugal analysis revealed peaks at 72.5S, 101S, and 135S, with the 101S peak being the most prominent. By lowering the Mg++ concentration to 10−3m, the ribosome preparation was dissociated to give 40S, 31S, and 54S peaks. These in turn were reassociated in the presence of 10−2m Mg++ to give the larger 73S and 135S particles. When heated in TM buffer, Escherichia coli ribosomes began a gradual dissociation at 58 C, and at 70 C underwent a large hyperchromic shift with a Tm at 72.8 C. In contrast, B. stearothermophilus ribosomes did not show a hyperchromic shift below 70 C; they had a Tm of 77.9 C. The thermal denaturation curves of the 4S, 16S, and 23S RNA from both organisms were virtually identical. The gross amino acid composition of B. stearothermophilus ribosomes showed no marked differences from that reported for E. coli ribosomes. These data suggest that the unusual thermal stability of B. stearothermophilus ribosomes may reflect either an unusual packing arrangement of the protein to the RNA or differences in the primary structure of the ribosomal proteins. Images PMID:5903099

Introducing Molecular Biology to Environmental Engineers through Development of a New Course.

ERIC Educational Resources Information Center

Oerther, Daniel B.

2002-01-01

Introduces a molecular biology course designed for environmental engineering majors using 16S ribosomal ribonucleic acid-targeted technology that allows students to identify and study microorganisms in bioreactor environments. (Contains 17 references.) (YDS)

5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

NASA Technical Reports Server (NTRS)

Van den Eynde, H.; De Baere, R.; Shah, H. N.; Gharbia, S. E.; Fox, G. E.; Michalik, J.; Van de Peer, Y.; De Wachter, R.

1989-01-01

The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

Small interfering ribonucleic acid induces liquid-to-ripple phase transformation in a phospholipid membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choubey, Amit; Nomura, Ken-ichi; Kalia, Rajiv K.

Small interfering ribonucleic acid (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. A key limitation to the widespread implementation of siRNA therapeutics is the difficulty of delivering siRNA-based drugs to cells. Here, we examine changes in the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer in the presence of a siRNA molecule and mechanical barriers to siRNA transfection in the bilayer. Our all-atom molecular dynamics simulation shows that siRNA induces a liquid crystalline-to-ripple phase transformation in the bilayer. The ripple phase consists of a major region of non-interdigitated and a minor region of interdigitatedmore » lipid molecules with an intervening kink. In the ripple phase, hydrocarbon chains of lipid molecules have large compressive stresses, which present a considerable barrier to siRNA transfection.« less

42 CFR 71.54 - Import regulations for infectious biological agents, infectious substances, and vectors.

Code of Federal Regulations, 2014 CFR

2014-10-01

... segmented configuration and may be positive sense (same polarity as mRNA), negative sense, or ambisense... material. Deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA) comprising the genome or organism's... threat to public health and safety as listed in 42 CFR 73.3 and 73.4. Vector. Any animals (vertebrate or...

42 CFR 71.54 - Import regulations for infectious biological agents, infectious substances, and vectors.

Code of Federal Regulations, 2013 CFR

2013-10-01

... segmented configuration and may be positive sense (same polarity as mRNA), negative sense, or ambisense... material. Deoxyribonucleic acid (DNA) or Ribonucleic acid (RNA) comprising the genome or organism's... threat to public health and safety as listed in 42 CFR 73.3 and 73.4. Vector. Any animals (vertebrate or...

New insights into the molecular mechanism of Boletus edulis ribonucleic acid fraction (BE3) concerning antiproliferative activity on human colon cancer cells.

PubMed

Lemieszek, Marta Kinga; Ribeiro, Miguel; Marques, Guilhermina; Nunes, Fernando Milheiro; Pożarowski, Piotr; Rzeski, Wojciech

2017-05-24

One of the relatively new and promising strategies of cancer treatment is chemoprevention, which involves the use of natural or synthetic compounds to block, inhibit or reverse carcinogenesis. A valuable and still untapped source of chemopreventive compounds seems to be edible mushrooms belonging to higher Basidiomycetes. Boletus edulis biopolymers extracted with hot water and purified by anion-exchange chromatography showed antiproliferative activity in colon cancer cells, but only fraction BE3, mostly composed of ribonucleic acids, was able to inhibit DNA synthesis in HT-29 cells. The present work aims to elucidate the molecular mechanism of this Boletus edulis ribonucleic acid fraction and in this sense flow cytometry and western blotting were applied to cell cycle analysis in HT-29 cells. We found that the antiproliferative ability of fraction BE3 observed in HT-29 cells was associated with the modulation of expression of cell cycle regulatory proteins (Cyclin D1, Cyclin A, p21 and p27) leading to cell accumulation in the S phase of the cell cycle. Furthermore, the BE3 fraction showed effective silencing of the signal transduction in an MAPK/Erk pathway in HT-29 and LS180 colon cancer cell lines. Thus, the previously and currently obtained results indicate that the BE3 fraction from Boletus edulis has great potential and needs to be further exploited through animal and clinical studies in order to develop a new efficient and safe therapeutic strategy for people who have been threatened by or suffered from colon cancer.

EPA Registers Innovative Tool to Control Corn Rootworm

EPA Pesticide Factsheets

Ribonucleic acid interference (RNAi) based Plant Incorporated Protectant (PIP) technology is a new and innovative scientific tool utilized by U.S. growers. Learn more about RNAi technology and the 4 new products containing the RNAi based PIP called SMARTST

Problems in aerial application. I, Some biochemical effects of lindane and dieldrin on vertebrates.

DOT National Transportation Integrated Search

1962-05-01

Chronic exposure to the chlorinated hydrocarbon, lindane, elicited alterations in several biochemical values of rat tissues. These included ribonucleic acid quantities, water content, and the cytochrome oxidase activity of heart sarcosomes. The chlor...

Calciviruses

USDA-ARS?s Scientific Manuscript database

The Caliciviridae is a family of small, nonenveloped viruses containing a single-stranded, plus-sense genomic ribonucleic acid (RNA) that is polyadenylated at its 3’-end. Most, but not all, caliciviruses have distinctive cup-shaped depressions (L. calix, cup) on their surface, giving them their char...

COMMISSION ON EPIDEMIOLOGICAL SURVEY

DTIC Science & Technology

Ribonucleic Acid Metabolism during Infection; Mechanisms of Endotoxin Tolerance; Typhoid Fever: Pathogenesis and Prevention; Studies on Rocky Mountain Spotted Fever : Serologic...Response in Man to Vaccination with Combined Epidemic Typhus, Rocky Mountain Spotted Fever and Q Fever Vaccine; and Influence of Tularemia on Insulin Secretion.

COMMISSION ON EPIDEMIOLOGICAL SURVEY

DTIC Science & Technology

Ribonucleic Acid Metabolism during Infection; Mechanisms of Endotoxin Tolerance; Typhoid Fever: Pathogenesis and Prevention; Studies on Rocky Mountain Spotted Fever : Serologic...Response in Man to Vaccination with Combined Epidemic Typhus, Rocky Mountain Spotted Fever and Q Fever Vaccine; and Influence of Tularemia on Tularemia on Insulin Secretion.

The diversity of the orthoreoviruses: molecular taxonomy and phylogentic divides.

USDA-ARS?s Scientific Manuscript database

The family Reoviridae is a diverse group of viruses with double-stranded ribonucleic acid (RNA) genomes contained within icosahedral, layered protein capsids. Within the Reoviridae, the Orthoreovirus genus includes viruses that infect reptiles, birds and mammals (including humans). Recent sequencing...

Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down.

PubMed Central

Jacobson, L A; Jen-Jacobson, L

1980-01-01

We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down. PMID:6155375

Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down.

PubMed

Jacobson, L A; Jen-Jacobson, L

1980-06-01

We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down.

Annual Research Report 1 October 1978-30 September 1979.

DTIC Science & Technology

1979-01-01

Roeder, R. G. and Rutter, W. J. Multiple acid polymerases in ribonucleic acid synthesis during sea urchin development. Biochemistry 9: 2543-2554...with ultrastructural transmission electron microscopy (TEM) studies and scanning electron microscopy ( SEM ) stud- ies of lateral ventricular lining and...1I alterations in animals about 100 days after Silastic implantation. SEM studies show flattening and stretching of ependymal cells in the dorsomedial

Molecular Recognition Directed Self-Assembly of Supramolecular Architectures

DTIC Science & Technology

1994-06-30

TMV-Like SupraxlecuiarArchiteturc _ TMV is a simple virus consisting only of a single type of protein molecule and of a strand of ribonucleic acid (RNA...experiments have demonstrated that various substituted gallic acid derivatives can be used to construct exo-receptors with a tapered shape. 1.2...with 3,4,5-tris(p - dodecyloxybenzyloxy)benzoic acid {12-ABCr) resulted in the taper shaped structural units. 12- ABG-B1SC5 and 12-ABG-15C5

Life or death? A Physiogenomic Approach to Understand Individual Variation in Responses to Hemorrhagic Shock

DTIC Science & Technology

2011-01-01

Olaomi, O.; Olldashi, F.; Perel, P.; Peto, R.; Ramana, P. V.; Ravi, R. R.; Yutthakasemsunt, S. Effects of tranexamic acid on death, vascular...Glutamine [197-199] Yes Histone deacetylase inhibitors (e.g., valproic acid ) [168-169, 200-201] No Naloxone [202] Yes 430 Current Genomics, 2011, Vol...regulation and dynamics of ribonucleic Acid . Endocrinology, 2010, 151, 1391-1397. [44] Spriggs, K. A.; Bushell, M.; Willis, A. E. Translational regulation

Asian citrus psyllid RNAi pathway - RNAi evidence

USDA-ARS?s Scientific Manuscript database

In silico analyses of the draft genome of Diaphorina citri, the Asian citrus psyllid, for genes within the Ribonucleic acid interference(RNAi), pathway was successful. The psyllid is the vector of the plant-infecting bacterium, Candidatus Liberibacter asiaticus (CLas), which is linked to citrus gree...

Discovery of huanglongbing (HLB) pre-symptomatic Ribonucleic acid (RNA) biomarkers

USDA-ARS?s Scientific Manuscript database

Huanglongbing (HLB) is the most devastating citrus disease and is associated with vector-borne Liberibacter. Currently there is no cure for huanglongbing. Visual disease symptoms appear in only a few leaves months after initial Liberibacter exposure compromising disease management by tree removal. S...

GENETIC INDICATORS OF ENVIRONMENTAL STRESS IN HERMATYPIC CORALS

EPA Science Inventory

An efficient, low cost method was developed for the detection of rapid changes in coral gene expression at the messenger ribonucleic acid (mRNA) level. Hermatypic (stony) corals were exposed to a variety of organic and inorganic toxicants and physical stressors at several concent...

Non-transgenic RNAi technology to control insects on citrus

USDA-ARS?s Scientific Manuscript database

This research demonstrated a non-transgenic delivery method for ribonucleic acid interference, RNAi, that reduced fitness as measured in increased mortality over time, of two insect pests of citrus, ie. psyllids and leafhoppers. The Asian citrus psyllid transmits a deadly plant-infecting bacterium o...

Current Research on Non-Coding Ribonucleic Acid (RNA).

PubMed

Wang, Jing; Samuels, David C; Zhao, Shilin; Xiang, Yu; Zhao, Ying-Yong; Guo, Yan

2017-12-05

Non-coding ribonucleic acid (RNA) has without a doubt captured the interest of biomedical researchers. The ability to screen the entire human genome with high-throughput sequencing technology has greatly enhanced the identification, annotation and prediction of the functionality of non-coding RNAs. In this review, we discuss the current landscape of non-coding RNA research and quantitative analysis. Non-coding RNA will be categorized into two major groups by size: long non-coding RNAs and small RNAs. In long non-coding RNA, we discuss regular long non-coding RNA, pseudogenes and circular RNA. In small RNA, we discuss miRNA, transfer RNA, piwi-interacting RNA, small nucleolar RNA, small nuclear RNA, Y RNA, single recognition particle RNA, and 7SK RNA. We elaborate on the origin, detection method, and potential association with disease, putative functional mechanisms, and public resources for these non-coding RNAs. We aim to provide readers with a complete overview of non-coding RNAs and incite additional interest in non-coding RNA research.

Detection of miRNA using a double-strand displacement biosensor with a self-complementary fluorescent reporter.

PubMed

Larkey, Nicholas E; Almlie, C Kyle; Tran, Victoria; Egan, Marianne; Burrows, Sean M

2014-02-04

Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.

Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

PubMed Central

Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

2014-01-01

Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

Phosphodiester models for cleavage of nucleic acids

PubMed Central

2018-01-01

Nucleic acids that store and transfer biological information are polymeric diesters of phosphoric acid. Cleavage of the phosphodiester linkages by protein enzymes, nucleases, is one of the underlying biological processes. The remarkable catalytic efficiency of nucleases, together with the ability of ribonucleic acids to serve sometimes as nucleases, has made the cleavage of phosphodiesters a subject of intensive mechanistic studies. In addition to studies of nucleases by pH-rate dependency, X-ray crystallography, amino acid/nucleotide substitution and computational approaches, experimental and theoretical studies with small molecular model compounds still play a role. With small molecules, the importance of various elementary processes, such as proton transfer and metal ion binding, for stabilization of transition states may be elucidated and systematic variation of the basicity of the entering or departing nucleophile enables determination of the position of the transition state on the reaction coordinate. Such data is important on analyzing enzyme mechanisms based on synergistic participation of several catalytic entities. Many nucleases are metalloenzymes and small molecular models offer an excellent tool to construct models for their catalytic centers. The present review tends to be an up to date summary of what has been achieved by mechanistic studies with small molecular phosphodiesters. PMID:29719577

PHAGE FORMATION IN STAPHYLOCOCCUS MUSCAE CULTURES

PubMed Central

Price, Winston H.

1949-01-01

1. The total nucleic acid synthesized by normal and by infected S. muscae suspensions is approximately the same. This is true for either lag phase cells or log phase cells. 2. The amount of nucleic acid synthesized per cell in normal cultures increases during the lag period and remains fairly constant during log growth. 3. The amount of nucleic acid synthesized per cell by infected cells increases during the whole course of the infection. 4. Infected cells synthesize less RNA and more DNA than normal cells. The ratio of RNA/DNA is larger in lag phase cells than in log phase cells. 5. Normal cells release neither ribonucleic acid nor desoxyribonucleic acid into the medium. 6. Infected cells release both ribonucleic acid and desoxyribonucleic acid into the medium. The time and extent of release depend upon the physiological state of the cells. 7. Infected lag phase cells may or may not show an increased RNA content. They release RNA, but not DNA, into the medium well before observable cellular lysis and before any virus is liberated. At virus liberation, the cell RNA content falls to a value below that initially present, while DNA, which increased during infection falls to approximately the original value. 8. Infected log cells show a continuous loss of cell RNA and a loss of DNA a short time after infection. At the time of virus liberation the cell RNA value is well below that initially present and the cells begin to lyse. PMID:18139006

Developing ribonucleic acid interference technology to manage whitefly-transmitted begomoviruses

USDA-ARS?s Scientific Manuscript database

Of nearly 300 viruses transmitted by the whitefly (Bemisia tabaci), 90% of them belong to the genus Begomovirus. Begomoviruses are efficiently transmitted by whiteflies to a range of agricultural crops. This results in billions of dollars lost annually, while jeopardizing food security worldwide. Se...

Molecular characterization of true morels (Morchella) in Turkey

USDA-ARS?s Scientific Manuscript database

A collection of 247 true morels (Morchella spp.) was made from 10 different provinces of Turkey during the 2007-2008 growing season. This collection was analyzed for species diversity using phylogenetic analyses of partial Ribonucleic acid (RNA) polymerase I (RPB1) and nuclear ribosomal large subuni...

Identification and initial characterization of a novel turkey-origin picobirnavirus using a metagenomic approach

USDA-ARS?s Scientific Manuscript database

Using the Genome Sequencer FLX Titanium technology (Roche, 454 Life Sciences), a ribonucleic acid (RNA) virus-specific metagenome was prepared using the pooled intestinal contents collected from North Carolina turkey flocks experiencing enteric disease signs. This analysis produced 6526 contigs rang...

The Meduza experiment: An orbital complex ten weeks in flight

NASA Technical Reports Server (NTRS)

Ovcharov, V.

1979-01-01

The newspaper article discusses the contribution of space research to understanding the origin of life on Earth. Part of this basic research involves studying amino acids, ribonucleic acid and DNA molecules subjected to cosmic radiation. The results from the Meduza experiment are not all analyzed as yet. The article also discusses the psychological changes in cosmonauts as evidenced by their attitude towards biology experiments in space.

Fine-structure mapping of the firA gene, a locus involved in the phenotypic expression of rifampin resistance in Escherichia.

PubMed

Lathe, R

1977-09-01

The firA (Ts)200 mutation not only eliminates the resistance to rifampin of certain genetically resistant strains, but, moreover, renders ribonucleic acid synthesis thermolabile. The firA gene has been mapped by P1 tranduction and is located extremely close to the structural gene for deoxyribonucleic acid polymerase III at 4 min on the Escherichia coli linkage map.

Ghrelin, MicroRNAs, and Critical Limb Ischemia: Hungering for a Novel Treatment Option.

PubMed

Neale, Joshua P H; Pearson, James T; Katare, Rajesh; Schwenke, Daryl O

2017-01-01

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease. It is characterized by chronic pain at rest, skin ulcerations, and gangrene tissue loss. CLI is a highly morbid condition, resulting in a severely diminished quality of life and a significant risk of mortality. The primary goal of therapy for CLI is to restore blood flow to the affected limb, which is only possible by surgery, but is inadvisable in up to 50% of patients. This subset of patients who are not candidates for revascularisation are referred to as "no-option" patients and are the focus of investigation for novel therapeutic strategies. Angiogenesis, arteriogenesis and vasculogenesis are the processes whereby new blood vessel networks form from the pre-existing vasculature and primordial cells, respectively. In therapeutic angiogenesis, exogenous stimulants are administered to promote angiogenesis and augment limb perfusion, offering a potential treatment option for "no option" patients. However, to date, very few clinical trials of therapeutic angiogenesis in patients with CLI have reported clinically significant results, and it remains a major challenge. Ghrelin, a 28-amino acid peptide, is emerging as a potential novel therapeutic for CLI. In pre-clinical models, exogenous ghrelin has been shown to induce therapeutic angiogenesis, promote muscle regeneration, and reduce oxidative stress via the modulation of microRNAs (miRs). miRs are endogenous, small, non-coding ribonucleic acids of ~20-22 nucleotides which regulate gene expression at the post-transcriptional level by either translational inhibition or by messenger ribonucleic acid cleavage. This review focuses on the mounting evidence for the use of ghrelin as a novel therapeutic for CLI, and highlights the miRs which orchestrate these physiological events.

NUCLEIC ACID CONCENTRATION AND RADIOSENSITIVITY OF THE SCORPION ANDROCTONUS AMOREUXI AUD. AND SAV (in French)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pascaud, X.; Niaussat, P.

1963-01-01

The concentration of desoxyribonucleic acid and of ribonucleic acid in the soft tissues was determined for the two invertebrates of the arid zone, the scorpion Androctonus amoreuxi Aud. and Sav. and the tenebrionide Pimelia angulata expiata Peyer. The radiosensitivity to gamma rays had been previously determined: LD/sub 50/30// days is 100,000 r for Androctonus and 40,000 for Pimelia. The mean rate of nucleic acids determined in the scorpion was relatively low. A possible relation between the high radioresistance of the scorpion and the low nucleic acid concentration was discussed. (J.S.R.)

Nucleic Acid-Induced Resistance to Viral Infection

PubMed Central

Takano, Kouichi; Warren, Joel; Jensen, Keith E.; Neal, Alan L.

1965-01-01

Takano, Kouichi (Chas. Pfizer & Co., Inc., Terre Haute, Ind.), Joel Warren, Keith E. Jensen, and Alan L. Neal. Nucleic acid resistance to viral infection. J. Bacteriol. 90:1542–1547. 1965.—Administration of nonviral nucleic acids to mice increased their resistance to a subsequent infection with influenza or encephalomyocarditis viruses. Injection of ribonucleic acid or deoxyribonucleic acid by peripheral routes did not modify susceptibility to intranasal infection. Lung tissue extracts from animals previously treated with yeast nucleic acid inhibited the growth of vaccinia and influenza viruses. The protective effect of exogenous nucleic acids persisted in mice for several days, but gradually diminished to undetectable levels. PMID:4285332

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

NASA Astrophysics Data System (ADS)

Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

2012-05-01

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

Fine-structure mapping of the firA gene, a locus involved in the phenotypic expression of rifampin resistance in Escherichia.

PubMed Central

Lathe, R

1977-01-01

The firA (Ts)200 mutation not only eliminates the resistance to rifampin of certain genetically resistant strains, but, moreover, renders ribonucleic acid synthesis thermolabile. The firA gene has been mapped by P1 tranduction and is located extremely close to the structural gene for deoxyribonucleic acid polymerase III at 4 min on the Escherichia coli linkage map. PMID:330494

Polysome Turnover During Amino Acid Starvation in Escherichia coli

PubMed Central

Ron, Eliora Z.

1971-01-01

The experiments presented in this paper support earlier evidence that ribosomes are released from polysomes when they encounter a codon for which no charged transfer ribonucleic acid is available. However, it is further shown that these ribosomes then reinitiate and resume translation. The size and the level of polysomes during deprival of an amino acid is a function of the frequency with which that particular amino acid appears in cellular proteins. Polysomes from starved cells are more stable than those from growing cells, and, moreover, polysomes from starved relaxed strains are more stable than those from starved stringent strains. PMID:4941559

Understanding LiP Promoters from Phanerochaete chrysosporium: A Bioinformatic Analysis

Treesearch

Sergio Lobos; Rubén Polanco; Mario Tello; Dan Cullen; Daniela Seelenfreund; Rafael Vicuña

2011-01-01

DNA contains the coding information for the entire set of proteins produced by an organism. The specific combination of proteins synthesized varies with developmental, metabolic and environmental circumstances. This variation is generated by regulatory mechanisms that direct the production of messenger ribonucleic acid (mRNA) and subsequent translation of the...

Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for Citrus Huanglongbing Disease

USDA-ARS?s Scientific Manuscript database

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus Candidatus Liberibacter(Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small Ribonucleic acid (sRNA) play a vital role in regulating host responses to pa...

Composition of ribonucleic acid from various parts of spider oocytes.

PubMed

EDSTROM, J E

1960-09-01

Microphoretic purine-pyrimidine analyses of the ribonucleic acid (RNA) in nucleoli, nucleoplasm, cytoplasm, and yolk nuclei of spider oocytes have been carried out. The material necessary for the analyses was isolated by micromanipulation. Determinations of the amounts of RNA in the different parts of the cell were also performed. No differences between the composition of RNA in the nucleolus and the cytoplasm could be disclosed. Nucleoplasmic RNA was, on the other hand, distinctly different from that in the nucleolus and in the cytoplasm. The difference lies in the content of adenine, which is highest in nucleoplasmic RNA. The few analyses carried out on yolk nuclei showed their RNA to be variable in composition with a tendency to high purine values. The cytoplasm contains about 99 per cent of the total RNA in these cells, the nucleoplasm about 1 per cent, and the nucleolus not more than 0.3 per cent, although the highest concentrations are found in these latter structures. When considered in the light of other recent findings the results are compatible with the view that nucleolar RNA is the precursor of cytoplasmic RNA.

Evaluating the reproducibility of quantifying modified nucleosides from ribonucleic acids by LC–UV–MS

PubMed Central

Russell, Susan P.; Limbach, Patrick A.

2013-01-01

Post-transcriptional chemical covalent modification of adenosine, guanosine, uridine and cytidine occurs frequently in all types of ribonucleic acids (RNAs). In ribosomal RNA (rRNA) and transfer RNA (tRNA) these modifications make important contributions to RNA structure and stability and to the accuracy and efficiency of protein translation. The functional dynamics, synergistic nature and regulatory roles of these posttranscriptional nucleoside modifications within the cell are not well characterized. These modifications are present at very low levels and isolation of individual nucleosides for analysis requires a complex multi-step approach. The focus of this study is to characterize the reproducibility of a liquid chromatography method used to isolate and quantitatively characterize modified nucleosides in tRNA and rRNA when nucleoside detection is performed using ultraviolet and mass spectrometric detection (UV and MS, respectively). Despite the analytical challenges of sample isolation and dynamic range, quantitative profiling of modified nucleosides obtained from bacterial tRNAs and rRNAs is feasible at relative standard deviations of 5% RSD or less. PMID:23500350

Strain of Escherichia coli with a temperature-sensitive mutation affecting ribosomal ribonucleic acid accumulation.

PubMed Central

Frey, T; Newlin, L L; Atherly, A G

1975-01-01

A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis. When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time. Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag. However, the phenotype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected. This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Böck and Neidhardt, 1966). However, the mutation occurred in a different position on the E. coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157. In this paper we present a study of macromolecular biosynthesis in this mutant. PMID:1090609

Regulation of ribonucleic acid synthesis by polyamines. Reversal by spermine of inhibition by methylglyoxal bis(guanylhydrazone) of ribonucleic acid synthesis and histone acetylation in rabbit heart.

PubMed

Caldarera, C M; Casti, A; Guarnier, C; Moruzzi, G

1975-10-01

The relationship between polyamines and RNA synthesis was studied by considering the action of spermine on histone acetylation in perfused heart. In addition, the effect of methylglyoxal bis(guanylhydrazone), inhibitor of putrescine-activated S-adenosylmethionine decarboxylase activity, on RNA and polyamine specific radioactivity and on acetylation of histone fractions was also investigated in perfused heart. Different concentrations of spermine and/or methylglyoxas bis(guanylhydrazone) were injected into the heart, 15 min after beginning the perfusion. The results demonstrate that spermine stimulates the specific radioactivity of RNA of subcellular fractions. Acetylation of the arginine-rich histone fractions, involved in the regulation of RNA transcription, is enhanced by spermine. The perfusion with methylglyoxal bis(guanylhydrazone) causes a decrease in the specific radioactivity of polyamines and RNA, and in acetylation of histone fractions. However, spermine is able to reverse the methylglyoxal bis(guanylhydrazone) inhibition when injected simultaneously. From these results we may assume a possible role for spermine in the regulation of RNA transcription.

Regulation of ribonucleic acid synthesis by polyamines. Reversal by spermine of inhibition by methylglyoxal bis(guanylhydrazone) of ribonucleic acid synthesis and histone acetylation in rabbit heart.

PubMed Central

Caldarera, C M; Casti, A; Guarnier, C; Moruzzi, G

1975-01-01

The relationship between polyamines and RNA synthesis was studied by considering the action of spermine on histone acetylation in perfused heart. In addition, the effect of methylglyoxal bis(guanylhydrazone), inhibitor of putrescine-activated S-adenosylmethionine decarboxylase activity, on RNA and polyamine specific radioactivity and on acetylation of histone fractions was also investigated in perfused heart. Different concentrations of spermine and/or methylglyoxas bis(guanylhydrazone) were injected into the heart, 15 min after beginning the perfusion. The results demonstrate that spermine stimulates the specific radioactivity of RNA of subcellular fractions. Acetylation of the arginine-rich histone fractions, involved in the regulation of RNA transcription, is enhanced by spermine. The perfusion with methylglyoxal bis(guanylhydrazone) causes a decrease in the specific radioactivity of polyamines and RNA, and in acetylation of histone fractions. However, spermine is able to reverse the methylglyoxal bis(guanylhydrazone) inhibition when injected simultaneously. From these results we may assume a possible role for spermine in the regulation of RNA transcription. PMID:1212228

BACTERIOPHAGE FORMATION WITHOUT BACTERIAL GROWTH

PubMed Central

Price, Winston H.

1947-01-01

1. Iodoacetate, fluoride, and azide have been found to prevent the formation of phage and to inhibit the synthesis of ATP by Staphylococcus muscae. It is suggested that energy-rich phosphate is needed for the synthesis of phage. 2. Gramicidin prevented the formation of phage. 3. No differences were found between normal bacteria and phage-infected bacteria in the inorganic phosphate, adenosinetriphosphate, ribonucleic acid, and desoxyribonucleic acid content of the cells. 4. The mechanism of phage formation is discussed. PMID:18896936

Tenascin in meningioma: expression is correlated with anaplasia, vascular endothelial growth factor expression, and peritumoral edema but not with tumor border shape.

PubMed

Kiliç, Türker; Bayri, Yaşar; Ozduman, Koray; Acar, Melih; Diren, Semin; Kurtkaya, Ozlem; Ekinci, Gazanfer; Buğra, Kuyaş; Sav, Aydin; Ozek, M Memet; Pamir, M Necmettin

2002-07-01

Tenascin is an extracellular matrix glycoprotein that is expressed during embryogenesis, inflammation, angiogenesis, and carcinogenesis. The aim of this study was to investigate how tenascin expression relates to histological grade, angiogenesis, and radiological findings in meningiomas. Twenty typical, 20 atypical, and 5 malignant meningiomas were studied retrospectively. Tenascin expression and vascular endothelial growth factor (VEGF) expression in the tumor tissue were investigated by immunohistochemistry. Tenascin messenger ribonucleic acid expression was also studied by comparative reverse transcriptase-polymerase chain reaction. Magnetic resonance images from each case were assessed for peritumoral edema and tumor border shape. The atypical and malignant meningiomas showed higher levels of tenascin expression than the typical meningiomas. The more sensitive messenger ribonucleic acid-based methods confirmed this finding. Tenascin expression was correlated with peritumoral edema and VEGF expression but not with tumor border shape. In the 13 tumors with marked tenascin expression, peritumoral edema was Grade 0 in one, Grade 1 in three, and Grade 2 in nine specimens. In the same 13 tumors, VEGF expression was Grade 1 in five and Grade 2 in eight specimens, and the findings for tumor border shape were Grade 0 in seven, Grade 1 in four, and Grade 2 in two specimens. In meningiomas, tenascin expression is correlated with anaplasia, tumor-associated edema, and VEGF expression but not with tumor border shape. This protein may play a role in the neoplastic and/or angiogenic processes in atypical and malignant meningiomas and may thus be a potential target for meningioma therapy.

Preparation of poly(ethylene glycol)/polylactide hybrid fibrous scaffolds for bone tissue engineering.

PubMed

Ni, PeiYan; Fu, ShaoZhi; Fan, Min; Guo, Gang; Shi, Shuai; Peng, JinRong; Luo, Feng; Qian, ZhiYong

2011-01-01

Polylactide (PLA) electrospun fibers have been reported as a scaffold for bone tissue engineering application, however, the great hydrophobicity limits its broad application. In this study, the hybrid amphiphilic poly(ethylene glycol) (PEG)/hydrophobic PLA fibrous scaffolds exhibited improved morphology with regular and continuous fibers compared to corresponding blank PLA fiber mats. The prepared PEG/PLA fibrous scaffolds favored mesenchymal stem cell (MSC) attachment and proliferation by providing an interconnected porous extracellular environment. Meanwhile, MSCs can penetrate into the fibrous scaffold through the interstitial pores and integrate well with the surrounding fibers, which is very important for favorable application in tissue engineering. More importantly, the electrospun hybrid PEG/PLA fibrous scaffolds can enhance MSCs to differentiate into bone-associated cells by comprehensively evaluating the representative markers of the osteogenic procedure with messenger ribonucleic acid quantitation and protein analysis. MSCs on the PEG/PLA fibrous scaffolds presented better differentiation potential with higher messenger ribonucleic acid expression of the earliest osteogenic marker Cbfa-1 and mid-stage osteogenic marker Col I. The significantly higher alkaline phosphatase activity of the PEG/PLA fibrous scaffolds indicated that these can enhance the differentiation of MSCs into osteoblast-like cells. Furthermore, the higher messenger ribonucleic acid level of the late osteogenic differentiation markers OCN (osteocalcin) and OPN (osteopontin), accompanied by the positive Alizarin red S staining, showed better maturation of osteogenic induction on the PEG/PLA fibrous scaffolds at the mineralization stage of differentiation. After transplantation into the thigh muscle pouches of rats, and evaluating the inflammatory cells surrounding the scaffolds and the physiological characteristics of the surrounding tissues, the PEG/PLA scaffolds presented good biocompatibility. Based on the good cellular response and excellent osteogenic potential in vitro, as well as the biocompatibility with the surrounding tissues in vivo, the electrospun PEG/PLA fibrous scaffolds could be one of the most promising candidates in bone tissue engineering.

Transcriptome analysis of Bemisia tabaci during tomato yellow leaf curl virus acquisition and ribonucleic acid interference to manage whitefly-transmitted viruses

USDA-ARS?s Scientific Manuscript database

Over 300 viruses are transmitted by the whitefly, Bemisia tabaci, with 90% of them belonging to the genus, Begomovirus. Begomoviruses are obligately transmitted by whiteflies to a wide range of agriculture crops, resulting in billions of dollars lost annually, while jeopardizing food security worldw...

Use of novel RT-PCR assay to detect picobirnavirus in Poultry Intestinal samples collected from the field

USDA-ARS?s Scientific Manuscript database

Recent metagenomic analyses of the turkey gut ribonucleic acid (RNA) virus community in our laboratory have identified novel enteric RNA viruses that may play roles in the poultry enteric diseases and in performance problems noted in the field. This has lead to new molecular diagnostic assays for ce...

Complete genome sequence of a novel genotype of squash mosaic virus

USDA-ARS?s Scientific Manuscript database

Complete genome sequence of a novel genotype of Squash mosaic virus (SqMV) infecting squash plants in Spain was obtained using deep sequencing of small ribonucleic acids and assembly. The low nucleotide sequence identities, with 87-88% on RNA1 and 84-86% on RNA2 to known SqMV isolates, suggest a new...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Science Inventory

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...

DETECTION OF GYNECOLOGICAL CANCER—Use of Fluorescence Microscopy to Show Nucleic Acids in Malignant Growth

PubMed Central

Von Bertalanffy, Ludwig; Masin, Marianna; Masin, Francis; Kaplan, Leo

1957-01-01

Early detection of malignant lesions of the cervix, a major problem in gynecology, has been made possible in more cases by the development of exfoliative cytology. Mass-screening programs have been impeded, however, by the demands on time and skill of the examiner as posed by conventional techniques. A new method in exfoliative cytology, using fluorescence microscopy, essentially reduces the time of processing as well as of scanning of specimens. Suspicious cells show flaming orange-red fluorescence of the cytoplasm on a black background, impressively distinct from normal cells and giving a warning signal to the examiner. This color reaction is based upon cytochemical changes—namely, the abundance of ribonucleic acid in vividly growing and especially malignant cells. Besides gynecological material, the method is applicable to other forms of malignant disease. ImagesFigure 1.Figure 2.Figure 3.Figure 4.Figure 5.Figure 6. PMID:13460741

Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.

2012-05-15

Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized bymore » interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.« less

Evidence for the absence of the terminal adenine nucleotide at the amino acid-acceptor end of transfer ribonucleic acid in non-lactating bovine mammary gland and its inhibitory effect on the aminoacylation of rat liver transfer ribonucleic acid

PubMed Central

Herrington, M. D.; Hawtrey, A. O.

1970-01-01

1. tRNA isolated from non-lactating bovine mammary gland competitively inhibits the formation of aminoacyl-tRNA in the rat liver system. 2. Non-lactating bovine mammary gland tRNA and twice-pyrophosphorolysed rat liver tRNA are unable to accept amino acids in a reaction catalysed by aminoacyl-tRNA synthetases from either rat liver or bovine mammary gland. Deacylated rat liver tRNA can however be aminoacylated in the presence of either enzyme. 3. Bovine mammary gland tRNA lacks the terminal adenine nucleotide at the 3′-terminus amino acid acceptor end, which can be replaced by incubation in the presence of rat liver nucleotide-incorporating enzyme, ATP and CTP. 4. The enzymically modified bovine tRNA (tRNApCpCpA) can bind labelled amino acids to form aminoacyl-tRNA, which can then transfer its labelled amino acids to growing polypeptide chains on ribosomes. 5. Molecules of rat liver tRNA or bovine mammary gland tRNA that lack the terminal adenine nucleotide or the terminal cytosine and adenine nucleotides inhibit the aminoacylation of normal rat liver tRNA to varying degrees. tRNA molecules lacking the terminal −pCpCpA nucleotide sequence exhibit the major inhibitory effect. 6. The enzyme fraction from bovine mammary gland corresponding to that containing the nucleotide-incorporating enzyme in rat liver is unable to catalyse the incorporation of cytosine and adenine nucleotides in pyrophosphorolysed rat liver tRNA and deacylated bovine tRNA. This fraction also markedly inhibits the action of the rat liver nucleotide-incorporating enzyme. PMID:5435687

Pediatric tuberculosis-human immunodeficiency virus co-infection in the United Kingdom highlights the need for better therapy monitoring tools: a case report.

PubMed

Evangelopoulos, Dimitrios; Whittaker, Elizabeth; Honeyborne, Isobella; McHugh, Timothy D; Klein, Nigel; Shingadia, Delane

2017-02-26

Tuberculosis is an infection that requires at least 6 months of chemotherapy in order to clear the bacteria from the patient's lungs. Usually, therapeutic monitoring is dependent on smear microscopy where a decline in acid-fast bacilli is observed. However, this might not be indicative of the actual decline of bacterial load and thus other tools such as culture and molecular assays are required for patient management. Here, we report the case of a 12-year-old Black African boy co-infected with tuberculosis and human immunodeficiency virus who remained smear culture positive and liquid culture negative for a prolonged period of time following chemotherapy. In order to determine whether there was any live bacteria present in his specimens, we applied the newly developed molecular bacterial load assay that detects the presence of 16S ribosomal ribonucleic acid derived from the bacteria. Using this methodology, we were able to quantify his bacterial load and inform the management of his treatment in order to reduce the disease burden. Following this intervention he went on to make a complete recovery. This case report highlights the value of improved biomarkers for monitoring the treatment of tuberculosis and the role of molecular assays such as the molecular bacterial load assay applied here. The molecular bacterial load assay detects bacterial ribonucleic acid which corresponds closely with the number of live bacilli as compared with polymerase chain reaction that detects deoxyribonucleic acid and may include dead bacteria.

A duplex real-time RT-PCR system with an internal control offers sensitive and reliable broad spectrum detection of Squash mosaic virus variants

USDA-ARS?s Scientific Manuscript database

Squash mosaic virus (SqMV) is a seed-borne virus, belonging to the genus Commovirus in the subfamily Comoviridae of family Secoviridae. SqMV has a bipartite single-stranded ribonucleic acid (RNA) genome (RNA1 and RNA2) encapsidated separately with two capsid proteins. Two serotypes (genotypes) of ...

Comparative transcriptome analysis in Bemisia tabaci in response to tomato yellow leaf curl virus and development of ribonucleic acid interference to manage whitefly-transmitted viruses

USDA-ARS?s Scientific Manuscript database

The whitefly, Bemisia tabaci transmits over 300 plant viruses, with the majority of them belonging to the Begomovirus genus. Begomoviruses are obligately transmitted to a wide range of agriculture crops, resulting in the loss of billions of dollars annually, while jeopardizing food security worldwid...

EFFECT OF ULTRA-VIOLET IRRADIATION OF RIBONUCLEIC ACID ON ITS CHROMATOGRAPHIC BEHAVIOUR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubinski, H.

1963-11-01

Experiments were made to illustrate the effects of ultraviolet radiation on RNA isolated from uninfected mammalian cells as well as those from those infected with polio virus. The chromatographic recovery of irradiated RNA, as judged by ultraviolet adsorbance and radioactivity (no plaque formers were found after irradiation), was considerably lower than that of unirradiated RNA. (P.C.H.)

RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids

PubMed Central

Sample, Paul J.; Gaston, Kirk W.; Alfonzo, Juan D.; Limbach, Patrick A.

2015-01-01

Ribosomal ribonucleic acid (RNA), transfer RNA and other biological or synthetic RNA polymers can contain nucleotides that have been modified by the addition of chemical groups. Traditional Sanger sequencing methods cannot establish the chemical nature and sequence of these modified-nucleotide containing oligomers. Mass spectrometry (MS) has become the conventional approach for determining the nucleotide composition, modification status and sequence of modified RNAs. Modified RNAs are analyzed by MS using collision-induced dissociation tandem mass spectrometry (CID MS/MS), which produces a complex dataset of oligomeric fragments that must be interpreted to identify and place modified nucleosides within the RNA sequence. Here we report the development of RoboOligo, an interactive software program for the robust analysis of data generated by CID MS/MS of RNA oligomers. There are three main functions of RoboOligo: (i) automated de novo sequencing via the local search paradigm. (ii) Manual sequencing with real-time spectrum labeling and cumulative intensity scoring. (iii) A hybrid approach, coined ‘variable sequencing’, which combines the user intuition of manual sequencing with the high-throughput sampling of automated de novo sequencing. PMID:25820423

Effect of the mutation (C3435T) at exon 26 of the MDR1 gene on expression level of MDR1 messenger ribonucleic acid in duodenal enterocytes of healthy Japanese subjects.

PubMed

Nakamura, Tsutomu; Sakaeda, Toshiyuki; Horinouchi, Masanori; Tamura, Takao; Aoyama, Nobuo; Shirakawa, Toshiro; Matsuo, Masafumi; Kasuga, Masato; Okumura, Katsuhiko

2002-04-01

The effect of the C3435T mutation at exon 26 of the MDR1 gene on the expression levels of MDR1 messenger ribonucleic acid (mRNA) was evaluated by means of real-time polymerase chain reaction in 51 biopsy specimens of duodenum obtained from 13 healthy Japanese subjects. The mRNA levels of MDR1 were 0.38 +/- 0.15, 0.56 +/- 0.14, and 1.13 +/- 0.42 (mean value +/- SE) in the subjects with the homozygote of wild-type allele (C/C), compound heterozygote with mutant T allele (C/T), and the homozygote of the mutant allele (T/T), respectively, reasonably explaining the lower digoxin serum concentration after administration of a single oral dose to subjects harboring a mutant T allele. Good correlation (r =.797; P <.01) was observed between the mRNA concentrations of MDR1 and CYP3A4 in the individual biopsy specimens. This finding suggested a lower plasma concentration of the substrates for CYP3A4 in subjects harboring the C3435T mutation of the MDR1 gene.

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

PubMed

Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

2017-04-01

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

Simplified Identification of mRNA or DNA in Whole Cells

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

A recently invented method of detecting a selected messenger ribonucleic acid (mRNA) or deoxyribonucleic acid (DNA) sequence offers two important advantages over prior such methods: it is simpler and can be implemented by means of compact equipment. The simplification and miniaturization achieved by this invention are such that this method is suitable for use outside laboratories, in field settings in which space and power supplies may be limited. The present method is based partly on hybridization of nucleic acid, which is a powerful technique for detection of specific complementary nucleic acid sequences and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes.

Inhibition of protein synthesis in intact HeLa cells by Shigella dysenteriae 1 toxin.

PubMed

Brown, J E; Rothman, S W; Doctor, B P

1980-07-01

Shiga toxin purified to near homogeneity from cell lysates of Shigella dysenteriae 1 inhibited protein and deoxyribonucle acid syntheses in intact HeLa cells. Inhibition was dependent on toxin concentration and time of incubation. A minimal latent period of 30 min was observed with saturating doses of toxin. Ribonucleic acid synthesis, uptake of alpha-aminoisobutyric acid, and maintenance of intracellular K+ concentrations were not affected until well after maximal inhibition of protein and deoxyribonucleic acid syntheses. The inhibitory effect of toxin was sensitive to heat inactivation and was prevented by antibody neutralization. Several cytotoxic components were separated by polyacrylamide gel electrophoresis of the purified toxin preparations; all inhibited protein and deoxyribonucleic acid syntheses equally.

The Small Intestinal Epithelia of Beef Steers Differentially Express Sugar Transporter Messenger Ribonucleic Acid in Response to Abomasal Versus Ruminal Infusion of Starch Hydrolysate

USDA-ARS?s Scientific Manuscript database

In mammals, the absorption of mono¬saccharides from small intestinal lumen involves at least 3 sugar transporters (SugT): sodium-dependent glucose transporter 1 (SGLT1; gene SLC5A1) transports glucose and galactose, whereas glucose transporter (GLUT) 5 (GLUT5; gene SLC2A5) transports fructose, acros...

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis.

PubMed

Peeters, M; Huang, C L; Vonk, L A; Lu, Z F; Bank, R A; Helder, M N; Doulabi, B Zandieh

2016-11-01

Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised.Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560-568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. © 2016 Peeters et al.

Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis

PubMed Central

Peeters, M.; Huang, C. L.; Vonk, L. A.; Lu, Z. F.; Bank, R. A.; Doulabi, B. Zandieh

2016-01-01

Objectives Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3. PMID:27881439

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

PubMed Central

Donini, Pierluigi

1970-01-01

Starvation for a required amino acid of normal or RCstrEscherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RCrelE. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RCstr phenotype but not in cells of RCrel phenotype. Inhibition of phage DNA synthesis in amino acid-starved RCstr host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought. PMID:4914067

Discovery of a new genotype of Squash mosaic virus through deep sequencing of small RNAs and development of a qRT-PCR for broad spectrum detection

USDA-ARS?s Scientific Manuscript database

Squash mosaic virus (SqMV), a seed-borne virus belonging to the genus Commovirus in the family Comoviridae, could cause a serious yield loss on cucurbit crops worldwide. SqMV has a bipartite single-stranded ribonucleic acid (RNA) genome (RNA-1 and RNA-2) encapsidated separately with two capsid prote...

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

PubMed Central

Friedman, Robert M.; Fantes, Karl H.; Levy, Hilton B.; Carter, William B.

1967-01-01

Actinomycin D-treated chick fibroblasts were infected with purified 32P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 μg/ml) or cycloheximide (200 μg/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of 32P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA. PMID:5621488

Dietary ribonucleic acid suppresses inflammation of adipose tissue and improves glucose intolerance that is mediated by immune cells in C57BL/6 mice fed a high-fat diet.

PubMed

Sakai, Tohru; Taki, Tomoyo; Nakamoto, Akiko; Tazaki, Shiho; Arakawa, Mai; Nakamoto, Mariko; Tsutsumi, Rie; Shuto, Emi

2015-01-01

Recent evidence suggests that immune cells play an important role in differentiation of inflammatory macrophages in adipose tissue, which contributes to systemic chronic inflammation. Dietary ribonucleic acid (RNA) has been shown to modulate immune function. We hypothesized that RNA affects immune cell function in adipose tissue and then improves inflammatory response in adipose tissue. C57/BL6 mice and recombination activating gene-1 (RAG-1) knockout mice on a C57BL/6 mice background were fed a high-fat diet containing 1% RNA for 12 wk. An oral glucose tolerance test was performed. Supplementation of dietary RNA in C57BL/6 mice fed a high-fat diet resulted in a smaller area under the curve (AUC) after oral glucose administration than that for control mice. The mRNA expression levels of inflammation-related cytokines in adipose tissue and serum interleukin-6 levels were reduced by dietary RNA supplementation. Interestingly, reduction of the AUC value by RNA supplementation was abolished in T and B cell-deficient RAG-1 knockout mice. These results indicate that RNA improves inflammation in adipose tissue and reduces the AUC value following oral glucose administration in a T and B cell-dependent manner.

Label-free serum ribonucleic acid analysis for colorectal cancer detection by surface-enhanced Raman spectroscopy and multivariate analysis

NASA Astrophysics Data System (ADS)

Chen, Yanping; Chen, Gang; Feng, Shangyuan; Pan, Jianji; Zheng, Xiongwei; Su, Ying; Chen, Yan; Huang, Zufang; Lin, Xiaoqian; Lan, Fenghua; Chen, Rong; Zeng, Haishan

2012-06-01

Studies with circulating ribonucleic acid (RNA) not only provide new targets for cancer detection, but also open up the possibility of noninvasive gene expression profiling for cancer. In this paper, we developed a surface-enhanced Raman scattering (SERS), platform for detection and differentiation of serum RNAs of colorectal cancer. A novel three-dimensional (3-D), Ag nanofilm formed by dry MgSO4 aggregated silver nanoparticles, Ag NP, as the SERS-active substrate was presented to effectively enhance the RNA Raman signals. SERS measurements were performed on two groups of serum RNA samples. One group from patients, n=55 with pathologically diagnosed colorectal cancer and the other group from healthy controls, n=45. Tentative assignments of the Raman bands in the normalized SERS spectra demonstrated that there are differential expressions of cancer-related RNAs between the two groups. Linear discriminate analysis, based on principal component analysis, generated features can differentiate the colorectal cancer SERS spectra from normal SERS spectra with sensitivity of 89.1 percent and specificity of 95.6 percent. This exploratory study demonstrated great potential for developing serum RNA SERS analysis into a useful clinical tool for label-free, noninvasive screening and detection of colorectal cancers.

TNF-α messenger ribonucleic acid (mRNA) in patients with nonalcoholic steatohepatitis.

PubMed

Alaaeddine, Nada; Sidaoui, Joseph; Hilal, George; Serhal, Reem; Abedelrahman, Abir; Khoury, Salem

2012-01-01

tumor necrosis factor (TNF)-α plays a significant role in the pathogenesis of nonalcoholic steatohepatitis (NASH). A few studies have confirmed high TNF-α plasma protein levels in patients with NASH compared to healthy volunteers. We herein aimed to revisit these findings using other molecular techniques. a cross-sectional evaluation of patients newly diagnosed with NASH. A quantitative assay for the measurement of TNF-α messenger ribonucleic acid (mRNA) was performed for NASH patients and controls using real-time reverse transcription polymerase chain reaction (RT-PCR). in 39 patients with NASH (mean age 38.6 ± 9.4 years, range 28-60 years; 79% males), the mean TNF-α mRNA level was significantly higher than that found for controls (137.6 ± 102.3 ng/mL versus 83.5 ± 43.8 ng/mL, respectively; P = 0.012). A TNF-α mRNA cut-off of 100 ng/mL predicted NASH most optimally (AUC 0.685 ± 0.066, P = 0.01; with 66.7% sensitivity and 74.1% specificity). Serum TNF-α and soluble TNF-α receptor II (sTNFRII) levels were significantly higher in patients compared to controls using ELISA. high TNF-α mRNA levels, determined by RT-PCR, characterize patients with NASH.

In Vitro Product of a Ribonucleic Acid Polymerase Induced by Influenza Virus

PubMed Central

Mahy, B. W. J.; Bromley, P. A.

1970-01-01

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8S region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18S. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11S, but molecules which may be replicative intermediate, sedimenting at 14 to 20S, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18S region. An 11S double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus. PMID:5480408

Macromolecular Synthesis During the Germination of Saccharomyces cerevisiae Spores

PubMed Central

Rousseau, Paul; Halvorson, Harlyn O.

1973-01-01

After the dormancy of Saccharomyces cerevisiae ascospores had been broken, the synthesis of proteins was observed first, followed rapidly by synthesis of ribonucleic acid (RNA) and much later by deoxyribonucleic acid (DNA) synthesis. Phosphoglucomutase activity increased in a periodic (step) fashion, whereas the activity of five other enzymes increased linearly during germination and outgrowth. The rate of synthesis of these enzymes was highest at about the period of DNA replication. The amino acid pools of dormant spores contained high levels of proline, glutamic acid, and histidine. At 2 h after onset of germination, the pools of phenylalanine and methionine had disappeared and the other components had decreased significantly. By 3.5 h, with the exception of proline and cystine, most amino acid pool components had significantly increased. PMID:4570780

Antibiotic Sensitivity of Micrococcus radiodurans

PubMed Central

Hawiger, J.; Jeljaszewicz, J.

1967-01-01

A wild-type strain of Micrococcus radiodurans and its nonpigmented mutant W1 were tested for sensitivity to 10 antibiotics selected from the standpoint of their mechanism of action. Representatives of groups of antibiotics inhibiting deoxyribonucleic acid (DNA) synthesis, DNA-dependent ribonucleic acid synthesis, protein synthesis, and cell wall synthesis were selected. M. radiodurans and its mutant exhibited full susceptibility to all antibiotics tested (mitomycin C, actinomycin D, chloramphenicol, dihydrostreptomycin, erythromycin, neomycin, kanamycin, benzylpenicillin, bacitracin, and vancomycin), the degree of susceptibility being of the same order as that of a standard strain of Staphylococcus aureus 209 P, with the exception of dihydrostreptomycin. PMID:4166078

Microbiological and chemical quality of ground water used as a source of public supply in southern Missouri : Phase II, April-July, 1998

USGS Publications Warehouse

Femmer, Suzanne R.

2000-01-01

The protection of public health through quality public ground-water systems is the responsibility of the U. S. Environmental Protection Agency and the State of Missouri, through the Missouri Department of Natural Resources, Public Drinking Water Program. Approximately 95 percent of the public-water supplies in Missouri use ground water as their source of drinking water through more than 3,700 public wells. Karst terrain, intensive agricultural operations, extensive numbers of on-site sewage systems, and poor well construction can lead to chemical and microbiological contamination of the contributing aquifers. Sitespecific studies and routine regulatory monitoring have produced information on the overall quality and potability of the State's public-drinking-water supplies, but little is known about the presence of viruses. The U.S. Geological Survey, in cooperation with the Missouri Department of Natural Resources, sampled 109 public-water supplies to characterize the physical, chemical, bacterial, and viral conditions in southern Missouri. During April to July 1998, these wells were sampled for nutrients, total organic carbon, optical brighteners, indicator bacteria, enteric viruses, and ribonucleic acid and somatic coli phages. These constituents indicate possible surface contamination of the sampled aquifer. Selection of the wells to be sampled depended on the age of the well (pre-1970), land use, geohydrology, and well construction. None of the physical or chemical constituents measured or analyzed exceeded Missouri's Drinking Water Standards set by the Public Drinking Water Program of the Missouri Department of Natural Resources. The majority of ammonia plus organic nitrogen, nitrite, and phosphorus concentrations were below the laboratory's minimum reporting levels. There were a greater number of detects above the minimum reporting level with respect to the nitrite plus nitrate, ammonia, orthophosphate, and total organic carbon concentrations. Analyses included comparing and contrasting the data by grouping according to well age and construction, karst type, geohydrology, soil type, and land use. There was little variation in well construction between selected wells. The results indicated several groupings of similar and dissimilar concentrations, most expected because of hydrological, physical, or land use differences. Dissolved oxygen values indicated distinct variation in the different groupings. There were significant differences in dissolved oxygen values between the secondary and non-karst areas, the Ozark confined and Ozark unconfined geohydrologic groups, and between agricultural and other land uses. In groupings by soil and geohydrology, the Missouri bootheel region differed with respect to ammonia, total organic carbon, and phosphorus when compared with the other groups. Less than 10 percent of the wells sampled tested positive for bacterial contamination. E. coli was the most frequently detected bacterium. The public wells at Monett and West Plains, Missouri, had plates with colonies too numerous to count for all three indicator bacteria. Further analyses by rRNA (ribosomal RiboNucleic Acid) hybridization techniques detennined that much of the bacteria present were from ruminant and human sources. No enteric viruses were detected in the 109 samples. Both ribonucleic acid and somatic coliphage were detected at two wells. One additional well had ribonucleic acid coliphage and another had somatic coliphage for a total of four wells with coliphage selects.

Characterization of a Fluorescent Protein Reporter System

DTIC Science & Technology

2008-03-01

pathways are initiated with the binding of a small molecule to a catalytic ribonucleic acid molecule (RNA), called a ribozyme (Thodima et al., 2006). The... ribozyme is part of a larger RNA construct, called a riboswitch, which initiates translation of a specific genetic sequence on a plasmid (circular...protein gene. Yen et al. (2004) reported insertion of a self-cleaving ribozyme upstream of the reporter gene. In the absence of a regulator (“off

RNA therapeutics: RNAi and antisense mechanisms and clinical applications.

PubMed

Chery, Jessica

2016-07-01

RNA therapeutics refers to the use of oligonucleotides to target primarily ribonucleic acids (RNA) for therapeutic efforts or in research studies to elucidate functions of genes. Oligonucleotides are distinct from other pharmacological modalities, such as small molecules and antibodies that target mainly proteins, due to their mechanisms of action and chemical properties. Nucleic acids come in two forms: deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Although DNA is more stable, RNA offers more structural variety ranging from messenger RNA (mRNA) that codes for protein to non-coding RNAs, microRNA (miRNA), transfer RNA (tRNA), short interfering RNAs (siRNAs), ribosomal RNA (rRNA), and long-noncoding RNAs (lncRNAs). As our understanding of the wide variety of RNAs deepens, researchers have sought to target RNA since >80% of the genome is estimated to be transcribed. These transcripts include non-coding RNAs such as miRNAs and siRNAs that function in gene regulation by playing key roles in the transfer of genetic information from DNA to protein, the final product of the central dogma in biology 1 . Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegeneration. In fact, ASOs targeting spinal muscular atrophy and amyotrophic lateral sclerosis have shown positive results in clinical trials 2 . Advantages of ASOs include higher affinity due to the development of chemical modifications that increase affinity, selectivity while decreasing toxicity due to off-target effects. This review will highlight the major therapeutic approaches of RNA medicine currently being applied with a focus on RNAi and ASOs.

Mechanisms for the formation of thymine under astrophysical conditions and implications for the origin of life

NASA Astrophysics Data System (ADS)

Bera, Partha P.; Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.; Lee, Timothy J.

2016-04-01

Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H2O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantum chemical studies confirmed that the reaction pathways were favorable provided that several H2O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H2O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth and have implications for the role of thymine and DNA in the origin of life.

Antiviral Activity of Polyacrylic and Polymethacrylic Acids

PubMed Central

De Somer, P.; De Clercq, E.; Billiau, A.; Schonne, E.; Claesen, M.

1968-01-01

A marked virus-inhibiting potency is obtained in the serum after intraperitoneal injection of polyacrylic acid (PAA) and polymethacrylic acid (PMAA) in mice. Much higher antiviral levels were reached than for other related polymers including dextran sulfate, heparin, polyvinyl sulfate, pyran copolymer, polystyrene sulfonate, and macrodex. The broad antiviral action of PAA and PMAA was attributed both to a direct interference with the virus-cell interaction and the viral ribonucleic acid metabolism and to the formation of an interferon-like factor. Both polyanions differed in interferon-inducing ability: highest serum interferon titer was obtained 18 hr after the intraperitoneal injection of PAA. The mechanism of interferon production by PAA and PMAA is discussed. As described previously for Sindbis virus and endotoxin, the animals also became hyporeactive after injection of PAA. PMID:5725320

Nuclear Synthesis of Cytoplasmic Ribonucleic Acid in Amoeba proteus

PubMed Central

Prescott, David M.

1959-01-01

The enucleation technique has been applied to Amoeba proteus by several laboratories in attempts to determine whether the cytoplasm is capable of nucleus-independent ribonucleic acid synthesis. This cell is very convenient for micrurgy, but its use requires a thorough starvation period to eliminate the possibility of metabolic influence by food vacuoles and frequent washings and medium renewal to maintain asepsis. In the experiments described here, amoebae were starved for periods of 24 to 96 hours, cut into nucleated and enucleated halves, and exposed to either C-14 uracil, C-14 adenine, C-14 orotic acid, or a mixture of all three. When the starvation period was short (less than 72 hours), organisms (especially yeast cells) contained within amoeba food vacuoles frequently showed RNA synthesis in both nucleated and enucleated amoebae. When the preperiod of starvation was longer than 72 hours, food vacuole influence was apparently negligible, and a more meaningful comparison between enucleated and nucleated amoebae was possible. Nucleated cells incorporated all three precursors into RNA; enucleated cells were incapable of such incorporation. The experiments indicate a complete dependence on the nucleus for RNA synthesis. The conflict with the experimental results of others on this problem could possibly stem from differences in culture conditions, starvation treatment, or experimental conditions. For an unequivocal answer in experiments of this design, ideally the cells should be capable of growth on an entirely synthetic medium under aseptic conditions. The use of a synthetic medium (experiments with A. proteus are done under starvation conditions) would permit, moreover, a more realistic comparison of metabolic capacities of nucleated and enucleated cells. PMID:14434750

Nuclear synthesis of cytoplasmic ribonucleic acid in Amoeba proteus.

PubMed

PRESCOTT, D M

1959-10-01

The enucleation technique has been applied to Amoeba proteus by several laboratories in attempts to determine whether the cytoplasm is capable of nucleus-independent ribonucleic acid synthesis. This cell is very convenient for micrurgy, but its use requires a thorough starvation period to eliminate the possibility of metabolic influence by food vacuoles and frequent washings and medium renewal to maintain asepsis. In the experiments described here, amoebae were starved for periods of 24 to 96 hours, cut into nucleated and enucleated halves, and exposed to either C-14 uracil, C-14 adenine, C-14 orotic acid, or a mixture of all three. When the starvation period was short (less than 72 hours), organisms (especially yeast cells) contained within amoeba food vacuoles frequently showed RNA synthesis in both nucleated and enucleated amoebae. When the preperiod of starvation was longer than 72 hours, food vacuole influence was apparently negligible, and a more meaningful comparison between enucleated and nucleated amoebae was possible. Nucleated cells incorporated all three precursors into RNA; enucleated cells were incapable of such incorporation. The experiments indicate a complete dependence on the nucleus for RNA synthesis. The conflict with the experimental results of others on this problem could possibly stem from differences in culture conditions, starvation treatment, or experimental conditions. For an unequivocal answer in experiments of this design, ideally the cells should be capable of growth on an entirely synthetic medium under aseptic conditions. The use of a synthetic medium (experiments with A. proteus are done under starvation conditions) would permit, moreover, a more realistic comparison of metabolic capacities of nucleated and enucleated cells.

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

PubMed Central

Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P.; Forst, Christian V.; Roth, Michael G.; Levy, David E.; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A.

2012-01-01

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. PMID:22312003

Biochips Containing Arrays of Carbon-Nanotube Electrodes

NASA Technical Reports Server (NTRS)

Li, Jun; Meyyappan, M.; Koehne, Jessica; Cassell, Alan; Chen, Hua

2008-01-01

Biochips containing arrays of nanoelectrodes based on multiwalled carbon nanotubes (MWCNTs) are being developed as means of ultrasensitive electrochemical detection of specific deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) biomarkers for purposes of medical diagnosis and bioenvironmental monitoring. In mass production, these biochips could be relatively inexpensive (hence, disposable). These biochips would be integrated with computer-controlled microfluidic and microelectronic devices in automated hand-held and bench-top instruments that could be used to perform rapid in vitro genetic analyses with simplified preparation of samples. Carbon nanotubes are attractive for use as nanoelectrodes for detection of biomolecules because of their nanoscale dimensions and their chemical properties.

Staining of Tissue Sections for Electron Microscopy with Heavy Metals

PubMed Central

Watson, Michael L.

1958-01-01

Descriptions of three heavy metal stains and methods of application to tissue sections for electron microscopy are presented. Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles. Barium hydroxide emphasizes certain bodies within vesicles of the Golgi region of hepatic cells. Alkalized lead acetate is useful as a general stain, as are also lead and barium hydroxides. PMID:13610936

Noxa/Mcl-1 Balance Regulates Susceptibility of Cells to Camptothecin-Induced Apoptosis1

PubMed Central

Mei, Yide; Xie, Chongwei; Xie, Wei; Tian, Xu; Li, Mei; Wu, Mian

2007-01-01

Although camptothecin (CPT) has been reported to induce apoptosis in various cancer cells, the molecular details of this regulation remain largely unknown. In this study, we demonstrate that BH3-only protein Noxa is upregulated during CPT-induced apoptosis, which is independent of p53. In addition, we show that phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is responsible for Noxa's induction. Luciferase assay and cAMP response element binding protein (CREB) knockdown experiments further demonstrate that CREB is involved in the transcriptional upregulation of Noxa. Moreover, blocking Noxa expression using specific small interfering ribonucleic acid (siRNA) significantly reduces the apoptosis in response to CPT, indicating that Noxa is an essential mediator for CPT-induced apoptosis. Interestingly, antiapoptotic Mcl-1 was also upregulated through PI3K/Akt signaling pathway upon CPT treatment. Using immunoprecipitation assay, Noxa was found to interact with Mcl-1 in the presence or absence of CPT. Knockdown of Mcl-1 expression by short hairpin ribonucleic acid (shRNA) was shown to potentiate CPT-induced apoptosis. Consistently, ectopic overexpression of Mcl-1 rescued cells from apoptosis induced by CPT. Cells coexpressing Noxa and Mcl-1 at different ratio correlates well with the extent of apoptosis, suggesting that the balance between Noxa and Mcl-1 may determine the susceptibility of HeLa cells to CPT-induced apoptosis. PMID:17971907

AMPK regulates energy metabolism through the SIRT1 signaling pathway to improve myocardial hypertrophy.

PubMed

Dong, H-W; Zhang, L-F; Bao, S-L

2018-05-01

We investigated the correlations of adenosine monophosphate-activated protein kinase (AMPK), Silence information regulator 1 (SIRT1) and energy metabolism with myocardial hypertrophy. Myocardial hypertrophy experimental model was established via transverse aortic constriction (TAC)-induced myocardial hypertrophy and phenylephrine (PE)-induced hypertrophic myocardial cell culture. After activation of AMPK, the messenger ribonucleic acid (mRNA) expressions in myocardial tissue- and myocardial cell hypertrophy-related genes, atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC), were detected. The production rate of 14C-labeled 14CO2 from palmitic acid was quantitatively determined to detect the fatty acid and glucose oxidation of hypertrophic myocardial tissues or cells, and the glucose uptake of myocardial cells was studied using [14C] glucose. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were performed to detect the changes in SIRT1 mRNA and protein expressions in hypertrophic myocardial tissues. Moreover, SIRT1 small interfering ribonucleic acid (siRNA) was used to interfere in SIRT1 expression to further investigate the role of SIRT1 in the effect of AMPK activation on myocardial hypertrophy. AMPK activation could significantly reduce the mRNA expressions of ANP and β-MHC in vitro and in vivo. AMPK could increase the ejection fraction (EF) and decrease the protein synthesis rate in myocardial cells in mice with myocardial hypertrophy. Besides, AMPK activation could increase the fatty acid oxidation, improve the glucose uptake and reduce the glucose oxidation. After AMPK activation, both SIRT1 mRNA and protein expressions in hypertrophic myocardial tissues and myocardial cells were increased. After SIRT1 siRNA was further used to interfere in SIRT1 expression in myocardial cells, it was found that mRNA expressions and protein synthesis rates of ANP and β-MHC were increased. The activation of AMPK can inhibit the myocardial hypertrophy, which may be realized through regulating the myocardial energy metabolism via SIRT1 signaling pathway.

Insights into the phylogenetic positions of photosynthetic bacteria obtained from 5S rRNA and 16S rRNA sequence data

NASA Technical Reports Server (NTRS)

Fox, G. E.

1985-01-01

Comparisons of complete 16S ribosomal ribonucleic acid (rRNA) sequences established that the secondary structure of these molecules is highly conserved. Earlier work with 5S rRNA secondary structure revealed that when structural conservation exists the alignment of sequences is straightforward. The constancy of structure implies minimal functional change. Under these conditions a uniform evolutionary rate can be expected so that conditions are favorable for phylogenetic tree construction.

Ribosomal vaccines. I. Immunogenicity of ribosomal fractions isolated from Salmonella typhimurium and Yersinia pestis.

PubMed

Johnson, W

1972-06-01

The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein.

Ribosomal Vaccines I. Immunogenicity of Ribosomal Fractions Isolated from Salmonella typhimurium and Yersinia pestis

PubMed Central

Johnson, William

1972-01-01

The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein. Images PMID:4564407

Asymmetry and Extent of In Vivo Transcripition of R-Plasmid Deoxyribonucleic Acid in Escherichia coli

PubMed Central

Vapnek, Daniel; Spingler, Elizabeth

1974-01-01

Deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization studies have been performed with R-plasmid DNA (R538-1drd) and in vivo-synthesized RNA. R-plasmid DNA was isolated from Escherichia coli K-12, and the complementary strands were separated in cesium chloride-polyuridylic acid-polyguanylic acid gradients. DNA-RNA hybridization was performed with the separated DNA strands and RNA purified from R-plasmid-carrying cells. The results demonstrated that an asymmetric transcription of the R-plasmid DNA occurs in vivo. Hybridization was only detected with the H strand (denser strand in cesium chloride-polyuridylic acid-polyguanylic acid). By determining the density of the RNA-DNA hybrid in CsCl gradients, it was estimated that greater than 60% of the nucleotide sequences in the R-plasmid DNA are transcribed in logarithmically growing E. coli cells. No R-plasmid-specific RNA was detected in E. coli cells that did not carry the plasmid. PMID:4612013

The lost language of the RNA World

PubMed Central

Nelson, James W.; Breaker, Ronald R.

2018-01-01

The possibility of an RNA World is based on the notion that life on Earth passed through a primitive phase without proteins, at a time when all genomes and enzymes were composed of ribonucleic acid. Numerous apparent vestiges of this ancient RNA World remain today, including many nucleotide-derived coenzymes, self-processing ribozymes, metabolite-binding riboswitches, and even ribosomes. Intriguingly, many of the most common signaling molecules and second messengers used by modern organisms are also formed from RNA. For example, nucleotide derivatives such as cAMP, ppGpp, and ZTP, as well as the cyclic dinucleotides c-di-GMP and c-di-AMP, are intimately involved in signaling diverse physiological or metabolic changes in bacteria and other organisms. Herein we describe the potential diversity of this ‘lost language’ of the RNA World, and speculate on whether additional components of this ancient communication machinery might remain hidden though still very much relevant to modern cells. PMID:28611182

Reagentless Measurement of Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic Acid Aptamer-Based Biosensor

PubMed Central

Rowe, Aaron A.; Miller, Erin A.; Plaxco, Kevin W.

2011-01-01

Biosensors built using ribonucleic acid (RNA) aptamers show promise as tools for point-of-care medical diagnostics, but they remain vulnerable to nuclease degradation when deployed in clinical samples. To explore methods for protecting RNA-based biosensors from such degradation we have constructed and characterized an electrochemical, aptamer-based sensor for the detection of aminoglycosidic antibiotics. We find that while this sensor achieves low micromolar detection limits and subminute equilibration times when challenged in buffer, it deteriorates rapidly when immersed directly in blood serum. In order to circumvent this problem, we have developed and tested sensors employing modified versions of the same aptamer. Our first effort to this end entailed the methylation of all of the 2′-hydroxyl groups outside of the aptamer’s antibiotic binding pocket. However, while devices employing this modified aptamer are as sensitive as those employing an unmodified parent, the modification fails to confer greater stability when the sensor is challenged directly in blood serum. As a second potentially naive alternative, we replaced the RNA bases in the aptamer with their more degradation-resistant deoxyribonucleic acid (DNA) equivalents. Surprisingly and unlike control DNA-stem loops employing other sequences, this DNA aptamer retains the ability to bind aminoglycosides, albeit with poorer affinity than the parent RNA aptamer. Unfortunately, however, while sensors fabricated using this DNA aptamer are stable in blood serum, its lower affinity pushes their detection limits above the therapeutically relevant range. Finally, we find that ultrafiltration through a low-molecular-weight-cutoff spin column rapidly and efficiently removes the relevant nucleases from serum samples spiked with gentamicin, allowing the convenient detection of this aminoglycoside at clinically relevant concentrations using the original RNA-based sensor. PMID:20687587

Mechanisms for the formation of thymine under astrophysical conditions and implications for the origin of life

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bera, Partha P., E-mail: Partha.P.Bera@nasa.gov, E-mail: Timothy.J.Lee@nasa.gov; Nuevo, Michel; Materese, Christopher K.

Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H{sub 2}O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantummore » chemical studies confirmed that the reaction pathways were favorable provided that several H{sub 2}O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H{sub 2}O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth and have implications for the role of thymine and DNA in the origin of life.« less

Mechanisms for the Formations of the Thymine Under Astrophysical Conditions and Implications for the Origin of Life

NASA Technical Reports Server (NTRS)

Bera, Partha P.; Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.; Lee, Timothy J.

2016-01-01

Nucleobases are the carriers of the genetic information in ribonucleic acid and deoxyribonucleic acid (DNA) for all life on Earth. Their presence in meteorites clearly indicates that compounds of biological importance can form via non-biological processes in extraterrestrial environments. Recent experimental studies have shown that the pyrimidine-based nucleobases uracil and cytosine can be easily formed from the ultraviolet irradiation of pyrimidine in H2O-rich ice mixtures that simulate astrophysical processes. In contrast, thymine, which is found only in DNA, is more difficult to form under the same experimental conditions, as its formation usually requires a higher photon dose. Earlier quantum chemical studies confirmed that the reaction pathways were favorable provided that several H2O molecules surrounded the reactants. However, the present quantum chemical study shows that the formation of thymine is limited because of the inefficiency of the methylation of pyrimidine and its oxidized derivatives in an H2O ice, as supported by the laboratory studies. Our results constrain the formation of thymine in astrophysical environments and thus the inventory of organic molecules delivered to the early Earth and have implications for the role of thymine and DNA in the origin of life.

Water avoidance stress induces frequency through cyclooxygenase-2 expression: a bladder rat model.

PubMed

Yamamoto, Keisuke; Takao, Tetsuya; Nakayama, Jiro; Kiuchi, Hiroshi; Okuda, Hidenobu; Fukuhara, Shinichiro; Yoshioka, Iwao; Matsuoka, Yasuhiro; Miyagawa, Yasushi; Tsujimura, Akira; Nonomura, Norio

2012-02-01

Water avoidance stress is a potent psychological stressor and it is associated with visceral hyperalgesia, which shows degeneration of the urothelial layer mimicking interstitial cystitis. Cyclooxygenase-2 inhibitors have been recognized to ameliorate frequency both in clinical and experimental settings. We investigated the voiding pattern and cyclooxygenase-2 expression in a rat bladder model of water avoidance stress. After being subjected to water avoidance stress or a sham procedure, rats underwent metabolic cage analysis and cystometrography. Real time reverse transcription polymerase chain reaction was carried out to examine cyclooxygenase-2 messenger ribonucleic acid in bladders of rats. Protein expression of cyclooxygenase-2 was analyzed with immunohistochemistry and western blotting. Furthermore, the effects of the cyclooxygenase-2 inhibitor, etodolac, were investigated by carrying out cystometrography, immunohistochemistry and western blotting. Metabolic cage analysis and cystometrography showed significantly shorter intervals and less volume of voiding in water avoidance stress rats. Significantly higher expression of cyclooxygenase-2 messenger ribonucleic acid was verified by reverse transcription polymerase chain reaction. Immunohistochemistry and western blotting showed significantly higher cyclooxygenase-2 protein levels in water avoidance stress bladders. Furthermore, immunohistochemistry showed high cyclooxygenase-2 expression exclusively in smooth muscle cells. All water avoidance stress-induced changes were reduced by cyclooxygenase-2 inhibitor pretreatment. Chronic stress might cause frequency through cyclooxygenase-2 gene upregulation in bladder smooth muscle cells. Further study of cyclooxygenase-2 in the water avoidance stress bladder might provide novel therapeutic modalities for interstitial cystitis. © 2011 The Japanese Urological Association.

Gradient enhanced-fluidity liquid hydrophilic interaction chromatography of ribonucleic acid nucleosides and nucleotides: A "green" technique.

PubMed

Beilke, Michael C; Beres, Martin J; Olesik, Susan V

2016-03-04

A "green" hydrophilic interaction liquid chromatography (HILIC) technique for separating the components of mixtures with a broad range of polarities is illustrated using enhanced-fluidity liquid mobile phases. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquid CO2 to conventional liquid mobile phases. Decreased mobile phase viscosity and increased analyte diffusivity results when a liquefied gas is dissolved in common liquid mobile phases. The impact of CO2 addition to a methanol:water (MeOH:H2O) mobile phase was studied to optimize HILIC gradient conditions. For the first time a fast separation of 16 ribonucleic acid (RNA) nucleosides/nucleotides was achieved (16min) with greater than 1.3 resolution for all analyte pairs. By using a gradient, the analysis time was reduced by over 100% compared to similar separations conducted under isocratic conditions. The optimal separation using MeOH:H2O:CO2 mobile phases was compared to MeOH:H2O and acetonitrile:water (ACN:H2O) mobile phases. Based on chromatographic performance parameters (efficiency, resolution and speed of analysis) and an assessment of the environmental impact of the mobile phase mixtures, MeOH:H2O:CO2 mixtures are preferred over ACN:H2O or MeOH:H2O mobile phases for the separation of mixtures of RNA nucleosides and nucleotides. Copyright © 2016 Elsevier B.V. All rights reserved.

Further investigation of the biosynthesis of caffeine in tea plants (Camellia sinensis L.). Methylation of transfer ribonucleic acid by tea leaf extracts.

PubMed Central

Suzuki, T; Takahashi, E

1976-01-01

1. The tRNA methyltransferase activity in vitro of leaves, cotyledons and roots of 85-day-old tea seedlings was studied. 2. The activity of extracts prepared from tea leaves with Polycar AT (insoluble polyvinylpyrrolidine) had optimum pH7.7 and was greatly influenced by thiol compounds, but only slightly by metal ions and ammonium acetate. 3. The activities of extracts, expressed per mg of protein, were as follows: roots greater than leaves greater than cotyledons. The only methylated base isolated after incubation with these preparations was 1-methyladenine. 4. The results did not support the view of involvement of methylation of nucleic acids in caffeine biosynthesis in tea plants. In contrast, it is suggested that theophylline is synthesized from the specific methylated precursor in nucleic acids, namely 1-methyladenylic acid, via 1-methylxanthine. PMID:12749

Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas.

PubMed

Yabuuchi, E; Yano, I; Oyaizu, H; Hashimoto, Y; Ezaki, T; Yamamoto, H

1990-01-01

Based on the partial nucleotide sequence analysis of 16S ribosomal ribonucleic acid (rRNA), presence of unique sphingoglycolipids in cellular lipid, and the major type of ubiquinone (Q10), we propose Sphingomonas gen. nov. with the type species Sphingomonas paucimobilis (Holmes et al, 1977) comb. nov. From the homology values of deoxyribonucleic acid-deoxyribonucleic acid hybridization and the phenotypic characteristics, three new species, Sphingomonas parapaucimobilis, Sphingomonas yanoikuyae, Sphingomonas adhaesiva, and one new combination, Sphingomonas capsulata, are described. S. parapaucimobilis JCM 7510 (= GIFU 11387), S. yanoikuyae JCM 7371 (= GIFU 9882), and S. adhaesiva JCM 7370 (= GIFU 11458) are designated as the type strains of the three new species. Emended description of the type strain of S. capsulata is presented.

Micro- and nanoscale devices for the investigation of epigenetics and chromatin dynamics

NASA Astrophysics Data System (ADS)

Aguilar, Carlos A.; Craighead, Harold G.

2013-10-01

Deoxyribonucleic acid (DNA) is the blueprint on which life is based and transmitted, but the way in which chromatin -- a dynamic complex of nucleic acids and proteins -- is packaged and behaves in the cellular nucleus has only begun to be investigated. Epigenetic modifications sit 'on top of' the genome and affect how DNA is compacted into chromatin and transcribed into ribonucleic acid (RNA). The packaging and modifications around the genome have been shown to exert significant influence on cellular behaviour and, in turn, human development and disease. However, conventional techniques for studying epigenetic or conformational modifications of chromosomes have inherent limitations and, therefore, new methods based on micro- and nanoscale devices have been sought. Here, we review the development of these devices and explore their use in the study of DNA modifications, chromatin modifications and higher-order chromatin structures.

Development of a Single-Step Subtraction Method for Eukaryotic 18S and 28S Ribonucleic Acids

DTIC Science & Technology

2011-01-01

ng of total RNA (92.5% rat thymus RNA/7.41% E . coli RNA). The reactions were performed according to the manufacture’s instruction. For the low target...capture conditions. The input RNA used was either 500 ng or 1000 ng of rat thymus RNA or total RNA (92.5% rat thymus RNA/7.41% E . coli RNA). 2.7. Real...concentrations. Rat thymus RNA (mammalian components) with E . coli RNA (bacterial target) was used as a model system to test the capture efficiency and monitor

Cytoplasmic Incorporation of a Ribonucleic Acid Precursor in Amoeba proteus

PubMed Central

Plaut, Walter; Rustad, Ronald C.

1957-01-01

The question of RNA synthesis in enucleate cytoplasm of Amoeba has been approached experimentally by incubating enucleate amoebae in a labelled RNA precursor and determining the incorporation into RNA autoradiographically. The results indicate that there is a cytoplasmic incorporation mechanism which can operate in the absence of the nucleus. A comparison is made between Acetabularia and Amoeba with respect to the origins of cytoplasmic RNA. It is concluded that the existing data are consistent with the assumption that some cytoplasmic RNA is of nuclear origin in both organisms. PMID:13449107

Cytoplasmic incorporation of a ribonucleic acid precursor in Amoeba proteus.

PubMed

PLAUT, W; RUSTAD, R C

1957-07-25

The question of RNA synthesis in enucleate cytoplasm of Amoeba has been approached experimentally by incubating enucleate amoebae in a labelled RNA precursor and determining the incorporation into RNA autoradiographically. The results indicate that there is a cytoplasmic incorporation mechanism which can operate in the absence of the nucleus. A comparison is made between Acetabularia and Amoeba with respect to the origins of cytoplasmic RNA. It is concluded that the existing data are consistent with the assumption that some cytoplasmic RNA is of nuclear origin in both organisms.

Helicase-dependent amplification of nucleic acids.

PubMed

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-10-11

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

Influence of the stringent control system on the transcription of ribosomal ribonucleic acid and ribosomal protein genes in Escherichia coli.

PubMed Central

Dennis, P P

1977-01-01

The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relA1 relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.5 degrees C to the semipermissive temperature of 35.5 degrees C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorpoarted 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages gammaspc1, which carries about 15 r-protein genes and lambdailv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is r protein mRNA and rRNA; in the rel+ strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate. PMID:320185

Ultrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice.

PubMed

Suzuki, Jun-ichi; Ogawa, Masahito; Takayama, Kiyoshi; Taniyama, Yoshiaki; Morishita, Ryuichi; Hirata, Yasunobu; Nagai, Ryozo; Isobe, Mitsuaki

2010-03-02

The purpose of this study was to investigate the efficiency of small interfering ribonucleic acid (siRNA) in murine arteries. We transfected it using a nonviral ultrasound-microbubble-mediated in vivo gene delivery system. siRNA is an effective methodology to suppress gene function. The siRNA can be synthesized easily; however, a major obstacle in the use of siRNA as therapeutics is the difficulty involved in effective in vivo delivery. To investigate the efficiency of nonviral ultrasound-microbubble-mediated in vivo siRNA delivery, we used a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA in murine arteries. Murine femoral arteries were injured using flexible wires to establish arterial injury. The fluorescein-labeled siRNA and GFP siRNA showed that this nonviral approach could deliver siRNA into target arteries effectively without any tissue damage and systemic adverse effects. ICAM-1 siRNA transfection into murine injured arteries significantly suppressed the development of neointimal formation in comparison to those in the control group. Immunohistochemistry revealed that accumulation of T cells and adhesion molecule positive cells was observed in nontreated injured arteries, whereas siRNA suppressed accumulation. The nonviral ultrasound-microbubble delivery of siRNA ensures effective transfection into target arteries. ICAM-1 siRNA has the potential to suppress arterial neointimal formation. Transfection of siRNA can be beneficial for the clinical treatment of cardiovascular and other inflammatory diseases. Copyright 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II. Directing Influence of RNA in the Reaction

PubMed Central

Leis, Jonathan P.; Hurwitz, Jerard

1972-01-01

The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg2+, and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qβ; and synthetic homopolymers such as polyadenylate·polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3′ hydroxyl ends of primer strands. The product is an RNA·DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S. PMID:4333539

Structures and functions of proteins and nucleic acids in protein biosynthesis

NASA Astrophysics Data System (ADS)

Miyazawa, Tatsuo; Yokoyama, Shigeyuki

Infrared and Raman spectroscopy is useful for studying helical conformations of polypeptides, which are determined by molecular structure parameters. Nuclear magnetic resonance spectroscopy, as well as X-ray analysis, is now established to be important for conformation studies of proteins and nucleic acids in solution. This article is mainly concerned with the conformational aspect and function regulation in protein biosynthesis. The strict recognition of transfer ribonucleic acid (tRNA) by aminoacyl-tRNA synthetase (ARS) is achieved by multi-step mutual adaptation. The conformations of ARS-bound amino acids have been elucidated by transferred nuclear Overhauser effect analysis. Aminoacyl-tRNA takes the 3‧-isomeric form in the polypeptide chain elongation cycle. The regulation of codon recognition by post-transcriptional modification is achieved by conversion of the conformational characteristic of the anticodon of tRNA. The cytidine → lysidine modification of the anticodon of minor isoleucine tRNA concurrently converts the amino acid specificity and the codon specificity. As novel protein engineering, a basic strategy has been established for in vivo biosynthesis of proteins that are substituted with unnatural amino acids (alloproteins).

Structures Containing Polyphosphate in Micrococcus lysodeikticus1

PubMed Central

Friedberg, Ilan; Avigad, Gad

1968-01-01

Granular structures containing inorganic polyphosphate were found in Micrococcus lysodeikticus. These structures were isolated by fractionation of the bacterial extract obtained by lysing the organisms with lysozyme. The composition of the fraction which was enriched with these structures was found to be: protein, 24%; lipids, 30%; and polyphosphate, 27%. This fraction also contained small amounts of ribonucleic acids, carbohydrate, and polyvalent cations. The effect of different reagents and enzymes on the integrity of the granules was examined. It was noticed that they accumulate in the bacteria during the logarithmic phase of growth but disappear gradually during the stationary phase. Images PMID:5674060

Selective probing of mRNA expression levels within a living cell.

PubMed

Nawarathna, D; Turan, T; Wickramasinghe, H Kumar

2009-08-24

We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high ( approximately 2500) and extremely low (11 0) copy number mRNA from a living cell mRNA in less than 10 s.

Non-viral gene therapy for bone tissue engineering.

PubMed

Wegman, Fiona; Oner, F Cumhur; Dhert, Wouter J A; Alblas, Jacqueline

2013-01-01

The possibilities of using gene therapy for bone regeneration have been extensively investigated. Improvements in the design of new transfection agents, combining vectors and delivery/release systems to diminish cytotoxicity and increase transfection efficiencies have led to several successful in vitro, ex vivo and in vivo strategies. These include growth factor or short interfering ribonucleic acid (siRNA) delivery, or even enzyme replacement therapies, and have led to increased osteogenic differentiation and bone formation in vivo. These results provide optimism to consider use in humans with some of these gene-delivery strategies in the near future.

5S ribosomal RNA database Y2K

PubMed Central

Szymanski, Maciej; Barciszewska, Miroslawa Z.; Barciszewski, Jan; Erdmann, Volker A.

2000-01-01

This paper presents the updated version (Y2K) of the database of ribosomal 5S ribonucleic acids (5S rRNA) and their genes (5S rDNA), http://rose.man/poznan. pl/5SData/index.html . This edition of the database contains 1985 primary structures of 5S rRNA and 5S rDNA. They include 60 archaebacterial, 470 eubacterial, 63 plastid, nine mitochondrial and 1383 eukaryotic sequences. The nucleotide sequences of the 5S rRNAs or 5S rDNAs are divided according to the taxonomic position of the source organisms. PMID:10592212

5S ribosomal RNA database Y2K.

PubMed

Szymanski, M; Barciszewska, M Z; Barciszewski, J; Erdmann, V A

2000-01-01

This paper presents the updated version (Y2K) of the database of ribosomal 5S ribonucleic acids (5S rRNA) and their genes (5S rDNA), http://rose.man/poznan.pl/5SData/index.html. This edition of the database contains 1985primary structures of 5S rRNA and 5S rDNA. They include 60 archaebacterial, 470 eubacterial, 63 plastid, nine mitochondrial and 1383 eukaryotic sequences. The nucleotide sequences of the 5S rRNAs or 5S rDNAs are divided according to the taxonomic position of the source organisms.

Roles of tRNA in cell wall biosynthesis

PubMed Central

Dare, Kiley; Ibba, Michael

2013-01-01

Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. PMID:22262511

Ebolavirus and Haemorrhagic Syndrome

PubMed Central

Matua, Gerald A.; Van der Wal, Dirk M.; Locsin, Rozzano C.

2015-01-01

The Ebola virus is a highly virulent, single-stranded ribonucleic acid virus which affects both humans and apes and has fast become one of the world’s most feared pathogens. The virus induces acute fever and death, with haemorrhagic syndrome occurring in up to 90% of patients. The known species within the genus Ebolavirus are Bundibugyo, Sudan, Zaïre, Reston and Taï Forest. Although endemic in Africa, Ebola has caused worldwide anxiety due to media hype and concerns about its international spread, including through bioterrorism. The high fatality rate is attributed to unavailability of a standard treatment regimen or vaccine. The disease is frightening since it is characterised by rapid immune suppression and systemic inflammatory response, causing multi-organ and system failure, shock and often death. Currently, disease management is largely supportive, with containment efforts geared towards mitigating the spread of the virus. This review describes the classification, morphology, infective process, natural ecology, transmission, epidemic patterns, diagnosis, clinical features and immunology of Ebola, including management and epidemic containment strategies. PMID:26052448

Ebolavirus and Haemorrhagic Syndrome.

PubMed

Matua, Gerald A; Van der Wal, Dirk M; Locsin, Rozzano C

2015-05-01

The Ebola virus is a highly virulent, single-stranded ribonucleic acid virus which affects both humans and apes and has fast become one of the world's most feared pathogens. The virus induces acute fever and death, with haemorrhagic syndrome occurring in up to 90% of patients. The known species within the genus Ebolavirus are Bundibugyo, Sudan, Zaïre, Reston and Taï Forest. Although endemic in Africa, Ebola has caused worldwide anxiety due to media hype and concerns about its international spread, including through bioterrorism. The high fatality rate is attributed to unavailability of a standard treatment regimen or vaccine. The disease is frightening since it is characterised by rapid immune suppression and systemic inflammatory response, causing multi-organ and system failure, shock and often death. Currently, disease management is largely supportive, with containment efforts geared towards mitigating the spread of the virus. This review describes the classification, morphology, infective process, natural ecology, transmission, epidemic patterns, diagnosis, clinical features and immunology of Ebola, including management and epidemic containment strategies.

Probing RNA Native Conformational Ensembles with Structural Constraints.

PubMed

Fonseca, Rasmus; van den Bedem, Henry; Bernauer, Julie

2016-05-01

Noncoding ribonucleic acids (RNA) play a critical role in a wide variety of cellular processes, ranging from regulating gene expression to post-translational modification and protein synthesis. Their activity is modulated by highly dynamic exchanges between three-dimensional conformational substates, which are difficult to characterize experimentally and computationally. Here, we present an innovative, entirely kinematic computational procedure to efficiently explore the native ensemble of RNA molecules. Our procedure projects degrees of freedom onto a subspace of conformation space defined by distance constraints in the tertiary structure. The dimensionality reduction enables efficient exploration of conformational space. We show that the conformational distributions obtained with our method broadly sample the conformational landscape observed in NMR experiments. Compared to normal mode analysis-based exploration, our procedure diffuses faster through the experimental ensemble while also accessing conformational substates to greater precision. Our results suggest that conformational sampling with a highly reduced but fully atomistic representation of noncoding RNA expresses key features of their dynamic nature.

Simplified Microarray Technique for Identifying mRNA in Rare Samples

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings. Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps: 1. Purification of cellular RNA, 2. Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR), 3. Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye), 4. Hybridization to a microarray chip, 5. Fluorescence scanning the array(s) with dual excitation wavelengths, and 6. Analysis of the resulting images. This six-step procedure must be performed in a laboratory because it requires bulky equipment.

Representation mutations from standard genetic codes

NASA Astrophysics Data System (ADS)

Aisah, I.; Suyudi, M.; Carnia, E.; Suhendi; Supriatna, A. K.

2018-03-01

Graph is widely used in everyday life especially to describe model problem and describe it concretely and clearly. In addition graph is also used to facilitate solve various kinds of problems that are difficult to be solved by calculation. In Biology, graph can be used to describe the process of protein synthesis in DNA. Protein has an important role for DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). Proteins are composed of amino acids. In this study, amino acids are related to genetics, especially the genetic code. The genetic code is also known as the triplet or codon code which is a three-letter arrangement of DNA nitrogen base. The bases are adenine (A), thymine (T), guanine (G) and cytosine (C). While on RNA thymine (T) is replaced with Urasil (U). The set of all Nitrogen bases in RNA is denoted by N = {C U, A, G}. This codon works at the time of protein synthesis inside the cell. This codon also encodes the stop signal as a sign of the stop of protein synthesis process. This paper will examine the process of protein synthesis through mathematical studies and present it in three-dimensional space or graph. The study begins by analysing the set of all codons denoted by NNN such that to obtain geometric representations. At this stage there is a matching between the sets of all nitrogen bases N with Z 2 × Z 2; C=(\\overline{0},\\overline{0}),{{U}}=(\\overline{0},\\overline{1}),{{A}}=(\\overline{1},\\overline{0}),{{G}}=(\\overline{1},\\overline{1}). By matching the algebraic structure will be obtained such as group, group Klein-4,Quotien group etc. With the help of Geogebra software, the set of all codons denoted by NNN can be presented in a three-dimensional space as a multicube NNN and also can be represented as a graph, so that can easily see relationship between the codon.

The involvement of mRNA processing factors TIA-1, TIAR, and PABP-1 during mammalian hibernation.

PubMed

Tessier, Shannon N; Audas, Timothy E; Wu, Cheng-Wei; Lee, Stephen; Storey, Kenneth B

2014-11-01

Mammalian hibernators survive low body temperatures, ischemia-reperfusion, and restricted nutritional resources via global reductions in energy-expensive cellular processes and selective increases in stress pathways. Consequently, studies that analyze hibernation uncover mechanisms which balance metabolism and support survival by enhancing stress tolerance. We hypothesized processing factors that influence messenger ribonucleic acid (mRNA) maturation and translation may play significant roles in hibernation. We characterized the amino acid sequences of three RNA processing proteins (T cell intracellular antigen 1 (TIA-1), TIA1-related (TIAR), and poly(A)-binding proteins (PABP-1)) from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), which all displayed a high degree of sequence identity with other mammals. Alternate Tia-1 and TiaR gene variants were found in the liver with higher expression of isoform b versus a in both cases. The localization of RNA-binding proteins to subnuclear structures was assessed by immunohistochemistry and confirmed by subcellular fractionation; TIA-1 was identified as a major component of subnuclear structures with up to a sevenfold increase in relative protein levels in the nucleus during hibernation. By contrast, there was no significant difference in the relative protein levels of TIARa/TIARb in the nucleus, and a decrease was observed for TIAR isoforms in cytoplasmic fractions of torpid animals. Finally, we used solubility tests to analyze the formation of reversible aggregates that are associated with TIA-1/R function during stress; a shift towards the soluble fraction (TIA-1a, TIA-1b) was observed during hibernation suggesting enhanced protein aggregation was not present during torpor. The present study identifies novel posttranscriptional regulatory mechanisms that may play a role in reducing translational rates and/or mRNA processing under unfavorable environmental conditions.

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma.

PubMed

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named "hTERT messenger ribonucleic acid (mRNA)" the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0-160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control-untreated group. The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent ( P < 0.05) and not in time-dependent manner ( P > 0.05), in vitro . Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.

Differentiating benign from malignant thyroid nodules using micro ribonucleic acid amplification in residual cells obtained by fine needle aspiration biopsy.

PubMed

Mazeh, Haggi; Levy, Yair; Mizrahi, Ido; Appelbaum, Liat; Ilyayev, Nadia; Halle, David; Freund, Herbert R; Nissan, Aviram

2013-04-01

Fine needle aspiration biopsy (FNAB) is the most commonly used diagnostic tool to differentiate benign from malignant thyroid nodules. Nevertheless, some FNAB cytology results are not definite. In such cases diagnostic thyroid lobectomy is performed with malignancy rate on final histopathology ranging at 15%-75%. The aim of this study was to improve on the accuracy of FNAB-based cytology by amplification of microRNAs (micro ribonucleic acids [miRs]) from the residual cells left in the FNAB needle after submission for cytology. Residual cells were collected from the needle cup after FNAB cytology of 77 consecutive patients with thyroid nodules. miR-enriched RNA was extracted for all patients with cytology showing either follicular lesion or suspicion for malignancy (n=11). The expression of miR-21, -31, -146b, -187, -221, and -222 was determined using real-time polymerase chain reaction. Results were compared with final surgical histopathology. RNA was successfully extracted from all FNAB specimens. Five patients had FNAB cytology suspicious for malignancy. The miR panel was positive in all five (100%). Six patients had follicular lesions on FNAB. The miR panel was positive in three of four patients (75%) with confirmed malignancy and was negative in two of two (0%) patients with benign pathology results. This corresponded to a specificity of 100%, sensitivity of 88%, and accuracy of 90%. RNA extraction from FNAB residual cells is feasible, and a miR panel amplified from the extracted RNA seems like a promising diagnostic tool in this limited number of patients. Copyright © 2013 Elsevier Inc. All rights reserved.

Prevalence and molecular characterization of Cryptosporidium spp. and Giardia duodenalis in deer in Henan and Jilin, China.

PubMed

Huang, Jianying; Zhang, Zhenjie; Zhang, Yiqi; Yang, Yong; Zhao, Jinfeng; Wang, Rongjun; Jian, Fuchun; Ning, Changshen; Zhang, Wanyu; Zhang, Longxian

2018-04-12

Little is known about the prevalence and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in deer in China. In this study, 662 fecal samples were collected from 11 farms in Henan and Jilin Provinces between July 2013 and August 2014, and were screened for the presence of Cryptosporidium and G. duodenalis with genotyping and subtyping methods. Cryptosporidium spp. and G. duodenalis were detected in 6.80% (45/662) and 1.21% (5/662) of samples, respectively. Six Cryptosporidium species/genotypes were identified based on the small subunit ribosomal ribonucleic acid (SSU rRNA) gene: C. parvum (n = 11); C. andersoni (n = 5); C. ubiquitum (n = 3); C. muris (n = 1); C. suis-like (n = 1); and Cryptosporidium deer genotype (n = 24). When five of the 11 C. parvum isolates were subtyped by sequencing the 60 kDa glycoprotein (gp60) gene, zoonotic subtypes IIaA15G2R2 (n = 4) and IIdA19G1 (n = 1) were found. According to a subtype analysis, three C. ubiquitum isolates belonged to XIIa subtype 2. In contrast, only assemblage E was detected in the five Giardia-positive samples with small subunit ribosomal ribonucleic acid (SSU rRNA) gene sequencing. To our knowledge, this is the first study to report C. andersoni, as well as C. parvum zoonotic subtypes IIaA15G2R2 and IIdA19G1 in cervids. These data, though limited, suggest that cervids may be a source of zoonotic Cryptosporidium and Giardia. Cervids in the present study are likely to be of low zoonotic potential to humans, and more molecular epidemiological studies are required to clarify the prevalence and public health significance of Cryptosporidium and G. duodenalis in cervids throughout China.

Saw palmetto extract enhances erectile responses by inhibition of phosphodiesterase 5 activity and increase in inducible nitric oxide synthase messenger ribonucleic acid expression in rat and rabbit corpus cavernosum.

PubMed

Yang, Surong; Chen, Changrui; Li, Yiying; Ren, Zhenghua; Zhang, Yungang; Wu, Gantong; Wang, Hao; Hu, Zhenzhen; Yao, Minghui

2013-06-01

To evaluate whether saw palmetto extract (SPE) relaxes corpus cavernosum and explore the underlying mechanisms. Forty Sprague-Dawley rats and 30 New Zealand rabbits were randomly allocated into 3 SPE-treated groups (low-, middle-, and high-dose) and 1 saline-treated control group. SPE was administered intragastrically for 7 consecutive days. Another 23 rats treated with sildenafil were used to appraise the erectile response to electrical stimulation of nerves in the corpus cavernosum. The erectile functions of rats and rabbits were evaluated 24 hours after the last SPE administration or 15 minutes after intragastric sildenafil. Outcome measures included corpus cavernosum electrical activity recording, phosphodiesterase 5 (PDE5) activity detected by the colorimetric quantitative method, and messenger ribonucleic acid (mRNA) expression level for PDE5 and inducible nitric oxide synthase (iNOS) determined using real-time polymerase chain reaction. In the SPE-treated animals, the relaxant response to electrical stimulation of nerves in the corpus cavernosum, reflected by the amplitude of the electrical activity within the cavernosum, was significantly and dose-dependently augmented. Similar effects were observed in the sildenafil-treated rats. PDE5 activity in rat and rabbit corpus cavernosum tissues was significantly and dose-dependently inhibited in SPE-treated animals, whereas the iNOS mRNA level increased compared with the saline group. PDE5 mRNA, however, was only significantly enhanced in the rats treated with the middle dose of SPE. The results suggest that SPE may have potential application value for the prevention or treatment of erectile dysfunction through an increase in iNOS mRNA expression and inhibition of PDE5 activity in corpus cavernosum smooth muscles. Copyright © 2013 Elsevier Inc. All rights reserved.

Fundamental approaches in molecular biology for communication sciences and disorders.

PubMed

Bartlett, Rebecca S; Jetté, Marie E; King, Suzanne N; Schaser, Allison; Thibeault, Susan L

2012-08-01

This contemporary tutorial will introduce general principles of molecular biology, common deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein assays and their relevance in the field of communication sciences and disorders. Over the past 2 decades, knowledge of the molecular pathophysiology of human disease has increased at a remarkable pace. Most of this progress can be attributed to concomitant advances in basic molecular biology and, specifically, the development of an ever-expanding armamentarium of technologies for analysis of DNA, RNA, and protein structure and function. Details of these methodologies, their limitations, and examples from the communication sciences and disorders literature are presented. Results/Conclusions The use of molecular biology techniques in the fields of speech, language, and hearing sciences is increasing, facilitating the need for an understanding of molecular biology fundamentals and common experimental assays.

Regulation of Bacteriophage T5 Development by ColI Factors

PubMed Central

Moyer, R. W.; Fu, A. S.; Szabo, C.

1972-01-01

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb+ cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb+ cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis. Images PMID:4554465

Biodegradation of methyl t-butyl ether by aerobic granules under a cosubstrate condition.

PubMed

Zhang, L L; Chen, J M; Fang, F

2008-03-01

Aerobic granules efficient at degrading methyl tert-butyl ether (MTBE) with ethanol as a cosubstrate were successfully developed in a well-mixed sequencing batch reactor (SBR). Aerobic granules were first observed about 100 days after reactor startup. Treatment efficiency of MTBE in the reactor during stable operation exceeded 99.9%, and effluent MTBE was in the range of 15-50 microg/L. The specific MTBE degradation rate was observed to increase with increasing MTBE initial concentration from 25 to 500 mg/L, which peaked at 22.7 mg MTBE/g (volatile suspended solids).h and declined with further increases in MTBE concentration as substrate inhibition effects became significant. Microbial-community deoxyribonucleic acid profiling was carried out using denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S ribosomal ribonucleic acid. The reactor was found to be inhabited by several diverse bacterial species, most notably microorganisms related to the genera Sphingomonas, Methylobacterium, and Hyphomicrobium vulgare. These organisms were previously reported to be associated with MTBE biodegradation. A majority of the bands in the reactor represented a group of organisms belonging to the Flavobacteria-Proteobacteria-Actinobacteridae class of bacteria. This study demonstrates that MTBE can be effectively degraded by aerobic granules under a cosubstrate condition and gives insight into the microorganisms potentially involved in the process.

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

PubMed Central

Soheilypour, M.; Mofrad, M. R. K.

2016-01-01

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

PubMed

Soheilypour, M; Mofrad, M R K

2016-11-02

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

Idiopathic Hypogonadotropic Hypogonadism Caused by Inactivating Mutations in SRA1

PubMed Central

Kotan, Leman Damla; Cooper, Charlton; Darcan, Şükran; Carr, Ian M.; Özen, Samim; Yan, Yi; Hamedani, Mohammad K.; Gürbüz, Fatih; Mengen, Eda; Turan, İhsan; Ulubay, Ayça; Akkuş, Gamze; Yüksel, Bilgin; Topaloğlu, A. Kemal; Leygue, Etienne

2016-01-01

Objective: What initiates the pubertal process in humans and other mammals is still unknown. We hypothesized that gene(s) taking roles in triggering human puberty may be identified by studying a cohort of idiopathic hypogonadotropic hypogonadism (IHH). Methods: A cohort of IHH cases was studied based on autozygosity mapping coupled with whole exome sequencing. Results: Our studies revealed three independent families in which IHH/delayed puberty is associated with inactivating SRA1 variants. SRA1 was the first gene to be identified to function through its protein as well as noncoding functional ribonucleic acid products. These products act as co-regulators of nuclear receptors including sex steroid receptors as well as SF-1 and LRH-1, the master regulators of steroidogenesis. Functional studies with a mutant SRA1 construct showed a reduced co-activation of ligand-dependent activity of the estrogen receptor alpha, as assessed by luciferase reporter assay in HeLa cells. Conclusion: Our findings strongly suggest that SRA1 gene function is required for initiation of puberty in humans. Furthermore, SRA1 with its alternative products and functionality may provide a potential explanation for the versatility and complexity of the pubertal process. PMID:27086651

Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.

PubMed

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E; Przytycka, Teresa M

2012-06-15

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.

Compositions and Methods for Inhibiting Gene Expressions

NASA Technical Reports Server (NTRS)

Williams, Loren D. (Inventor); Hsiao, Chiaolong (Inventor); Fang, Po-Yu (Inventor); Williams, Justin (Inventor)

2018-01-01

A combined packing and assembly method that efficiently packs ribonucleic acid (RNA) into virus like particles (VLPs) has been developed. The VLPs can spontaneously assemble and load RNA in vivo, efficiently packaging specifically designed RNAs at high densities and with high purity. In some embodiments the RNA is capable of interference activity, or is a precursor of a RNA capable of causing interference activity. Compositions and methods for the efficient expression, production and purification of VLP-RNAs are provided. VLP-RNAs can be used for the storage of RNA for long periods, and provide the ability to deliver RNA in stable form that is readily taken up by cells.

[The experimental evaluation of the possibility of the penetration of enteric viruses from the surface into the pulp of contaminated fruits and vegetables].

PubMed

Sergevnin, V I; Ladeyshchikova, Yu I; Sarmometov, E V; Podgorunskaya, I L; Kudrevatykh, E V

2014-01-01

According to the results of complex microbiological examination of samples of vegetables, fruits and grapes there was established significant contamination of them with opportunistic bacteria, antigens of intestinal viruses and cysts of intestinal Protozoa, that confirms the epidemiological role of these products as factors in transmission of acute intestinal infections. There was revealed ribonucleic acid of enteric viruses in experimentally infected pulp from the surface of tomatoes and apples, that indicates to the possibility of penetration of these pathogens into the fruits and vegetables through intact (having no visible damages) surface.

SEMIANNUAL REPORT TO THE ATOMIC ENERGY COMMISSION

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobson, L.O. ed.

Progress is reported in the following studies: carbohydrate metabolism in human erythrocytes; the development of a system of electron therapy employing a linear electron accelerator and an electromagnetic beann deflector; the capacity of progesterone to inhibit the sodium-retaining influence of aldosterone and desoxycorticosterone; use of tritium-labeled cholesterol in measuring the lability of cholesterol in human atherosclerotic plaques; the capacity of a number of steroid hormone metabelites to provoke fever in man; the failure of human placental tissue homogenates to convent progesterone to estrogens; and tracer studies on enzyme requirements for the incorporation of cytidine triphosphate into ribonucleic acid in amore » mammalian system. (L.H.)« less

Selective probing of mRNA expression levels within a living cell

PubMed Central

Nawarathna, D.; Turan, T.; Wickramasinghe, H. Kumar

2009-01-01

We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high (∼2500) and extremely low (11¯0) copy number mRNA from a living cell mRNA in less than 10 s. PMID:19777090

A focus on polarity: Investigating the role of orientation cues in mediating student performance on mRNA synthesis tasks in an introductory cell and molecular biology course.

PubMed

Olimpo, Jeffrey T; Quijas, Daniel A; Quintana, Anita M

2017-11-01

The central dogma has served as a foundational model for information flow, exchange, and storage in the biological sciences for several decades. Despite its continued importance, however, recent research suggests that novices in the domain possess several misconceptions regarding the aforementioned processes, including those pertaining specifically to the formation of messenger ribonucleic acid (mRNA) transcripts. In the present study, we sought to expand upon these observations through exploration of the influence of orientation cues on students' aptitude at synthesizing mRNAs from provided deoxyribonucleic acid (DNA) template strands. Data indicated that participants (n = 45) were proficient at solving tasks of this nature when the DNA template strand and the mRNA molecule were represented in an antiparallel orientation. In contrast, participants' performance decreased significantly on items in which the mRNA was depicted in a parallel orientation relative to the DNA template strand. Furthermore, participants' Grade Point Average, self-reported confidence in understanding the transcriptional process, and spatial ability were found to mediate their performance on the mRNA synthesis tasks. Collectively, these data reaffirm the need for future research and pedagogical interventions designed to enhance students' comprehension of the central dogma in a manner that makes transparent its relevance to real-world scientific phenomena. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):501-508, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Specific Effect of Guanidine in the Programming of Poliovirus Inhibition of Deoxyribonucleic Acid Synthesis

PubMed Central

Powers, C. D.; Miller, B. A.; Kurtz, H.; Ackermann, W. W.

1969-01-01

Inhibition of HeLa cell deoxyribonucleic acid (DNA) synthesis, which occurred by the 4th to 5th hr after infection with poliovirus, could be blocked completely by guanidine only when it was present before the 2nd hr. At the 2nd hr, there was no significant ribonucleic acid (RNA)-replicase activity, and addition of guanidine inhibited all production of virus but allowed 57% of maximal DNA inhibition to develop. Maximum DNA inhibition developed in cells infected for 4 hr in the presence of guanidine when the guanidine was removed for a 10-min interval. RNA-replicase activity was not enzymatically detectable and viral multiplication did not develop in these cells unless the interval without guanidine was extended to 60 min. The interpretation of the data was that the effect of guanidine on viral-induced inhibition of DNA synthesis was distinct and not a consequence of the inhibition of RNA-replicase. PMID:4305675

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

PubMed Central

Johnson, John L.; Anderson, Robert S.; Ordal, Erling J.

1970-01-01

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA–DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA–DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid. PMID:5413826

Computational Approaches to Nucleic Acid Origami.

PubMed

Jabbari, Hosna; Aminpour, Maral; Montemagno, Carlo

2015-10-12

Recent advances in experimental DNA origami have dramatically expanded the horizon of DNA nanotechnology. Complex 3D suprastructures have been designed and developed using DNA origami with applications in biomaterial science, nanomedicine, nanorobotics, and molecular computation. Ribonucleic acid (RNA) origami has recently been realized as a new approach. Similar to DNA, RNA molecules can be designed to form complex 3D structures through complementary base pairings. RNA origami structures are, however, more compact and more thermodynamically stable due to RNA's non-canonical base pairing and tertiary interactions. With all these advantages, the development of RNA origami lags behind DNA origami by a large gap. Furthermore, although computational methods have proven to be effective in designing DNA and RNA origami structures and in their evaluation, advances in computational nucleic acid origami is even more limited. In this paper, we review major milestones in experimental and computational DNA and RNA origami and present current challenges in these fields. We believe collaboration between experimental nanotechnologists and computer scientists are critical for advancing these new research paradigms.

ISSOL Meeting, 7th, Barcelona, Spain, July 4-9, 1993. [Abstracts only

NASA Technical Reports Server (NTRS)

Ferris, James P. (Editor)

1994-01-01

The journal issue consists of abstracts presented at the International Society for the Study of the Origins of Life (ISSOL) conference. Topics include research on biological and chemical evolution including prebiotic evolution: cosmic and terrestrial; mechanisms of abiogenesis including synthesis and reactions of biomonomers; and analysis of cometary matter and its possible relationship to organic compounds on Earth. Theories and research on origins of ribonucleic acids (RNA), deoxyribonucleic acid (DNA), and other amino acids and complex proteins including their autocatalysis, replication, and translation are presented. Abiotic synthesis of biopolymers, mechanisms of the Genetic Code, precellular membrane systems and energetics are considered. Earth planetary evolution including early microfossils and geochemical conditions and simulations to study these conditions are discussed. The role of chirality in precellular evolution and the taxonomy and phylogeny of very simple organisms are reported. Past and future explorations in exobiology and space research directed toward study of the origins of life and solar system evolution are described.

Comparison of the activities of extracts of Escherichia coli and Salmonella typhimurium in amino acid incorporation.

PubMed

Bassel, B A; Curry, M E

1973-11-01

We have compared the amino acid incorporating activities of extracts of Escherichia coli and Salmonella typhimurium in in vitro protein-synthesizing systems directed by bacterial messenger ribonucleic acid (mRNA) of both species and by the genomes of coliphages Qbeta and f2. E. coli and S. typhimurium extracts translate both homologous and heterologous bacterial mRNAs at comparable rates. S. typhimurium extracts translate phage RNAs only 10 to 15% as fast as E. coli extracts do. The presence of glucose in the growth medium increases the activity of S. typhimurium extracts three- to fourfold in the phage RNA-directed systems. Glucose has a much more limited effect on the activities of E. coli extracts. We show that similar amounts of phage RNA-ribosome complexes are formed in both the E. coli and the S. typhimurium systems, indicating that the different activities observed may be attributed to different rates of peptide elongation or to the formation of complexes at different sites on the RNA strand.

EFFECTS OF X IRRADIATION ON THE LEVEL OF NUCLEIC ACIDS AND PROTEINS OF THE UTERUS AND VAGINA OF PREPUBERTAL MICE SUBMITTED TO INDUCED HYPERPLASIA (in French)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ledoux, L.; Charles, P.

1961-12-01

In 20-day-old mice injected subcutaneously with a single 10 ug. dose of estradiol benzoate the deoxyribonucleic acid (DNA) content rose 100% in uterus and 50% in vagina 72 hr later. Ribonucleic acid (RNA) content rose 250 and 100%, respectively, and protein content rose in parallel with DNA. Similarly treated mice were exposed to x rays (150 to 850 r) 24 hr before, simultaneously with, or 24 to 48 hr after injection of estradiol. Irradiation with 750 r before or with the injection inhibited the rise of DNA and RNA 50 to 80% but did not affect the increase in proteins.more » Irradiation of uninjected control mice diminished DNA and RNA. Irradiation 24 to 38 hr after the injection immediately suppressed the increases of DNA and RNA in uterus and vagina and of protein in uterus. Protein content of vagina was not influenced. (H.H.D.)« less

Methionine-Mediated Repression in Saccharomyces cerevisiae: a Pleiotropic Regulatory System Involving Methionyl Transfer Ribonucleic Acid and the Product of Gene eth2

PubMed Central

Cherest, H.; Surdin-Kerjan, Y.; De Robichon-Szulmajster, H.

1971-01-01

Detailed study of methionine-mediated repression of enzymes involved in methionine biosynthesis in Saccharomyces cerevisiae led to classification of these enzymes into two distinct regulatory groups. Group I comprises four enzymes specifically involved in different parts of methionine biosynthesis, namely, homoserine-O-transacetylase, homocysteine synthetase, adenosine triphosphate sulfurylase, and sulfite reductase. Repressibility of these enzymes is greatly decreased in strains carrying a genetically impaired methionyl-transfer ribonucleic acid (tRNA) synthetase (mutation ts− 296). Conditions leading to absence of repression in the mutant strain have been correlated with a sharp decrease in bulk tRNAmet charging, whereas conditions which restore repressibility of group I enzymes also restore tRNAmet charging. These findings implicate methionyl-tRNA in the regulatory process. However, the absence of a correlation in the wild type between methionyl-tRNA charging and the levels of methionine group I enzymes suggests that only a minor iso accepting species of tRNAmet may be devoted with a regulatory function. Repressibility of the same four enzymes (group I) was also decreased in strains carrying the regulatory mutation eth2r. Although structural genes coding for two of these enzymes, as well as mutations ts− 296 and eth2r segregate independently to each other, synthesis of group I enzymes is coordinated. The pleiotropic regulatory system involved seems then to comprise beside a “regulatory methionyl tRNAmet,” another element, product of gene eth2, which might correspond either to an aporepressor protein or to the “regulatory tRNAmet” itself. Regulation of group II enzymes is defined by response to exogenous methionine, absence of response to either mutations ts− 296 and eth2r, and absence of coordinacy with group I enzymes. However, the two enzymes which belong to this group and are both involved in threonine and methionine biosynthesis undergo distinct regulatory patterns. One, aspartokinase, is subject to a bivalent repression exerted by threonine and methionine, and the other, homoserine dehydrogenase, is subject only to methionine-mediated repression. Participation of at least another aporepressor and another corepressor, different from the ones involved in regulation of group I enzymes, is discussed. PMID:5557593

Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

PubMed

Quay, S C; Oxender, D L

1977-06-01

Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur.

Therapeutic nucleic acids: current clinical status

PubMed Central

Sridharan, Kannan

2016-01-01

Abstract Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are simple linear polymers that have been the subject of considerable research in the last two decades and have now moved into the realm of being stand‐alone therapeutic agents. Much of this has stemmed from the appreciation that they carry out myriad functions that go beyond mere storage of genetic information and protein synthesis. Therapy with nucleic acids either uses unmodified DNA or RNA or closely related compounds. From both a development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. This review addresses therapeutic uses of DNA based on antisense oligonucleotides, DNA aptamers and gene therapy; and therapeutic uses of RNA including micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs. With their specificity, functional diversity and limited toxicity, therapeutic nucleic acids hold enormous promise. However, challenges that need to be addressed include targeted delivery, mass production at low cost, sustaining efficacy and minimizing off‐target toxicity. Technological developments will hold the key to this and help accelerate drug approvals in the years to come. PMID:27111518

Formulation/preparation of functionalized nanoparticles for in vivo targeted drug delivery.

PubMed

Gu, Frank; Langer, Robert; Farokhzad, Omid C

2009-01-01

Targeted cancer therapy allows the delivery of therapeutic agents to cancer cells without incurring undesirable side effects on the neighboring healthy tissues. Over the past decade, there has been an increasing interest in the development of advanced cancer therapeutics using targeted nanoparticles. Here we describe the preparation of drug-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D: ,L: -lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG) copolymer and surface functionalized with the A10 2-fluoropyrimidine ribonucleic acid aptamers that recognize the extracellular domain of prostate-specific membrane antigen (PSMA), a well-characterized antigen expressed on the surface of prostate cancer cells. We show that the self-assembled nanoparticles can selectively bind to PSMA-targeted prostate cancer cells in vitro and in vivo. This formulation method may contribute to the development of highly selective and effective cancer therapeutic and diagnostic devices.

[Acatalasemia and type 2 diabetes mellitus].

PubMed

Góth, László; Nagy, Teréz; Káplár, Miklós

2015-03-08

The catalase enzyme decomposes the toxic concentrations of hydrogen peroxide into oxygen and water. Hydrogen peroxide is a highly reactive small molecule and its excessive concentration may cause significant damages to proteins, deoxyribonucleic acid, ribonucleic acid and lipids. Acatalasemia refers to inherited deficiency of the catalase enzyme. In this review the authors discuss the possible role of the human catalase enzyme, the metabolism of hydrogen peroxide, and the phenomenon of hydrogen peroxide paradox. In addition, they review data obtained from Hungarian acatalasemic patients indicating an increased frequency of type 2 diabetes mellitus, especially in female patients, and an early onset of type 2 diabetes in these patients. There are 10 catalase gene variants which appear to be responsible for decreased blood catalase activity in acatalasemic patients with type 2 diabetes. It is assumed that low levels of blood catalase may cause an increased concentration of hydrogen peroxide which may contribute to the pathogenesis of type 2 diabetes mellitus.

Methanohalophilus zhilinae sp. nov., an alkaliphilic, halophilic, methylotrophic methanogen

NASA Technical Reports Server (NTRS)

Mathrani, I. M.; Boone, D. R.; Mah, R. A.; Fox, G. E.; Lau, P. P.

1988-01-01

Methanohalophilus zhilinae, a new alkaliphilic, halophilic, methylotrophic species of methanogenic bacteria, is described. Strain WeN5T (T = type strain) from Bosa Lake of the Wadi el Natrun in Egypt was designated the type strain and was further characterized. This strain was nonmotile, able to catabolize dimethylsulfide, and able to grow in medium with a methyl group-containing substrate (such as methanol or trimethylamine) as the sole organic compound added. Sulfide (21 mM) inhibited cultures growing on trimethylamine. The antibiotic susceptibility pattern of strain WeN5T was typical of the pattern for archaeobacteria, and the guanine-plus-cytosine content of the deoxyribonucleic acid was 38 mol%. Characterization of the 16S ribosomal ribonucleic acid sequence indicated that strain WeN5T is phylogenetically distinct from members of previously described genera other than Methanohalophilus and supported the partition of halophilic methanogens into their own genus.

CONSIDERATIONS OF THE HISTOLOGIC AND HISTOCHEMICAL INJURY INDUCED BY TOTAL ROENTGEN IRRADIATION IN THE FEMALE GENITAL ORGANS OF PUBERTAL AND PREPUBERTAL MICE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cecio, A.; Cerasuolo, G.

1961-07-15

Changes in uterus and ovaries were studied in mice exposed to 700 r whole-body irradiation. Endometrial glands showed an increase in mitotic figures after irradiation and somewhat decreased numbers of cells although histochemical tests for polysaccharides and ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) were intensified. The results were similar in adult and immature mice. In ovary, irradiation caused degeneration of follicles with separation of granulosa cells, which was followed by hyperplasia during the reparative phase. Here also polysaccharides were increased in amounts, especially in the zona pellucida of the oocytes. Epithelium of Fallopiant tube also showed increased histochemical reactionsmore » for polysaccharides, RNA, and DNA. Most of these changes were evident within 24 hr after irradiation; they increased in intensity up to the 2nd day, then tended to decrease. (H.H.D.)« less

Insights into the RNA quadruplex binding specificity of DDX21.

PubMed

McRae, Ewan K S; Davidson, David E; Dupas, Steven J; McKenna, Sean A

2018-06-12

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 specifically binds to TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2'OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2'OH of loop nucleotides. Copyright © 2018. Published by Elsevier B.V.

Adaptation of Ribonucleic Acid Metabolism to Anoxia in Rice Embryos 1

PubMed Central

Aspart, Lorette; Got, Alain; Delseny, Michel; Mocquot, Bernard; Pradet, Alain

1983-01-01

Rice (Oryza sativa var. Cigalon) is a plant which can adapt to very stringent anoxic conditions. It has previously been shown that during the adaptation period the energy charge can be used as a marker for metabolic activity. We have studied RNA metabolism during this period and correlated it with changes in the energy charge. Uptake of labeled precursor, UTP-specific activity, and incorporation were measured. Immediately after transfer to anaerobic conditions, the UTP pool size is reduced and the overall rate of incorporation drops. During adaptation, the rate of incorporation increases and stabilizes at about half of its value in aerobic conditions. Analysis of RNA shows that rRNA and mRNA are synthesized and that the processing of ribosomal RNA precursor is altered. Polyribosomes are present throughout the adaptation period although their amount is reduced during the first hour of anoxia. Changes in poly(A) content were noticed, indicating that some mRNA are rapidly degraded. Taken together, the results show that the RNA metabolism can be modulated during adaptation to anoxia in a parallel manner with energy charge changes. PMID:16662943

Profiling of modified nucleosides from ribonucleic acid digestion by supercritical fluid chromatography coupled to high resolution mass spectrometry.

PubMed

Laboureur, Laurent; Guérineau, Vincent; Auxilien, Sylvie; Yoshizawa, Satoko; Touboul, David

2018-02-16

A method based on supercritical fluid chromatography coupled to high resolution mass spectrometry for the profiling of canonical and modified nucleosides was optimized, and compared to classical reverse-phase liquid chromatography in terms of separation, number of detected modified nucleosides and sensitivity. Limits of detection and quantification were measured using statistical method and quantifications of twelve nucleosides of a tRNA digest from E. coli are in good agreement with previously reported data. Results highlight the complementarity of both separation techniques to cover the largest view of nucleoside modifications for forthcoming epigenetic studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Factors Affecting the Extraction of Intact Ribonucleic Acid from Plant Tissues Containing Interfering Phenolic Compounds

PubMed Central

Newbury, H. John; Possingham, John V.

1977-01-01

Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions. PMID:16660134

Prosthetic Valve Endocarditis Caused by Bartonella henselae: A Case Report of Molecular Diagnostics Informing Nonsurgical Management

PubMed Central

Bartley, Patricia; Angelakis, Emmanouil; Raoult, Didier; Sampath, Rangarajan; Bonomo, Robert A.

2016-01-01

Identifying the pathogen responsible for culture-negative valve endocarditis often depends on molecular studies performed on surgical specimens. A patient with Ehlers-Danlos syndrome who had an aortic graft, a mechanical aortic valve, and a mitral anulloplasty ring presented with culture-negative prosthetic valve endocarditis and aortic graft infection. Research-based polymerase chain reaction (PCR)/electrospray ionization mass spectrometry on peripheral blood samples identified Bartonella henselae. Quantitative PCR targeting the16S-23S ribonucleic acid intergenic region and Western immunoblotting confirmed this result. This, in turn, permitted early initiation of pathogen-directed therapy and subsequent successful medical management of B henselae prosthetic valve endocarditis and aortic graft infection. PMID:27844027

Studies on transfer ribonucleic acids and related compounds. XXXII. Synthesis of ribonucleotides corresponding to residues 1-5 and 6-10 of tRNAfMet from E. coli and their base conversion analogs.

PubMed Central

Ohtsuka, E; Tanaka, T; Ikehara, M

1979-01-01

E. Coli tRNAfMet fragments, C-G-C-G-Gp (bases 1-5), U-G-C-G-Gp (base 1 transition, analog) pG-G-C-G-Gp (base 1 transversion analog) and pG-G-s4U-G-Gp (bases 6-10) were synthesized by triester methods using 2'-O-(o-nitrobenzyl) nucleotides including a 3',5'-bisphosphorylated guanosine derivative. The s4U containing pentanucleotide was derived from the pG-G-C-G-Gp by treatment with liquid hydrogen sulfide. Images PMID:390499

Evolution of early life inferred from protein and ribonucleic acid sequences

NASA Technical Reports Server (NTRS)

Dayhoff, M. O.; Schwartz, R. M.

1978-01-01

The chemical structures of ferredoxin, 5S ribosomal RNA, and c-type cytochrome sequences have been employed to construct a phylogenetic tree which connects all major photosynthesizing organisms: the three types of bacteria, blue-green algae, and chloroplasts. Anaerobic and aerobic bacteria, eukaryotic cytoplasmic components and mitochondria are also included in the phylogenetic tree. Anaerobic nonphotosynthesizing bacteria similar to Clostridium were the earliest organisms, arising more than 3.2 billion years ago. Bacterial photosynthesis evolved nearly 3.0 billion years ago, while oxygen-evolving photosynthesis, originating in the blue-green algal line, came into being about 2.0 billion years ago. The phylogenetic tree supports the symbiotic theory of the origin of eukaryotes.

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

PubMed Central

Woodard, John; Rasch, Ellen; Swift, Hewson

1961-01-01

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C14 and uridine-H3 were used as ribonucleic acid (RNA) precursors, thymidine-H3 as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C14 as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. PMID:13786522

The effect of tRNA levels on decoding times of mRNA codons.

PubMed

Dana, Alexandra; Tuller, Tamir

2014-08-01

The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (-0.38 to -0.66, all P values <0.006); in addition, we show that when considering tRNA concentrations, codons decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of sequence–structure RNA binding motifs for SELEX-derived aptamers

PubMed Central

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E.; Przytycka, Teresa M.

2012-01-01

Motivation: Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. Results: To close this gap we developed, Aptamotif, a computational method for the identification of sequence–structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process. Contact: przytyck@ncbi.nlm.nih.gov, Zuben.Sauna@fda.hhs.gov PMID:22689764

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma

PubMed Central

Mirzazadeh, Azin; Kheirollahi, Majid; Farashahi, Ehsan; Sadeghian-Nodoushan, Fatemeh; Sheikhha, Mohammad Hasan; Aflatoonian, Behrouz

2017-01-01

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named “hTERT messenger ribonucleic acid (mRNA)” the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression. Materials and Methods: U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0–160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control–untreated group. Results: The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent (P < 0.05) and not in time-dependent manner (P > 0.05), in vitro. Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression. Conclusion: With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM. PMID:28706881

(Pro)renin receptor: Involvement in diabetic retinopathy and development of molecular targeted therapy.

PubMed

Kanda, Atsuhiro; Ishida, Susumu

2018-03-25

The renin-angiotensin system (RAS), a crucial regulator of systemic blood pressure (circulatory RAS), plays distinct roles in pathological angiogenesis and inflammation in various organs (tissue RAS), such as diabetic microvascular complications. Using ocular clinical samples and animal disease models, we elucidated molecular mechanisms in which tissue RAS excites the expression of vascular endothelial growth factor (VEGF)-A responsible for retinal inflammation and angiogenesis, the two major pathological events in diabetic retinopathy (DR). Furthermore, we showed the involvement of (pro)renin receptor [(P)RR] in retinal RAS activation and its concurrent intracellular signal transduction (e.g., extracellular signal-regulated kinase); namely, the (P)RR-induced dual pathogenic bioactivity referred to as the receptor-associated prorenin system. Indeed, neovascular endothelial cells in the fibrovascular tissue collected from eyes with proliferative DR were immunoreactive for the receptor-associated prorenin system components including prorenin, (P)RR, phosphorylated extracellular signal-regulated kinase and VEGF-A. Protein levels of soluble (P)RR increased with its positive correlations with prorenin, renin enzymatic activity and VEGF in the vitreous of proliferative DR eyes, suggesting a close link between (P)RR and VEGF-A-driven angiogenic activity. Furthermore, we revealed an unsuspected, PAPS-independent role of (P)RR in glucose-induced oxidative stress. Recently, we developed an innovative single-strand ribonucleic acid interference molecule selectively targeting human and mouse (P)RR, and confirmed its efficacy in suppressing diabetes-induced retinal inflammation in mice. Our data using clinical samples and animal models suggested the significant implication of (P)RR in the pathogenesis of DR, and the potential usefulness of the ribonucleic acid interference molecule as a therapeutic agent to attenuate ocular inflammation and angiogenesis. © 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Muscarinic receptors mediate cold stress-induced detrusor overactivity in type 2 diabetes mellitus rats.

PubMed

Imamura, Tetsuya; Ishizuka, Osamu; Ogawa, Teruyuki; Yamagishi, Takahiro; Yokoyama, Hitoshi; Minagawa, Tomonori; Nakazawa, Masaki; Gautam, Sudha Silwal; Nishizawa, Osamu

2014-10-01

This study determined if muscarinic receptors could mediate the cold stress-induced detrusor overactivity induced in type 2 diabetes mellitus rats. Ten-week-old female Goto-Kakizaki diabetic rats (n = 12) and Wister Kyoto non-diabetic rats (n = 12) were maintained on a high-fat diet for 4 weeks. Cystometric investigations of the unanesthetized rats were carried out at room temperature (27 ± 2°C) for 20 min. They were intravenously administered imidafenacin (0.3 mg/kg, n = 6) or vehicle (n = 6). After 5 min, the rats were transferred to a low temperature (4 ± 2°C) for 40 min where the cystometry was continued. The rats were then returned to room temperature for the final cystometric measurements. Afterwards, expressions of bladder muscarinic receptor M3 and M2 messenger ribonucleic acids and proteins were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. In non-diabetic Wister Kyoto rats, imidafenacin did not reduce cold stress-induced detrusor overactivity. In diabetic Goto-Kakizaki rats, just after transfer to a low temperature, the cold stress-induced detrusor overactivity in imidafenacin-treated rats was reduced compared with vehicle-treated rats. Within the urinary bladders, the ratio of M3 to M2 receptor messenger ribonucleic acid in the diabetic Goto-Kakizaki rats was significantly higher than that of the non-diabetic Wister Kyoto rats. The proportion of muscarinic M3 receptor-positive area within the detrusor in diabetic Goto-Kakizaki rats was also significantly higher than that in non-diabetic Wister Kyoto rats. Imidafenacin partially inhibits cold stress-induced detrusor overactivity in diabetic Goto-Kakizaki rats. In this animal model, muscarinic M3 receptors partially mediate cold stress-induced detrusor overactivity. © 2014 The Japanese Urological Association.

A comparison of cell-free placental messenger ribonucleic acid and color Doppler ultrasound for the prediction of placental invasion in patients with placenta accreta

PubMed Central

Naghshineh, Elham; Khorvash, Elahe; Kamali, Sara

2015-01-01

Background: The aim of the present study was to comparison between cell-free placental messenger ribonucleic acid (mRNA) and Doppler ultrasound for the prediction of placental invasion in women with placenta accreta. Materials and Methods: In this cross-sectional study, 50 pregnant women at risk for placenta accreta underwent color Doppler and assessment of cell-free placental mRNA. Real-time reverse-transcription polymerase chain reaction was used for measurement of cell-free placental mRNA in maternal plasma. Based on the findings at cesarean delivery and histological examination, patients were divided into two groups of women with and without placenta accrete. To compare of the mean of mRNA levels between the two groups we used independent t-test and to compare of the mean of age and gestational age at sonography we used Mann-Whitney test. For determination of sensitivity and specificity and the cut-off point of mRNA levels we used the receiver operating characteristic curve. Results: A total of 50 women with a mean age of 30.24 ± 4.905 years entered the study and 12 (24%) patients were diagnosed with placenta accreta. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Doppler ultrasound were 83.3%, 78.9%, 56% and 94%, respectively. Results of our study showed if we consider a cut-off point equal to 3.325, with sensitivity and specificity of 0.917 and 0.789, respectively and the sensitivity, specificity, PPV and NPV of mRNA with were cut-off point of 3.325 were 91.7%, 78.9%, 57.9% and 96.8%, respectively. Conclusions: Cell-free mRNA is an acceptable, easy made, functional test with sensitivity, specificity, PPV and NPV more than Doppler ultrasound for diagnosis and prediction of incidence of placenta accrete and we recommend the use of cell-free mRNA test for diagnosis of placenta accreta. PMID:25709996

A comparison of cell-free placental messenger ribonucleic acid and color Doppler ultrasound for the prediction of placental invasion in patients with placenta accreta.

PubMed

Naghshineh, Elham; Khorvash, Elahe; Kamali, Sara

2015-01-01

The aim of the present study was to comparison between cell-free placental messenger ribonucleic acid (mRNA) and Doppler ultrasound for the prediction of placental invasion in women with placenta accreta. In this cross-sectional study, 50 pregnant women at risk for placenta accreta underwent color Doppler and assessment of cell-free placental mRNA. Real-time reverse-transcription polymerase chain reaction was used for measurement of cell-free placental mRNA in maternal plasma. Based on the findings at cesarean delivery and histological examination, patients were divided into two groups of women with and without placenta accrete. To compare of the mean of mRNA levels between the two groups we used independent t-test and to compare of the mean of age and gestational age at sonography we used Mann-Whitney test. For determination of sensitivity and specificity and the cut-off point of mRNA levels we used the receiver operating characteristic curve. A total of 50 women with a mean age of 30.24 ± 4.905 years entered the study and 12 (24%) patients were diagnosed with placenta accreta. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Doppler ultrasound were 83.3%, 78.9%, 56% and 94%, respectively. Results of our study showed if we consider a cut-off point equal to 3.325, with sensitivity and specificity of 0.917 and 0.789, respectively and the sensitivity, specificity, PPV and NPV of mRNA with were cut-off point of 3.325 were 91.7%, 78.9%, 57.9% and 96.8%, respectively. Cell-free mRNA is an acceptable, easy made, functional test with sensitivity, specificity, PPV and NPV more than Doppler ultrasound for diagnosis and prediction of incidence of placenta accrete and we recommend the use of cell-free mRNA test for diagnosis of placenta accreta.

Effects of Phenethyl Alcohol on Phospholipid Metabolism in Escherichia coli

PubMed Central

Nunn, William D.; Tropp, Burton E.

1972-01-01

The incorporation of labeled precursors into the deoxyribonucleic acid, ribonucleic acid (RNA), proteins, and phospholipids of Escherichia coli cultured in the presence of phenethyl alcohol (PEA) was determined. PEA inhibited the uptake of labeled uracil to the same extent in cells exhibiting relaxed and stringent control of RNA synthesis. This indicates that PEA does not primarily affect amino acid synthesis or activation. Uptake of labeled acetate into the phospholipid fraction was more sensitive to inhibition by low concentrations of PEA than was the uptake of labeled precursors into the macromolecules. Thymine starvation or the addition of nalidixic acid (10 μg/ml) had no effect on acetate incorporation. Chloramphenicol (25 μg/ml) was a much less effective inhibitor of acetate incorporation than was PEA. The distribution of labeled acetate incorporated into phospholipids was markedly affected by the presence of PEA. The uptake of acetate into phosphatidylethanolamine and phosphatidylglycerol was inhibited, whereas the uptake of acetate into the cardiolipin fraction was unaffected. Since acetate incorporation into phospholipid was quite sensitive to PEA, we suggest that the PEA-sensitive component required for the initiation of replication may be a phospholipid(s). PMID:4550658

Chemical composition and biological activities of a phenol-water extract from Haemophilus influenzae type a.

PubMed Central

Raichvarg, D; Brossard, C; Agneray, J

1979-01-01

Ribonucleic acid was removed from a phenol-water extract of Haemophilus influenzae type a by streptomycin sulfate. This preparation was called purified preparation or PP. It contained neutral sugars (glucose, galactose, mannose, pentose), glucosamine, amino acids, and fatty acids. Heptose and 2-keto-3-deoxyoctonic acid were not present. The biological properties and immunogenicity were compared with the activities of lipopolysaccharide of Escherichia coli or Salmonella typhimurium. Higher doses were necessary to obtain lethality in mice and Sanarelli and Shwartzman reactions with our preparations than were necessary with lipopolysaccharide. The Limulus test and pyrogen assay in rabbits gave the same results with purified preparation and lipopolysaccharide, but pyrogenicity of purified preparation was not destroyed by NaOH treatment. Purified preparation was not as immunogenic at low doeses for rabbits as lipopolysaccharide. The results were different from those obtained with lipopolysaccharide but similar to those known from peptidoglycan studies. The contamination of purified preparation with peptidoglycan was negligible and cannot explain the biological activities of purified preparation. We suggest that the phenol-water extract from H. influenzae is not a classical endotoxin, but rather an endotoxin-like substance. PMID:317593

Improved monoclonal antibodies to halodeoxyuridine

DOEpatents

Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

1983-10-18

The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

Association of the Bacillus subtilis Chromosome with the Cell Membrane: Resolution of Free and Bound Deoxyribonucleic Acid on Renografin Gradients

PubMed Central

Ivarie, Robert D.; Pène, Jacques J.

1970-01-01

Linear density gradients of Renografin have resolved two components of bacterial deoxyribonucleic acid (DNA) in sheared lysates. Component 1, at equilibrium density after 5 hr of centrifugation, is enriched for newly synthesized DNA and markers near the origin and terminus of replication. It contains 5% of total cellular protein, 25% of the phospholipids, 30 to 50% of the DNA, 4 to 11% of unstable ribonucleic acid (RNA), RNA polymerase, and low amounts of DNA polymerase. The material is sensitive to Pronase and Sarkosyl. In unsheared lysates, all of the DNA forms a band at this position. Shearing the lysate generates a slow-sedimenting fraction of DNA (component 2) which contains more uniformly labeled than newly synthesized DNA. These observations suggest that replicating DNA and DNA at the origin and possibly the terminus of replication are associated with membrane. The amount of uniformly labeled DNA in component 1 and an estimate of the number of chromosomal fragments suggest that other parts of the chromosome are possibly associated with the membrane. PMID:4992373

Haloarcula hispanica CRISPR authenticates PAM of a target sequence to prime discriminative adaptation

PubMed Central

Li, Ming; Wang, Rui; Xiang, Hua

2014-01-01

The prokaryotic immune system CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) adapts to foreign invaders by acquiring their short deoxyribonucleic acid (DNA) fragments as spacers, which guide subsequent interference to foreign nucleic acids based on sequence matching. The adaptation mechanism avoiding acquiring ‘self’ DNA fragments is poorly understood. In Haloarcula hispanica, we previously showed that CRISPR adaptation requires being primed by a pre-existing spacer partially matching the invader DNA. Here, we further demonstrate that flanking a fully-matched target sequence, a functional PAM (protospacer adjacent motif) is still required to prime adaptation. Interestingly, interference utilizes only four PAM sequences, whereas adaptation-priming tolerates as many as 23 PAM sequences. This relaxed PAM selectivity explains how adaptation-priming maximizes its tolerance of PAM mutations (that escape interference) while avoiding mis-targeting the spacer DNA within CRISPR locus. We propose that the primed adaptation, which hitches and cooperates with the interference pathway, distinguishes target from non-target by CRISPR ribonucleic acid guidance and PAM recognition. PMID:24803673

Prevention of 5-Fluorouracil-Caused Growth Inhibition in Sordaria fimicola

PubMed Central

Schoen, Howard F.; Berech, John

1977-01-01

Growth (dry weight accumulation) of Sordaria fimicola in standing liquid culture (sucrose-nitrate-salts-vitamins) is inhibited by the presence of 5 μM 5-fluorouracil in the medium. This inhibition is completely prevented by uracil, deoxyuridine, and 5-bromouracil, partly prevented (40 to 90% of growth observed without 5-fluorouracil) by uridine, thymidine, and 5-bromodeoxyuridine, and slightly prevented by trifluorothymine, cytosine, cytidine, deoxycytidine, and 5-methylcytosine (all at 0.5 to 1 mM). Thymidine and thymine riboside were without any apparent effect. Growth is also inhibited by 0.2 mM 6-azauracil, and this inhibition was completely prevented by uracil and uridine, partly prevented by deoxyuridine, 5-bromouracil, cytidine, and 5-methylcytosine, and slightly prevented by thymine, thymidine, 5-bromodeoxyuridine, cytosine, and deoxycytidine. The data suggest that the observed inhibition of growth by 5-fluorouracil is due to inhibition of both ribonucleic acid and deoxyribonucleic acid synthesis. The data also allow inferences concerning pyrimidine interconversions in S. fimicola; i.e., thymine can be anabolized to thymidylic acid without first being demethylated, although demethylation appears to occur also. PMID:848926

Prevention of 5-fluorouracil-caused growth inhibition in Sordaria fimicola.

PubMed

Schoen, H F; Berech, J

1977-02-01

Growth (dry weight accumulation) of Sordaria fimicola in standing liquid culture (sucrose-nitrate-salts-vitamins) is inhibited by the presence of 5 muM 5-fluorouracil in the medium. This inhibition is completely prevented by uracil, deoxyuridine, and 5-bromouracil, partly prevented (40 to 90% of growth observed without 5-fluorouracil) by uridine, thymidine, and 5-bromodeoxyuridine, and slightly prevented by trifluorothymine, cytosine, cytidine, deoxycytidine, and 5-methylcytosine (all at 0.5 to 1 mM). Thymidine and thymine riboside were without any apparent effect. Growth is also inhibited by 0.2 mM 6-azauracil, and this inhibition was completely prevented by uracil and uridine, partly prevented by deoxyuridine, 5-bromouracil, cytidine, and 5-methylcytosine, and slightly prevented by thymine, thymidine, 5-bromodeoxyuridine, cytosine, and deoxycytidine. The data suggest that the observed inhibition of growth by 5-fluorouracil is due to inhibition of both ribonucleic acid and deoxyribonucleic acid synthesis. The data also allow inferences concerning pyrimidine interconversions in S. fimicola; i.e., thymine can be anabolized to thymidylic acid without first being demethylated, although demethylation appears to occur also.

Prevalence of HCV infection and associated factors among illicit drug users in Breves, State of Pará, northern Brazil.

PubMed

Pacheco, Suzy Danielly Barbosa; Silva-Oliveira, Gláucia Caroline; Maradei-Pereira, Luciana Maria Cunha; Crescente, José Ângelo Barletta; Lemos, José Alexandre Rodrigues de; Oliveira-Filho, Aldemir Branco de

2014-01-01

Illicit drug users (DUs) are vulnerable to hepatitis C virus (HCV) infection. The shared use of illicit drugs is the main method of HCV transmission. A cross-sectional study was conducted in Breves, in northern Brazil. We surveyed 187 DUs to determine the prevalence of and factors associated with HCV infection. The prevalence of anti-HCV antibodies was 36.9%, and the prevalence of hepatitis C virus-ribonucleic acid (HCV-RNA) was 31%. Hepatitis C virus infection was associated with tattoos, intravenous drug use, shared use of equipment for drug use, drug use for longer than 3 years, and daily drug use. Strategies for preventing and controlling HCV transmission should be implemented among DUs.

Early Clinical Diagnosis of PC1/3 Deficiency in a Patient With a Novel Homozygous PCSK1 Splice-Site Mutation.

PubMed

Härter, Bettina; Fuchs, Irene; Müller, Thomas; Akbulut, Ulas Emre; Cakir, Murat; Janecke, Andreas R

2016-04-01

Autosomal recessive proprotein convertase 1/3 (PC1/3) deficiency, caused by mutations in the PCSK1 gene, is characterized by severe congenital malabsorptive diarrhea, early-onset obesity, and certain endocrine abnormalities. We suspected PC1/3 deficiency in a 4-month-old girl based on the presence of congenital diarrhea and polyuria. Sequencing the whole coding region and splice sites detected a novel homozygous PCSK1 splice-site mutation, c.544-2A>G, in the patient. The mutation resulted in the skipping of exon 5, the generation of a premature termination codon, and nonsense-mediated PCSK1 messenger ribonucleic acid decay, which was demonstrated in complementary DNA derived from fibroblasts.

Topochemical differences in the amount of RNA in the motoneurons of the spinal chord in hypoxia and hypokinesia

NASA Technical Reports Server (NTRS)

Brumberg, V. A.; Gazenko, O. G.; Demin, N. N.; Malkin, V. B.; Pevzner, L. Z.

1980-01-01

Reactions to hypoxia and hypoknesia were compared by measuring charges in the amount of ribonucleic acid (RNA) in the cytoplasm of neurons of the intumescentia cervicalis and lumbalis. Animals were subjected to hypoxia, hypokinesia and both combined and a control group to neither. A total of two groups of motoneurons were compared, one innervating the respiratory musculature, the other the musculature of the lower extremities, so that hypoxic hypoxia would probably affect the first group primarily and hypokinesia the second. Results indicate that neither affect the amount of RNA in the neurons of the first group but a significant increase is noted in neurons of the second group. Other significant results are reported.

Origin and identity of Fejervarya (Anura: Dicroglossidae) on Guam

USGS Publications Warehouse

Wostl, Elijah; Smith, Eric N.; Reed, Robert

2016-01-01

We used morphological and molecular data to infer the identity and origin of frogs in the genus Fejervarya that have been introduced to the island of Guam. Mensural and meristic data were collected from 96 specimens from throughout their range on the island and a principal component analysis was used to investigate the distribution of these data in morphological space. We also amplified a fragment of the 16S ribosomal ribonucleic acid mitochondrial gene from 27 of these specimens and compared it to 63 published sequences of Fejervarya and the morphologically similar Zakerana. All examined Fejervarya from Guam are morphologically indistinguishable and share an identical haplotype. The molecular data identify them as Fejervarya cancrivora with a haplotype identical to F. cancrivora from Taiwan.

Silence of the transcripts: RNA interference in medicine.

PubMed

Barik, Sailen

2005-10-01

Silencing of gene expression by ribonucleic acid (RNA), known as RNA interference (RNAi), is now recognized as a major means of gene regulation in biology. In this mechanism, small noncoding double-stranded RNA molecules knock down gene expression through a variety of mechanisms that include messenger RNA (mRNA) degradation, inhibition of mRNA translation, or chromatin remodeling. The posttranscriptional mechanism of RNAi has been embraced by researchers as a powerful tool for generating deficient phenotypes without mutating the gene. In parallel, exciting recent results have promised its application in disease therapy. This review aims to summarize the current knowledge in this area and provide a roadmap that may eventually launch RNAi from the research bench to the medicine chest.

Effect of restricted mobility on RNA content and nucleotide composition and on protein content in motoneurons of spinal cord anterior horns

NASA Technical Reports Server (NTRS)

Gorbunova, A. V.

1980-01-01

An investigation into the effect of hypokinesia on the ribonucleic acid (RNA) content, the nucleotide composition, and dynamics of protein content in the motoneuron of the rat spinal cord anterior horns is described. Methodology and findings are presented. The study results showed that the nucleotide composition of the total cellular RNA at all the studied periods of hypokinesia remained unchanged and is characteristic for the cytoplasmic, high polymer ribosomal RNA. This means that with a change in the functional state of the neuron the newly formed RNA of the nerve cell has the same composition of bases as the original RNA that belongs to the ribosomal type.

Nanotechnology in the targeted drug delivery for bone diseases and bone regeneration

PubMed Central

Gu, Wenyi; Wu, Chengtie; Chen, Jiezhong; Xiao, Yin

2013-01-01

Nanotechnology is a vigorous research area and one of its important applications is in biomedical sciences. Among biomedical applications, targeted drug delivery is one of the most extensively studied subjects. Nanostructured particles and scaffolds have been widely studied for increasing treatment efficacy and specificity of present treatment approaches. Similarly, this technique has been used for treating bone diseases including bone regeneration. In this review, we have summarized and highlighted the recent advancement of nanostructured particles and scaffolds for the treatment of cancer bone metastasis, osteosarcoma, bone infections and inflammatory diseases, osteoarthritis, as well as for bone regeneration. Nanoparticles used to deliver deoxyribonucleic acid and ribonucleic acid molecules to specific bone sites for gene therapies are also included. The investigation of the implications of nanoparticles in bone diseases have just begun, and has already shown some promising potential. Further studies have to be conducted, aimed specifically at assessing targeted delivery and bioactive scaffolds to further improve their efficacy before they can be used clinically. PMID:23836972

Versatile RNA tetra-U helix linking motif as a toolkit for nucleic acid nanotechnology.

PubMed

Bui, My N; Brittany Johnson, M; Viard, Mathias; Satterwhite, Emily; Martins, Angelica N; Li, Zhihai; Marriott, Ian; Afonin, Kirill A; Khisamutdinov, Emil F

2017-04-01

RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community. Copyright © 2017 Elsevier Inc. All rights reserved.

Nanotechnology in the targeted drug delivery for bone diseases and bone regeneration.

PubMed

Gu, Wenyi; Wu, Chengtie; Chen, Jiezhong; Xiao, Yin

2013-01-01

Nanotechnology is a vigorous research area and one of its important applications is in biomedical sciences. Among biomedical applications, targeted drug delivery is one of the most extensively studied subjects. Nanostructured particles and scaffolds have been widely studied for increasing treatment efficacy and specificity of present treatment approaches. Similarly, this technique has been used for treating bone diseases including bone regeneration. In this review, we have summarized and highlighted the recent advancement of nanostructured particles and scaffolds for the treatment of cancer bone metastasis, osteosarcoma, bone infections and inflammatory diseases, osteoarthritis, as well as for bone regeneration. Nanoparticles used to deliver deoxyribonucleic acid and ribonucleic acid molecules to specific bone sites for gene therapies are also included. The investigation of the implications of nanoparticles in bone diseases have just begun, and has already shown some promising potential. Further studies have to be conducted, aimed specifically at assessing targeted delivery and bioactive scaffolds to further improve their efficacy before they can be used clinically.

Preparation and Analysis of RNA Crystals

NASA Technical Reports Server (NTRS)

Todd, Paul

2000-01-01

The crystallization of RiboNucleic Acids (RNA) was studied from the standpoint of mechanisms of crystal growth in three tasks: (1) preparation of high-quality crystals of oligonuclotides for X-ray diffraction, (2) finding pathways to the growth of high-quality crystals for X-ray diffraction and (3) investigation of mechanisms of action of inertial acceleration on crystal growth. In these tasks: (1) RNA crystals were prepared and studied by X-ray diffraction; (2) a pathway to high-quality crystals was discovered and characterized; a combination of kinetic and equilibrium factors could be optimized as described below; and (3) an interplay between purity and gravity was found in a combination of space and ground experiments with nucleic acids and proteins. Most significantly, the rate of concentration of precipitant and RNA can be controlled by membrane-based methods of water removal or by diffusion of multivalent cations across an interface stabilized by a membrane. Oligonucleotide solutions are electrokinetically stabilized colloids, and crystals can form by the controlled addition of multivalent cations.

Microfluidic integrated acoustic waving for manipulation of cells and molecules.

PubMed

Barani, Alireza; Paktinat, Hossein; Janmaleki, Mohsen; Mohammadi, Aminollah; Mosaddegh, Peiman; Fadaei-Tehrani, Alireza; Sanati-Nezhad, Amir

2016-11-15

Acoustophoresis with its simple and low-cost fabrication, rapid and localized fluid actuation, compatibility with microfluidic components, and biocompatibility for cellular studies, has been extensively integrated into microfluidics to provide on-chip microdevices for a variety of applications in biology, bioengineering and chemistry. Among different applications, noninvasive manipulation of cells and biomolecules are significantly important, which are addressed by acoustic-based microfluidics. Here in this paper, we briefly explain the principles and different configurations of acoustic wave and acoustic streaming for the manipulation of cells and molecules and overview its applications for single cell isolation, cell focusing and sorting, cell washing and patterning, cell-cell fusion and communication, and tissue engineering. We further discuss the application of acoustic-based microfluidic systems for the mixing and transport of liquids, manipulation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules, followed by explanation on the present challenges of acoustic-based microfluidics for the handling of cells and molecules, and highlighting the future directions. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Aptamer-modified nanoparticles and their use in cancer diagnostics and treatment.

PubMed

Reinemann, Christine; Strehlitz, Beate

2014-01-06

Aptamers are single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) oligonucleotides, which are able to bind their target with high selectivity and affinity. Owing to their multiple talents, aptamers combined with nanoparticles are nanosystems well qualified for the development of new biomedical devices for analytical, imaging, drug delivery and many other medical applications. Because of their target affinity, aptamers can direct the transport of aptamer-nanoparticle conjugates. The binding of the aptamers to the target "anchors" the nanoparticle-aptamer conjugates at their site of action. In this way, nanoparticle-based bioimaging and smart drug delivery are enabled, especially by use of systematically developed aptamers for cancer-associated biomarkers. This review article gives a brief overview of recent relevant research into aptamers and trends in their use in cancer diagnostics and therapy. A concise description of aptamers, their development and functionalities relating to nanoparticle modification is given. The main part of the article is dedicated to current developments of aptamer-modified nanoparticles and their use in cancer diagnostics and treatment.

Self-Organizing Hidden Markov Model Map (SOHMMM).

PubMed

Ferles, Christos; Stafylopatis, Andreas

2013-12-01

A hybrid approach combining the Self-Organizing Map (SOM) and the Hidden Markov Model (HMM) is presented. The Self-Organizing Hidden Markov Model Map (SOHMMM) establishes a cross-section between the theoretic foundations and algorithmic realizations of its constituents. The respective architectures and learning methodologies are fused in an attempt to meet the increasing requirements imposed by the properties of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein chain molecules. The fusion and synergy of the SOM unsupervised training and the HMM dynamic programming algorithms bring forth a novel on-line gradient descent unsupervised learning algorithm, which is fully integrated into the SOHMMM. Since the SOHMMM carries out probabilistic sequence analysis with little or no prior knowledge, it can have a variety of applications in clustering, dimensionality reduction and visualization of large-scale sequence spaces, and also, in sequence discrimination, search and classification. Two series of experiments based on artificial sequence data and splice junction gene sequences demonstrate the SOHMMM's characteristics and capabilities. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of lipid-lowering pharmaceuticals bezafibrate and clofibric acid on lipid metabolism in fathead minnow (Pimephales promelas).

PubMed

Weston, Anna; Caminada, Daniel; Galicia, Hector; Fent, Karl

2009-12-01

The lipid-lowering agents bezafibrate and clofibric acid, which occur at concentrations up to 3.1 and 1.6 microg/L, respectively, are among the most frequently found human pharmaceuticals in the aquatic environment. In contrast to knowledge about their environmental occurrence, little is known about their effects in the environment. The aim of the present study was to analyze effects of these lipid-lowering agents in fish by focusing on their modes of action, lipid metabolism. Fathead minnows were exposed in aquaria to measured concentrations of 0.1, 1.27, 10.18, 101.56, and 106.7 mg/L bezafibrate and to 1.07, 10.75, and 108.91 mg/L clofibric acid for 14 and 21 d, respectively. After exposure, fish liver was analyzed for expression of peroxisome proliferator-activated receptor alpha (PPARalpha) by quantitative polymerase chain reaction (PCR), and the PPAR-regulated enzyme fatty acyl-coenzyme-A oxidase (FAO) involved in fatty acid oxidation. Bezafibrate had no effect, either on PPARalpha expression or on FAO activity, at all concentrations. In contrast, clofibric acid induced FAO activity in male fathead minnows at 108.91 mg/L. No increase in expression of PPARalpha messenger ribonucleic acid was observed. Egg production was apparently decreased after 21 d of exposure to 108.91 mg/L clofibric acid. The present study demonstrates that bezafibrate has very little or no effect on PPARalpha expression and FAO activity, but clofibric acid affects FAO activity.

Properties and substrate specificities of the phenylalanyl-transfer-ribonucleic acid synthetases of Aesculus species

PubMed Central

Anderson, J. W.; Fowden, L.

1970-01-01

1. Phenylalanyl-tRNA synthetases have been partially purified from cotyledons of seeds of Aesculus californica, which contains 2-amino-4-methylhex-4-enoic acid, and from four other species of Aesculus that do not contain this amino acid. The A. californica preparation was free from other aminoacyl-tRNA synthetases, and the contaminating synthetase activity in preparations from A. hippocastanum was decreased to acceptable limits by conducting assays of pyrophosphate exchange activity in 0.5m-potassium chloride. 2. The phenylalanyl-tRNA synthetase from each species activated 2-amino-4-methylhex-4-enoic acid with Km 30–40 times that for phenylalanine. The maximum velocity for 2-amino-4-methylhex-4-enoic acid was only 30% of that for phenylalanine with the A. californica enzyme, but the maximum velocities for the two substrates were identical for the other four species. 3. 2-Amino-4-methylhex-4-enoic acid was not found in the protein of A. californica, so discrimination against this amino acid probably occurs in the step of transfer to tRNA, though subcellular localization, or subsequent steps of protein synthesis could be involved. 4. Crotylglycine, methallylglycine, ethallylglycine, 2-aminohex-4,5-dienoic acid, 2-amino-5-methylhex-4-enoic acid, 2-amino-4-methylhex-4-enoic acid, β-(thien-2-yl)alanine, β-(pyrazol-1-yl)alanine, phenylserine and m-fluorophenylalanine were substrates for pyrophosphate exchange catalysed by the phenylalanyl-tRNA synthetases of A. californica or A. hippocastanum. Allylglycine, phenylglycine and 2-amino-4-phenylbutyric acid were inactive. PMID:5493504

Stratospheric microbiology at 20 km over the Pacific Ocean

USGS Publications Warehouse

Smith, David J.; Griffin, Dale W.; Schuerger, Andrew C.

2010-01-01

An aerobiology sampling flight at 20 km was conducted on 28 April 2008 over the Pacific Ocean (36.5° N, 118–149° W), a period of time that coincided with the movement of Asian dust across the ocean. The aim of this study was to confirm the presence of viable bacteria and fungi within a transoceanic, atmospheric bridge and to improve the resolution of flight hardware processing techniques. Isolates of the microbial strains recovered were analyzed with ribosomal ribonucleic acid (rRNA) sequencing to identify bacterial species Bacillus sp., Bacillus subtilis, Bacillus endophyticus, and the fungal genus Penicillium. Satellite imagery and ground-based radiosonde observations were used to measure dust movement and characterize the high-altitude environment at the time of collection. Considering the atmospheric residency time (7–10 days), the extreme temperature regime of the environment (-75°C), and the absence of a mechanism that could sustain particulates at high altitude, it is unlikely that our samples indicate a permanent, stratospheric ecosystem. However, the presence of viable fungi and bacteria in transoceanic stratosphere remains relevant to understanding the distribution and extent of microbial life on Earth.

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

PubMed Central

Zheng, Chenkang; Black, Katherine A.; Dos Santos, Patricia C.

2017-01-01

Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes. PMID:28327539

Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

PubMed Central

Elbediwy, Ahmed; Zihni, Ceniz; Terry, Stephen J.; Clark, Peter

2012-01-01

Epithelial cell–cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell–cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin–capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics. PMID:22891260

Large cryoconite aggregates on a Svalbard glacier support a diverse microbial community including ammonia-oxidizing archaea

NASA Astrophysics Data System (ADS)

Zarsky, Jakub D.; Stibal, Marek; Hodson, Andy; Sattler, Birgit; Schostag, Morten; Hansen, Lars H.; Jacobsen, Carsten S.; Psenner, Roland

2013-09-01

The aggregation of surface debris particles on melting glaciers into larger units (cryoconite) provides microenvironments for various microorganisms and metabolic processes. Here we investigate the microbial community on the surface of Aldegondabreen, a valley glacier in Svalbard which is supplied with carbon and nutrients from different sources across its surface, including colonies of seabirds. We used a combination of geochemical analysis (of surface debris, ice and meltwater), quantitative polymerase chain reactions (targeting the 16S ribosomal ribonucleic acid and amoA genes), pyrosequencing and multivariate statistical analysis to suggest possible factors driving the ecology of prokaryotic microbes on the surface of Aldegondabreen and their potential role in nitrogen cycling. The combination of high nutrient input with subsidy from the bird colonies, supraglacial meltwater flow and the presence of fine, clay-like particles supports the formation of centimetre-scale cryoconite aggregates in some areas of the glacier surface. We show that a diverse microbial community is present, dominated by the cyanobacteria, Proteobacteria, Bacteroidetes, and Actinobacteria, that are well-known in supraglacial environments. Importantly, ammonia-oxidizing archaea were detected in the aggregates for the first time on an Arctic glacier.

Calculation of density functional theory (DFT) vibrational parameters of nucleotides for use in theoretical optical calculations: Herein applied to circular dichroism (CD) and absorption of polynucleotides

NASA Astrophysics Data System (ADS)

Ferber, Steven Dwight

2005-11-01

The Vibrational Circular Dichroism (VCD) of Nucleic Acids is a sensitive function of their conformation. DeVoe's classically derived polarizability theory allows the calculation of polymer absorption and circular dichroism spectra in any frequency range. Following the approach of Tinoco and Cech as modified by Moore and Self, calculations were done in the infrared (IR) region with theoretically derived monomer input parameters. Presented herein are calculated absorption and CD spectra for nucleic acid oligomers and polymers. These calculations improve upon earlier attempts, which utilized frequencies, intensities and normal modes from empirical analysis of the nitrogenous base of the monomers. These more complete input polarizability parameters include all contributions to specific vibrational normal modes for the entire nucleotide structure. They are derived from density functional theory (DFT) vibrational analysis on quasi-nucleotide monomers using the GAUSSIAN '98/'03 program. The normal modes are "integrated" for the first time into single virtual (DeVoe) oscillators by incorporating "fixed partial charges" in the manner of Schellman. The results include the complete set of monomer normal modes. All of these modes may be analyzed, in a manner similar to those demonstrated here (for the 1500-1800 cm-1 region). A model is utilized for the polymer/oligomer monomers which maintains the actual electrostatic charge on the adjacent protonated phosphoryl groups (hydrogen phosphate, a mono-anion). This deters the optimization from "collapsing" into a hydrogen-bonded "ball" and thereby maintains the extended (polymer-like) conformation. As well, the precise C2 "endo" conformation of the sugar ring is maintained in the DNA monomers. The analogous C3 "endo" conformation is also maintained for the RNA monomers, which are constrained by massive "anchors" at the phosphates. The complete IR absorbance spectra (0-4,000 cm-1) are calculated directly in Gaussian. Calculated VCD and Absorbance Spectra for the eight standard Ribonucleic and Deoxy-ribonucleic acid homo-polymers in the nitrogenous base absorbing region 1550-1750 cm-1 are presented. These spectra match measured spectra at least as well as spectra calculated from empirical parameters. These results demonstrate that the purely theoretical calculation, an example given herein, should serve to provide more transferable, universal parameters for the polarizability treatment of the optical properties of oligomers and polymers.

The Marine Fungi Rhodotorula sp. (Strain CNYC4007) as a Potential Feed Source for Fish Larvae Nutrition

PubMed Central

Barra, M.; Llanos-Rivera, A.; Cruzat, F.; Pino-Maureira, N.; González-Saldía, R. R.

2017-01-01

Fish oil is used in the production of feed for cultured fish owing to its high polyunsaturated fatty acid content (PUFA). The over-exploitation of fisheries and events like “El Niño” are reducing the fish oil supply. Some marine microorganisms are considered potentially as alternative fatty acid sources. This study assesses a strain of Rhodotorula sp. (strain CNYC4007; 27% docosahexaenoic acid (DHA) of total fatty acids), as feed for fish larvae. The total length and ribonucleic acid (RNA)/deoxyribonucleic acid (DNA) ratio of Danio rerio larvae was determined at first feeding at six and 12 days old (post-yolk absorption larvae). Larvae fed with microencapsulated Rhodotorula sp. CNYC4007 had a significantly higher RNA/DNA ratio than control group (C1). At six days post-yolk absorption group, the RNA/DNA ratio of larvae fed with Rhodotorula sp. bioencapsulated in Brachionus sp. was significantly higher than control group fed with a commercial diet high in DHA (C2-DHA). Finally, at 12 days post-yolk absorption, the RNA/DNA ratio was significantly higher in larvae fed with Rhodotorula sp. CNYC4007 and C2-DHA (both bioencapsulated in Artemia sp. nauplii) than in control group (C1). These results suggest that Rhodotorula sp. CNYC4007 can be an alternative source of DHA for feeding fish at larval stage, providing a sustainable source of fatty acids. PMID:29194350

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

PubMed

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K

2015-12-15

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear-cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. © 2015 Wu et al.; Published by Cold Spring Harbor Laboratory Press.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics

PubMed Central

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K.

2015-01-01

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. PMID:26680305

Morphology, antigenicity, and nucleic acid content of the Bacteroides sp. used in the culture of Entamoeba histolytica.

PubMed

Albach, R A; Shaffer, J G; Watson, R H

1965-10-01

Albach, Richard A. (Lutheran General Hospital, Park Ridge, Ill.), James G. Shaffer, and Robert H. Watson. Morphology, antigenicity, and nucleic acid content of the Bacteroides sp. used in the culture of Entamoeba histolytica. J. Bacteriol. 90:1045-1053. 1965.-Certain changes in morphology, antigenicity, and nucleic acid content that occur in a culture of Bacteroides sp. in the presence of penicillin G in CLG medium are described. This "variant" is one of seven recovered in several laboratories, all of which are descendants of the original Bacteroides isolated by Shaffer and Frye. Penicillin-inhibited cells of this culture are currently being used in the routine propagation of Entamoeba histolytica in CLG medium. Evidence is presented for the loss of ability to react with antibody in these penicillin-inhibited bacteria in CLG medium, when studied by fluorescent-antibody techniques. The implications of the antigenic changes observed as they pertain to similar antigenic studies of the amoebas are discussed. A pronounced reduction in the ribonucleic acid (RNA) content of such penicillin-inhibited cells was also observed. The potential importance of the changes that occur in the RNA of these cells with respect to considerations of the growth requirements of the amoebas is also discussed.

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

PubMed Central

Kalghatgi, K K; Subba Rao, P V

1976-01-01

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate. Images PMID:1262311

Insulin in UW solution exacerbates hepatic ischemia / reperfusion injury by energy depletion through the IRS-2 / SREBP-1c pathway.

PubMed

Li, Xian Liang; Man, Kwan; Ng, Kevin T; Lee, Terence K; Lo, Chung Mau; Fan, Sheung Tat

2004-09-01

Ischemia / reperfusion (I / R) injury is related to tissue graft energy status. Insulin, which is currently used in the University of Wisconsin (UW) preservation solution with insulin (UWI), is an anabolic hormone and was shown to exacerbate the hepatic I / R injury in our previous study. In this study, the energy status and regulation of metabolism genes by insulin were investigated in liver grafts preserved by UW solution. Insulin could significantly decrease adenosine triphosphate (ATP) level after 3 hours of preservation, as well as total adenine nucleotides (TANs) and energy charge (EC) levels. Energy regeneration deteriorated in the grafts preserved by insulin in terms of ATP and EC levels at 24 hours after transplantation. The insulin signal was transduced through the insulin receptor substrate-2 (IRS-2) pathway and the activity of IRS-2 was decreased gradually at the messenger ribonucleic acid (mRNA) level during cold preservation. Downstream targeting genes such as sterol regulatory element-binding protein-1c (SREBP-1c), glucokinase (GKC), and fatty acid synthase (FAS) genes, as well as phospho-glycogen synthase kinase-3beta (GSK-3beta) were activated and they showed the similar expression profiles during cold preservation. Lipoprotein metabolism was accelerated by insulin through upregulation of the activity of apolipoprotein C-III (Apo C-III) during cold preservation. The insulin-like growth factor-binding protein-1 pathway was inhibited during cold preservation. In conclusion, insulin in UW solution exacerbates hepatic I / R injury by energy depletion as the graft maintains its anabolic activity. The key enzyme activities of the energy-consuming process of glycogen and fatty acid synthesis as well as lipoprotein metabolism were accelerated by insulin through the IRS-2 / SREBP-1c pathway.

Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

PubMed Central

Burge, Boyce W.; Pfefferkorn, E. R.

1967-01-01

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes. PMID:5630228

Theoretical analysis of the distribution of isolated particles in totally asymmetric exclusion processes: Application to mRNA translation rate estimation

NASA Astrophysics Data System (ADS)

Dao Duc, Khanh; Saleem, Zain H.; Song, Yun S.

2018-01-01

The Totally Asymmetric Exclusion Process (TASEP) is a classical stochastic model for describing the transport of interacting particles, such as ribosomes moving along the messenger ribonucleic acid (mRNA) during translation. Although this model has been widely studied in the past, the extent of collision between particles and the average distance between a particle to its nearest neighbor have not been quantified explicitly. We provide here a theoretical analysis of such quantities via the distribution of isolated particles. In the classical form of the model in which each particle occupies only a single site, we obtain an exact analytic solution using the matrix ansatz. We then employ a refined mean-field approach to extend the analysis to a generalized TASEP with particles of an arbitrary size. Our theoretical study has direct applications in mRNA translation and the interpretation of experimental ribosome profiling data. In particular, our analysis of data from Saccharomyces cerevisiae suggests a potential bias against the detection of nearby ribosomes with a gap distance of less than approximately three codons, which leads to some ambiguity in estimating the initiation rate and protein production flux for a substantial fraction of genes. Despite such ambiguity, however, we demonstrate theoretically that the interference rate associated with collisions can be robustly estimated and show that approximately 1% of the translating ribosomes get obstructed.

Deoxyribonucleic Acid Replication and Expression of Early and Late Bacteriophage Functions in Bacillus subtilis

PubMed Central

Pène, Jacques J.; Marmur, Julius

1967-01-01

The role of deoxyribonucleic acid (DNA) replication in the control of the synthesis of deoxycytidylate (dCMP) deaminase and lysozyme in Bacillus subtilis infected with bacteriophage 2C has been studied. These phage-induced enzymes are synthesized at different times during the latent period. It was shown by actinomycin inhibition that the formation of the late enzyme (lysozyme) required messenger ribonucleic acid (mRNA) synthesized de novo after the initiation of translation of mRNA which specifies the early function (dCMP deaminase). The inhibition of phage DNA synthesis by mitomycin C prevented the synthesis of lysozyme only when added before the onset of phage DNA replication, but it did not affect the synthesis or action of dCMP deaminase when added at any time during the latent period. Treatment of infected cells with mitomycin C after phage DNA synthesis had reached 8 to 10% of its maximal rate resulted in the production of normal amounts of lysozyme. These observations suggest that mRNA specifying early enzymes can be transcribed from parental (and probably also from progeny) DNA, whereas late functional messengers can be transcribed only after the formation of progeny DNA. PMID:4990039

Morphokinetic Reaction of Cells of Streptococcus faecalis (ATCC 9790) to Specific Inhibition of Macromolecular Synthesis: Dependence of Mesosome Growth on Deoxyribonucleic Acid Synthesis

PubMed Central

Higgins, Michael L.; Daneo-Moore, Lolita

1972-01-01

The application of quantitative electron microscopy to thin sections of cells of Streptococcus faecalis specifically inhibited for deoxyribonucleic acid (DNA), ribonucleic acid, and protein synthesis shows that septal mesosomes (i) increase in size when protein synthesis is inhibited by at least 80% while DNA synthesis proceeds at no less than 50% of the control rate and (ii) decrease in size when DNA synthesis is inhibited 50% or more during the initial 10 min of treatment. This indicates that fluctuations in mesosome size are dependent on the extent of DNA synthesis. The fluctuations in mesosome areas observed on treatment do not correlate with the kinetics of glycerol incorporation per milliliter of a culture. However, when glycerol incorporation is placed on a per cell basis, a strong correlation is observed between increases in (i) the thickness of the electron-transparent layer of the cytoplasmic membrane and (ii) the amount of glycerol incorporated per cell. It seems that the electron-transparent membrane layer may thicken to accommodate changes in lipid content when protein and lipid synthesis are uncoupled. Images PMID:4110926

Model systems: how chemical biologists study RNA

PubMed Central

Rios, Andro C.; Tor, Yitzhak

2009-01-01

Ribonucleic acids are structurally and functionally sophisticated biomolecules and the use of models, frequently truncated or modified sequences representing functional domains of the natural systems, is essential to their exploration. Functional non-coding RNAs such as miRNAs, riboswitches, and, in particular, ribozymes, have changed the view of RNA’s role in biology and its catalytic potential. The well-known truncated hammerhead model has recently been refined and new data provide a clearer molecular picture of the elements responsible for its catalytic power. A model for the spliceosome, a massive and highly intricate ribonucleoprotein, is also emerging, although its true utility is yet to be cemented. Such catalytic model systems could also serve as “chemo-paleontological” tools, further refining the RNA world hypothesis and its relevance to the origin and evolution of life. PMID:19879179

[Ribonucleic acids and proteins of influenza A/USSR/90/77 viruses].

PubMed

Vorkunova, G K; Dotsenko, G N; Bukrinskaia, A G; Zhdanov, V M

1979-01-01

The "return" of influenza A (HINI) virus after 20 years of absence raised the question of the sources and mechanisms of emergence of epidemic influenza viruses and, particularly, of a new HINI virus (A/USSR/90/77). Two alternative hypotheses answer this question differently: the new HINI virus is the progeny of old HINI viruses retained in the human population or is a newly arising recombinant between numerous human and animal influenza viruses circulation in the biosphere. For the acceptance of one or the other hypothesis further accumulation of facts is required and, first of all, comparative investigations of RNAs and proteins of various influenza viruses. This paper presents the results of comparative studies of RNAs and proteins of old and new influenza A (HINI) viruses.

Properties and substrate specificity of the leucyl-, the threonyl- and the valyl-transfer-ribonucleic acid synthetases from Aesculus species

PubMed Central

Anderson, J. W.; Fowden, L.

1970-01-01

1. Leucyl- and threonyl-tRNA synthetases were partially purified up to 100-fold and 30-fold respectively from cotyledons of Aesculus hippocastanum and were largely separated from the other aminoacyl-tRNA synthetases. Valyl-tRNA synthetase was purified 25-fold from cotyledons of Aesculus californica. 2. Some properties are reported for the three enzymes when assayed by the [32P]pyrophosphate-ATP exchange technique. 3. β-(Methylenecyclopropyl)alanine, isoleucine, azaleucine, norleucine and γ-hydroxynorvaline acted as alternative substrates for the leucyl-tRNA synthetase; the enzyme's affinity for β-(methylenecyclopropyl)-alanine and for isoleucine was about 80-fold less than that exhibited for leucine. 4. α-Cyclopropylglycine and α-cyclobutylglycine acted as alternative substrates for the valyl-tRNA synthetase. PMID:5493505

Molecular mimicry between protein and tRNA.

PubMed

Nakamura, Y

2001-01-01

Mimicry is a sophisticated development in animals, fish, and plants that allows them to fool others by imitating a shape or color for diverse purposes, such as to prey, evade, lure, pollinate, or threaten. This is not restricted to the macro-world, but extends to the micro-world as molecular mimicry. Recent advances in structural and molecular biology uncovered a set of translation factors that resembles a tRNA shape and, in one case, even mimics a tRNA function for deciphering the genetic code. Nature must have evolved this art of molecular mimicry between protein and ribonucleic acid by using different protein structures until the translation factors sat in the cockpit of a ribosome machine, on behalf of tRNA, and achieved diverse actions. Structural, functional, and evolutionary aspects of molecular mimicry will be discussed.

[The role of miRNA in endometrial cancer in the context of miRNA 205].

PubMed

Wilczyński, Miłosz; Danielska, Justyna; Dzieniecka, Monika; Malinowski, Andrzej

2015-11-01

MiRNAs are small, non-coding molecules of ribonucleic acids of approximately 22 bp length, which serve as regulators of gene expression and protein translation due to interference with messenger RNA (mRNA). MiRNAs, which take part in the regulation of cell cycle and apoptosis, may be associated with carcinogenesis. Aberrant expression of miRNAs in endometrial cancer might contribute to the endometrial cancer initiation or progression, as well as metastasis formation, and may influence cancer invasiveness. Specific-miRNAs expressed in endometrial cancer tissues may serve as diagnostic markers of the disease, prognostic biomarkers, or play an important part in oncological therapy We aimed to describe the role of miRNAs in endometrial cancer with special consideration of miRNA 205.

Evolution of the transcription complex during sporulation of Bacillus subtilis.

PubMed

Brevet, J

1976-01-01

Ribonucleic acid polymerase activity in partially purified extract of cells of Bacillus subtilis harvested at different times (t-1, to, t1, and t2) was studied by zone centrifugation. During the course of sporulation, vegetative sigma-factor activity decreased and the transcription complex lost some of its affinity for active sigma factor. The complex underwent a two-stage change in sedimentation value, from 14.5S in vegetative growth phase to a 13S species very early in sporulation to a 16S species at later times. Two SpoO mutants have been studied by zone centrifugation. One strain, a rifampin-resistant (RfmR) mutant, failed to show any modification of the transcription complex, whereas the other, a Rfms strain, underwent a partial evolution of the transcription complex after to.

Transcription of exogenous and endogenous deoxyribonucleic acid templates in cold-shocked Bacillus subtilis.

PubMed Central

Kuhl, S J; Brown, L R

1980-01-01

Ribonucleic acid (RNA) synthesis was examined in cold-shocked Bacillus subtilis cells. The cells were grown to mid-log stage, harvested, and cold shocked. RNA synthesis was monitored by the incorporation of [3H]uridine triphosphate or [alpha 32P]adenosine triphosphate into trichloroacetic acid-precipitable material in the presence of all four nucleoside triphosphates. The inhibition of RNA synthesis in cold-shocked cells by lipiarmycin, ethidium bromide, rifampin. or streptolydigin was analyzed using mutant or wild-type cells. Also examined were the effects of temperature, salt concentration, and the addition of polyamines or highly phosphorylated nucleotides. In ultraviolet-irradiated and cold-shocked cells, RNA wynthesis decreased to low levels. The addition of exogenous phi 29 or TSP-1 template to these cells caused a 13- to 20-fold increase in RNA synthesis, as monitored by trichloroacetic acid-precipitable counts. RNA synthesized in the presence of phi 29 deoxyribonucleic acid (DNA) hybridizes mainly to EcoRI fragments A and C of phi 29 DBA, These two fragments direct transcription by purified RNA polymerase in vitro and hybridize to early phi 29 DNA produced in vivo. Our results with TSP-1 DNA in this system indicated that the RNA produced hybridizes to the same fragments as early RNA produced in vivo. Plasmic pUB110 DNA was not transcribed in this system. Images PMID:6157674

The expression of 11β-hydroxysteroid dehydrogenase type 1 and 2 in nasal polyp-derived epithelial cells and its possible contribution to glucocorticoid activation in nasal polyp.

PubMed

Kook, Jin Ho; Kim, Hyun Jin; Kim, Kyung Won; Park, Se Jin; Kim, Tae Hoon; Lim, Sae Hee; Kang, Sung Hoon; Lee, Sang Hag

2015-01-01

The actions of glucocorticoids in target tissues depend on the local metabolism of glucocorticoids catalyzed by 11β hydroxysteroid dehydrogenase (HSD) 1 and 2. Glucocorticoids are the most effective anti-inflammatory drugs in the treatment of nasal polyps. However, the mechanisms that underlie the anti-inflammatory effects are unclear. The present study analyzed the expression of 11β-HSD1, 11β-HSD2, and steroidogenic enzymes (cytochrome P450, family 11, subfamily B, polypeptide 1 [CYP11B1]; cytochrome P450, family 11, subfamily A, polypeptide 1 [CYP11A1]) in nasal polyp tissues, and endogenous cortisol levels in nasal polyp-derived epithelial cells. The expression levels and distribution pattern of 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1 were determined in nasal polyp tissues or nasal polyp-derived epithelial cells by using real-time polymerase chain reaction, Western blot, and immunohistochemistry testing. The expression levels of cortisol by using enzyme-linked immunosorbent assay were determined in cultured polyp-derived epithelial cells treated with adrenocorticotrophic hormone (ACTH), 11β-HSD1 inhibitor, or small interfering ribonucleic acid technique. The effect of glucocorticoids on the expression levels of these enzymes was investigated in cultured cells. Expressed in nasal polyp tissues and nasal polyp-derived epithelial cells were 11β-HSD1, 11β-HSD2, CYP11B1, and CYP11A1. Cortisol production in cultured epithelial cells was decreased in cells treated with 11β-HSD1 small interfering ribonucleic acid or inhibitor, compared with nontreated cells. Cultured cells treated with adrenocorticotropic hormone induced increased cortisol production. 11β-HSD1 expression levels were upregulated in cells treated with glucocorticoid. Analysis of these results indicated that 11β-HSD1 expressed in polyp-derived epithelial cells may be involved in the anti-inflammatory function of glucocorticoid in the treatment of nasal polyps, which contributes to increased levels of endogenous cortisol.

Influence of continuous intervention on growth and metastasis of human cervical cancer cells and expression of RNAmiR-574-5p.

PubMed

Ma, D L; Li, J Y; Liu, Y E; Liu, C M; Li, J; Lin, G Z; Yan, J

2016-01-01

This study was carried out to acquire solid evidence that some common treatments could affect micro ribonucleic acids (miRNAs) by revealing the regulatory effect of genes, so as to provide a reference for further exploration of the prevention and treatment of cervical cancer. Nude mouse tumorigenicity assay was used to study the effect of inhibiting miR-574-5p on development and tumorigenic ability of Henrietta Lacks (HeLa) tumor. Cell wound scratch assay, flow cytometry and real-time quantitative polymerase chain reaction (RT-qPCR) were adopted to study the effects of anoxia and temperature, etc., on expression of miR-574-5p and QKI in HeLa as well as on the clone and migration ability of cells, to provide prevention and treatment of cervical cancer with new ideas and evidence. The results demonstrated that cervical cancer tissues had a significantly increased miR-574-5p expression compared with para-carcinoma tissues; conversely, Gomafu, overall QKI (pan-QKI) and QKI-5 messenger ribonucleic acid (mRNA) and protein expression all decreased. Part of the common nursing methods had a certain influence on miR-574-5p expression, HeLa reproduction and metastasis, and even cell cycle. For example, ultraviolet (UV) irradiation was effective in decreasing miR-574-5p expression of HeLa and inhibiting cell migration; severe hypoxia significantly decreased the survival rate of HeLa, leading to the increase of programmed death percentage and cell ratio in G2/M phase as well as the decrease of cell ratio in G1 phase. Incubation at different temperatures also affected miR-574-5p expression and cell proliferation. Thus, it can be known that miR-574-5p, Gomafu and QKI expression in cervical cancer tissues and para-carcinoma tissues are significantly up-regulated or down-regulated. Some treatments, such as UV irradiation, hypoxia, incubation temperatures, etc., can affect miR-574-5p expression and HeLa proliferation as well as metastases in different degrees. These findings provide a reference and basis for further study.

Spermatozoa micro ribonucleic acid-34c level is correlated with intracytoplasmic sperm injection outcomes.

PubMed

Cui, Long; Fang, Li; Shi, Biwei; Qiu, Sunquan; Ye, Yinghui

2015-08-01

To assess the effects of micro ribonucleic acid (miR)-34b/c expression levels in human spermatozoa on intracytoplasmic sperm injection (ICSI) outcomes. Retrospective observational study. In vitro fertilization center. A total of 162 patients with idiopathic male infertility who had undergone first ICSI cycles. None. The levels of miR-34b/c in spermatozoa were measured using real-time polymerase chain reaction. Fertilization, early cleavage, day-3 good-quality embryo, pregnancy, implantation, and live birth rate were assessed. A receiver operating characteristic curve was employed to analyze the cutoff values. No correlation was found between the spermatozoa miR-34b/c levels and the 2 pronuclei early cleavage rate. A correlation was seen between an increased level of miR-34c and a higher percentage of good-quality embryos on day 3. Although miR-34b and miR-34c levels were higher in the pregnancy group, compared with the nonpregnancy group, receiver operating characteristic curve analysis showed that miR-34c levels in spermatozoa were more strongly correlated with ICSI treatment outcomes, compared with miR-34b (area under the curve = 0.75). Patients in the miR-34c-positive group were more likely to exhibit higher rates of good-quality embryos, implantation, pregnancy, and live birth. A multivariable logistic regression analysis showed that miR-34c in spermatozoa (odds ratio: 5.699, with 95% confidence interval [CI]: 2.687-12.088) and woman's age (odds ratio: 0.843, with 95% CI: 0.736-0.966) were the 2 parameters that were significantly correlated with pregnancy. Our results demonstrate that miR-34c levels in spermatozoa are correlated with ICSI outcomes, suggesting that paternal miR-34c may play a role in the early phases of embryonic development. Levels of MiR-34c in human spermatozoa may be used as an indicator for ICSI outcomes. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Boletus edulis ribonucleic acid - a potent apoptosis inducer in human colon adenocarcinoma cells.

PubMed

Lemieszek, Marta Kinga; Ribeiro, Miguel; Guichard Alves, Helena; Marques, Guilhermina; Nunes, Fernando Milheiro; Rzeski, Wojciech

2016-07-13

Despite the large popularity of the Boletus edulis mushroom, little is known about its influence on human health and the possibilities of its therapeutic use. Nevertheless, several reports revealed the usefulness of biopolymers isolated from it in cancer treatment. Our previous studies have shown that B. edulis water soluble biopolymers are not toxic against normal colon epithelial cells (CCD841 CoTr) and at the same concentration range elicited a very prominent antiproliferative effect in colon cancer cells (LS180) which was accompanied with cell cycle arrest in the G0/G1 phase. The purpose of the present study was to verify the proapoptotic properties of a selected fraction from B. edulis - BE3, as well as determine its chemical nature. The BE3 fraction was extracted with hot water and purified by anion-exchange chromatography. Further chemical examinations revealed that BE3 consists mainly of ribonucleic acid (59.1%). The ability of BE3 to induce programmed cell death was examined in human colon cancer cell lines LS180 and HT-29 by measuring caspase activation, DNA fragmentation and expression of BAX, BCL2, TP53 and CDKN1A genes. The sensitivity of colon cancer cells with silenced BAX, TP53 and CDKN1A expression to BE3 treatment was also evaluated. We have demonstrated for the first time that the BE3 fraction is a potent apoptosis inducer in human colon cancer cells. The revealed mechanism of apoptosis triggering was dependent on the presence of functional p53 and consequently was a little different in investigated cell lines. Our results indicated that BE3 stimulated proapoptotic genes BAX (LS180, HT-29), TP53 (LS180) and CDKN1A (HT-29) while at the same time silenced the expression of the key prosurvival gene BCL2 (LS180, HT-29). The obtained results indicate the high therapeutic potential of the BE3 fraction against colon cancer, yet it is necessary to further confirm fraction efficacy and safety in animal and clinical studies.

Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes.

PubMed

Ma, Melissa; Crump, Doug; Farmahin, Reza; Kennedy, Sean W

2015-02-01

A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression. Bisphenol A (BPA) and 1 of its replacement alternatives, bisphenol S (BPS), were also screened for effects. Both TBBPA and BPA decreased CEH viability with calculated median lethal concentration (LC50) values of 40.6 μM and 61.7 μM, respectively. However, the replacement alternatives, TBBPA-DBPE and BPS, did not affect cell viability (up to 300 μM). Effects on mRNA expression were determined using an Avian ToxChip polymerse chain reaction (PCR) array and a real-time (RT)-PCR assay for the estrogen-responsive genes, apolipoproteinII (ApoII) and vitellogenin (Vtg). A luciferase reporter gene assay was used to assess dioxin-like effects. Tetrabromobisphenol-A altered mRNA levels of 4 genes from multiple toxicity pathways and increased luciferase activity in the luciferase reporter gene assay, whereas its alternative, TBBPA-DBPE, only altered 1 gene on the array, Cyp1a4, and increased luciferase activity. At 300 μM, a concentration that decreased cell viability for TBBPA and BPA, the BPA replacement, BPS, altered the greatest number of transcripts, including both ApoII and Vtg. Bisphenol A exposure did not alter any genes on the array but did up-regulate Vtg at 10 μM. Characterization of the potential toxicological and molecular-level effects of these compounds will ideally be useful to chemical regulators tasked with assessing the risk of new and existing chemicals. © 2014 SETAC.

FlindersTechnology Associates (FTA) filter paper-based DNA extraction with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii from respiratory specimens of immunocompromised patients.

PubMed

Nuchprayoon, Surang; Saksirisampant, Wilai; Jaijakul, Siraya; Nuchprayoon, Issarang

2007-01-01

We evaluated the diagnostic value of Flinders Technology Associates (FTA) filter paper together with polymerase chain reaction (PCR) for detection of Pneumocystis jirovecii (carinii) from induced sputum (IS) and bronchoalveolar lavage fluid (BALF) samples. The study involved 162 patients with clinical diagnosis of pneumocystis pneumonia (PcP) of human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients and other immunocompromised patients. P. jirovecii cysts or trophozoites were detected in IS and BALF by cytological method. The mitochondrial 5S ribosomal ribonucleic acid (rRNA) gene of P. jirovecii was amplified from these samples by using FTA filters together with a one-step PCR method (FTA-PCR). With the FTA-PCR method, the sensitivity and specificity of the test compared to microscopic examination were 67% and 90% for IS, while they were 67% and 91% for BALF, respectively. The sensitivity and specificity of the FTA-PCR test was also comparable to PCR with the conventional deoxyribonucleic acid (DNA) extraction method. We concluded that FTA-PCR is useful to detect P. jirovecii in noninvasive IS.

[Effects of bushen yinao tablet on physiology and cerebral gene expression in senescence-accelerated mice].

PubMed

Zhang, Chong; Wang, Jin-gang; Yang, Ting

2006-06-01

To study the effects of Bushen Yin' ao Tablet (BSYNT) on physiology and cerebral gene expression in senescence-accelerated mice (SAM). The change of cerebral tissues mRNA expression in SAM was analyzed and compared by messenger ribonucleic acids reverse transcription differential display polymerase chain reaction (mRNA DDRT-PCR) between the medicated group and the control group. BSYNT could increase the level of hemoglobin (Hb) and amount of erythrocyte (RBC) of blood deficiency mice, improve the spatial learning and memory function and the escape response by conditional stimulus. In this study, 14 differential display bands had been discerned, and three of them had been sequenced. The sequence of the three fragments was similar to fatty acid binding protein 7, ubiquinol-cytochrome C reductase complex (7. 2 kD) and 60S ribosomal protein L21 respectively. And the homogeneity was 97% , 100% , and 99% , respectively. BSYNT has effect on the physiological changing of mice, and its effect on cerebral tissues mRNA expression maybe play an important role in anti-aging on the molecular level.

Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70

PubMed Central

Bases, Robert

2005-01-01

Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation. PMID:15832946

Metabolism of 4-N-Hydroxy-Cytidine in Escherichia coli

PubMed Central

Trimble, R. B.; Maley, Frank

1971-01-01

4-N-hydroxy-cytidine was found to substitute for uridine as a pyrimidine supplement for the growth of Escherichia coli Bu−. Measurement of the incorporation of 4-N-hydroxy-cytidine-2-14C into ribonucleic acid and deoxyribonucleic acid revealed that this compound was converted to cytidine or uridine before utilization. Two pathways for metabolism were considered: (i) the reduction of 4-N-hydroxy-cytidine to cytidine followed by deamination, (ii) the direct hydrolysis of hydroxylamine from 4-N-hydroxy-cytidine to yield uridine. A threefold increase in cytidine (deoxycytidine) deaminase (EC 3.5.4.5) activity, when the cells were grown on 4-N-hydroxy-cytidine, suggested the involvement of this enzyme. More direct proof was obtained by purifying the deaminase 185-fold and finding that it released hydroxylamine from 4-N-hydroxy-cytidine at one-fiftieth the rate at which ammonia was removed from cytidine. This result is consistent with the slower rate of growth of the Bu− cells on 4-N-hydroxy-cytidine than cytidine and suggests that the second pathway is the major route for utilization of this compound. PMID:4941553

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts.

PubMed

Wrobel, Dominika; Kolanowska, Katarzyna; Gajek, Arkadiusz; Gomez-Ramirez, Rafael; de la Mata, Javier; Pedziwiatr-Werbicka, Elżbieta; Klajnert, Barbara; Waczulikova, Iveta; Bryszewska, Maria

2014-03-01

We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon-silicon bonds, but NN16 possesses some oxygen-silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1. By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA-dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation. Copyright © 2013 Elsevier B.V. All rights reserved.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

RNA Viruses: ROS-Mediated Cell Death

PubMed Central

Reshi, Mohammad Latif; Su, Yi-Che; Hong, Jiann-Ruey

2014-01-01

Reactive oxygen species (ROS) are well known for being both beneficial and deleterious. The main thrust of this review is to investigate the role of ROS in ribonucleic acid (RNA) virus pathogenesis. Much evidences has accumulated over the past decade, suggesting that patients infected with RNA viruses are under chronic oxidative stress. Changes to the body's antioxidant defense system, in relation to SOD, ascorbic acid, selenium, carotenoids, and glutathione, have been reported in various tissues of RNA-virus infected patients. This review focuses on RNA viruses and retroviruses, giving particular attention to the human influenza virus, Hepatitis c virus (HCV), human immunodeficiency virus (HIV), and the aquatic Betanodavirus. Oxidative stress via RNA virus infections can contribute to several aspects of viral disease pathogenesis including apoptosis, loss of immune function, viral replication, inflammatory response, and loss of body weight. We focus on how ROS production is correlated with host cell death. Moreover, ROS may play an important role as a signal molecule in the regulation of viral replication and organelle function, potentially providing new insights in the prevention and treatment of RNA viruses and retrovirus infections. PMID:24899897

In vitro evidence for UV-protection of the eye by the corneal epithelium mediated by the cytoplasmic protein, RNA, and ascorbate.

PubMed

Ringvold, A

1997-10-01

(1) To evaluate the effect of ribonucleic acid (RNA) and ascorbate on UV-absorption and their impact on ultraviolet-induced (UV) fluorescence from various proteins, and (2) to compare RNA and DNA reduction of protein fluorescence. These informations will be useful for later work on the UV-filtering effect of the corneal epithelium. Spectrophotometry and spectrofluorimetry. (1) RNA and ascorbate caused a significant UV-absorption and reduced the fluorescence from various water-soluble proteins, the degree of reduction varying independently from one protein to the other. (2) RNA and DNA showed protein fluorescence reduction of roughly the same order. The results are discussed both in the context of UV-protection of the cell nucleus in general, and the possible UV-filtering effect for the eye of bovine corneal epithelium.

Osteosarcoma: Diagnostic dilemmas in histopathology and prognostic factors

PubMed Central

Wadhwa, Neelam

2014-01-01

Osteosarcoma (OS), the commonest malignancy of osteoarticular origin, is a very aggressive neoplasm. Divergent histologic differentiation is common in OS; hence triple diagnostic approach is essential in all cases. 20% cases are atypical owing to lack of concurrence among clinicoradiologic and pathologic features necessitating resampling. Recognition of specific anatomic and histologic variants is essential in view of better outcome. Traditional prognostic factors of OS do stratify patients for short term outcome, but often fail to predict their long term outcome. Considering the negligible improvement in the patient outcome during the last 20 years, search for novel prognostic factors is in progress like ezrin vascular endothelial growth factor, chemokine receptors, dysregulation of various micro ribonucleic acid are potentially promising. Their utility needs to be validated by long term followup studies before they are incorporated in routine clinical practice. PMID:24932029

Development of Solid-State Nanopore Technology for Life Detection

NASA Technical Reports Server (NTRS)

Bywaters, K. B.; Schmidt, H.; Vercoutere, W.; Deamer, D.; Hawkins, A. R.; Quinn, R. C.; Burton, A. S.; Mckay, C. P.

2017-01-01

Biomarkers for life on Earth are an important starting point to guide the search for life elsewhere. However, the search for life beyond Earth should incorporate technologies capable of recognizing an array of potential biomarkers beyond what we see on Earth, in order to minimize the risk of false negatives from life detection missions. With this in mind, charged linear polymers may be a universal signature for life, due to their ability to store information while also inherently reducing the tendency of complex tertiary structure formation that significantly inhibit replication. Thus, these molecules are attractive targets for biosignature detection as potential "self-sustaining chemical signatures." Examples of charged linear polymers, or polyelectrolytes, include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as synthetic polyelectrolytes that could potentially support life, including threose nucleic acid (TNA) and other xenonucleic acids (XNAs). Nanopore analysis is a novel technology that has been developed for singlemolecule sequencing with exquisite single nucleotide resolution which is also well-suited for analysis of polyelectrolyte molecules. Nanopore analysis has the ability to detect repeating sequences of electrical charges in organic linear polymers, and it is not molecule- specific (i.e. it is not restricted to only DNA or RNA). In this sense, it is a better life detection technique than approaches that are based on specific molecules, such as the polymerase chain reaction (PCR), which requires that the molecule being detected be composed of DNA.

Removal of bacteria, protozoa and viruses through a multiple-barrier household water disinfection system.

PubMed

Espinosa-García, A C; Díaz-Ávalos, C; Solano-Ortiz, R; Tapia-Palacios, M A; Vázquez-Salvador, N; Espinosa-García, S; Sarmiento-Silva, R E; Mazari-Hiriart, M

2014-03-01

Municipal water disinfection systems in some areas are not always able to meet water consumer needs, such as ensuring distributed water quality, because household water management can be a contributing factor in water re-contamination. This fact is related to the storage options that are common in places where water is scarce or is distributed over limited time periods. The aim of this study is to assess the removal capacity of a multiple-barrier water disinfection device for protozoa, bacteria, and viruses. Water samples were taken from households in Mexico City and spiked with a known amount of protozoa (Giardia cyst, Cryptosporidium oocyst), bacteria (Escherichia coli), and viruses (rotavirus, adenovirus, F-specific ribonucleic acid (FRNA) coliphage). Each inoculated sample was processed through a multiple-barrier device. The efficiency of the multiple-barrier device to remove E. coli was close to 100%, and more than 87% of Cryptosporidium oocysts and more than 98% of Giardia cysts were removed. Close to 100% of coliphages were removed, 99.6% of the adenovirus was removed, and the rotavirus was almost totally removed. An effect of site by zone was detected; this observation is important because the water characteristics could indicate the efficiency of the multiple-barrier disinfection device.

RNase activity in erythroid cell lysates.

PubMed

Burka, E R

1969-09-01

The characteristics of degradation of reticulocyte ribonucleic acid (RNA) and ribosomes were studied in a whole erythroid cell lysate system. The process followed Michaelis-Menten kinetics, and indicated that RNA degradation in the erythroid cell is mediated by an enzyme previously isolated from reticulocyte hemolysates. Erythroid cell RNase activity had a temperature optimum of 50 degrees C, a pH optimum of 7.0, was not energy dependent, was heat labile at physiologic pH, and was inhibited by Mg(++), Ca(++), and exposure to bentonite and deoxycholate. Free sulfhydryl groups were not essential for RNase activity. Of the substrates occurring naturally within the erythroid cell, isolated ribosomal RNA was most susceptible to the action of the enzyme, intact ribosomes least susceptible, and transfer RNA intermediate between them. Natural substrates were degraded completely to nucleotides in cell lysates. Competitive inhibition studies indicate that one enzyme system is capable of degrading both RNA and ribosomes, although the existence of more than one enzyme has not been excluded. Erythroid cell lysates quickly broke down polyribosomes into single ribosomes. The more rapid degradation of ribosomes, as compared with transfer RNA, which occurs in vivo, as opposed to findings in vitro, suggests that there is a special intracellular mechanism responsible for ribosome degradation in the maturing erythroid cell.

Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1

PubMed Central

Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2016-01-01

Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5′-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). PMID:27292014

Evaluation of the process performance of a down-flow hanging sponge reactor for direct treatment of domestic wastewater in Bangkok, Thailand.

PubMed

Miyaoka, Yuma; Yoochatchaval, Wilasinee; Sumino, Haruhiko; Banjongproo, Pathan; Yamaguchi, Takashi; Onodera, Takashi; Okadera, Tomohiro; Syutsubo, Kazuaki

2017-08-24

This study assesses the performance of an aerobic trickling filter, down-flow hanging sponge (DHS) reactor, as a decentralized domestic wastewater treatment technology. Also, the characteristic eukaryotic community structure in DHS reactor was investigated. Long-term operation of a DHS reactor for direct treatment of domestic wastewater (COD = 150-170 mg/L and BOD = 60-90 mg/L) was performed under the average ambient temperature ranged from 28°C to 31°C in Bangkok, Thailand. Throughout the evaluation period of 550 days, the DHS reactor at a hydraulic retention time of 3 h showed better performance than the existing oxidation ditch process in the removal of organic carbon (COD removal rate = 80-83% and BOD removal rate = 91%), nitrogen compounds (total nitrogen removal rate = 45-51% and NH 4 + -N removal rate = 95-98%), and low excess sludge production (0.04 gTS/gCOD removed). The clone library based on the 18S ribosomal ribonucleic acid gene sequence revealed that phylogenetic diversity of 18S rRNA gene in the DHS reactor was higher than that of the present oxidation ditch process. Furthermore, the DHS reactor also demonstrated sufficient COD and NH 4 + -N removal efficiency under flow rate fluctuation conditions that simulates a small-scale treatment facility. The results show that a DHS reactor could be applied as a decentralized domestic wastewater treatment technology in tropical regions such as Bangkok, Thailand.

RNA isolation from mammalian cells using porous polymer monoliths: an approach for high-throughput automation.

PubMed

Chatterjee, Anirban; Mirer, Paul L; Zaldivar Santamaria, Elvira; Klapperich, Catherine; Sharon, Andre; Sauer-Budge, Alexis F

2010-06-01

The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.

Biochemical effect of chocolate colouring and flavouring like substances on thyroid function and protein biosynthesis.

PubMed

el-Saadany, S S

1991-01-01

Synthetic chocolate colourant, flavourant and the mixture of both were administered to healthy adult male albino rats to evaluate their effect on the nucleic acids metabolism, i.e. deoxyribonucleic and ribonucleic acids (DNA and RNA), total serum protein, thyroid hormones (T4 and T3) and nuclease enzymes, i.e. cytoplasmic- and mitochondrial deoxyribonuclease and ribonuclease (DNase and RNase) in brain, liver, and kidneys. Also, the activity of the fundamental enzymes of the oxidative pentose phosphate pathway, i.e. cytoplasmic and mitochondrial glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (G-6-PD and 6-PGD), as well as total lipids and cholesterol contents in the same organs were studied. Ingestion of the studied food additives significantly increased serum protein, RNA and T4 hormone, while, DNA and T3 hormone were insignificantly elevated. In connection with this, the hydrolytic enzymes of nucleic acids (DNase and RNase activities) were stimulated by all studied food additives and in all mentioned organs. The activity of G-6-PD and 6-PGD in both cytoplasmic and mitochondrial fractions of all studied organs were increased. The highest increase was noticed in rats fed on diets supplemented with the mixture of both colourant and flavourant followed by colourant then flavourant, respectively.

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

PubMed Central

Glasser, S. R.; Chytil, F.; Spelsberg, T. C.

1972-01-01

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se. PMID:4656807

An R132H Mutation in Isocitrate Dehydrogenase 1 Enhances p21 Expression and Inhibits Phosphorylation of Retinoblastoma Protein in Glioma Cells

PubMed Central

Miyata, Satsuki; Urabe, Masashi; Gomi, Akira; Nagai, Mutsumi; Yamaguchi, Takashi; Tsukahara, Tomonori; Mizukami, Hiroaki; Kume, Akihiro; Ozawa, Keiya; Watanabe, Eiju

2013-01-01

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1R132H-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1R132H-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progressionof the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation. PMID:24077277

An R132H mutation in isocitrate dehydrogenase 1 enhances p21 expression and inhibits phosphorylation of retinoblastoma protein in glioma cells.

PubMed

Miyata, Satsuki; Urabe, Masashi; Gomi, Akira; Nagai, Mutsumi; Yamaguchi, Takashi; Tsukahara, Tomonori; Mizukami, Hiroaki; Kume, Akihiro; Ozawa, Keiya; Watanabe, Eiju

2013-01-01

Cytosolic isocitrate dehydrogenase 1 (IDH1) with an R132H mutation in brain tumors loses its enzymatic activity for catalyzing isocitrate to α-ketoglutarate (α-KG) and acquires new activity whereby it converts α-KG to 2-hydroxyglutarate. The IDH1 mutation induces down-regulation of tricarboxylic acid cycle intermediates and up-regulation of lipid metabolism. Sterol regulatory element-binding proteins (SREBPs) regulate not only the synthesis of cholesterol and fatty acids but also acyclin-dependent kinase inhibitor p21 that halts the cell cycle at G1. Here we show that SREBPs were up-regulated in U87 human glioblastoma cells transfected with an IDH1(R132H)-expression plasmid. Small interfering ribonucleic acid (siRNA) for SREBP1 specifically decreased p21 messenger RNA (mRNA) levels independent of the p53 pathway. In IDH1(R132H)-expressing U87 cells, phosphorylation of Retinoblastoma (Rb) protein also decreased. We propose that metabolic changes induced by the IDH1 mutation enhance p21 expression via SREBP1 and inhibit phosphorylation of Rb, which slows progression of the cell cycle and may be associated with non-aggressive features of gliomas with an IDH1 mutation.

Enhancing the anaerobic digestion potential of dairy waste activated sludge by two step sono-alkalization pretreatment.

PubMed

Rani, R Uma; Kumar, S Adish; Kaliappan, S; Yeom, Ick-Tae; Banu, J Rajesh

2014-05-01

High efficiency resource recovery from dairy waste activated sludge (WAS) has been a focus of attention. An investigation into the influence of two step sono-alkalization pretreatment (using different alkaline agents, pH and sonic reaction times) on sludge reduction potential in a semi-continuous anaerobic reactor was performed for the first time in literature. Firstly, effect of sludge pretreatment was evaluated by COD solubilization, suspended solids reduction and biogas production. At optimized condition (4172 kJ/kg TS of supplied energy for NaOH - pH 10), COD solubilization, suspended solids reduction and biogas production was 59%, 46% and 80% higher than control. In order to clearly describe the hydrolysis of waste activated sludge during sono-alkalization pretreatment by a two step process, concentrations of ribonucleic acid (RNA) and bound extracellular polymeric substance (EPS) were also measured. Secondly, semi-continuous process performance was studied in a lab-scale semi-continuous anaerobic reactor (5L), with 4 L working volume. With three operated SRTs, the SRT of 15 d was found to be most appropriate for economic operation of the reactor. Combining pretreatment with anaerobic digestion led to 58% and 62% of suspended solids and volatile solids reduction, respectively, with an improvement of 83% in biogas production. Thus, two step sono-alkalization pretreatment laid the basis in enhancing the anaerobic digestion potential of dairy WAS. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of the Catalytic Subunits of the RNA Exosome-like Complex in Plasmodium falciparum.

PubMed

Jiang, Ning; Yu, Shengchao; Yang, Na; Feng, Ying; Sang, Xiaoyu; Wang, Yao; Wahlgren, Mats; Chen, Qijun

2018-04-17

The eukaryotic ribonucleic acid (RNA) exosome is a versatile multiribonuclease complex that mediates the processing, surveillance, and degradation of virtually all classes of RNA in both the nucleus and cytoplasm. The complex, composed of 10 to 11 subunits, has been widely described in many organisms. Bioinformatic analyses revealed that there may be also an exosome-like complex in Plasmodium falciparum, a parasite of great importance in public health, with eight predicted subunits having high sequence similarity to their counterparts in yeast and human. In this work, the putative RNA catalytic components, designated as PfRrp4, PfRrp41, PfDis3, and PfRrp6, were identified and systematically analyzed. Quantitative polymerase chain reaction (QPCR) analyses suggested that all of them were transcribed steadily throughout the asexual stage. The expression of these proteins was determined by Western blot, and their localization narrowed to the cytoplasm of the parasite by indirect immunofluorescence. The recombinant proteins of PfRrp41, PfDis3, and PfRrp6 exhibited catalytic activity for single-stranded RNA (ssRNA), whereas PfRrp4 showed no processing activity of both ssRNA and dsRNA. The identification of these putative components of the RNA exosome complex opens up new perspectives for a deep understanding of RNA metabolism in the malarial parasite P. falciparum. © 2018 The Authors Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

Ground-water quality and discharge to Chincoteague and Sinepuxent Bays adjacent to Assateague Island National Seashore, Maryland

USGS Publications Warehouse

Dillow, Jonathan J.A.; Banks, William S.L.; Smigaj, Michael J.

2002-01-01

The U.S. Geological Survey, in cooperation with the Maryland Department of the Environment and the Wisconsin State Laboratory of Hygiene, conducted a study to characterize the occurrence and distribution of viral contamination in small (withdrawing less than 10,000 gallons per day) public water-supply wells screened in the shallow aquifer in the Piedmont Physiographic Province in Baltimore and Harford Counties, Maryland. Two hundred sixty-three small public water-supply wells were in operation in these counties during the spring of 2000. Ninety-one of these sites were selected for sampling using a methodology that distributed the samples evenly over the population and the spatial extent of the study area. Each site, and its potential susceptibility to microbiological contamination, was evaluated with regard to hole depth, casing interval, and open interval. Each site was evaluated using characteristics such as on-site geology and on-site land use.Samples were collected by pumping between 200 and 400 gallons of untreated well water through an electropositive cartridge filter. Water concentrates were subjected to cell-culture assay for the detection of culturable viruses and reverse-transcription polymerase chain reaction/gene probe assays to detect viral ribonucleic acid; grab samples were analyzed for somatic and male-specific coliphages, Bacteroides fragilis, Clostridium perfringens, enterococci, Escherichia coli, total coliforms, total oxidized nitrogen, nitrite, organic nitrogen, total phosphate, ortho-phosphate, calcium, magnesium, sodium, potas-sium, chloride, sulfate, iron, acid-neutralizing capacity, pH, specific conductance, temperature, and dissolved oxygen.One sample tested positive for the presence of the ribonucleic acid of rotavirus through poly-merase chain-reaction analysis. Twenty-nine per-cent of the samples (26 of 90) had bacterial con-tamination. About 7 percent of the samples (6 of 90) were contaminated with either male-specific coliphage, somatic coliphage, or bacteriophages of Bacteroides fragilis. About 3 percent of the sam-ples (3 of 87) had oxidized nitrogen concentra-tions that exceeded the U.S. Environmental Protection Agency?s Maximum Contaminant Level of 10.0 milligrams per liter. A statistical analysis showed that no significant relation exists between the presence of bacteria or coliphage and all variables, except the mean temperature of the water sample as measured in the field. Additionally, the concentration of total coliform bacteria had a statistically significant, moderately strong cor-relation with the concentration of sulfate and sample pH as measured at the U.S. Geological Survey National Water-Quality Laboratory in Denver, Colorado.

Occurrence and distribution of microbiological contamination and enteric viruses in shallow ground water in Baltimore and Harford counties, Maryland

USGS Publications Warehouse

Banks, William S.L.; Battigelli, David A.

2002-01-01

The U.S. Geological Survey, in cooperation with the Maryland Department of the Environment and the Wisconsin State Laboratory of Hygiene, conducted a study to characterize the occurrence and distribution of viral contamination in small (withdrawing less than 10,000 gallons per day) public water-supply wells screened in the shallow aquifer in the Piedmont Physiographic Province in Baltimore and Harford Counties, Maryland. Two hundred sixty-three small public water-supply wells were in operation in these counties during the spring of 2000. Ninety-one of these sites were selected for sampling using a methodology that distributed the samples evenly over the population and the spatial extent of the study area. Each site, and its potential susceptibility to microbiological contamination, was evaluated with regard to hole depth, casing interval, and open interval. Each site was evaluated using characteristics such as on-site geology and on-site land use.Samples were collected by pumping between 200 and 400 gallons of untreated well water through an electropositive cartridge filter. Water concentrates were subjected to cell-culture assay for the detection of culturable viruses and reverse-transcription polymerase chain reaction/gene probe assays to detect viral ribonucleic acid; grab samples were analyzed for somatic and male-specific coliphages, Bacteroides fragilis, Clostridium perfringens, enterococci, Escherichia coli, total coliforms, total oxidized nitrogen, nitrite, organic nitrogen, total phosphate, ortho-phosphate, calcium, magnesium, sodium, potas-sium, chloride, sulfate, iron, acid-neutralizing capacity, pH, specific conductance, temperature, and dissolved oxygen.One sample tested positive for the presence of the ribonucleic acid of rotavirus through poly-merase chain-reaction analysis. Twenty-nine per-cent of the samples (26 of 90) had bacterial con-tamination. About 7 percent of the samples (6 of 90) were contaminated with either male-specific coliphage, somatic coliphage, or bacteriophages of Bacteroides fragilis. About 3 percent of the sam-ples (3 of 87) had oxidized nitrogen concentra-tions that exceeded the U.S. Environmental Protection Agency?s Maximum Contaminant Level of 10.0 milligrams per liter. A statistical analysis showed that no significant relation exists between the presence of bacteria or coliphage and all variables, except the mean temperature of the water sample as measured in the field. Additionally, the concentration of total coliform bacteria had a statistically significant, moderately strong cor-relation with the concentration of sulfate and sample pH as measured at the U.S. Geological Survey National Water-Quality Laboratory in Denver, Colorado.

The Hepatitis Testing and Linkage-to-Care Data Review Process: An Approach to Ensuring the Quality of Program Data.

PubMed

Mezzo, Jennifer L; Lamia, Tamara L; Danelski, Lisa L; Schipani, Anne Marie; Stokes, Scott A; Jacobs-Ware, Elizabeth D

2016-01-01

CDC's 2012 Hepatitis Testing and Linkage to Care (HepTLC) initiative was a nationally coordinated effort to conduct hepatitis B and hepatitis C screening, posttest counseling, and linkage to care at 34 U.S. sites. This project provided support for data management and monthly data reviews between awardees and a data manager, which facilitated monitoring of awardee progress and regular program improvement opportunities. CDC provided technical assistance to awardees for testing processes and program improvement, including Internet-based data submission, reporting software and data management to awardees, offering assistance with submitting, and reviewing data in real time. We describe how one awardee, AIDS Resource Center of Wisconsin (ARCW), used the data management process to improve data quality, inform testing processes and implementation, and measure and report missing variables from an online database. From October 2012 through July 2014, ARCW performed 2,255 HCV antibody (anti-HCV) tests and 244 HCV ribonucleic acid (RNA) tests as part of the HepTLC initiative. Participants who tested HCV RNA positive (n=189) were referred to medical care. At the end of the study, no records were missing for the anti-HCV test result or HCV RNA test result variables, and only one record was missing for those who were referred to medical care. Regular data review and monitoring by awardees and CDC-supported data managers provided opportunities for data quality and program improvement. Through regular data review, ARCW reduced the amount of missing data and promoted timely follow-up with participants testing positive for HCV to ensure receipt of results and linkage to care. Other programs can adopt a similar data management model.

Organic acid production from potato starch waste fermentation by rumen microbial communities from Dutch and Thai dairy cows.

PubMed

Palakawong Na Ayudthaya, Susakul; van de Weijer, Antonius H P; van Gelder, Antonie H; Stams, Alfons J M; de Vos, Willem M; Plugge, Caroline M

2018-01-01

Exploring different microbial sources for biotechnological production of organic acids is important. Dutch and Thai cow rumen samples were used as inocula to produce organic acid from starch waste in anaerobic reactors. Organic acid production profiles were determined and microbial communities were compared using 16S ribosomal ribonucleic acid gene amplicon pyrosequencing. In both reactors, lactate was the main initial product and was associated with growth of Streptococcus spp. (86% average relative abundance). Subsequently, lactate served as a substrate for secondary fermentations. In the reactor inoculated with rumen fluid from the Dutch cow, the relative abundance of Bacillus and Streptococcus increased from the start, and lactate, acetate, formate and ethanol were produced. From day 1.33 to 2, lactate and acetate were degraded, resulting in butyrate production. Butyrate production coincided with a decrease in relative abundance of Streptococcus spp. and increased relative abundances of bacteria of other groups, including Parabacteroides , Sporanaerobacter , Helicobacteraceae, Peptostreptococcaceae and Porphyromonadaceae. In the reactor with the Thai cow inoculum, Streptococcus spp. also increased from the start. When lactate was consumed, acetate, propionate and butyrate were produced (day 3-4). After day 3, bacteria belonging to five dominant groups, Bacteroides, Pseudoramibacter _ Eubacterium , Dysgonomonas , Enterobacteriaceae and Porphyromonadaceae, were detected and these showed significant positive correlations with acetate, propionate and butyrate levels. The complexity of rumen microorganisms with high adaptation capacity makes rumen fluid a suitable source to convert organic waste into valuable products without the addition of hydrolytic enzymes. Starch waste is a source for organic acid production, especially lactate.

First report of Sapajus cay naturally infected by Trypanosoma cruzi in San Pedro Department, Paraguay.

PubMed

Acosta, Nidia; Miret, Jorge; López, Elsa; Schinini, Alicia

2016-08-29

To verify the occurrence of natural Trypanosoma cruzi infection in non-human primates from a rural endemic area of the east region of Paraguay, xenodiagnosis was performed in 35 animals belonging to two species. For genotyping and T. cruzi discrete typing unit (DTU) assignment, a combination of four markers was used, including amplification products of the small (18S) and large (24Sα) subunits of ribosomal ribonucleic acid gene, the intergenic region of mini-exon gene and the heat shock protein 60 Eco-RV polymerase chain reaction-restriction fragment length polymorphism (HSP60/EcoRV-PCR-RFLP). One specimen of Sapajus cay was found positive and infected by the DTU TcII. This result constitutes the first record of natural T. cruzi infection in a sylvatic monkey in Paraguay, harbouring a DTU associated with severe Chagas disease in humans.

Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

NASA Astrophysics Data System (ADS)

Lin, Chenxiang; Jungmann, Ralf; Leifer, Andrew M.; Li, Chao; Levner, Daniel; Church, George M.; Shih, William M.; Yin, Peng

2012-10-01

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by ˜40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

Synergistic Effect of Auto-Activation and Small RNA Regulation on Gene Expression

NASA Astrophysics Data System (ADS)

Xiong, Li-Ping; Ma, Yu-Qiang; Tang, Lei-Han

2010-09-01

Auto-activation and small ribonucleic acid (RNA)-mediated regulation are two important mechanisms in controlling gene expression. We study the synergistic effect of these two regulations on gene expression. It is found that under this combinatorial regulation, gene expression exhibits bistable behaviors at the transition regime, while each of these two regulations, if working solely, only leads to monostability. Within the stochastic framework, the base pairing strength between sRNA and mRNA plays an important role in controlling the transition time between on and off states. The noise strength of protein number in the off state approaches 1 and is smaller than that in the on state. The noise strength also depends on which parameters, the feedback strength or the synthesis rate of small RNA, are tuned in switching the gene expression on and off. Our findings may provide a new insight into gene-regulation mechanism and can be applied in synthetic biology.

New Insights Into “Plant Memories”

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanbonmatsu, Karissa

A special stretch of ribonucleic acid (RNA) called COOLAIR is revealing its inner structure and function to scientists, displaying a striking resemblance to an RNA molecular machine, territory previously understood to be limited to the cells’ protein factory (the ‘ribosome’) and not a skill set given to mere strings of RNA. “We are uncovering the nuts and bolts of plant memories,” said Karissa Sanbonmatsu of Los Alamos National Laboratory, lead author on a new article this week in the journal Cell Reports. In the past 5 years or so, material in the cell known as “junk DNA” had actually turnedmore » out not to be junk at all. Instead, it was shown to produce RNA molecules that play key roles in the development of organs in the embryo, as well as affecting cancer, brain function and plant biology.« less

Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight

DOE Office of Scientific and Technical Information (OSTI.GOV)

Touré, B. Barry; Giraldes, John; Smith, Troy

2016-05-26

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency.more » These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.« less

Galactosylated magnetic nanovectors for regulation of lipid metabolism based on biomarker-specific RNAi and MR imaging.

PubMed

Heo, Dan; Lee, Chanjoo; Ku, Minhee; Haam, Seungjoo; Suh, Jin-Suck; Huh, Yong-Min; Park, Sahng Wook; Yang, Jaemoon

2015-08-21

The specific delivery of ribonucleic acid (RNA) interfering molecules to disease-related cells is still a critical blockade for in vivo systemic treatment. Here, this study suggests a robust delivery carrier for targeted delivery of RNA-interfering molecules using galactosylated magnetic nanovectors (gMNVs). gMNVs are an organic-inorganic polymeric nanomaterial composed of polycationics and magnetic nanocrystal for delivery of RNA-interfering molecules and tracking via magnetic resonance (MR) imaging. In particular, the surface of gMNVs was modified by galactosylgluconic groups for targeted delivering to asialoglycoprotein receptor (ASGPR) of hepatocytes. Moreover, the small interfering RNAs were used to regulate target proteins related with low-density lipoprotein level and in vivo MR imaging was conducted for tracking of nanovectors. The obtained results show that the prepared gMNVs demonstrate potential as a systemic theragnostic nanoplatform for RNA interference and MR imaging.

A PAR-1–dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte

PubMed Central

Parton, Richard M.; Hamilton, Russell S.; Ball, Graeme; Yang, Lei; Cullen, C. Fiona; Lu, Weiping; Ohkura, Hiroyuki

2011-01-01

Cytoskeletal organization is central to establishing cell polarity in various cellular contexts, including during messenger ribonucleic acid sorting in Drosophila melanogaster oocytes by microtubule (MT)-dependent molecular motors. However, MT organization and dynamics remain controversial in the oocyte. In this paper, we use rapid multichannel live-cell imaging with novel image analysis, tracking, and visualization tools to characterize MT polarity and dynamics while imaging posterior cargo transport. We found that all MTs in the oocyte were highly dynamic and were organized with a biased random polarity that increased toward the posterior. This organization originated through MT nucleation at the oocyte nucleus and cortex, except at the posterior end of the oocyte, where PAR-1 suppressed nucleation. Our findings explain the biased random posterior cargo movements in the oocyte that establish the germline and posterior. PMID:21746854

Practical diagnostic testing for human immunodeficiency virus.

PubMed Central

Jackson, J B; Balfour, H H

1988-01-01

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures. Images PMID:3060241

Activation of the innate immune system provides broad-spectrum protection against influenza A viruses with pandemic potential in mice

PubMed Central

Lau, Yuk-Fai; Tang, Lay-Hoon; Ooi, Eng-Eong; Subbarao, Kanta

2010-01-01

The efficacy of a stabilized chemical analog of double-stranded ribonucleic acid (RNA), PIKA, as prophylaxis against infection with 5 different influenza A virus subtypes, including the 2009 swine-origin pandemic H1N1 virus, was evaluated in mice. Intranasal treatment with PIKA resulted in significant reduction of viral replication in the respiratory tract. The inhibitory effect was mediated by rapid infiltration of immune cells into the lungs, and production of inflammatory cytokines. While TLR3 is important for the optimal production of these inflammatory cytokines, inhibition of viral replication was still observed in TLR3−/− mice. In addition, a significant synergistic effect in inhibiting H5N1 virus replication was observed when PIKA was co-administered with oseltamivir. The broad-spectrum protection provided by PIKA makes it an attractive option for prophylaxis from infection with influenza A viruses. PMID:20667572

Tackling both the player and the ball: lessons from crystallographic studies on the N-utilization substance B (NusB) from Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

Haire, L. F.; Gopal, B.

2001-11-01

The N-utilization substance B (NusB) from Mycobacterium tuberculosis is an important element in a complex assembly of other proteins and ribonucleic acid effecting transcription antitermination in this organism. The cloning and overexpression of the protein in E. coli, followed by the purification, crystallization, and use of selenomethionine samples to obtain phase information by anomalous dispersion techniques, allows us to investigate the fine interplay of sample engineering and modification of crystallization parameters leading to successful structure determination. Knowledge of the crystal structure and the surface properties of the protein allows an analysis of the packing of the NusB dimers in the crystal lattice. This exercise, albeit post facto, helps to demonstrate how biophysical and functional information could help 'rationalize' the course of obtaining protein crystals suitable for structural studies.

Photoacoustic imaging with an acoustic lens detects prostate cancer cells labeled with PSMA-targeting near-infrared dye-conjugates

NASA Astrophysics Data System (ADS)

Dogra, Vikram; Chinni, Bhargava; Singh, Shalini; Schmitthenner, Hans; Rao, Navalgund; Krolewski, John J.; Nastiuk, Kent L.

2016-06-01

There is an urgent need for sensitive and specific tools to accurately image early stage, organ-confined human prostate cancers to facilitate active surveillance and reduce unnecessary treatment. Recently, we developed an acoustic lens that enhances the sensitivity of photoacoustic imaging. Here, we report the use of this device in conjunction with two molecular imaging agents that specifically target the prostate-specific membrane antigen (PSMA) expressed on the tumor cell surface of most prostate cancers. We demonstrate successful imaging of phantoms containing cancer cells labeled with either of two different PSMA-targeting agents, the ribonucleic acid aptamer A10-3.2 and a urea-based peptidomimetic inhibitor, each linked to the near-infrared dye IRDye800CW. By specifically targeting cells with these agents linked to a dye chosen for optimal signal, we are able to discriminate prostate cancer cells that express PSMA.

A 20-basepair duplication in the human thyroid peroxidase gene results in a total iodide organification defect and congenital hypothyroidism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bikker, H.; Hartog, M.T. den; Gons, M.H.

1994-07-01

In this study, the authors present the molecular basis of a total iodide organification defect causing severe congenital hypothyroidism. In the thyroid gland of the patient, thyroid peroxidase (TPO) activity and the iodination degree of thyroglobulin were below detection limits, and no TPO messenger ribonucleic acid was detectable by Northern blot analysis. Denaturing gradient gel electrophoretic analysis of the TPO gene of the patient revealed a homozygous mutation in exon 2. Sequence analysis showed the presence of a 20-basepair duplication, 47 basepairs down-stream of the ATG start codon. This duplication generates a frame shift, resulting in a termination signal inmore » exon 3, compatible with the complete absence of TPO. Both parents of the patient are heterozygous for the same duplication, confirming the recessive mode of inheritance of the mutation. 32 refs., 4 figs.« less

Genetic analysis of an Escherichia coli syndrome.

PubMed

Lennette, E T; Apirion, D

1971-12-01

A mutant strain of Escherichia coli that fails to recover from prolonged (72 hr) starvation also fails to grow at 43 C. Extracts of this mutant strain show an increased ribonuclease II activity as compared to extracts of the parental strain, and stable ribonucleic acid is degraded to a larger extent in this strain during starvation. Ts(+) transductants and revertants were tested for all the above-mentioned phenotypes. All the Ts(+) transductants and revertants tested behaved like the Ts(+) parental strain, which suggests that all the observed phenotypes are caused by a single sts (starvation-temperature sensitivity) mutation. The reversion rate from sts(-) to sts(+) is rather low but is within the range of reversion rates for other single-site mutations. Three-point transduction crosses located this sts mutation between the ilv and rbs genes. The properties of sts(+)/sts(-) merozygotes suggested that the Ts(-) phenotype of this mutation is recessive.

Toxicity of nalidixic acid on candida albicans, Saccharomyces cerevisiae, and Kluyveromyces lactis.

PubMed

Sobieski, R J; Brewer, A R

1976-03-01

The antibacterial drug nalidixic acid (Nal) can suppress the growth of Candida albicans at levels of the drug normally found in urine. Growth suppression increases as drug levels are increased, and Nal also causes a similar proportional inhibition of the synthesis of all cellular macromolecules. However, growth temperature (25 versus 37 C) and the divalent cations Mg(2+) and Mn(2+) can increase C. albicans resistance to Nal. Also, nitrogen depletion of Candida shows that Nal-treated and untreated cells exhibit no difference in leucine uptake during readaptation to nitrogen. In Nal-treated, nitrogen-starved cells, ribonucleic acid and deoxyribonucleic acid (DNA) biosynthesis are less affected than in unstarved Nal-treated cells, but of the two nucleic acids DNA synthesis is the most affected. Nal-resistant strains of C. albicans exhibit a slight toxicity for macromolecular synthesis. Nal treatment of a synchronized population of Saccharomyces cerevisiae results in an increase in the culture mean doubling time of, at most, 20%, but Nal causes the loss of synchronous cell division. With a synchronized population of Kluyveromyces lactis, Nal causes an increase in the mean doubling time of upwards of 300%, with synchrony of cell division being maintained. It is known that S. cerevisiae asynchronously synthesizes mitochondrial DNA during the cell cycle, whereas with K. lactis it is synchronous. Thus, with C. albicans Nal toxicity is dependent both on the dose and the physiological state of the cell. Furthermore, Nal inhibits growth of yeast with synchronous mitochondrial DNA synthesis more adversely than yeast with asynchronous mitochondrial DNA synthesis.

Biological Uptake of Phosphorus by Activated Sludge 1

PubMed Central

Yall, Irving; Boughton, William H.; Knudsen, Richard C.; Sinclair, Norval A.

1970-01-01

The ability of activated sludge to remove phosphates was studied by adding carrier-free 32P to raw sewage and measuring incorporation of the radioactivity into the cells over a period of time. Radioisotope determinations indicated that 48% of the 32P radioactivity was removed by 12 hr. However, chemical methods indicated that only 30% of the orthophosphate apparently disappeared from the sewage during this period. Experiments with sludge prelabeled with 32P indicated that considerable phosphate turnover occurred. The cells released large amounts of radioactivity as they were incorporating fresh phosphates. Starvation in isotonic saline for 18 hr caused the sludge to dump phosphate. When introduced into fresh sewage containing 32P, the starved sludge removed about 60% of the radioactivity in 6 hr with little phosphate turnover. The ability of sludge to remove 32P was inhibited approximately 83% by 10−3m 2,4-dinitrophenol. This inhibition was at the expense of the cell fraction that contained ribonucleic acid and deoxyribonucleic acid. The sludge cells released orthophosphate when exposed to the chemical agent. Experiments using 45Ca indicated that calcium phosphate precipitation plays a minor role in phosphate removal under our experimental conditions. PMID:5456935

Biological uptake of phosphorus by activated sludge.

PubMed

Yall, I; Boughton, W H; Knudsen, R C; Sinclair, N A

1970-07-01

The ability of activated sludge to remove phosphates was studied by adding carrier-free (32)P to raw sewage and measuring incorporation of the radioactivity into the cells over a period of time. Radioisotope determinations indicated that 48% of the (32)P radioactivity was removed by 12 hr. However, chemical methods indicated that only 30% of the orthophosphate apparently disappeared from the sewage during this period. Experiments with sludge prelabeled with (32)P indicated that considerable phosphate turnover occurred. The cells released large amounts of radioactivity as they were incorporating fresh phosphates. Starvation in isotonic saline for 18 hr caused the sludge to dump phosphate. When introduced into fresh sewage containing (32)P, the starved sludge removed about 60% of the radioactivity in 6 hr with little phosphate turnover. The ability of sludge to remove (32)P was inhibited approximately 83% by 10(-3)m 2,4-dinitrophenol. This inhibition was at the expense of the cell fraction that contained ribonucleic acid and deoxyribonucleic acid. The sludge cells released orthophosphate when exposed to the chemical agent. Experiments using (45)Ca indicated that calcium phosphate precipitation plays a minor role in phosphate removal under our experimental conditions.

Label-free, direct localization and relative quantitation of the RNA nucleobase methylations m6A, m5C, m3U, and m5U by top-down mass spectrometry.

PubMed

Glasner, Heidelinde; Riml, Christian; Micura, Ronald; Breuker, Kathrin

2017-07-27

Nucleobase methylations are ubiquitous posttranscriptional modifications of ribonucleic acids (RNA) that can substantially increase the structural diversity of RNA in a highly dynamic fashion with implications for gene expression and human disease. However, high throughput, deep sequencing does not generally provide information on posttranscriptional modifications (PTMs). A promising alternative approach for the characterization of PTMs, i.e. their identification, localization, and relative quantitation, is top-down mass spectrometry (MS). In this study, we have investigated how specific nucleobase methylations affect RNA ionization in electrospray ionization (ESI), and backbone cleavage in collisionally activated dissociation (CAD) and electron detachment dissociation (EDD). For this purpose, we have developed two new approaches for the characterization of RNA methylations in mixtures of either isomers of RNA or nonisomeric RNA forms. Fragment ions from dissociation experiments were analyzed to identify the modification type, to localize the modification sites, and to reveal the site-specific, relative extent of modification for each site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Web-accessible molecular modeling with Rosetta: The Rosetta Online Server that Includes Everyone (ROSIE).

PubMed

Moretti, Rocco; Lyskov, Sergey; Das, Rhiju; Meiler, Jens; Gray, Jeffrey J

2018-01-01

The Rosetta molecular modeling software package provides a large number of experimentally validated tools for modeling and designing proteins, nucleic acids, and other biopolymers, with new protocols being added continually. While freely available to academic users, external usage is limited by the need for expertise in the Unix command line environment. To make Rosetta protocols available to a wider audience, we previously created a web server called Rosetta Online Server that Includes Everyone (ROSIE), which provides a common environment for hosting web-accessible Rosetta protocols. Here we describe a simplification of the ROSIE protocol specification format, one that permits easier implementation of Rosetta protocols. Whereas the previous format required creating multiple separate files in different locations, the new format allows specification of the protocol in a single file. This new, simplified protocol specification has more than doubled the number of Rosetta protocols available under ROSIE. These new applications include pK a determination, lipid accessibility calculation, ribonucleic acid redesign, protein-protein docking, protein-small molecule docking, symmetric docking, antibody docking, cyclic toxin docking, critical binding peptide determination, and mapping small molecule binding sites. ROSIE is freely available to academic users at http://rosie.rosettacommons.org. © 2017 The Protein Society.

Ribonucleic Acid Synthesis by Cucumber Chromatin

PubMed Central

Johnson, Kenneth D.; Purves, William K.

1970-01-01

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A7 (GA7), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA7 also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA7 solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment. Age of the seedling tissue had a profound influence on the chromatin characteristics. With progression of development from the 2-day to the 4-day stage, the endogenous chromatin activity declined while the DNA template activity increased. PMID:16657509

The use of a sensitive equilibrium dialysis method for the measurement of free testosterone levels in healthy, cycling women and in human immunodeficiency virus-infected women.

PubMed

Sinha-Hikim, I; Arver, S; Beall, G; Shen, R; Guerrero, M; Sattler, F; Shikuma, C; Nelson, J C; Landgren, B M; Mazer, N A; Bhasin, S

1998-04-01

Measurements of total and free testosterone levels in women have lacked precision and accuracy because of limited assay sensitivity. The paucity of normative data on total and free testosterone levels in healthy women has confounded interpretation of androgen levels in women with human immunodeficiency virus (HIV) infection and other disease states. Therefore, the objectives of this study were to develop sensitive assays for the measurement of the low total and free testosterone levels in women to define the range for these hormones during the normal menstrual cycle and assess the total and free testosterone levels in HIV-infected women. By using a larger volume of serum, increasing the incubation time, and reducing the antibody concentration, the sensitivity of the total testosterone assay was increased to 0.008 nmol/L, and that of the free testosterone assay was increased to 2 pmol/L. The mean percent free testosterone was 1.0 +/- 0.1% of the total testosterone. Serum total and free testosterone levels in the follicular and luteal phases were not significantly different, but both demonstrated a modest preovulatory increase, 3 days before the LH peak. Serum total [0.50 +/- 0.32 (14.60 +/- 9.22) vs. 1.2 +/- 0.7 nmol/L (34.3 +/- 21.0 ng/dL); P < 0.0001] and free testosterone levels (5.56 +/- 2.70 (1.58 +/- 0.80) vs. 12.8 +/- 5.5 pmol/L (3.4 +/- 1.7 pg/mL); P < 0.0001) were significantly lower in HIV-infected women (n = 37) than in healthy women (n = 34). Serum total and free testosterone levels were also significantly lower in HIV-infected women who were menstruating normally. There were no significant differences in serum total and free testosterone levels between those who had lost weight and those who had not. Testosterone levels correlated inversely with plasma HIV ribonucleic acid copy number. Serum FSH, but not LH, levels were significantly higher in HIV-infected women than in controls. Using assays with sufficient sensitivity, we defined the range for total and free testosterone levels during the normal menstrual cycle. Serum total and free testosterone levels are lower in HIV-infected women and correlate inversely with plasma HIV ribonucleic acid levels. The hypothesis that androgen deficiency contributes to wasting in HIV-infected women remains to be tested.

Schisandra chinensis regulates drug metabolizing enzymes and drug transporters via activation of Nrf2-mediated signaling pathway

PubMed Central

He, Jin-Lian; Zhou, Zhi-Wei; Yin, Juan-Juan; He, Chang-Qiang; Zhou, Shu-Feng; Yu, Yang

2015-01-01

Drug metabolizing enzymes (DMEs) and drug transporters are regulated via epigenetic, transcriptional, posttranscriptional, and translational and posttranslational modifications. Phase I and II DMEs and drug transporters play an important role in the disposition and detoxification of a large number of endogenous and exogenous compounds. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a critical regulator of a variety of important cytoprotective genes that are involved in disposition and detoxification of xenobiotics. Schisandra chinensis (SC) is a commonly used traditional Chinese herbal medicine that has been primarily used to protect the liver because of its potent antioxidative and anti-inflammatory activities. SC can modulate some DMEs and drug transporters, but the underlying mechanisms are unclear. In this study, we aimed to explore the role of Nrf2 in the regulatory effect of SC extract (SCE) on selected DMEs and drug transporters in human hepatocellular liver carcinoma cell line (HepG2) cells. The results showed that SCE, schisandrin A, and schisandrin B significantly increased the expression of NAD(P)H: Nicotinamide Adenine Dinucleotide Phosphate-oxidase or:quinone oxidoreductase 1, heme oxygenase-1, glutamate–cysteine ligase, and glutathione S-transferase A4 at both transcriptional and posttranscriptional levels. Incubation of HepG2 cells with SCE resulted in a significant increase in the intracellular level of glutathione and total glutathione S-transferase content. SCE significantly elevated the messenger ribonucleic acid and protein levels of P-glycoprotein and multidrug resistance-associated protein 2 and 4, whereas the expression of organic anion transporting peptide 1A2 and 1B1 was significantly downregulated by SCE. Knockdown of Nrf2 by small interfering ribonucleic acid attenuated the regulatory effect of SCE on these DMEs and drug transporters. SCE significantly upregulated Nrf2 and promoted the translocation of Nrf2 from cytoplasm to the nuclei. Additionally, SCE significantly suppressed the expression of cytosolic Kelch-like ECH-associated protein 1 (the repressor of Nrf2) and remarkably increased Nrf2 stability in HepG2 cells. Taken together, our findings suggest that the hepatoprotective effects of SCE may be partially ascribed to the modulation of DMEs and drug transporters via Nrf2-mediated signaling pathway. SCE may alter the pharmacokinetics of other coadministered drugs that are substrates of these DMEs and transporters and thus cause unfavorable herb–drug interactions. PMID:25552902

Eubacterium rangiferina, a novel usnic acid-resistant bacterium from the reindeer rumen

NASA Astrophysics Data System (ADS)

Sundset, Monica A.; Kohn, Alexandra; Mathiesen, Svein D.; Præsteng, Kirsti E.

2008-08-01

Reindeer are able to eat and utilize lichens as an important source of energy and nutrients. In the current study, the activities of antibiotic secondary metabolites including usnic, antranoric, fumarprotocetraric, and lobaric acid commonly found in lichens were tested against a collection of 26 anaerobic rumen bacterial isolates from reindeer ( Rangifer tarandus tarandus) using the agar diffusion method. The isolates were identified based on their 16S ribosomal ribonucleic acid (rRNA) gene sequences. Usnic acid had a potent antimicrobial effect against 25 of the isolates, belonging to Clostridiales, Enterococci, and Streptococci. Isolates of Clostridia and Streptococci were also susceptible to atranoric and lobaric acid. However, one isolate (R3_91_1) was found to be resistant to usnic, antranoric, fumarprotocetraric, and lobaric acid. R3_91_1 was also seen invading and adhering to lichen particles when grown in a liquid anaerobic culture as demonstrated by transmission electron microscopy. This was a Gram-negative, nonmotile rod (0.2-0.7 × 2.0-3.5 μm) with a deoxyribonucleic acid G + C content of 47.0 mol% and main cellular fatty acids including 15:0 anteiso-dimethyl acetal (DMA), 16:0 iso-fatty acid methyl ester (FAME), 13:0 iso-3OH FAME, and 17:0 anteiso-FAME, not matching any of the presently known profiles in the MIDI database. Combined, the phenotypic and genotypic traits including the 16S rRNA gene sequence show that R3_91_1 is a novel species inside the order Clostridiales within the family Lachnospiraceae, for which we propose the name Eubacterium rangiferina. This is the first record of a rumen bacterium able to tolerate and grow in the presence of usnic acid, indicating that the rumen microorganisms in these animals have adapted mechanisms to deal with lichen secondary metabolites, well known for their antimicrobial and toxic effects.

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

NASA Technical Reports Server (NTRS)

Khaoustov, V. I.; Risin, D.; Pellis, N. R.; Yoffe, B.; McIntire, L. V. (Principal Investigator)

2001-01-01

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture.

PubMed

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P H

2014-05-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. © 2014 The Author(s). Published by Oxford University Press [on behalf of Nucleic Acids Research].

CPU-GPU hybrid accelerating the Zuker algorithm for RNA secondary structure prediction applications.

PubMed

Lei, Guoqing; Dou, Yong; Wan, Wen; Xia, Fei; Li, Rongchun; Ma, Meng; Zou, Dan

2012-01-01

Prediction of ribonucleic acid (RNA) secondary structure remains one of the most important research areas in bioinformatics. The Zuker algorithm is one of the most popular methods of free energy minimization for RNA secondary structure prediction. Thus far, few studies have been reported on the acceleration of the Zuker algorithm on general-purpose processors or on extra accelerators such as Field Programmable Gate-Array (FPGA) and Graphics Processing Units (GPU). To the best of our knowledge, no implementation combines both CPU and extra accelerators, such as GPUs, to accelerate the Zuker algorithm applications. In this paper, a CPU-GPU hybrid computing system that accelerates Zuker algorithm applications for RNA secondary structure prediction is proposed. The computing tasks are allocated between CPU and GPU for parallel cooperate execution. Performance differences between the CPU and the GPU in the task-allocation scheme are considered to obtain workload balance. To improve the hybrid system performance, the Zuker algorithm is optimally implemented with special methods for CPU and GPU architecture. Speedup of 15.93× over optimized multi-core SIMD CPU implementation and performance advantage of 16% over optimized GPU implementation are shown in the experimental results. More than 14% of the sequences are executed on CPU in the hybrid system. The system combining CPU and GPU to accelerate the Zuker algorithm is proven to be promising and can be applied to other bioinformatics applications.

Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs

NASA Astrophysics Data System (ADS)

Wetzel, Collin; Li, Siwei; Limbach, Patrick A.

2014-07-01

Mapping, sequencing, and quantifying individual noncoding ribonucleic acids (ncRNAs), including post-transcriptionally modified nucleosides, by mass spectrometry is a challenge that often requires rigorous sample preparation prior to analysis. Previously, we have described a simplified method for the comparative analysis of RNA digests (CARD) that is applicable to relatively complex mixtures of ncRNAs. In the CARD approach for transfer RNA (tRNA) analysis, two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. This approach allows one to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging because of convoluted mass spectra arising from the natural 13C and 15 N isotopes present in the ribonuclease-digested tRNA samples. Here, we demonstrate that culturing in 12C-enriched/13C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality yield a 35 % improvement in detection of tRNA digestion products that can be uniquely assigned to particular tRNAs. These mass spectral improvements lead to a significant reduction in data processing attributable to the ease of spectral identification of labeled digestion products and will enable improvements in the relative quantification of modified RNAs by the 16O/18O differential labeling approach.

Vascular endothelial growth factor is upregulated by l-dopa in the parkinsonian brain: implications for the development of dyskinesia

PubMed Central

Francardo, Veronica; Lindgren, Hanna S.; Sillivan, Stephanie E.; O’Sullivan, Sean S.; Luksik, Andrew S.; Vassoler, Fair M.; Lees, Andrew J.; Konradi, Christine

2011-01-01

Angiogenesis and increased permeability of the blood–brain barrier have been reported to occur in animal models of Parkinson’s disease and l-dopa-induced dyskinesia, but the significance of these phenomena has remained unclear. Using a validated rat model of l-dopa-induced dyskinesia, this study demonstrates that chronic treatment with l-dopa dose dependently induces the expression of vascular endothelial growth factor in the basal ganglia nuclei. Vascular endothelial growth factor was abundantly expressed in astrocytes and astrocytic processes in the proximity of blood vessels. When co-administered with l-dopa, a small molecule inhibitor of vascular endothelial growth factor signalling significantly attenuated the development of dyskinesia and completely blocked the angiogenic response and associated increase in blood–brain barrier permeability induced by the treatment. The occurrence of angiogenesis and vascular endothelial growth factor upregulation was verified in post-mortem basal ganglia tissue from patients with Parkinson’s disease with a history of dyskinesia, who exhibited increased microvascular density, microvascular nestin expression and an upregulation of vascular endothelial growth factor messenger ribonucleic acid. These congruent findings in the rat model and human patients indicate that vascular endothelial growth factor is implicated in the pathophysiology of l-dopa-induced dyskinesia and emphasize an involvement of the microvascular compartment in the adverse effects of l-dopa pharmacotherapy in Parkinson’s disease. PMID:21771855

Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages.

PubMed Central

Lolait, S J; Clements, J A; Markwick, A J; Cheng, C; McNally, M; Smith, A I; Funder, J W

1986-01-01

We have previously demonstrated low levels of immunoreactive (ir)-beta-endorphin (beta-EP) and ir-ACTH in a subpopulation of mouse spleen macrophages, which is consistent with an involvement of opioid peptides in modulation of immune responses. Gel chromatography studies suggested the presence of an approximately 3.5,000-molecular weight (mol wt) species, putatively beta-EP, an approximately 11.5,000-mol-wt species, putatively beta-lipotropin, and a higher molecular weight species (putative beta-EP precursor, pro-opiomelanocortin (POMC). In this study we have extended our original findings by demonstrating the presence of messenger RNA for POMC by the use of a complementary DNA probe and Northern blot analysis of extracts of mouse and rat spleen. In addition, using high performance liquid chromatography (HPLC), we have shown that the major endorphin species in mouse spleen macrophages is beta-EP1-31, and that there are smaller amounts of each of the acetylated forms, N-acetyl-beta-EP1-16 (alpha-endorphin), N-acetyl-beta-EP1-17 (gamma-endorphin), N-acetyl-beta-EP1-27, and N-acetyl-beta-EP1-31. We interpret these studies as showing that (a) the spleen is an organ of POMC synthesis and that (b) the predominant COOH-terminal product of macrophage POMC is the opiate-receptor active species beta-EP1-31. Images PMID:2423557

Aldolase as a Chirality Intersection of L-Amino Acids and D-Sugars

NASA Astrophysics Data System (ADS)

Munegumi, Toratane

2015-06-01

Aldolase plays an important role in glycolysis and gluconeogenesis to produce D-fructose-1,6-bisphosphate (D-FBP) from dihydroxyacetone phosphate (DHP) and D-glyceraldehyde-3-phosphate (D-GAP). This reaction is stereoselective and retains the D-GAP 2R configuration and yields D-FBP (with the configuration: 3S, 4S, 5R). The 3- and 4-position carbons are the newly formed chiral carbons because the 5-position carbon of D-FBP comes from the 2-position of D-GAP. Although four diastereomeric products, ( 3S, 4R, 5R), ( 3R, 4R, 5R), ( 3R, 4S, 5R), ( 3S, 4S, 5R), are expected in the nonenzymatic reaction, only the ( 3S, 4S, 5R) diastereomer (D-FBP) is obtained. Therefore, the chirality in the 3- and 4-positions is induced by the chirality of the enzyme composed of L-amino acid residues. D-Glucose-6-phosphate (D-G6P), which is generated from D-FBP in the gluconeogenesis pathway, produces D-ribose-5-phosphate (D-R5P) in the pentose phosphate pathway. D-R5P is converted to PRPP (5-phosphoribosyl-α-pyrophosphate), which is used for the de novo synthesis of nucleotides. Ribonucleic acid (RNA) uses the nucleotides as building blocks. The configurations of the 4R-carbon and of the 3S-carbon are retained. The stereochemical structure of RNA is based on 3S as well as 4R (D). The consideration above suggests that aldolase is a key enzyme that determines the 3S configuration in D-R5P. It is thus a chirality intersection between amino acids and sugars, because the sugar chirality is determined by the chiral environment of an L-amino acid protein, aldolase, to produce D-FBP.

Rope-based oral fluid sampling for early detection of classical swine fever in domestic pigs at group level.

PubMed

Dietze, Klaas; Tucakov, Anna; Engel, Tatjana; Wirtz, Sabine; Depner, Klaus; Globig, Anja; Kammerer, Robert; Mouchantat, Susan

2017-01-05

Non-invasive sampling techniques based on the analysis of oral fluid specimen have gained substantial importance in the field of swine herd management. Methodological advances have a focus on endemic viral diseases in commercial pig production. More recently, these approaches have been adapted to non-invasive sampling of wild boar for transboundary animal disease detection for which these effective population level sampling methods have not been available. In this study, a rope-in-a-bait based oral fluid sampling technique was tested to detect classical swine fever virus nucleic acid shedding from experimentally infected domestic pigs. Separated in two groups treated identically, the course of the infection was slightly differing in terms of onset of the clinical signs and levels of viral ribonucleic acid detection in the blood and oral fluid. The technique was capable of detecting classical swine fever virus nucleic acid as of day 7 post infection coinciding with the first detection in conventional oropharyngeal swab samples from some individual animals. Except for day 7 post infection in the "slower onset group", the chances of classical swine fever virus nucleic acid detection in ropes were identical or higher as compared to the individual sampling. With the provided evidence, non-invasive oral fluid sampling at group level can be considered as additional cost-effective detection tool in classical swine fever prevention and control strategies. The proposed methodology is of particular use in production systems with reduced access to veterinary services such as backyard or scavenging pig production where it can be integrated in feeding or baiting practices.

Circulating nucleic acids as possible damage-associated molecular patterns in different stages of renal failure.

PubMed

Kocić, Gordana; Radenkovic, Sonja; Cvetkovic, Tatjana; Cencic, Avrelija; Carluccio, Francesco; Musovic, Dijana; Nikolić, Goran; Jevtović-Stoimenov, Tatjana; Sokolović, Dusan; Milojkovic, Boban; Basic, Jelena; Veljkovic, Andrej; Stojanović, Svetlana

2010-05-01

Chronic renal failure (CRF) is a condition associated with the risk of cardiovascular complications. Systemic inflammatory response, initiated by the pathogen-associated molecular-pattern (PAMP) molecules, exerts many similarities with the damage-associated molecular-pattern (DAMP) molecule-induced systemic response. Up to now, a number of DAMP molecules were identified. We hypothesized that the available circulating nucleic acids, acting as DAMPs, may modulate immunoinflammatory reaction in CRF. Patients with the different stages of chronic kidney disease, kidney transplantation, and patients on dialysis were included in the study. Obtained results about higher concentration of circulating ribonucleic acid (RNA), according to the stages of kidney diseases, may contribute to the hypothesis that damaged kidney tissue releases nucleic acids. Circulating RNAs expressed maximal absorbance peak at 270 nm in spectrophotometric scan analysis, which corresponded to polyC, compared to different standard samples. During in vitro conditions, by using the culture of human residential macrophages, circulating RNA isolated from patients with IV-V-stage renal diseases, patients on hemodialysis, and patients who underwent renal transplantation were able to significantly change signal transduction proteins related to inflammation and antiviral response. They significantly increased the intracellular concentration of active nuclear transcription factor nuclear factor kappa B (NF-kappaB), interferon regulatory factors (IRF)-3, and IRF-7 and significantly decreased melanoma differentiation-associated protein-5 (MDA-5) and p38. In this way, it seems that circulating RNA, acting as DAMP, may contribute to the mechanisms of additional inflammatory reaction, possible immune destruction, and decreased antiviral response, related to complications in kidney diseases.

Spectroscopic studies on the binding interaction of phenothiazinium dyes, azure A and azure B to double stranded RNA polynucleotides

NASA Astrophysics Data System (ADS)

Khan, Asma Yasmeen; Suresh Kumar, Gopinatha

2016-01-01

This manuscript presents spectroscopic characterization of the interaction of two phenothiazinium dyes, azure A and azure B with double stranded (ds) ribonucleic acids, poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C). Absorbance and fluorescence studies revealed that these dyes bind to the RNAs with binding affinities of the order 106 M-1 to poly(A).poly(U), and 105 M-1 to poly(C).poly(G) and poly(I).poly(C), respectively. Fluorescence quenching and viscosity data gave conclusive evidence for the intercalation of the dyes to these RNA duplexes. Circular dichroism results suggested that the conformation of the RNAs was perturbed on interaction and the dyes acquired strong induced optical activity on binding. Azure B bound to all the three RNAs stronger than azure A and the binding affinity varied as poly(A).poly(U) > poly(C).poly(G) > poly(I).poly(C) for both dyes.

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

PubMed Central

Gaston, Kirk W; Limbach, Patrick A

2014-01-01

The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems. PMID:25616408

Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

PubMed Central

Chao, Julie; Weathersbee, Carolyn J.

1974-01-01

Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76.

PubMed

Di Bartolo, Vincenzo; Montagne, Benjamin; Salek, Mogjiborahman; Jungwirth, Britta; Carrette, Florent; Fourtane, Julien; Sol-Foulon, Nathalie; Michel, Frédérique; Schwartz, Olivier; Lehmann, Wolf D; Acuto, Oreste

2007-03-19

The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.

Ultra-low input transcriptomics reveal the spore functional content and phylogenetic affiliations of poorly studied arbuscular mycorrhizal fungi

PubMed Central

Beaudet, Denis; Chen, Eric C H; Mathieu, Stephanie; Yildirir, Gokalp; Ndikumana, Steve; Dalpé, Yolande; Séguin, Sylvie; Farinelli, Laurent; Stajich, Jason E; Corradi, Nicolas

2018-01-01

Abstract Arbuscular mycorrhizal fungi (AMF) are a group of soil microorganisms that establish symbioses with the vast majority of land plants. To date, generation of AMF coding information has been limited to model genera that grow well axenically; Rhizoglomus and Gigaspora. Meanwhile, data on the functional gene repertoire of most AMF families is non-existent. Here, we provide primary large-scale transcriptome data from eight poorly studied AMF species (Acaulospora morrowiae, Diversispora versiforme, Scutellospora calospora, Racocetra castanea, Paraglomus brasilianum, Ambispora leptoticha, Claroideoglomus claroideum and Funneliformis mosseae) using ultra-low input ribonucleic acid (RNA)-seq approaches. Our analyses reveals that quiescent spores of many AMF species harbour a diverse functional diversity and solidify known evolutionary relationships within the group. Our findings demonstrate that RNA-seq data obtained from low-input RNA are reliable in comparison to conventional RNA-seq experiments. Thus, our methodology can potentially be used to deepen our understanding of fungal microbial function and phylogeny using minute amounts of RNA material. PMID:29211832

Laboratory Surveillance of Rabies in Humans, Domestic Animals, and Bats in Madagascar from 2005 to 2010

PubMed Central

Reynes, Jean-Marc; Andriamandimby, Soa Fy; Razafitrimo, Girard Marcelin; Razainirina, Josette; Jeanmaire, Elisabeth Marie; Bourhy, Hervé; Heraud, Jean-Michel

2011-01-01

Background. Rabies virus (RABV) has circulated in Madagascar at least since the 19th century. Objectives. To assess the circulation of lyssavirus in the island from 2005 to 2010. Materials and Methods. Animal (including bats) and human samples were tested for RABV and other lyssavirus using antigen, ribonucleic acid (RNA), and antibodies detection and virus isolation. Results. Half of the 437 domestic or tame wild terrestrial mammal brains tested were found RABV antigen positive, including 54% of the 341 dogs tested. This percentage ranged from 26% to 75% across the period. Nine of the 10 suspected human cases tested were laboratory confirmed. RABV circulation was confirmed in 34 of the 38 districts sampled. No lyssavirus RNA was detected in 1983 bats specimens. Nevertheless, antibodies against Lagos bat virus were detected in the sera of 12 among 50 Eidolon dupreanum specimens sampled. Conclusion. More than a century after the introduction of the vaccine, rabies still remains endemic in Madagascar. PMID:21991442

New Insights Into “Plant Memories”

ScienceCinema

Sanbonmatsu, Karissa

2018-01-16

A special stretch of ribonucleic acid (RNA) called COOLAIR is revealing its inner structure and function to scientists, displaying a striking resemblance to an RNA molecular machine, territory previously understood to be limited to the cells’ protein factory (the ‘ribosome’) and not a skill set given to mere strings of RNA. “We are uncovering the nuts and bolts of plant memories,” said Karissa Sanbonmatsu of Los Alamos National Laboratory, lead author on a new article this week in the journal Cell Reports. In the past 5 years or so, material in the cell known as “junk DNA” had actually turned out not to be junk at all. Instead, it was shown to produce RNA molecules that play key roles in the development of organs in the embryo, as well as affecting cancer, brain function and plant biology.

Deciphering the Role of the Barr Body in Malignancy: An insight into head and neck cancer.

PubMed

Sharma, Deepti; Koshy, George; Gupta, Shruti; Sharma, Bhushan; Grover, Sonal

2017-11-01

X chromosome inactivation is the epitome of epigenetic regulation and long non-coding ribonucleic acid function. The differentiation status of cells has been ascribed to X chromosome activity, with two active X chromosomes generally only observed in undifferentiated or poorly differentiated cells. Recently, several studies have indicated that the reactivation of an inactive X chromosome or X chromosome multiplication correlates with the development of malignancy; however, this concept is still controversial. This review sought to shed light on the role of the X chromosome in cancer development. In particular, there is a need for further exploration of the expression patterns of X-linked genes in cancer cells, especially those in head and neck squamous cell carcinoma (HNSCC), in order to identify different prognostic subpopulations with distinct clinical implications. This article proposes a functional relationship between the loss of the Barr body and the disproportional expression of X-linked genes in HNSCC development.

Emerging Bacterial Infection: Identification and Clinical Significance of Kocuria Species

PubMed Central

Palange, Padmavali; Vaish, Ritu; Bhatti, Adnan Bashir; Kale, Vinod; Kandi, Maheshwar Reddy; Bhoomagiri, Mohan Rao

2016-01-01

Recently there have been reports of gram-positive cocci which are morphologically similar to both Staphylococci and the Micrococci. These bacteria have been identified as Kocuria species with the help of automated identification system and other molecular methods including 16S rRNA (ribosomal ribonucleic acid) evaluation. Kocuria belongs to the family Micrococcaceae which also includes Staphylococcus species and Micrococcus species. Isolation and clinical significance of these bacteria from human specimens warrant great caution as it does not necessarily confirm infection due to their ubiquitous presence, and as a normal flora of skin and mucous membranes in human and animals. Most clinical microbiology laboratories ignore such bacteria as laboratory and specimen contaminants. With increasing reports of infections associated with these bacteria, it is now important for clinical microbiologists to identify and enumerate the virulence and antibiotic susceptibility patterns of such bacteria and assist clinicians in improving the patient care and management. We review the occurrence and clinical significance of Kocuria species. PMID:27630804

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry.

PubMed

Gaston, Kirk W; Limbach, Patrick A

2014-01-01

The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

Community Structure Analysis and Biodegradation Potential of Aniline-Degrading Bacteria in Biofilters.

PubMed

Hou, Luanfeng; Wu, Qingping; Gu, Qihui; Zhou, Qin; Zhang, Jumei

2018-07-01

Aniline has aroused general concern owing to its strong toxicity and widespread distribution in water and soil. In the present study, the bacterial community composition before and after aniline acclimation was investigated. High-throughput Illumina MiSeq sequencing analysis illustrated a large shift in the structure of the bacterial community during the aniline acclimation period. Bacillus, Lactococcus, and Enterococcus were the dominant bacteria in biologically activated carbon before acclimation. However, the proportions of Pseudomonas, Thermomonas, and Acinetobacter increased significantly and several new bacterial taxa appeared after aniline acclimation, indicating that aniline acclimation had a strong impact on the bacterial community structure of biological activated carbon samples. Strain AN-1 accounted for the highest number of colonies on incubation plates and was identified as Acinetobacter sp. according to phylogenetic analysis of the 16S ribosomal ribonucleic acid gene sequence. Strain AN-1 was able to grow on aniline at pH value 4.0-10.0 and showed high aniline-degrading ability at neutral pH.

The discriminatory power of ribotyping as automatable technique for differentiation of bacteria.

PubMed

Schumann, Peter; Pukall, Rüdiger

2013-09-01

Since the introduction of ribonucleic acid gene restriction patterns as taxonomic tools in 1986, ribotyping has become an established method for systematics, epidemiological, ecological and population studies of microorganisms. In the last 25 years, several modifications have improved the convenience, reproducibility and turn-around time of this technique. The technological development culminated in the automation of ribotyping which allowed for high-throughput applications e.g. in the quality control of food production, pharmaceutical industry and culture collections. The capability of the fully automated RiboPrinter(®) System for the differentiation of bacteria below the species level is compared with the discriminatory power of traditional ribotyping, of molecular fingerprint techniques like PFGE, MLST and MLVA as well as of MALDI-TOF mass spectrometry. While automated RiboPrinting is advantageous with respect to standardization, ease and speed, PCR ribotyping has proved being a highly discriminatory, flexible, robust and cost-efficient routine technique which makes inter-laboratory comparison and build of ribotype databases possible, too. Copyright © 2013 Elsevier GmbH. All rights reserved.

Mass spectrometric detection of siRNA in plasma samples for doping control purposes.

PubMed

Kohler, Maxie; Thomas, Andreas; Walpurgis, Katja; Schänzer, Wilhelm; Thevis, Mario

2010-10-01

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25-1 nmol mL(-1)) and performance (precision 11-21%, recovery 23-67%) for typical antisense oligonucleotides currently used in clinical studies.

Analysis of Altered Micro RNA Expression Profiles in Focal Cortical Dysplasia IIB.

PubMed

Li, Lin; Liu, Chang-Qing; Li, Tian-Fu; Guan, Yu-Guang; Zhou, Jian; Qi, Xue-Ling; Yang, Yu-Tao; Deng, Jia-Hui; Xu, Zhi-Qing David; Luan, Guo-Ming

2016-04-01

Focal cortical dysplasia type IIB is a commonly encountered subtype of developmental malformation of the cerebral cortex and is often associated with pharmacoresistant epilepsy. In this study, to investigate the molecular etiology of focal cortical dysplasia type IIB, the authors performed micro ribonucleic acid (RNA) microarray on surgical specimens from 5 children (2 female and 3 male, mean age was 73.4 months, range 50-112 months) diagnosed of focal cortical dysplasia type IIB and matched normal tissue adjacent to the lesion. In all, 24 micro RNAs were differentially expressed in focal cortical dysplasia type IIB, and the microarray results were validated using quantitative real-time polymerase chain reaction (PCR). Then the putative target genes of the differentially expressed micro RNAs were identified by bioinformatics analysis. Moreover, biological significance of the target genes was evaluated by investigating the pathways in which the genes were enriched, and the Hippo signaling pathway was proposed to be highly related with the pathogenesis of focal cortical dysplasia type IIB. © The Author(s) 2015.

Finding new pathway-specific regulators by clustering method using threshold standard deviation based on DNA chip data of Streptomyces coelicolor.

PubMed

Yang, Yung-Hun; Kim, Ji-Nu; Song, Eunjung; Kim, Eunjung; Oh, Min-Kyu; Kim, Byung-Gee

2008-09-01

In order to identify the regulators involved in antibiotic production or time-specific cellular events, the messenger ribonucleic acid (mRNA) expression data of the two gene clusters, actinorhodin (ACT) and undecylprodigiosin (RED) biosynthetic genes, were clustered with known mRNA expression data of regulators from S. coelicolor using a filtering method based on standard deviation and clustering analysis. The result identified five regulators including two well-known regulators namely, SCO3579 (WlbA) and SCO6722 (SsgD). Using overexpression and deletion of the regulator genes, we were able to identify two regulators, i.e., SCO0608 and SCO6808, playing roles as repressors in antibiotics production and sporulation. This approach can be easily applied to mapping out new regulators related to any interesting target gene clusters showing characteristic expression patterns. The result can also be used to provide insightful information on the selection rules among a large number of regulators.

Direct-acting Antivirals and Host-targeting Agents against the Hepatitis A Virus

PubMed Central

Kanda, Tatsuo; Nakamoto, Shingo; Wu, Shuang; Nakamura, Masato; Jiang, Xia; Haga, Yuki; Sasaki, Reina; Yokosuka, Osamu

2015-01-01

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis and occasionally leads to acute liver failure in both developing and developed countries. Although effective vaccines for HAV are available, the development of new antivirals against HAV may be important for the control of HAV infection in developed countries where no universal vaccination program against HAV exists, such as Japan. There are two forms of antiviral agents against HAV: direct-acting antivirals (DAAs) and host-targeting agents (HTAs). Studies using small interfering ribonucleic acid (siRNA) have suggested that the HAV internal ribosomal entry site (IRES) is an attractive target for the control of HAV replication and infection. Among the HTAs, amantadine and interferon-lambda 1 (IL-29) inhibit HAV IRES-mediated translation and HAV replication. Janus kinase (JAK) inhibitors inhibit La protein expression, HAV IRES activity, and HAV replication. Based on this review, both DAAs and HTAs may be needed to control effectively HAV infection, and their use should continue to be explored. PMID:26623267

Prebiotic chemistry in eutectic solutions at the water-ice matrix.

PubMed

Menor-Salván, César; Marín-Yaseli, Margarita R

2012-08-21

A crystalline ice matrix at subzero temperatures can maintain a liquid phase where organic solutes and salts concentrate to form eutectic solutions. This concentration effect converts the confined reactant solutions in the ice matrix, sometimes making condensation and polymerisation reactions occur more favourably. These reactions occur at significantly high rates from a prebiotic chemistry standpoint, and the labile products can be protected from degradation. The experimental study of the synthesis of nitrogen heterocycles at the ice-water system showed the efficiency of this scenario and could explain the origin of nucleobases in the inner Solar System bodies, including meteorites and extra-terrestrial ices, and on the early Earth. The same conditions can also favour the condensation of monomers to form ribonucleic acid and peptides. Together with the synthesis of these monomers, the ice world (i.e., the chemical evolution in the range between the freezing point of water and the limit of stability of liquid brines, 273 to 210 K) is an under-explored experimental model in prebiotic chemistry.

Characterization of the ribonuclease activity on the skin surface

PubMed Central

Probst, Jochen; Brechtel, Sonja; Scheel, Birgit; Hoerr, Ingmar; Jung, Günther; Rammensee, Hans-Georg; Pascolo, Steve

2006-01-01

The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin® although not by the ribonuclease inhibitor SUPERase· In™. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development. PMID:16732888

Gene expression in the rectus abdominus muscle of patients with and without pelvic organ prolapse.

PubMed

Hundley, Andrew F; Yuan, Lingwen; Visco, Anthony G

2008-02-01

The objective of the study was to compare gene expression in a group of actin and myosin-related proteins in the rectus muscle of 15 patients with pelvic organ prolapse and 13 controls. Six genes previously identified by microarray GeneChip analysis were examined using real-time quantitative reverse transcriptase-polymerase chain reaction analysis, including 2 genes showing differential expression in pubococcygeus muscle. Samples and controls were run in triplicate in multiplexed wells, and levels of gene expression were analyzed using the comparative critical threshold method. One gene, MYH3, was 3.2 times overexpressed in patients with prolapse (P = .032), but no significant differences in expression were seen for the other genes examined. An age-matched subset of 9 patients and controls showed that MYH3 gene expression was no longer significantly different (P = .058). Differential messenger ribonucleic acid levels of actin and myosin-related genes in patients with pelvic organ prolapse and controls may be limited to skeletal muscle from the pelvic floor.

Alkaliphilic and halophilic hydrocarbon-utilizing bacteria from Kuwaiti coasts of the Arabian Gulf.

PubMed

Al-Awadhi, H; Sulaiman, Rasha H D; Mahmoud, Huda M; Radwan, S S

2007-11-01

Green animate materials from the intertidal zone of the Arabian Gulf coast accommodated more alkaliphilic and halophilic bacteria than inanimate materials. The alkaliphilic oil-utilizing bacteria, as identified by their 16S ribonucleic acid sequences, belonged to the following genera arranged in decreasing frequences: Marinobacter, Micrococcus, Dietzia, Bacillus, Oceanobacillus, and Citricoccus. The halophilic oil-utilizing bacteria belonged to the genera: Marinobacter, Georgenia, Microbacterium, Stappia, Bacillus, Isoptericola, and Cellulomonas. Most isolates could grow on a wide range of pure n-alkanes and aromatic compounds, as sole sources of carbon and energy. Quantitative gas liquid chromatographic analysis showed that individual isolates attenuated crude oil and representative pure hydrocarbons in culture. The optimum pH for most of the alkaliphilic genera was pH 10, and the optimum salinity for the halophiles ranged between 2.5 and 5% NaCl (w/v). It was concluded that as far as their microbial makeup is concerned, oily alkaline and saline intertidal areas of the Kuwaiti coasts have a self-cleaning potential.

Reverse engineering of gene regulatory networks.

PubMed

Cho, K H; Choo, S M; Jung, S H; Kim, J R; Choi, H S; Kim, J

2007-05-01

Systems biology is a multi-disciplinary approach to the study of the interactions of various cellular mechanisms and cellular components. Owing to the development of new technologies that simultaneously measure the expression of genetic information, systems biological studies involving gene interactions are increasingly prominent. In this regard, reconstructing gene regulatory networks (GRNs) forms the basis for the dynamical analysis of gene interactions and related effects on cellular control pathways. Various approaches of inferring GRNs from gene expression profiles and biological information, including machine learning approaches, have been reviewed, with a brief introduction of DNA microarray experiments as typical tools for measuring levels of messenger ribonucleic acid (mRNA) expression. In particular, the inference methods are classified according to the required input information, and the main idea of each method is elucidated by comparing its advantages and disadvantages with respect to the other methods. In addition, recent developments in this field are introduced and discussions on the challenges and opportunities for future research are provided.

Detection of Albumin Expression by RNA In Situ Hybridization Is a Sensitive and Specific Method for Identification of Hepatocellular Carcinomas and Intrahepatic Cholangiocarcinomas.

PubMed

Lin, Fan; Shi, Jianhui; Wang, Hanlin L; Ma, Xiao-Jun; Monroe, Robert; Luo, Yuling; Chen, Zongming; Liu, Haiyan

2018-05-09

Inconsistent data on detection of albumin expression by ribonucleic acid (RNA) in situ hybridization have been reported. We investigated the utility of RNAscope (Advanced Cell Diagnostics, Hayward, CA) in detection of albumin in hepatocellular carcinomas (HCCs), intrahepatic cholangiocarcinomas (ICCs), and carcinomas from various organs using manual and automated staining. RNAscope for albumin detection was performed on 482 cases on tissue microarray sections and on 22 cases of ICC, including 14 surgical resection and eight core biopsy specimens. Thirty-six of 37 (97%) HCCs had detectable mRNA, whereas all non-HCC and non-ICC cases, except one lung adenocarcinoma, were negative for albumin. Fourteen of 22 ICCs (64%) were positive for albumin. RNAscope for albumin is highly sensitive and specific for identifying HCCs and is highly specific and moderately sensitive for detection of ICCs; however, rare carcinomas (non-HCC, non-ICC, and those with no hepatoid histomorphology) can also have aberrant expression of albumin.

Oligodeoxynucleotide Probes for Detecting Intact Cells

NASA Technical Reports Server (NTRS)

Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

2004-01-01

A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of target bacteria in samples are usually amplified by overnight pre-enrichment growth. These tests are usually performed in laboratories by skilled technicians. The present test was designed to overcome the shortcomings of the commercial hybridization tests. The figure summarizes the major steps of the test. It is important to emphasize that the hybridization process used in this test differs from that of other hybridization tests in that it does not extract target nucleic acids. This process is based on intact-cell hybridization (so-called in situ hybridization ), wherein the intact cells act as immobilizing agents. The cells are identified and enumerated by measuring the chemiluminescence emitted from alkaline phosphatase-probe (AP-probe) hybridization; the chemiluminescence is detected or measured by use of photographic film or a luminometer, respectively.

Phylogenetic and structural response of heterotrophic bacteria to dissolved organic matter of different chemical composition in a continuous culture study.

PubMed

Landa, M; Cottrell, M T; Kirchman, D L; Kaiser, K; Medeiros, P M; Tremblay, L; Batailler, N; Caparros, J; Catala, P; Escoubeyrou, K; Oriol, L; Blain, S; Obernosterer, I

2014-06-01

Dissolved organic matter (DOM) and heterotrophic bacteria are highly diverse components of the ocean system, and their interactions are key in regulating the biogeochemical cycles of major elements. How chemical and phylogenetic diversity are linked remains largely unexplored to date. To investigate interactions between bacterial diversity and DOM, we followed the response of natural bacterial communities to two sources of phytoplankton-derived DOM over six bacterial generation times in continuous cultures. Analyses of total hydrolysable neutral sugars and amino acids, and ultrahigh resolution mass spectrometry revealed large differences in the chemical composition of the two DOM sources. According to 454 pyrosequences of 16S ribosomal ribonucleic acid genes, diatom-derived DOM sustained higher levels of bacterial richness, evenness and phylogenetic diversity than cyanobacteria-derived DOM. These distinct community structures were, however, not associated with specific taxa. Grazing pressure affected bacterial community composition without changing the overall pattern of bacterial diversity levels set by DOM. Our results demonstrate that resource composition can shape several facets of bacterial diversity without influencing the phylogenetic composition of bacterial communities, suggesting functional redundancy at different taxonomic levels for the degradation of phytoplankton-derived DOM. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Characterization of Seasonal Influenza Virus Type and Subtypes Isolated from Influenza Like Illness Cases of 2012.

PubMed

Upadhyay, B P; Ghimire, P; Tashiro, M; Banjara, M R

Background Seasonal influenza is one of the increasing public health burdens in Nepal. Objective The objective of this study was to isolate and characterize the influenza virus type and subtypes of Nepal. Method A total of 1536 throat swab specimens were collected from January to December 2012. Total ribonucleic acid was extracted using Qiagen viral nucleic acid extraction kit and polymerase chain reaction assay was performed following the US; CDC Real-time PCR protocol. Ten percent of positive specimens were inoculated onto Madin-Darby Canine Kidney cells. Isolates were characterized by using reference ferret antisera. Result Of the total specimens (n=1536), influenza virus type A was detected in 196 (22%) cases; of which 194 (99%) were influenza A (H1N1) pdm09 and 2 (1 %) were influenza A/H3 subtype. Influenza B was detected in 684 (76.9%) cases. Influenza A (H1N1) pdm09, A/H3 and influenza B virus were antigenically similar to the recommended influenza virus vaccine candidate of the year 2012. Although sporadic cases of influenza were observed throughout the year, peak was observed during July to November. Conclusion Similar to other tropical countries, A (H1N1) pdm09, A/H3 and influenza B viruses were co-circulated in Nepal.

Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy

NASA Astrophysics Data System (ADS)

Kunstar, Aliz; Leijten, Jeroen; van Leuveren, Stefan; Hilderink, Janneke; Otto, Cees; van Blitterswijk, Clemens A.; Karperien, Marcel; van Apeldoorn, Aart A.

2012-11-01

Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.

[1,10]Phenanthroline based cyanine dyes as fluorescent probes for ribonucleic acids in live cells

NASA Astrophysics Data System (ADS)

Kovalska, Vladyslava; Kuperman, Marina; Varzatskii, Oleg; Kryvorotenko, Dmytro; Kinski, Elisa; Schikora, Margot; Janko, Christina; Alexiou, Christoph; Yarmoluk, Sergiy; Mokhir, Andriy

2017-12-01

A series of monomethine, trimethine- and styrylcyanine dyes based on a [1,10]phenanthroline moiety was synthesized, characterized and investigated as potential fluorescent probes for nucleic acids in cell free settings and in cells. The dyes were found to be weakly fluorescent in the unbound state, whereas upon the binding to dsDNA or RNA their emission intensity raised up to 50 times (for monomethine benzothiazole derivative FT1 complexed with RNA). The strongest fluorescence intensity in assemblies with dsDNA and RNA was observed for the trimethine benzothiazole derivative FT4. The quantum yield of FT4 fluorescence in its complex with dsDNA was found to be 1.5% and the binding constant (K b) was estimated to be 7.9 × 104 M-1 that is a typical value for intercalating molecules. The FT4 dye was found to be cell membrane permeable. It stains RNA rich components—the nucleoli and most probably the cytoplasmic RNA. FT4 bound to RNAs delivers a very strong fluorescence signal, which makes this easily accessible dye a potentially useful alternative to known RNA stains, e.g. expensive SYTO® 83. The advantage of FT4 is its easy synthetic access including no chromatographic purification steps, which will be reflected in its substantially lower price.

Quantitative Raman spectral changes of the differentiation of mesenchymal stem cells into islet-like cells by biochemical component analysis and multiple peak fitting

NASA Astrophysics Data System (ADS)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; He, Yingtian; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-12-01

Mesenchymal stem cells (MSCs) differentiate into islet-like cells, providing a possible solution for type I diabetes treatment. To search for the precise molecular mechanism of the directional differentiation of MSC-derived islet-like cells, biomolecular composition, and structural conformation information during MSC differentiation, is required. Because islet-like cells lack specific surface markers, the commonly employed immunostaining technique is not suitable for their identification, physical separation, and enrichment. Combining Raman spectroscopic data, a fitting accuracy-improved biochemical component analysis, and multiple peaks fitting approach, we identified the quantitative biochemical and intensity change of Raman peaks that show the differentiation of MSCs into islet-like cells. Along with increases in protein and glycogen content, and decreases in deoxyribonucleic acid and ribonucleic acid content, in islet-like cells relative to MSCs, it was found that a characteristic peak of insulin (665 cm-1) has twice the intensity in islet-like cells relative to MSCs, indicating differentiation of MSCs into islet-like cells was successful. Importantly, these Raman signatures provide useful information on the structural and pathological states during MSC differentiation and help to develop noninvasive and label-free Raman sorting methods for stem cells and their lineages.

Reverse Transcriptase-Containing Particles Induced in Rous Sarcoma Virus-Transformed Rat Cells by Arginine Deprivation

PubMed Central

Kotler, Moshe; Weinberg, Eynat; Haspel, Osnat; Becker, Yechiel

1972-01-01

Incubation of rat cells transformed by Rous sarcoma virus (RSV) in an arginine-deficient medium resulted in accumulation of particles in the culture medium. Such particles did not appear when the transformed rat cells were incubated in a complete medium nor in the medium of primary rat cells which were incubated either in arginine-deficient or complete media. The particles which were released from the arginine-deprived transformed rat cells resemble C-type particles in their properties. These particles band in sucrose gradients at a density of 1.16 g/ml and contain 35S ribonucleic acid (RNA) molecules and a reverse transcriptase activity. Analysis of the cytoplasm of transformed and primary rat cells, deprived and undeprived of arginine, revealed the presence of reverse transcriptase-containing particles which banded in sucrose gradients at a density of 1.14 g/ml. These particles differed from the particles released into the medium by the arginine-deprived RSV-transformed rat cells. The deoxyribonucleic acid (DNA) molecules synthesized in vitro by the reverse transcriptase present in the particles isolated from the medium of arginine-deprived cells hybridized to RSV RNA, whereas the DNA synthesized by the cell-bound enzyme had no homology to RSV RNA. PMID:4116137

RNA as a stable polymer to build controllable and defined nanostructures for material and biomedical applications

PubMed Central

Li, Hui; Lee, Taek; Dziubla, Thomas; Pi, Fengmei; Guo, Sijin; Xu, Jing; Li, Chan; Haque, Farzin; Liang, Xing-Jie; Guo, Peixuan

2015-01-01

Summary The value of polymers is manifested in their vital use as building blocks in material and life sciences. Ribonucleic acid (RNA) is a polynucleic acid, but its polymeric nature in materials and technological applications is often overlooked due to an impression that RNA is seemingly unstable. Recent findings that certain modifications can make RNA resistant to RNase degradation while retaining its authentic folding property and biological function, and the discovery of ultra-thermostable RNA motifs have adequately addressed the concerns of RNA unstability. RNA can serve as a unique polymeric material to build varieties of nanostructures including nanoparticles, polygons, arrays, bundles, membrane, and microsponges that have potential applications in biomedical and material sciences. Since 2005, more than a thousand publications on RNA nanostructures have been published in diverse fields, indicating a remarkable increase of interest in the emerging field of RNA nanotechnology. In this review, we aim to: delineate the physical and chemical properties of polymers that can be applied to RNA; introduce the unique properties of RNA as a polymer; review the current methods for the construction of RNA nanostructures; describe its applications in material, biomedical and computer sciences; and, discuss the challenges and future prospects in this field. PMID:26770259

Hierarchical classification strategy for Phenotype extraction from epidermal growth factor receptor endocytosis screening.

PubMed

Cao, Lu; Graauw, Marjo de; Yan, Kuan; Winkel, Leah; Verbeek, Fons J

2016-05-03

Endocytosis is regarded as a mechanism of attenuating the epidermal growth factor receptor (EGFR) signaling and of receptor degradation. There is increasing evidence becoming available showing that breast cancer progression is associated with a defect in EGFR endocytosis. In order to find related Ribonucleic acid (RNA) regulators in this process, high-throughput imaging with fluorescent markers is used to visualize the complex EGFR endocytosis process. Subsequently a dedicated automatic image and data analysis system is developed and applied to extract the phenotype measurement and distinguish different developmental episodes from a huge amount of images acquired through high-throughput imaging. For the image analysis, a phenotype measurement quantifies the important image information into distinct features or measurements. Therefore, the manner in which prominent measurements are chosen to represent the dynamics of the EGFR process becomes a crucial step for the identification of the phenotype. In the subsequent data analysis, classification is used to categorize each observation by making use of all prominent measurements obtained from image analysis. Therefore, a better construction for a classification strategy will support to raise the performance level in our image and data analysis system. In this paper, we illustrate an integrated analysis method for EGFR signalling through image analysis of microscopy images. Sophisticated wavelet-based texture measurements are used to obtain a good description of the characteristic stages in the EGFR signalling. A hierarchical classification strategy is designed to improve the recognition of phenotypic episodes of EGFR during endocytosis. Different strategies for normalization, feature selection and classification are evaluated. The results of performance assessment clearly demonstrate that our hierarchical classification scheme combined with a selected set of features provides a notable improvement in the temporal analysis of EGFR endocytosis. Moreover, it is shown that the addition of the wavelet-based texture features contributes to this improvement. Our workflow can be applied to drug discovery to analyze defected EGFR endocytosis processes.

RNA sequencing analysis of transcriptional change in the freshwater mussel Elliptio complanata after environmentally relevant sodium chloride exposure

USGS Publications Warehouse

Robertson, Laura S.; Galbraith, Heather S.; Iwanowicz, Deborah; Blakeslee, Carrie J.; Cornman, Robert S.

2017-01-01

To identify potential biomarkers of salt stress in a freshwater sentinel species, we examined transcriptional responses of the common mussel Elliptio complanata to controlled sodium chloride (NaCl) exposures. Ribonucleic acid sequencing (RNA-Seq) of mantle tissue identified 481 transcripts differentially expressed in adult mussels exposed to 2 ppt NaCl (1.2 ppt chloride) for 7 d, of which 290 had nonoverlapping intervals. Differentially expressed gene categories included ion and transmembrane transport, oxidoreductase activity, maintenance of protein folding, and amino acid metabolism. The rate-limiting enzyme for synthesis of taurine, an amino acid frequently linked to osmotic stress in aquatic species, was upregulated, as was the transmembrane ion pump sodium/potassium adenosine 5′-triphosphatase. These patterns confirm a primary transcriptional response to the experimental dose, albeit likely overlapping with nonspecific secondary stress responses. Substantial involvement of the heat shock protein 70 chaperone family and the water-transporting aquaporin family was not detected, however, in contrast to some studies in other bivalves. A subset of the most significantly regulated genes was confirmed by quantitative polymerase chain reaction in an independent sample. Cluster analysis showed separation of mussels exposed to 2 ppt NaCl from control mussels in multivariate space, but mussels exposed to 1 ppt NaCl were largely indistinguishable from controls. Transcriptome-scale analysis of salt exposure under laboratory conditions efficiently identified candidate biomarkers for further functional analysis and field validation

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid.

PubMed

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation.

Modulation expression of tumor necrosis factor α in the radiation-induced lung injury by glycyrrhizic acid

PubMed Central

Refahi, Soheila; Pourissa, Masoud; Zirak, Mohammad Reza; Hadadi, GholamHassan

2015-01-01

To evaluate the ability of glycyrrhizic acid (GLA) to reduce the tumor necrosis factor α (TNF-α), release on messenger ribonucleic acid (mRNA) and protein production in the lungs using GLA in response to irradiation were studied. The animals were divided into four groups: No treatment (NT group), GLA treatment only (GLA group), irradiation only (XRT group), and GLA treatment plus irradiation (GLA/XRT group). Rats were killed at different time points. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the mRNA expression of TNF-α in the lungs (compared with non-irradiated lungs). An enzyme-linked immunosorbant assay (ELISA) assay was used to measure the TNF-α protein level. The TNF-α mRNA expression in the lungs of the XRT rats was clearly higher at all-time points compared to the NT rats. The TNF-α mRNA expression in the lungs of the GLA/XRT rats was lower at all-time points compared to the XRT rats. Release of the TNF-α on protein level in the lungs of the XRT rats increased at all-time points compared to the NT rats. In contrast to the XRT rats, the lungs of the GLA/XRT rats revealed a reduction on TNF-α protein level at 6 h after irradiation. This study has clearly showed the immediate down-regulation of the TNF-α mRNA and protein production in the lungs using GLA in response to irradiation. PMID:26170556

A PCR method based on 18S rRNA gene for detection of malaria parasite in Balochistan.

PubMed

Shahwani, Zubeda; Aleem, Abdul; Ahmed, Nazeer; Mushtaq, Muhammad; Afridi, Sarwat

2016-12-01

To establish a polymerase chain reaction method based on 18S ribosomal ribonucleic acid gene for the detection of plasmodium deoxyribonucleic acid in patients suffering from malaria symptoms. This cross-sectional study was conducted from September 2013 to October 2014 in district Quetta of Pakistan's Balochistan province. Blood samples were collected from patients suffering from general symptoms of malaria. A polymerase chain reaction-based technique was applied for the diagnosis of malaria and detection of responsible species in the patients who were suspected to carry the parasite. Performance of this polymerase chain reaction method was compared against the microscopy results. Parasite number was also calculated for microscopy positive samples.All samples after the genomic deoxyribonucleic acid isolation were subjected to polymerase chain reaction amplification and agarose gel electrophoresis. Of the 200 samples, 114(57%) were confirmed as positive and 86(43%) as negative for malaria by microscopy. Polymerase chain reaction identified 124(62%) samples as positive and 76(38%) as negative for malaria. The comparative analysis of both diagnostic methods confirmed 109(54.5%) samples as positive by both techniques. Besides, 5(6.58%) samples were identified as false positive and 15(12.1%) samples as false negative by polymerase chain reaction. Sensitivity, specificity and positive predictive values for polymerase chain reaction in comparison to microscopy were 87.98%, 93.42% and 96%, respectively. Polymerase chain reaction-based methods in malaria diagnosis and species identification were found to be more effective than other techniques.

Injectable Chitosan/β-Glycerophosphate System for Sustained Release: Gelation Study, Structural Investigation, and Erosion Tests.

PubMed

Dalmoro, Annalisa; Abrami, Michela; Galzerano, Barbara; Bochicchio, Sabrina; Barba, Anna Angela; Grassi, Mario; Larobina, Domenico

2017-01-01

Hydrogels can constitute reliable delivery systems of drugs, including those based on nucleic acids (NABDs) such as small interfering ribonucleic acid (siRNA). Their nature, structure, and response to physiological or external stimuli strongly influence the delivery mechanisms of entrapped active molecules, and, in turn, their possible uses in pharmacological and biomedical applications. In this study, a thermo-gelling chitosan/β-glycero-phosphate system has been optimized in order to assess its use as injectable system able to: i) gelling at physiological pH and temperature, and ii) modulate the release of included active ingredients. To this aim, we first analyzed the effect of acetic acid concentration on the gelation temperature. We then found the "optimized composition", namely, the one in which the Tgel is equal to the physiological temperature. The resulting gel was tested, by low field nuclear magnetic resonance (LF-NMR), to evaluate its average mesh-size, which can affect release kinetics of loaded drug. Finally, films of gelled chitosan, loaded with a model drug, have been tested in vitro to monitor their characteristic times, i.e. diffusion and erosion time, when they are exposed to a medium mimicking a physiological environment (buffer solution at pH 7.4). Results display that the optimized system is deemed to be an ideal candidate as injectable gelling material for a sustained release. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The use of topical honey in the treatment of corneal abrasions and endotoxin-induced keratitis in an animal model.

PubMed

Uwaydat, Sami; Jha, Purushottam; Tytarenko, Ruslana; Brown, Harry; Wiggins, Michael; Bora, Puran S; Bora, Nalini S

2011-09-01

 To investigate the effect of topically applied honey on intact corneas, surgically induced corneal abrasions and endotoxin induced keratitis. The effect of honey on the cornea was investigated by application of honey on intact corneas, wounded corneas and endotoxin-induced keratitis in Lewis rats. The corneas were wounded by creating an epithelial defect using a surgical blade, and the keratitis was induced by topically applying Pseudomonas aeruginosa endotoxin to scarified corneas. After treatment rats were sacrificed and cornea harvested in each case. Corneas were processed for paraffin embedding for histological and immuno-fluorescence staining. Corneas were also harvested and processed for total ribonucleic acid (RNA) isolation for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for various growth factors and inflammatory chemokines/cytokines).  Histological analysis revealed that no inflammation or morphological changes occurred after honey treatment in naive intact corneas. Vascular endothelial growth factor (VEGF) levels were also not altered after honey treatment. Topical application of honey to injured corneas resulted in faster epithelial healing and decreased expression of VEGF, transforming growth factor beta (TGF-β), interferon gamma (IFN-γ), interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-α) in injured corneas. Our results also established that honey treatment reduced the inflammation in endotoxin-induced keratitis by reducing the levels of angiogenic factors (VEGF and TGF-β), inflammatory cytokines (IL-12) and chemokines (CC chemokine receptor 5(CCR-5)).  Short term use of honey on intact corneas can be safe. Honey has anti-angiogenic and anti-inflammatory properties that can be explored in several corneal inflammatory and infectious conditions.

Structural and Functional Analysis of the Interaction Between the Nucleoporin Nup98 and the mRNA Export Facto Rae1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Ren; H Seo; G Blobel

The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 {angstrom} resolution. Rae1 forms a seven-bladed {beta}-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an {approx}50-{angstrom}-long hairpinmore » that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 {beta}-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1 {center_dot} Nup98{sup GLEBS} surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1 {center_dot} Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.« less

Changes in Global Transcriptional Profiling of Women Following Obesity Surgery Bypass.

PubMed

Pinhel, Marcela Augusta de Souza; Noronha, Natalia Yumi; Nicoletti, Carolina Ferreira; de Oliveira, Bruno Affonso Parente; Cortes-Oliveira, Cristiana; Pinhanelli, Vitor Caressato; Salgado Junior, Wilson; Machry, Ana Julia; da Silva Junior, Wilson Araújo; Souza, Dorotéia Rossi Silva; Marchini, Júlio Sérgio; Nonino, Carla Barbosa

2018-01-01

Differential gene expression in peripheral blood mononuclear cells (PBMCs) after Roux-en-Y gastric bypass (RYGB) is poorly characterized. Markers of these processes may provide a deeper understanding of the mechanisms that underlie these events. The main goal of this study was to identify changes in PBMC gene expression in women with obesity before and 6 months after RYGB-induced weight loss. The ribonucleic acid (RNA) of PBMCs from 13 obese women was analyzed before and 6 months after RYGB; the RNA of PBMCs from nine healthy women served as control. The gene expression levels were determined by microarray analysis. Significant differences in gene expression were validated by real-time quantitative polymerase chain reaction (RT-qPCR). Microarray analysis for comparison of the pre- and postoperative periods showed that 1366 genes were differentially expressed genes (DEGs). The main pathways were related to gene transcription; lipid, energy, and glycide metabolism; inflammatory and immunological response; cell differentiation; oxidative stress regulation; response to endogenous and exogenous stimuli; substrate oxidation; mTOR signaling pathway; interferon signaling; mitogen-activated protein kinases (MAPK), cAMP response element binding protein (CREB1), heat shock factor 1 (HSF1), and sterol regulatory element binding protein 1c (SREBP-1c) gene expression; adipocyte differentiation; and methylation. Six months after bariatric surgery and significant weight loss, many molecular pathways involved in obesity and metabolic diseases change. These findings are an important tool to identify potential targets for therapeutic intervention and clinical practice of nutritional genomics in obesity.

Characterization of cold-associated microRNAs in the freeze-tolerant gall fly Eurosta solidaginis using high-throughput sequencing.

PubMed

Lyons, Pierre J; Govaere, Louise; Crapoulet, Nicolas; Storey, Kenneth B; Morin, Pier Jr

2016-12-01

Significant physiological and biochemical changes are observed in freeze-tolerant insects when confronted with cold temperatures. These insects have adapted to winter by retreating into a hypometabolic state of diapause and implementing cryoprotective mechanisms that allow them to survive whole body freezing. MicroRNAs (miRNAs), a family of short ribonucleic acids, are emerging as likely molecular players underlying the process of cold adaptation. Unfortunately, the data is sparse concerning the signature of miRNAs that are modulated following cold exposure in the freeze-tolerant goldenrod gall fly Eurosta solidaginis. Leveraging for the first time a next-generation sequencing approach, differentially expressed miRNAs were evaluated in 5°C and -15°C-exposed E. solidaginis larvae. Next-generation sequencing expression data was subsequently validated by qRT-PCR for selected miRNA targets. Results demonstrate 24 differentially expressed freeze-responsive miRNAs. Notable, miR-1-3p, a miRNA modulated at low temperature in another cold-hardy insect, and miR-14-3p, a miRNA associated with stress response in the fruit fly, were shown to be significantly up-regulated in -15°C-exposed larvae. Overall, this work identifies, for the first time in a high-throughput manner, differentially expressed miRNAs in cold-exposed E. solidaginis larvae and further clarifies an emerging signature of miRNAs modulated at low temperatures in cold-hardy insects. Copyright © 2016 Elsevier Inc. All rights reserved.

The MADS-box transcription factor FgMcm1 regulates cell identity and fungal development in Fusarium graminearum.

PubMed

Yang, Cui; Liu, Huiquan; Li, Guotian; Liu, Meigang; Yun, Yingzi; Wang, Chenfang; Ma, Zhonghua; Xu, Jin-Rong

2015-08-01

In eukaryotic cells, MADS-box genes are known to play major regulatory roles in various biological processes by combinatorial interactions with other transcription factors. In this study, we functionally characterized the FgMCM1 MADS-box gene in Fusarium graminearum, the causal agent of wheat and barley head blight. Deletion of FgMCM1 resulted in the loss of perithecium production and phialide formation. The Fgmcm1 mutant was significantly reduced in virulence, deoxynivalenol biosynthesis and conidiation. In yeast two-hybrid assays, FgMcm1 interacted with Mat1-1-1 and Fst12, two transcription factors important for sexual reproduction. Whereas Fgmcm1 mutants were unstable and produced stunted subcultures, Fgmcm1 mat1-1-1 but not Fgmcm1 fst12 double mutants were stable. Furthermore, spontaneous suppressor mutations occurred frequently in stunted subcultures to recover growth rate. Ribonucleic acid sequencing analysis indicated that a number of sexual reproduction-related genes were upregulated in stunted subcultures compared with the Fgmcm1 mutant, which was downregulated in the expression of genes involved in pathogenesis, secondary metabolism and conidiation. We also showed that culture instability was not observed in the Fvmcm1 mutants of the heterothallic Fusarium verticillioides. Overall, our data indicate that FgMcm1 plays a critical role in the regulation of cell identity, sexual and asexual reproduction, secondary metabolism and pathogenesis in F. graminearum. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

PubMed

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

PubMed Central

Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

2015-01-01

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis. PMID:25987838

CPU-GPU hybrid accelerating the Zuker algorithm for RNA secondary structure prediction applications

PubMed Central

2012-01-01

Background Prediction of ribonucleic acid (RNA) secondary structure remains one of the most important research areas in bioinformatics. The Zuker algorithm is one of the most popular methods of free energy minimization for RNA secondary structure prediction. Thus far, few studies have been reported on the acceleration of the Zuker algorithm on general-purpose processors or on extra accelerators such as Field Programmable Gate-Array (FPGA) and Graphics Processing Units (GPU). To the best of our knowledge, no implementation combines both CPU and extra accelerators, such as GPUs, to accelerate the Zuker algorithm applications. Results In this paper, a CPU-GPU hybrid computing system that accelerates Zuker algorithm applications for RNA secondary structure prediction is proposed. The computing tasks are allocated between CPU and GPU for parallel cooperate execution. Performance differences between the CPU and the GPU in the task-allocation scheme are considered to obtain workload balance. To improve the hybrid system performance, the Zuker algorithm is optimally implemented with special methods for CPU and GPU architecture. Conclusions Speedup of 15.93× over optimized multi-core SIMD CPU implementation and performance advantage of 16% over optimized GPU implementation are shown in the experimental results. More than 14% of the sequences are executed on CPU in the hybrid system. The system combining CPU and GPU to accelerate the Zuker algorithm is proven to be promising and can be applied to other bioinformatics applications. PMID:22369626

Microarray expression profiling in adhesion and normal peritoneal tissues.

PubMed

Ambler, Dana R; Golden, Alicia M; Gell, Jennifer S; Saed, Ghassan M; Carey, David J; Diamond, Michael P

2012-05-01

To identify molecular markers associated with adhesion and normal peritoneal tissue using microarray expression profiling. Comparative study. University hospital. Five premenopausal women. Adhesion and normal peritoneal tissue samples were obtained from premenopausal women. Ribonucleic acid was extracted using standard protocols and processed for hybridization to Affymetrix Whole Transcript Human Gene Expression Chips. Microarray data were obtained from five different patients, each with adhesion tissue and normal peritoneal samples. Real-time polymerase chain reaction was performed for confirmation using standard protocols. Gene expression in postoperative adhesion and normal peritoneal tissues. A total of 1,263 genes were differentially expressed between adhesion and normal tissues. One hundred seventy-three genes were found to be up-regulated and 56 genes were down-regulated in the adhesion tissues compared with normal peritoneal tissues. The genes were sorted into functional categories according to Gene Ontology annotations. Twenty-six up-regulated genes and 11 down-regulated genes were identified with functions potentially relevant to the pathophysiology of postoperative adhesions. We evaluated and confirmed expression of 12 of these specific genes via polymerase chain reaction. The pathogenesis, natural history, and optimal treatment of postoperative adhesive disease remains unanswered. Microarray analysis of adhesions identified specific genes with increased and decreased expression when compared with normal peritoneum. Knowledge of these genes and ontologic pathways with altered expression provide targets for new therapies to treat patients who have or are at risk for postoperative adhesions. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

PubMed Central

Cherry, Jonathan J.; Kobayashi, Dione T.; Lynes, Maureen M.; Naryshkin, Nikolai N.; Tiziano, Francesco Danilo; Zaworski, Phillip G.; Rubin, Lee L.

2014-01-01

Abstract Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects ∼1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of high-throughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials. PMID:25147906

Control of the photocatalytic activity of bimetallic complexes of pyropheophorbide-a by nucleic acids.

PubMed

Arian, Dumitru; Kovbasyuk, Larisa; Mokhir, Andriy

2011-12-05

Photocatalytic activity of a photosensitizer (PS) in an oligodeoxyribonucleotide duplex 5'-PS~ODN1/ODN2~Q-3' is inhibited because of close proximity of a quencher Q. The ODN2 in this duplex is selected to be longer than the ODN1. Therefore, in the presence of a nucleic acid (analyte), which is fully complementary to the ODN2 strand, the duplex is decomposed with formation of an analyte/ODN2~Q duplex and a catalytically active, single stranded PS~ODN1. In this way the catalytic activity of the PS can be controlled by the specific nucleic acids. We applied this reaction earlier for the amplified detection of ribonucleic acids in live cells (Arian, D.; Cló, E.; Gothelf, K.; Mokhir, A. Chem.-Eur. J.2010, 16(1), 288). As a photosensitizer (PS) we used In(3+)(pyropheophorbide-a)chloride and as a quencher (Q)--Black-Hole-Quencher-3 (BHQ-3). The In(3+) complex is a highly active photocatalyst in aqueous solution. However, it can coordinate additional ligands containing thiols (e.g., proteins, peptides, and aminoacids), that modulate properties of the complex itself and of the corresponding bio- molecules. These possible interactions can lead to undesired side effects of nucleic acid controlled photocatalysts (PS~ODN1/ODN2∼Q) in live cells. In this work we explored the possibility to substitute the In(3+) complex for those ones of divalent metal ions, Zn(2+) and Pd(2+), which exhibit lower or no tendency to coordinate the fifth ligand. We found that one of the compounds tested (Pd(pyropheophorbide-a) is as potent and as stable photosensitizer as its In(3+) analogue, but does not coordinate additional ligands that makes it more suitable for cellular applications. When the Pd complex was introduced in the duplex PS~ODN1/ODN2~Q as a PS, its photocatalytic activity could be controlled by nucleic acids as efficiently as that of the corresponding In(3+) complex.

Far-red fluorescent probes for canonical and non-canonical nucleic acid structures: current progress and future implications.

PubMed

Suseela, Y V; Narayanaswamy, Nagarjun; Pratihar, Sumon; Govindaraju, Thimmaiah

2018-02-05

The structural diversity and functional relevance of nucleic acids (NAs), mainly deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are indispensable for almost all living organisms, with minute aberrations in their structure and function becoming causative factors in numerous human diseases. The standard structures of NAs, termed canonical structures, are supported by Watson-Crick hydrogen bonding. Under special physiological conditions, NAs adopt distinct spatial organisations, giving rise to non-canonical conformations supported by hydrogen bonding other than the Watson-Crick type; such non-canonical structures have a definite function in controlling gene expression and are considered as novel diagnostic and therapeutic targets. Development of molecular probes for these canonical and non-canonical DNA/RNA structures has been an active field of research. Among the numerous probes studied, probes with turn-on fluorescence in the far-red (600-750 nm) region are highly sought-after due to minimal autofluorescence and cellular damage. Far-red fluorescent probes are vital for real-time imaging of NAs in live cells as they provide good resolution and minimal perturbation of the cell under investigation. In this review, we present recent advances in the area of far-red fluorescent probes of DNA/RNA and non-canonical G-quadruplex structures. For the sake of continuity and completeness, we provide a brief overview of visible fluorescent probes. Utmost importance is given to design criteria, characteristic properties and biological applications, including in cellulo imaging, apart from critical discussion on limitations of the far-red fluorescent probes. Finally, we offer current and future prospects in targeting canonical and non-canonical NAs specific to cellular organelles, through sequence- and conformation-specific far-red fluorescent probes. We also cover their implications in chemical and molecular biology, with particular focus on decoding various disease mechanisms involving NAs.

The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

NASA Technical Reports Server (NTRS)

Nelson, Kevin E.; Miller, Stanley L.

1992-01-01

The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or polymers with related peptide backbones were the dominant information macromolecules.

In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency.

PubMed Central

Davis, R; Vapnek, D

1976-01-01

The amounts of plasmid deoxyribonucleic acid (DNA) and the levels of the in vivo transcription of the Escherichia coli plasmids R538-1 (repressed for conjugal transfer) and R538-1drd (derepressed for transfer) were determined by DNA-DNA hybridization and DNA-ribonucleic acid hybridization, respectively. The results demonstrate that the level of plasmid transcription is increased by two-fold in the strain carrying the derepressed plasmid, compared to an isogenic strain carrying the repressed plasmid, whereas the amount of plasmid DNA is approximately the same, suggesting that the transfer genes are under transcriptional control. Levels of plasmid DNA, plasmid DNA transcription, and chloramphenicol acetyltransferase activity were also compared in a mutant strain that carried the R538-1drd plasmid and was resistant to high levels of antibiotics. This strain produces about 13 copies of plasmid DNA per chromosome compared to five copies for the parent strain. The level of transcription of plasmid DNA was found to be twofold higher in the high-level resistant strain, whereas the level of chloramphenition, acetyltransferase activity was increased by 10-fold. In addition the levels of plasmid DNA transcription and chloramphenicol acetyltransferase activity in the high-level resistant strain were found to be further increased by the presence of high levels of chloramphenicol in the growth medium. The amount of plasmid DNA remained constant under these conditions, indicating that high levels of chloramphenicol can stimulate the expression of plasmid genes at the level of transcription in this strain. PMID:767321

Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

PubMed

Taha; Siddiqui, K S; Campanaro, S; Najnin, T; Deshpande, N; Williams, T J; Aldrich-Wright, J; Wilkins, M; Curmi, P M G; Cavicchioli, R

2016-09-01

TRAM domain proteins present in Archaea and Bacteria have a β-barrel shape with anti-parallel β-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli: Culturability, membrane permeability, metabolism, and genetic damage.

PubMed

Xu, Limei; Zhang, Chongmiao; Xu, Pengcheng; Wang, Xiaochang C

2018-03-01

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection. The selection of a strict method is crucial for the evaluation of disinfection performance. The actions of 2 typical disinfectants - ultraviolet (UV) and chlorine - on the fecal indicator Escherichia coli were investigated by the detection of culturability, membrane permeability, metabolic activity, deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA). During UV disinfection, the irreversible damages in the cell membrane and cellular adenosine triphosphate (ATP) were negligible at low UV doses (<80mJ/cm 2 ). However, membrane permeability was damaged at low doses of chlorine (<5mg/L), leading to leakage of cellular ATP. Our study showed that a slight lesion in DNA was detected even at high doses of UV (400mJ/cm 2 ) and chlorine (>5mg/L) treatments. The decay of mRNA was more rapid than that of DNA. The degradation level of mRNA depended on the choice of target genes. After exposure to 50mJ/cm 2 UV dose or 5mg/L chlorine for 30min, the DNA damage repair function (RecA mRNA) was inhibited. The mRNA involved in the DNA damage repair function can be a potential indicator of bacterial viability. Copyright © 2017. Published by Elsevier B.V.

Association of anatase (TiO2) and microbes: unusual fossilization effect or a potential biosignature?

USGS Publications Warehouse

Glamoclija, Mihaela; Andrew Steele,; Marc Fries,; Juergen Schieber,; Voytek, Mary A.; Charles S. Cockell,

2015-01-01

We combined microbial paleontology and molecular biology methods to study the Eyreville B drill core from the 35.3-Ma-old Chesapeake Bay impact structure,Virginia, USA. The investigated sample is a pyrite vein collected from the 1353.81-1353.89 m depth interval, located within a section of biotite granite. The granite is a pre-impact rock that was disrupted by the impact event. A search for inorganic (mineral) biosignatures revealed the presence of micron-size rod morphologies of anatase (TiO2) embedded in chlorite coatings on pyrite grains. Neither the Acridine Orange microbial probe nor deoxyribonucleic acid (DNA) extraction followed by polymerase chain reaction (PCR) amplifi cation showed the presence of DNA or ribonucleic acid (RNA) at the location of anatase rods, implying the absence of viable cells in the investigated area. A Nile Red microbial probe revealed the presence of lipids in the rods. Because most of the lipids are resistant over geologic time spans, they are good biomarkers, and they are an indicator of biogenicity for these possibly 35-Ma-old microbial fossils. The mineral assemblage suggests that rod morphologies are associated with low-temperature (<100 °C) hydrothermal alteration that involved aqueous fl uids. The temporal constraints on the anatase fossils are still uncertain because pre-impact alteration of the granite and postimpact heating may have provided identical conditions for anatase precipitation and microbial preservation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamdani, Hazrina Yusof, E-mail: hazrina@mfrlab.org; Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas; Artymiuk, Peter J., E-mail: p.artymiuk@sheffield.ac.uk

A fundamental understanding of the atomic level interactions in ribonucleic acid (RNA) and how they contribute towards RNA architecture is an important knowledge platform to develop through the discovery of motifs from simple arrangements base pairs, to more complex arrangements such as triples and larger patterns involving non-standard interactions. The network of hydrogen bond interactions is important in connecting bases to form potential tertiary motifs. Therefore, there is an urgent need for the development of automated methods for annotating RNA 3D structures based on hydrogen bond interactions. COnnection tables Graphs for Nucleic ACids (COGNAC) is automated annotation system using graphmore » theoretical approaches that has been developed for the identification of RNA 3D motifs. This program searches for patterns in the unbroken networks of hydrogen bonds for RNA structures and capable of annotating base pairs and higher-order base interactions, which ranges from triples to sextuples. COGNAC was able to discover 22 out of 32 quadruples occurrences of the Haloarcula marismortui large ribosomal subunit (PDB ID: 1FFK) and two out of three occurrences of quintuple interaction reported by the non-canonical interactions in RNA (NCIR) database. These and several other interactions of interest will be discussed in this paper. These examples demonstrate that the COGNAC program can serve as an automated annotation system that can be used to annotate conserved base-base interactions and could be added as additional information to established RNA secondary structure prediction methods.« less

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PubMed

Singh, Susheel Kumar; Yadav, Deepti; Lal, Raj Kishori; Gupta, Madan M; Dhawan, Sunita Singh

2017-04-01

To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

The 193-Kd Vault Protein, Vparp, Is a Novel Poly(Adp-Ribose) Polymerase

PubMed Central

Kickhoefer, Valerie A.; Siva, Amara C.; Kedersha, Nancy L.; Inman, Elisabeth M.; Ruland, Cristina; Streuli, Michel; Rome, Leonard H.

1999-01-01

Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of ∼350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle. PMID:10477748

Legionella Pneumophila and Dendrimers-Mediated Antisense Therapy.

PubMed

Pashaei-Asl, Roghiyeh; Khodadadi, Khodadad; Pashaei-Asl, Fatima; Haqshenas, Gholamreza; Ahmadian, Nasser; Pashaiasl, Maryam; Hajihosseini Baghdadabadi, Reza

2017-06-01

Finding novel and effective antibiotics for treatment of Legionella disease is a challenging field. Treatment with antibiotics usually cures Legionella infection; however, if the resultant disease is not timely recognized and treated properly, it leads to poor prognosis and high case fatality rate. Legionella pneumophila DrrA protein (Defects in Rab1 recruitment protein A)/also known as SidM affects host cell vesicular trafficking through modification of the activity of cellular small guanosine triphosphatase )GTPase( Rab (Ras-related in brain) function which facilitates intracellular bacterial replication within a supporter vacuole. Also, Legionella pneumophila LepA and LepB (Legionella effector protein A and B) proteins suppress host-cell Rab1 protein's function resulting in the cell lysis and release of bacteria that subsequently infect neighbour cells. Legionella readily develops resistant to antibiotics and, therefore, new drugs with different modes of action and therapeutic strategic approaches are urgently required among antimicrobial drug therapies;gene therapy is a novel approach for Legionnaires disease treatment. On the contrary to the conventional treatment approaches that target bacterial proteins, new treatment interventions target DNA (Deoxyribonucleic acid), RNA (Ribonucleic acid) species, and different protein families or macromolecular complexes of these components. The above approaches can overcome the problems in therapy of Legionella infections caused by antibiotics resistance pathogens. Targeting Legionella genes involved in manipulating cellular vesicular trafficking using a dendrimer-mediated antisense therapy is a promising approach to inhibit bacterial replication within the target cells.

Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

PubMed Central

Shiue, Chiou-Nan; Nematollahi-Mahani, Amir; Wright, Anthony P.H.

2014-01-01

Chromatin domain organization and the compartmentalized distribution of chromosomal regions are essential for packaging of deoxyribonucleic acid (DNA) in the eukaryotic nucleus as well as regulated gene expression. Nucleoli are the most prominent morphological structures of cell nuclei and nucleolar organization is coupled to cell growth. It has been shown that nuclear scaffold/matrix attachment regions often define the base of looped chromosomal domains in vivo and that they are thereby critical for correct chromosome architecture and gene expression. Here, we show regulated organization of mammalian ribosomal ribonucleic acid genes into distinct chromatin loops by tethering to nucleolar matrix via the non-transcribed inter-genic spacer region of the ribosomal DNA (rDNA). The rDNA gene loop structures are induced specifically upon growth stimulation and are dependent on the activity of the c-Myc protein. Matrix-attached rDNA genes are hypomethylated at the promoter and are thus available for transcriptional activation. rDNA genes silenced by methylation are not recruited to the matrix. c-Myc, which has been shown to induce rDNA transcription directly, is physically associated with rDNA gene looping structures and the intergenic spacer sequence in growing cells. Such a role of Myc proteins in gene activation has not been reported previously. PMID:24609384

The influence of femtosecond laser pulse wavelength on embryonic stem cell differentiation

NASA Astrophysics Data System (ADS)

Mthunzi, Patience

2012-10-01

Stem cells are rich in proteins, carbohydrates, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and various other cellular components which are responsible for a diversity of functions. Mostly the building blocks of these intracellular entities play an active role in absorbing ultra-violet (UV) and visible light sources. Light-matter interactions in biomaterials are a complex situation and subsequent damage may not always amount only from wavelength dependent effects but may also be driven by a wealth of other optical parameters which may lead to a variety photochemical reactions. Previously, literature has reported efficient photo-transfection and differentiation of pluripotent stem cells via near infrared (NIR) femtosecond (fs) laser pulses with minimum compromise to their viability. Therefore, in this study the influence of using different fs laser wavelengths on optical stem cell transfection and differentiation is investigated. A potassium titanyl phosphate (KTP) crystal was employed in frequency doubling a 1064 nm fs laser beam. The newly generated 532 nm fs pulsed beam was then utilized for the first time in transient photo-transfection of ES-E14TG2a mouse embryonic stem (mES) cells. Compared to using 1064 nm fs pulses which non-invasively introduce plasmid DNA and other macromolecules into mES cells, our results showed a significant decline in the photo-transfection efficiency following transfecting with a pulsed fs visible green beam.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

PubMed

Massana, Ramon; Gobet, Angélique; Audic, Stéphane; Bass, David; Bittner, Lucie; Boutte, Christophe; Chambouvet, Aurélie; Christen, Richard; Claverie, Jean-Michel; Decelle, Johan; Dolan, John R; Dunthorn, Micah; Edvardsen, Bente; Forn, Irene; Forster, Dominik; Guillou, Laure; Jaillon, Olivier; Kooistra, Wiebe H C F; Logares, Ramiro; Mahé, Frédéric; Not, Fabrice; Ogata, Hiroyuki; Pawlowski, Jan; Pernice, Massimo C; Probert, Ian; Romac, Sarah; Richards, Thomas; Santini, Sébastien; Shalchian-Tabrizi, Kamran; Siano, Raffaele; Simon, Nathalie; Stoeck, Thorsten; Vaulot, Daniel; Zingone, Adriana; de Vargas, Colomban

2015-10-01

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Partial purification, characterization and translation in vitro of rat liver metallothionein messenger ribonucleic acid.

PubMed Central

Andersen, R D; Weser, U

1978-01-01

Poly(A)+ (polyadenylated) mRNA coding for metallothioneins was purified 13-fold from rat liver polyribosomes and was identified by its ability to direct the biosynthesis of these proteins in a wheat-germ cell-free system. The carboxymethylated products of the protein-synthesizing system in vitro were analysed with sodium dodecyl sulphate/20% polyacrylamide-gel electrophoresis. The labelled compounds [3H]serine and [35S]cysteine were incorporated at high specific radioactivity into proteins that co-migrated with authentic metallothioneins. No [3H]leucine incorporation was found, in agreement with the amino acid composition of the metallothioneins. Metallothionein mRNA had a sedimentation coefficient of 9 S and carried a maximum of four ribosomes. At 5 h after a subcutaneous injection of ZnCl2 or CdCl2 (10 mumol/kg body wt.), the amount of this mRNA increased approx. 2- and 4-fold respectively, on the basis of translation in vitro. The increase in metallothionein mRNA (defined by translation in the wheat-germ system) was transient and, after CdCl2 treatment, fell back to control values by 17 h. Metallothioneins constituted a maximum of 0.8% of the total protein products synthesized in the wheat-germ system by total mRNA isolated from rat liver after CdCl2 treatment. Images Fig. 5. Fig. 6. PMID:743237

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

PubMed Central

Ledinko, Nada; Fong, Caroline K. Y.

1969-01-01

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of 3H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. 3H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much 3H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase. PMID:5806981

Facile Synthesis of Multivalent Folate-Block Copolymer Conjugates via Aqueous RAFT Polymerization: Targeted Delivery of siRNA and Subsequent Gene Suppression†

PubMed Central

York, Adam W.; Zhang, Yilin; Holley, Andrew C.; Guo, Yanlin; Huang, Faqing; McCormick, Charles L.

2009-01-01

Cell specific delivery of small interfering ribonucleic acid (siRNA) using well-defined multivalent folate-conjugated block copolymers is reported. Primary amine functional, biocompatible, hydrophilic-block-cationic copolymers were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. N-(2-hydroxypropyl)methacrylamide) (HPMA), a permanently hydrophilic monomer, was copolymerized with a primary amine containing monomer, N-(3-aminopropyl)methacrylamide (APMA). Poly(HPMA) confers biocompatibility while APMA provides amine functionality allowing conjugation of folate derivatives. (HPMA-stat-APMA) was chain extended with a cationic block, poly(N-[3-(dimethylamino)propyl]methacrylamide) in order to promote electrostatic complexation between the copolymer and the negatively charged phosphate backbone of siRNA. Notably, poly(HPMA) stabilizes the neutral complexes in aqueous solution while APMA allows the conjugation of a targeting moiety, thus, dually circumventing problems associated with the delivery of genes via cationically charged complexes (universal transfection). Fluorescence microscopy and gene down-regulation studies indicate that these neutral complexes can be specifically delivered to cancer cells that over-express folate receptors. PMID:19290625

Biochemical transformation of lignin for deriving valued commodities from lignocellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gall, Daniel L.; Ralph, John; Donohue, Timothy J.

The biochemical properties of lignin present major obstacles to deriving societally beneficial entities from lignocellulosic biomass, an abundant and renewable feedstock. Similar to other biopolymers such as polysaccharides, polypeptides, and ribonucleic acids, lignin polymers are derived from multiple types of monomeric units. However, lignin’s renowned recalcitrance is largely attributable to its racemic nature and the variety of covalent inter-unit linkages through which its aromatic monomers are linked. Indeed, unlike other biopolymers whose monomers are consistently inter-linked by a single type of covalent bond, the monomeric units in lignin are linked via non-enzymatic, combinatorial radical coupling reactions that give rise tomore » a variety of inter-unit covalent bonds in mildly branched racemic polymers. Yet, despite the chemical complexity and stability of lignin, significant strides have been made in recent years to identify routes through which valued commodities can be derived from it. This paper discusses emerging biological and biochemical means through which degradation of lignin to aromatic monomers can lead to the derivation of commercially valuable products.« less

Biochemical transformation of lignin for deriving valued commodities from lignocellulose

DOE PAGES

Gall, Daniel L.; Ralph, John; Donohue, Timothy J.; ...

2017-03-24

The biochemical properties of lignin present major obstacles to deriving societally beneficial entities from lignocellulosic biomass, an abundant and renewable feedstock. Similar to other biopolymers such as polysaccharides, polypeptides, and ribonucleic acids, lignin polymers are derived from multiple types of monomeric units. However, lignin’s renowned recalcitrance is largely attributable to its racemic nature and the variety of covalent inter-unit linkages through which its aromatic monomers are linked. Indeed, unlike other biopolymers whose monomers are consistently inter-linked by a single type of covalent bond, the monomeric units in lignin are linked via non-enzymatic, combinatorial radical coupling reactions that give rise tomore » a variety of inter-unit covalent bonds in mildly branched racemic polymers. Yet, despite the chemical complexity and stability of lignin, significant strides have been made in recent years to identify routes through which valued commodities can be derived from it. This paper discusses emerging biological and biochemical means through which degradation of lignin to aromatic monomers can lead to the derivation of commercially valuable products.« less

Ultra-low input transcriptomics reveal the spore functional content and phylogenetic affiliations of poorly studied arbuscular mycorrhizal fungi.

PubMed

Beaudet, Denis; Chen, Eric C H; Mathieu, Stephanie; Yildirir, Gokalp; Ndikumana, Steve; Dalpé, Yolande; Séguin, Sylvie; Farinelli, Laurent; Stajich, Jason E; Corradi, Nicolas

2017-12-02

Arbuscular mycorrhizal fungi (AMF) are a group of soil microorganisms that establish symbioses with the vast majority of land plants. To date, generation of AMF coding information has been limited to model genera that grow well axenically; Rhizoglomus and Gigaspora. Meanwhile, data on the functional gene repertoire of most AMF families is non-existent. Here, we provide primary large-scale transcriptome data from eight poorly studied AMF species (Acaulospora morrowiae, Diversispora versiforme, Scutellospora calospora, Racocetra castanea, Paraglomus brasilianum, Ambispora leptoticha, Claroideoglomus claroideum and Funneliformis mosseae) using ultra-low input ribonucleic acid (RNA)-seq approaches. Our analyses reveals that quiescent spores of many AMF species harbour a diverse functional diversity and solidify known evolutionary relationships within the group. Our findings demonstrate that RNA-seq data obtained from low-input RNA are reliable in comparison to conventional RNA-seq experiments. Thus, our methodology can potentially be used to deepen our understanding of fungal microbial function and phylogeny using minute amounts of RNA material. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Utility of COX1 phylogenetics to differentiate between locally acquired and imported Plasmodium knowlesi infections in Singapore

PubMed Central

Loh, Jin Phang; Gao, Qiu Han Christine; Lee, Vernon J; Tetteh, Kevin; Drakeley, Chris

2016-01-01

INTRODUCTION Although there have been several phylogenetic studies on Plasmodium knowlesi (P. knowlesi), only cytochrome c oxidase subunit 1 (COX1) gene analysis has shown some geographical differentiation between the isolates of different countries. METHODS Phylogenetic analysis of locally acquired P. knowlesi infections, based on circumsporozoite, small subunit ribosomal ribonucleic acid (SSU rRNA), merozoite surface protein 1 and COX1 gene targets, was performed. The results were compared with the published sequences of regional isolates from Malaysia and Thailand. RESULTS Phylogenetic analysis of the circumsporozoite, SSU rRNA and merozoite surface protein 1 gene sequences for regional P. knowlesi isolates showed no obvious differentiation that could be attributed to their geographical origin. However, COX1 gene analysis showed that it was possible to differentiate between Singapore-acquired P. knowlesi infections and P. knowlesi infections from Peninsular Malaysia and Sarawak, Borneo, Malaysia. CONCLUSION The ability to differentiate between locally acquired P. knowlesi infections and imported P. knowlesi infections has important utility for the monitoring of P. knowlesi malaria control programmes in Singapore. PMID:26805667

Analysis of antigen-specific B-cell memory directly ex vivo.

PubMed

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

PubMed

Thorne, James L; Ouboussad, Lylia; Lefevre, Pascal F

2012-09-01

IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.

Utility of COX1 phylogenetics to differentiate between locally acquired and imported Plasmodium knowlesi infections in Singapore.

PubMed

Loh, Jin Phang; Gao, Qiu Han Christine; Lee, Vernon J; Tetteh, Kevin; Drakeley, Chris

2016-12-01

Although there have been several phylogenetic studies on Plasmodium knowlesi (P. knowlesi), only cytochrome c oxidase subunit 1 (COX1) gene analysis has shown some geographical differentiation between the isolates of different countries. Phylogenetic analysis of locally acquired P. knowlesi infections, based on circumsporozoite, small subunit ribosomal ribonucleic acid (SSU rRNA), merozoite surface protein 1 and COX1 gene targets, was performed. The results were compared with the published sequences of regional isolates from Malaysia and Thailand. Phylogenetic analysis of the circumsporozoite, SSU rRNA and merozoite surface protein 1 gene sequences for regional P. knowlesi isolates showed no obvious differentiation that could be attributed to their geographical origin. However, COX1 gene analysis showed that it was possible to differentiate between Singapore-acquired P. knowlesi infections and P. knowlesi infections from Peninsular Malaysia and Sarawak, Borneo, Malaysia. The ability to differentiate between locally acquired P. knowlesi infections and imported P. knowlesi infections has important utility for the monitoring of P. knowlesi malaria control programmes in Singapore. Copyright: © Singapore Medical Association

Structure of the nucleoid in cells of Streptococcus faecalis.

PubMed Central

Daneo-Moore, L; Dicker, D; Higgins, M L

1980-01-01

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells. Images PMID:6767695

Mechanism of fever induction in rabbits.

PubMed Central

Siegert, R; Philipp-Dormston, W K; Radsak, K; Menzel, H

1976-01-01

Three exogenous pyrogens (Escherichia coli lipopolysaccharide, synthetic double-stranded ribonucleic acid. Newcastle disease virus) were compared with respect to their mechanisms of fever induction in rabbits. All inducers stimulated the production of an endogenous pyrogen demonstrated in the blood as well as prostaglandins of the E group, and of cyclic adenosine 3',5'-monophosphate in the cerebrospinal fluid. The concentrations of these compounds were elevated approximately twofold as compared to the controls. Independently of the mode of induction, the fever reaction could be prevented by pretreatment with 5 mg of cycloheximide per kg, although the three fever mediators were induced as in febrile animals. Consequently, at least one additional fever mediator that is sensitive to a 30 to 50% inhibition of protein synthesis by cycloheximide has to be postulated. The comparable reactions of the rabbits after administration of different pyrogens argues for a similar fever mechanism. In contrast to fever induction there was no stimulation of endogenous pyrogen, prostaglandins of the E group, and cyclic adenosine 3',5'-monophosphate in hyperthermia as a consequence of exposure of the animals to exogenous overheating. Furthermore, hyperthermia could not be prevented by cycloheximide. PMID:185148

Gene expression-based biological test for major depressive disorder: an advanced study.

PubMed

Watanabe, Shin-Ya; Numata, Shusuke; Iga, Jun-Ichi; Kinoshita, Makoto; Umehara, Hidehiro; Ishii, Kazuo; Ohmori, Tetsuro

2017-01-01

Recently, we could distinguished patients with major depressive disorder (MDD) from nonpsychiatric controls with high accuracy using a panel of five gene expression markers ( ARHGAP24, HDAC5, PDGFC, PRNP , and SLC6A4 ) in leukocyte. In the present study, we examined whether this biological test is able to discriminate patients with MDD from those without MDD, including those with schizophrenia and bipolar disorder. We measured messenger ribonucleic acid expression levels of the aforementioned five genes in peripheral leukocytes in 17 patients with schizophrenia and 36 patients with bipolar disorder using quantitative real-time polymerase chain reaction (PCR), and we combined these expression data with our previous expression data of 25 patients with MDD and 25 controls. Subsequently, a linear discriminant function was developed for use in discriminating between patients with MDD and without MDD. This expression panel was able to segregate patients with MDD from those without MDD with a sensitivity and specificity of 64% and 67.9%, respectively. Further research to identify MDD-specific markers is needed to improve the performance of this biological test.

Oxytocin receptor mRNA expression in rat brain: implications for behavioral integration and reproductive success.

PubMed

Ostrowski, N L

1998-11-01

The nonapeptide, oxytocin (OT), has been implicated in a wide range of physiological, behavioral and pharmacological effects related to learning and memory, parturition and lactation, maternal and sexual behavior, and the formation of social attachments. Specific G-protein linked membrane bound OT receptors mediate OTs effects. The unavailability of highly selective pharmacological ligands that discriminate the OT receptor from the highly homologous vasopressin receptors (V1a, V1b and V2 subtypes) has made it difficult to confirm specific effects of oxytocin, particularly in brain regions where OT and multiple AVP receptor subtypes may be coexpressed. Here, data on the oxytocin receptor (OTR) messenger ribonucleic acid (mRNA) localization in brain are presented in the context of a model that proposes a reproductive state-dependent role for steroid-hormone restructuring of neural circuits, and a role for oxytocin in the integration of neural transmission in pathways subserving: (1) steroid-sensitive reproductive behaviors; (2) learning; and (3) reinforcement. It is hypothesized that social attachments emerge as a consequence of a conditioned association between OT-related activity in these pathways and the eliciting stimulus.

Congenitally learned helpless rats show abnormalities in intracellular signaling.

PubMed

Kohen, Ruth; Neumaier, John F; Hamblin, Mark W; Edwards, Emmeline

2003-03-15

Affective disorders and the drugs used to treat them lead to changes in intracellular signaling. We used a genetic animal model to investigate to what extent changes in intracellular signal transduction confer a vulnerability to mood or anxiety disorders. Levels of gene expression in a selectively bred strain of rats with a high vulnerability to develop congenitally learned helplessness (cLH), a strain highly resistant to the same behavior (cNLH) and outbred Sprague-Dawley (SD) control animals were compared using quantitative reverse transcription polymerase chain reaction. Congenitally learned helpless animals had a 24%-30% reduced expression of the cyclic adenosine monophosphate response element binding protein messenger ribonucleic acid (mRNA) in the hippocampus and a 40%-41% increased level of the antiapoptotic protein bcl-2 mRNA in the prefrontal cortex compared to cNLH and SD rats. Other significant changes included changes in the expression levels of the alpha catalytic subunit of protein kinase A, glycogen synthase kinase 3beta, and protein kinase C epsilon. Congenitally learned helpless animals show evidence of altered signal transduction and regulation of apoptosis compared to cNLH and SD control animals.

Base modifications affecting RNA polymerase and reverse transcriptase fidelity.

PubMed

Potapov, Vladimir; Fu, Xiaoqing; Dai, Nan; Corrêa, Ivan R; Tanner, Nathan A; Ong, Jennifer L

2018-06-20

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

[Protein and ribonucleic acid metabolism in the central nervous system of rats during space flight in the "Kosmos-605" satellite].

PubMed

Gazenko, O G; Demin, N N; Panov, A N; Rubinskaia, N L; Tigranian, R A

1976-01-01

On the 2nd postflight day the activity of neutral protamine peptide hydrolase of different compartments of the rat brain did not differ from the control level. With respect to the protein and RNA content and concentration motoneurons of anterior horns of the spinal cord and their glial-cells-satellites of rats exposed to the ground-based synchronous experiment did not differ from those of vivarium controls, except cells of the supraoptic nucleus. That was found on the 2nd and 27th postflight days. On the 2nd postflight day the protein and RNA concentration in neurons decreased and the protein concentration and content in gliocytes lowered; the RNA concentration and content in Purkinje cells of the cerebellum became reduced. On the 27th postflight day the RNA concentration in neurons of the supraoptic nucleus remained diminished whereas the protein content increased; in spinal motoneurons the protein concentration decreased and in adjacent gliocytes the protein concentration and content lowered. All the above changes made no more than 15% of the control values.

The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay

PubMed Central

Garrido-Bailón, Encarna; Higes, Mariano; Martínez-Salvador, Amparo; Antúnez, Karina; Botías, Cristina; Meana, Aránzazu; Prieto, Lourdes; Martín-Hernández, Raquel

2013-01-01

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied. PMID:23919248

Comparison of the Virion Polymerase of Reovirus with the Enzyme Purified from Reovirus-Infected Cells

PubMed Central

Gomatos, Peter J.

1970-01-01

Reovirus has in its protein coat an enzyme which catalyzes the net synthesis of the three size classes of virus-specific, single-stranded ribonucleic acid (RNA). For synthesis of 24, 19, and 14S single-stranded RNA, Mn++ was the preferred divalent cation, and ammonium sulfate at an optimal concentration of 4.2% of saturation was an absolute requirement. During synthesis, the parental double-stranded RNA was conserved in the viral core and the newly synthesized completed RNA chains were released as free RNA. The viral cores synthesizing RNA had properties consistent with the presence of nascent RNA on their outer surface. The enzyme-template complex from the infected cells described in an earlier paper was comprised of viral cores already active in the in vivo synthesis of single-stranded RNA. This pool of viral cores was newly made during infection, and exponential increase in the number of particles in this pool, as detected by the increase in enzymatic activity, occurred 2 hr earlier than that in mature virus. PMID:5483438

Preparation, purification and analyses of thirteen alkali-stable dinucleotides from ribonucleic acid

PubMed Central

Trim, A. R.; Parker, Janet E.

1970-01-01

Of the 16 alkali-stable dinucleotides known to be obtained by hydrolysis of commercial yeast RNA with alkali, 13 were prepared in quantities of the order of 10mg or more. The samples, with only one exception, contain at least 90% of dinucleotide, and spectroscopic constants and nucleotide-sequence determinations, although not conclusive, indicate a high degree of purity of these products. The small dinucleotide fraction in 150g of RNA hydrolysed with alkali (1–2% of the total nucleotides) was separated from the mononucleotides by stepwise ion-exchange chromatography on DEAE-cellulose columns and resolved into seven fractions containing from one to four different dinucleotides by electrophoresis on paper at pH3.0. These fractions were resolved into their constituent dinucleotides by chromatography in ammonium sulphate. Contamination of the products by impurities from the paper was minimized by washing it before using it for chromatography or electrophoresis and, by using a thick grade of paper (Whatman no. 17), it was possible to handle and purify relatively large quantities of nucleotides. PMID:5435489

Preservation of Fine-Needle Aspiration Specimens for Future Use in RNA-Based Molecular Testing

PubMed Central

Ladd, Amy C.; O'Sullivan-Mejia, Emerald; Lea, Tasha; Perry, Jessica; Dumur, Catherine I.; Dragoescu, Ema; Garrett, Carleton T.; Powers, Celeste N.

2015-01-01

Background The application of ancillary molecular testing is becoming more important for the diagnosis and classification of disease. The use of fine-needle aspiration (FNA) biopsy as the means of sampling tumors in conjunction with molecular testing could be a powerful combination. FNA is minimally invasive, cost effective, and usually demonstrates accuracy comparable to diagnoses based on excisional biopsies. Quality control (QC) and test validation requirements for development of molecular tests impose a need for access to pre-existing clinical samples. Tissue banking of excisional biopsy specimens is frequently performed at large research institutions, but few have developed protocols for preservation of cytologic specimens. This study aimed to evaluate cryopreservation of FNA specimens as a method of maintaining cellular morphology and ribonucleic acid (RNA) integrity in banked tissues. Methods FNA specimens were obtained from fresh tumor resections, processed by using a cryopreservation protocol, and stored for up to 27 weeks. Upon retrieval, samples were made into slides for morphological evaluation, and RNA was extracted and assessed for integrity by using the Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif). Results Cryopreserved specimens showed good cell morphology and, in many cases, yielded intact RNA. Cases showing moderate or severe RNA degradation could generally be associated with prolonged specimen handling or sampling of necrotic areas. Conclusions FNA specimens can be stored in a manner that maintains cellular morphology and RNA integrity necessary for studies of gene expression. In addition to addressing quality control (QC) and test validation needs, cytology banks will be an invaluable resource for future molecular morphologic and diagnostic research studies. PMID:21287691

Planetary Organic Chemistry and the Origins of Biomolecules

PubMed Central

Benner, Steven A.; Kim, Hyo-Joong; Kim, Myung-Jung; Ricardo, Alonso

2010-01-01

Organic chemistry on a planetary scale is likely to have transformed carbon dioxide and reduced carbon species delivered to an accreting Earth. According to various models for the origin of life on Earth, biological molecules that jump-started Darwinian evolution arose via this planetary chemistry. The grandest of these models assumes that ribonucleic acid (RNA) arose prebiotically, together with components for compartments that held it and a primitive metabolism that nourished it. Unfortunately, it has been challenging to identify possible prebiotic chemistry that might have created RNA. Organic molecules, given energy, have a well-known propensity to form multiple products, sometimes referred to collectively as “tar” or “tholin.” These mixtures appear to be unsuited to support Darwinian processes, and certainly have never been observed to spontaneously yield a homochiral genetic polymer. To date, proposed solutions to this challenge either involve too much direct human intervention to satisfy many in the community, or generate molecules that are unreactive “dead ends” under standard conditions of temperature and pressure. Carbohydrates, organic species having carbon, hydrogen, and oxygen atoms in a ratio of 1:2:1 and an aldehyde or ketone group, conspicuously embody this challenge. They are components of RNA and their reactivity can support both interesting spontaneous chemistry as part of a “carbohydrate world,” but they also easily form mixtures, polymers and tars. We describe here the latest thoughts on how on this challenge, focusing on how it might be resolved using minerals containing borate, silicate, and molybdate, inter alia. PMID:20504964

Group B Streptococci Colonization in Pregnant Guatemalan Women: Prevalence, Risk Factors, and Vaginal Microbiome.

PubMed

Rick, Anne-Marie; Aguilar, Angie; Cortes, Rosita; Gordillo, Remei; Melgar, Mario; Samayoa-Reyes, Gabriela; Frank, Daniel N; Asturias, Edwin J

2017-01-01

Infection causes 1 of every 5 neonatal deaths globally. Group B Streptococcus (GBS) is the most significant pathogen, although little is known about its epidemiology and risk in low-income countries. A cross-sectional study in 2015 at a public hospital in Guatemala City enrolled women ≥35 weeks' gestation. Vaginal and rectal swabs were processed using Lim broth and GBS CHROMagar then agglutination testing. Risk factors were assessed using multivariate analysis. Vaginal microbiota were profiled by 16S ribosomal ribonucleic acid sequencing in a subset of 94 women. Of 896 pregnant women, 155 (17.3%; 95% confidence interval [CI], 14.9-19.9) were GBS colonized. Colonization was associated with history of previous infant with poor outcome (odds ratio [OR], 1.94; 95% CI, 1.15-3.27) and increasing maternal age (OR, 1.05; 95% CI, 1.02-1.09). Multiparity was protective (OR, .39; 95% CI, .21-.72). Four (6%) GBS-exposed infants had early-onset neonatal sepsis. Vaginal microbiome composition was associated with previous antibiotic exposure ( P = .003) and previous low birth weight infant ( P = .03), but not GBS colonization ( P = .72). Several individual taxa differed in abundance between colonized and noncolonized women. Group B Streptococcus is prevalent in pregnant women from Guatemala with different risk factors than previously described. Although the vaginal microbiome was not altered significantly in GBS-colonized women, use of antibiotics had an effect on its composition. © The Author 2017. Published by Oxford University Press on behalf of Infectious Diseases Society of America.

Secondary Structure Predictions for Long RNA Sequences Based on Inversion Excursions and MapReduce.

PubMed

Yehdego, Daniel T; Zhang, Boyu; Kodimala, Vikram K R; Johnson, Kyle L; Taufer, Michela; Leung, Ming-Ying

2013-05-01

Secondary structures of ribonucleic acid (RNA) molecules play important roles in many biological processes including gene expression and regulation. Experimental observations and computing limitations suggest that we can approach the secondary structure prediction problem for long RNA sequences by segmenting them into shorter chunks, predicting the secondary structures of each chunk individually using existing prediction programs, and then assembling the results to give the structure of the original sequence. The selection of cutting points is a crucial component of the segmenting step. Noting that stem-loops and pseudoknots always contain an inversion, i.e., a stretch of nucleotides followed closely by its inverse complementary sequence, we developed two cutting methods for segmenting long RNA sequences based on inversion excursions: the centered and optimized method. Each step of searching for inversions, chunking, and predictions can be performed in parallel. In this paper we use a MapReduce framework, i.e., Hadoop, to extensively explore meaningful inversion stem lengths and gap sizes for the segmentation and identify correlations between chunking methods and prediction accuracy. We show that for a set of long RNA sequences in the RFAM database, whose secondary structures are known to contain pseudoknots, our approach predicts secondary structures more accurately than methods that do not segment the sequence, when the latter predictions are possible computationally. We also show that, as sequences exceed certain lengths, some programs cannot computationally predict pseudoknots while our chunking methods can. Overall, our predicted structures still retain the accuracy level of the original prediction programs when compared with known experimental secondary structure.

Biomarkers of the Hedgehog/Smoothened pathway in healthy volunteers

PubMed Central

Kadam, Sunil K; Patel, Bharvin K R; Jones, Emma; Nguyen, Tuan S; Verma, Lalit K; Landschulz, Katherine T; Stepaniants, Sergey; Li, Bin; Brandt, John T; Brail, Leslie H

2012-01-01

The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA) levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with ≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays. Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5 (MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors of the Hh pathway. PMID:22611475

Changes in insulin-like growth factor-binding protein-3 messenger ribonucleic acid in endothelial cells of the human corpus luteum: a possible role in luteal development and rescue.

PubMed

Fraser, H M; Lunn, S F; Kim, H; Duncan, W C; Rodger, F E; Illingworth, P J; Erickson, G F

2000-04-01

In the human menstrual cycle, extensive angiogenesis accompanies luteinization; and the process is physiologically important for corpus luteum (CL) function. During luteolysis, the vasculature collapses, and the endothelial cells die. In a conceptual cycle, the CL persists both functionally and structurally beyond the luteoplacental shift. Although luteal rescue is not associated with increased angiogenesis, endothelial survival is extended. Despite the central role of the luteal vasculature in fertility, the mechanisms regulating its development and demise are poorly understood. There is increasing evidence that insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) may be important effectors of luteal function. Here, we have found that IGFBP-3 messenger RNA is expressed in the endothelium of the human CL and that the levels of message change during luteal development and rescue by human CG. The signal was strong during the early luteal phase, but it showed significant reduction during the mid- and late luteal phases. Interestingly, administration of human CG caused a marked increase in the levels of IGFBP-3 messenger RNA in luteal endothelial cells that was comparable to that observed during the early luteal phase. We conclude that endothelial cell IGFBP-3 expression is a physiological property of the CL of menstruation and pregnancy. These observations raise the intriguing possibility that the regulated expression of endothelial IGFBP-3 may play a role in controlling angiogenesis and cell responses in the human CL by autocrine/paracrine mechanisms.

RNA sequencing analysis of transcriptional change in the freshwater mussel Elliptio complanata after environmentally relevant sodium chloride exposure.

PubMed

Robertson, Laura S; Galbraith, Heather S; Iwanowicz, Deborah; Blakeslee, Carrie J; Cornman, R Scott

2017-09-01

To identify potential biomarkers of salt stress in a freshwater sentinel species, we examined transcriptional responses of the common mussel Elliptio complanata to controlled sodium chloride (NaCl) exposures. Ribonucleic acid sequencing (RNA-Seq) of mantle tissue identified 481 transcripts differentially expressed in adult mussels exposed to 2 ppt NaCl (1.2 ppt chloride) for 7 d, of which 290 had nonoverlapping intervals. Differentially expressed gene categories included ion and transmembrane transport, oxidoreductase activity, maintenance of protein folding, and amino acid metabolism. The rate-limiting enzyme for synthesis of taurine, an amino acid frequently linked to osmotic stress in aquatic species, was upregulated, as was the transmembrane ion pump sodium/potassium adenosine 5'-triphosphatase. These patterns confirm a primary transcriptional response to the experimental dose, albeit likely overlapping with nonspecific secondary stress responses. Substantial involvement of the heat shock protein 70 chaperone family and the water-transporting aquaporin family was not detected, however, in contrast to some studies in other bivalves. A subset of the most significantly regulated genes was confirmed by quantitative polymerase chain reaction in an independent sample. Cluster analysis showed separation of mussels exposed to 2 ppt NaCl from control mussels in multivariate space, but mussels exposed to 1 ppt NaCl were largely indistinguishable from controls. Transcriptome-scale analysis of salt exposure under laboratory conditions efficiently identified candidate biomarkers for further functional analysis and field validation. Environ Toxicol Chem 2017;36:2352-2366. © Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America. © 2017 SETAC.

Retinoic acid stimulates interstitial collagenase messenger ribonucleic acid in osteosarcoma cells

NASA Technical Reports Server (NTRS)

Connolly, T. J.; Clohisy, J. C.; Shilt, J. S.; Bergman, K. D.; Partridge, N. C.; Quinn, C. O.

1994-01-01

The rat osteoblastic osteosarcoma cell line UMR 106-01 secretes interstitial collagenase in response to retinoic acid (RA). The present study demonstrates by Northern blot analysis that RA causes an increase in collagenase messenger RNA (mRNA) at 6 h, which is maximal at 24 h (20.5 times basal) and declines toward basal level by 72 h. This stimulation is dose dependent, with a maximal response at 5 x 10(-7) M RA. Nuclear run-on assays show a greater than 20-fold increase in the rate of collagenase mRNA transcription between 12-24 h after RA treatment. Cycloheximide blocks RA stimulation of collagenase mRNA, demonstrating the need for de novo protein synthesis. RA not only causes an increase in collagenase secretion, but is known to decrease collagen synthesis in UMR 106-01 cells. In this study, the increase in collagenase mRNA is accompanied by a concomitant decrease in the level of alpha 1(I) procollagen mRNA, which is maximal at 24 h (70% decrease), with a return to near-control levels by 72 h. Nuclear run-on assays demonstrated that the decrease in alpha 1 (I) procollagen expression does not have a statistically significant transcriptional component. RA did not statistically decrease the stability of alpha 1 (I) procollagen mRNA (calculated t1/2 = 8.06 +/- 0.30 and 9.01 +/- 0.62 h in the presence and absence of RA, respectively). However, transcription and stability together probably contribute to the major decrease in stable alpha 1 (I) procollagen mRNA observed. Cycloheximide treatment inhibits basal level alpha 1 (I) procollagen mRNA accumulation, demonstrating the need for on-going protein synthesis to maintain basal expression of this gene.

Effects of Oleate Starvation in a Fatty Acid Auxotroph of Escherichia coli K-12

PubMed Central

Henning, U.; Dennert, G.; Rehn, K.; Deppe, Gisela

1969-01-01

The effects of oleate starvation on an oleate auxotroph of Escherichia coli K-12 were investigated. Following removal of oleate from the mutant growing in a minimal glycerol-peptone medium, the cells stopped making deoxyribonucleic acid, ribonucleic acid, protein, and phospholipids; they began to die exponentially and finally lysed. During oleate starvation in minimal medium minus peptone, inhibition of macromolecular syntheses and death occurred; however, lysis did not follow. When growth ceased, no further dying was observed. It is shown that none of the early effects (inhibition of macromolecular syntheses and death) can be due to leakiness of the cells, induction of a prophage or a colicin, or lack of energy sources. The cause of inhibition of macromolecular syntheses remained unknown. Since the rate of death was the same as the generation time under different conditions, it appears that death is due to the defective synthesis of some cellular structure (quite possibly, cytoplasmic membrane) during phospholipid deficiency. Lysis was found to require protein synthesis; electron microscopy revealed a peculiar type of “lysis from within”; i.e., the shape of the cells did not change but fragmentation of the inner layer of the cell envelope occurred. The murein was found to be unaltered. Most likely, lysis was a consequence of the cell's attempt to synthesize cytoplasmic membrane with altered phospholipid composition or during phospholipid deficiency. Several membrane functions (respiration, adenosine triphosphate formation, permeability) existing before oleate removal were not lost during starvation. Therefore, general damage to the membrane did not occur, and it could be that most, if not all, described effects were due to defective de novo membrane synthesis. Images PMID:4891268

Transcriptome analysis of the pectoral muscles of local chickens and commercial broilers using Ribo-Zero ribonucleic acid sequencing

PubMed Central

Han, Ruili; Wang, Yanbin; Li, Guoxi; Liu, Xiaojun; Tian, Yadong; Kang, Xiangtao

2017-01-01

Background The molecular mechanisms underlying meat quality and muscle growth are not clear. The meat quality and growth rates of local chickens and commercial broilers are very different. The Ribo-Zero RNA-Seq technology is an effective means of analyzing transcript groups to clarify molecular mechanisms. The aim of this study was to provide a reference for studies of the differences in the meat quality and growth of different breeds of chickens. Results Ribo-Zero RNA-Seq technology was used to analyze the pectoral muscle transcriptomes of Gushi chickens and AA broilers. Compared with AA broilers, 1649 genes with annotated information were significantly differentially expressed (736 upregulated and 913 downregulated) in Gushi chickens with Q≤0.05 (Q is the P-value corrected by multiple assumptions test) at a fold change ≥2 or ≤0.5. In addition, 2540 novel significantly differentially expressed (SDE) genes (1405 upregulated and 1135 downregulated) were discovered. The results showed that the main signal transduction pathways that differed between Gushi chickens and AA broilers were related to amino acid metabolism. Amino acids are important for protein synthesis, and they regulate key metabolic pathways to improve the growth, development and reproduction of organisms. Conclusion This study showed that differentially expressed genes in the pectoral tissues of Gushi chickens and AA broilers were related to fat metabolism, which affects meat. Additionally, a large number of novel genes were found that may be involved in fat metabolism and thus may affect the formation of meat, which requires further study. The results of this study provide a reference for further studies of the molecular mechanisms of meat formation. PMID:28863190

Transcriptome analysis of the pectoral muscles of local chickens and commercial broilers using Ribo-Zero ribonucleic acid sequencing.

PubMed

Zhang, Yanhua; Li, Donghua; Han, Ruili; Wang, Yanbin; Li, Guoxi; Liu, Xiaojun; Tian, Yadong; Kang, Xiangtao; Li, Zhuanjian

2017-01-01

The molecular mechanisms underlying meat quality and muscle growth are not clear. The meat quality and growth rates of local chickens and commercial broilers are very different. The Ribo-Zero RNA-Seq technology is an effective means of analyzing transcript groups to clarify molecular mechanisms. The aim of this study was to provide a reference for studies of the differences in the meat quality and growth of different breeds of chickens. Ribo-Zero RNA-Seq technology was used to analyze the pectoral muscle transcriptomes of Gushi chickens and AA broilers. Compared with AA broilers, 1649 genes with annotated information were significantly differentially expressed (736 upregulated and 913 downregulated) in Gushi chickens with Q≤0.05 (Q is the P-value corrected by multiple assumptions test) at a fold change ≥2 or ≤0.5. In addition, 2540 novel significantly differentially expressed (SDE) genes (1405 upregulated and 1135 downregulated) were discovered. The results showed that the main signal transduction pathways that differed between Gushi chickens and AA broilers were related to amino acid metabolism. Amino acids are important for protein synthesis, and they regulate key metabolic pathways to improve the growth, development and reproduction of organisms. This study showed that differentially expressed genes in the pectoral tissues of Gushi chickens and AA broilers were related to fat metabolism, which affects meat. Additionally, a large number of novel genes were found that may be involved in fat metabolism and thus may affect the formation of meat, which requires further study. The results of this study provide a reference for further studies of the molecular mechanisms of meat formation.

Yaba Tumor Poxvirus Synthesis In Vitro I. Cytopathological, Histochemical, and Immunofluorescent Studies

PubMed Central

Yohn, David S.; Haendiges, Victoria A.; Grace, James T.

1966-01-01

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Victoria A. Haendiges, and James T. Grace, Jr. Yaba tumor poxvirus synthesis in vitro. I. Cytopathological, histochemical, and immunofluorescent studies. J. Bacteriol. 91:1977–1985. 1966.—Yaba tumor poxvirus synthesis in BSC-1 cell culture was followed sequentially with light microscopy, immunofluorescent microscopy, and various histochemical stains. The first evidence of infection was detected at 24 hr when nucleoli became hypertrophic, reflecting enhanced ribonucleic acid (RNA) synthesis. At 36 hr, deoxyribonucleic acid synthesis was detected in the cytoplasm. This was immediately followed by or associated with antigen synthesis at paranuclear sites and enhanced RNA synthesis in the cytoplasm. Cytoplasmic inclusions were readily apparent at 4 days in initially infected cells. Contiguous spread of virus was judged to have occurred around the third day of infection. The time required to complete the synthetic cycle from time of infection to production of infectious progeny was estimated to be of the order of 60 hr. This cycle is 6 to 10 times longer than for vaccinia virus. By light microscopy, cytopathic effects (CPE) were detectable in 5 days in heavily infected cultures. With 100 units or less of infectious virus, CPE was not readily detected until the 10th to 14th day. At this time, focal areas of infection classified either as microtumors or microplaques were present. Secondary foci appeared during the 4th week of incubation. Images PMID:4287080

Relevance of feline calicivirus, feline immunodeficiency virus, feline leukemia virus, feline herpesvirus and Bartonella henselae in cats with chronic gingivostomatitis.

PubMed

Belgard, Sylvia; Truyen, Uwe; Thibault, Jean-Christophe; Sauter-Louis, Carola; Hartmann, Katrin

2010-01-01

Despite its common occurrence, the aetiology of chronic gingivostomatitis in cats remains uncertain. Aetiology is likely multifactorial, and several infectious agents may be associated with chronic gingivostomatitis. The purpose of this study was to investigate the prevalence of feline calicivirus (FCV), feline immunodeficiency virus (FIV), feline leukemia virus (FeLV), feline herpesvirus (FHV), and Bartonella henselae (B. henselae) in cats with chronic gingivostomatitis and in an age-matched control group. In addition, other factors, e. g., environmental conditions were investigated. In 52 cats with chronic gingivostomatitis and 50 healthy age-matched control cats, the presence of FCV ribonucleic acid (RNA), and FHV deoxyribonucleic acid (DNA) (polymerase chain reaction [PCR] from oropharyngeal swabs), and B. henselae DNA (PCR from oropharyngeal swabs and blood), as well as FeLV antigen (serum), and antibodies against FCV, B. henselae, and FIV (serum) were examined. FCV RNA was significantly more common in cats with chronic gingivostomatitis (53.8%, p < 0.001) than in controls (14.0%); a significant difference was also found in the prevalence of antibodies to FCV between the cats with chronic gingivostomatitis (78.8%, p = 0.023) and controls (58.0%). Of the other infectious agents investigated, there was no significant difference in the prevalence between the cats with chronic gingivostomatitis and the controls. The results of this study allow the conclusion that FCV, but no other infectious agents, is commonly associated with chronic gingivostomatitis in cats.

Detection and sequencing of rotavirus among sudanese children.

PubMed

Magzoub, Magzoub Abbas; Bilal, Naser Eldin; Bilal, Jalal Ali; Alzohairy, Mohammad Abdulrahman; Elamin, Bahaeldin Khalid; Gasim, Gasim Ibrahim

2017-01-01

Diarrheal diseases are a big public health problem worldwide, particularly among developing countries. The current study was conducted to detect and characterize group A rotavirus among admitted children with gastroenteritis to the pediatric hospitals, Sudan. A total of 755 stool samples were collected from Sudanese children with less than 5 years of age presenting with acute gastroenteritis during the period from April to September 2010. Enzyme-linked immunosorbent assay (ELISA) was used to Detection of Rotavirus antigens. Ribonucleic acid (RNAs) were extracted from rotavirus-positive stool samples using (QIAamp ® Viral RNA Mini Kit). (Omniscript ® Reverse Transcription kit) was used to convert RNA to complementary Deoxyribonucleic acid (cDNA). The cDNAs were used as template for detection of VP4-P (P for Protease-sensitive) and VP7-G (G for Glycoprotein) genotyping of Rotavirus using nested PCR and sequencing. Out of the 755 stool samples from children with acute gastroenteritis, 121 were positive for rotavirus A. Among 24 samples that were sequenced; the VP7 predominant G type was G1 (83.3%), followed by G9 (16.7%). Out of these samples, only one VP4 P[8] genotype was detected. As a conclusion the VP7 predominant G type was G1, followed by G9 whereas only one VP4 genotype was detected and showed similarity to P[8] GenBank strain. It appears that the recently approved rotavirus vaccines in Sudan are well matched to the rotavirus genotypes identified in this study, though more studies are needed.

Association of anatase (TiO2) and microbes: Unusual fossilization effect or a potential biosignature?

USGS Publications Warehouse

Glamoclija, M.; Steele, A.; Fries, M.; Schieber, J.; Voytek, M.A.; Cockell, C.S.

2009-01-01

We combined microbial paleontology and molecular biology methods to study the Eyreville B drill core from the 35.3-Ma-old Chesapeake Bay impact structure, Virginia, USA. The investigated sample is a pyrite vein collected from the 1353.81- 1353.89 m depth interval, located within a section of biotite granite. The granite is a pre-impact rock that was disrupted by the impact event. A search for inorganic (mineral) biosignatures revealed the presence of micron-size rod morphologies of anatase (TiO2) embedded in chlorite coatings on pyrite grains. Neither the Acridine Orange microbial probe nor deoxyribonucleic acid (DNA) extraction followed by polymerase chain reaction (PCR) amplifi cation showed the presence of DNA or ribonucleic acid (RNA) at the location of anatase rods, implying the absence of viable cells in the investigated area. A Nile Red microbial probe revealed the presence of lipids in the rods. Because most of the lipids are resistant over geologic time spans, they are good biomarkers, and they are an indicator of biogenicity for these possibly 35-Ma-old microbial fossils. The mineral assemblage suggests that rod morphologies are associated with low-temperature (<100 ??C) hydrothermal alteration that involved aqueous fluids. The temporal constraints on the anatase fossils are still uncertain because pre-impact alteration of the granite and postimpact heating may have provided identical conditions for anatase precipitation and microbial preservation. ?? 2009 The Geological Society of America.

Detection of genome, antigen, and antibodies in oral fluids from pigs infected with foot-and-mouth disease virus.

PubMed

Senthilkumaran, Chandrika; Yang, Ming; Bittner, Hilary; Ambagala, Aruna; Lung, Oliver; Zimmerman, Jeffrey; Giménez-Lirola, Luis G; Nfon, Charles

2017-04-01

Virus nucleic acids and antibody response to pathogens can be measured using swine oral fluids (OFs). Detection of foot-and-mouth disease virus (FMDV) genome in swine OFs has previously been demonstrated. Virus isolation and viral antigen detection are additional confirmatory assays for diagnosing FMDV, but these methods have not been evaluated using swine OF. The objectives of this study were to further validate the molecular detection of FMDV in oral fluids, evaluate antigen detection and FMDV isolation from swine OFs, and develop an assay for isotypic anti-FMDV antibody detection in OFs. Ribonucleic acid (RNA) from FMDV was detected in OFs from experimentally infected pigs by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) from 1 day post-infection (dpi) to 21 dpi. Foot-and-mouth disease virus (FMDV) was isolated from OFs at 1 to 5 dpi. Additionally, FMDV antigens were detected in OFs from 1 to 6 dpi using a lateral flow immunochromatographic strip test (LFIST), which is a rapid pen-side test, and from 2 to 3 dpi using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). Furthermore, FMDV-specific immunoglobulin A (IgA) was detected in OFs using an isotype-specific indirect ELISA starting at dpi 14. These results further demonstrated the potential use of oral fluids for detecting FMDV genome, live virus, and viral antigens, as well as for quantifying mucosal IgA antibody response.

Refining the Genetic Alphabet: A Late-Period Selection Pressure?

PubMed Central

Tor, Yitzhak

2012-01-01

Abstract The transition from genomic ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) in primitive cells may have created a selection pressure that refined the genetic alphabet, resulting from the global weakening of the N-glycosyl bonds. Hydrolytic rupture of these bonds, termed deglycosylation, leaves an abasic site that is the single greatest threat to the stability and integrity of genomic DNA. The rates of deglycosylation are highly dependent on the identity of the nucleobases. Modifications made to the bases, such as deamination, oxidation, and alkylation, can further increase deglycosylation reaction rates, suggesting that the native bases provide optimum N-glycosyl bond stability. To protect their genomes, cells have evolved highly specific enzymes called glycosylases, associated with DNA repair, that detect and remove these damaged bases. In RNA, however, the occurrence of many of these modified bases is deliberate. The dichotomous behavior that cells exhibit toward base modifications may have originated in the RNA world. Modified bases would have been advantageous for the functional and structural repertoire of catalytic RNAs. Yet in an early DNA world, the utility of these heterocycles was greatly diminished, and their presence posed a distinct liability to the stability of cells' genomes. A natural selection for bases exhibiting the greatest resistance to deglycosylation would have ensured the viability of early DNA life, along with the recruitment of DNA repair. Key Words: DNA—Nucleic acids—RNA world—Asteroid—Chemical evolution—Ribozymes. Astrobiology 12, 884–891. PMID:22984873

Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging

PubMed Central

Rockey, William M.; Huang, Ling; Kloepping, Kyle C.; Baumhover, Nicholas J.; Giangrande, Paloma H.; Schultz, Michael K.

2014-01-01

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g. copper-64, 64Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of 64Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10–3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g. pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with 64Cu. PMID:21658962

Stellate macroporous silica nanospheres in bio-macromolecules encapsulation and delivery

NASA Astrophysics Data System (ADS)

Chi, Hao-Hsin

This project focused on using mesoporous silica as a solid support to encapsulate enzymes for operating a highly economic, and recyclable biomass processing system. The main objective is to turn non-food biomass sources into food products. Enzymes are macromolecules with the structural backbone of proteins or ribonucleic acid sequences (RNAs) which work as catalysts in living organisms. Enzymes have the advantage of being the least contaminating catalyst due to normal catalyst might generate toxic by-product, and preferable to organic and inorganic catalysts, especially when used for product related to human used, which require biocompatibility of final product. However, there are several disadvantages in enzyme utilization. Their fabrication is time-consuming and requires elaborated molecular biology processes. Most of the enzymes need well-defined reaction conditions to be functional and operate at high yield. Unfortunately, although they are reusable as normal catalysts, it proves difficult to extract or reuse the enzymes from a reaction. Also, enzyme molecules are easily degradable and demand proper storage. To overcome some of the disadvantages, especially regarding stability to degradation, recovery, and reusability, immobilization of enzyme on solid support has become a thriving methodology. In recent years, mesoporous silica nanomaterials(MSN) have been at the forefront of enzyme immobilization given their extensive surface area, which provides capability to increase enzyme loading and for their demonstrate ability to protect enzyme from degradation, thus enabling high recyclability. Mesoporous silica is biocompatible and has already been used for several applications included. Catalysis, drug delivery, and Bio-imaging. Previously published research utilized mesoporous silica to deliver drugs, DNAs, RNAs or encapsulate single enzyme. The objective of this research is completed to develop a new porous silica platform that is unique in its porosity structure and develop it into a dual-enzyme platform with the scope of demonstrating a multi-reaction bio nanocatalyst. In regard to the further applications, the stellate MSN can be used as drug delivery or become a package of the biomacromolecule delivery system kit.

Assessment and Optimization of the GeneXpert Diagnostic Platform for Detection of Ebola Virus RNA in Seminal Fluid.

PubMed

Pettitt, James; Higgs, Elizabeth; Fallah, Mosoka; Nason, Martha; Stavale, Eric; Marchand, Jonathan; Reilly, Cavan; Jensen, Kenneth; Dighero-Kemp, Bonnie; Tuznik, Kaylie; Logue, James; Bolay, Fatorma; Hensley, Lisa

2017-02-15

Recent studies have suggested that Ebola virus (EBOV) ribonucleic acid (RNA) potentially present in the semen of a large number of survivors of Ebola virus disease (EVD) in Western Africa may contribute to sexual transmission of EVD and generate new clusters of cases in regions previously declared EVD-free. These findings drive the immediate need for a reliable, rapid, user-friendly assay for detection of EBOV RNA in semen that is deployable to multiple sites across Western Africa. In this study, we optimized the Xpert EBOV assay for semen samples by adding dithiothreitol. Compared to the assays currently in use in Liberia (including Ebola Zaire Target 1, major groove binder real-time-polymerase chain reaction assays, and original Xpert EBOV assay), the modified Xpert EBOV assay demonstrated greater sensitivity than the comparator assays. Thus, the modified Xpert EBOV assay is optimal for large-scale monitoring of EBOV RNA persistence in male survivors. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

5'-adenosine monophosphate-induced hypothermia attenuates brain ischemia/reperfusion injury in a rat model by inhibiting the inflammatory response.

PubMed

Miao, Yi-Feng; Wu, Hui; Yang, Shao-Feng; Dai, Jiong; Qiu, Yong-Ming; Tao, Zhen-Yi; Zhang, Xiao-Hua

2015-01-01

Hypothermia treatment is a promising therapeutic strategy for brain injury. We previously demonstrated that 5'-adenosine monophosphate (5'-AMP), a ribonucleic acid nucleotide, produces reversible deep hypothermia in rats when the ambient temperature is appropriately controlled. Thus, we hypothesized that 5'-AMP-induced hypothermia (AIH) may attenuate brain ischemia/reperfusion injury. Transient cerebral ischemia was induced by using the middle cerebral artery occlusion (MCAO) model in rats. Rats that underwent AIH treatment exhibited a significant reduction in neutrophil elastase infiltration into neuronal cells and matrix metalloproteinase 9 (MMP-9), interleukin-1 receptor (IL-1R), tumor necrosis factor receptor (TNFR), and Toll-like receptor (TLR) protein expression in the infarcted area compared to euthermic controls. AIH treatment also decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL-) positive neuronal cells. The overall infarct volume was significantly smaller in AIH-treated rats, and neurological function was improved. By contrast, rats with ischemic brain injury that were administered 5'-AMP without inducing hypothermia had ischemia/reperfusion injuries similar to those in euthermic controls. Thus, the neuroprotective effects of AIH were primarily related to hypothermia.

Studies on sex-organ development. Changes in chromatin structure during spermatogenesis in maturing rooster testis as demonstrated by the initiation pattern of ribonucleic acid synthesis in vitro.

PubMed Central

Mezquita, C; Teng, C S

1978-01-01

To probe the structural change in the genome of the differentiating germ cell of the maturing rooster testis, the chromatin from nuclei at various stages of differentiation were transcribed with prokaryotic RNA polymerase from Escherichia coli or with eukaryotic RNA polymerase II from wheat germ. The transcription was performed under conditions of blockage of RNA chain reinitiation in vitro with rifampicin or rifampicin AF/013. With the E. coli enzyme, the changes in (1) the titration curve for the enzyme-chromatin interaction, (2) the number of initiation sites, (3) the rate of elongation of RNA chains, and (4) the kinetics of the formation of stable initiation complexes revealed the unmasking of DNA in elongated spermatids and the masking of DNA in spermatozoa. In both cases the stability of the DNA duplex in the initiation region for RNA synthesis greatly increased. In contrast with the E. coli enzyme, the wheat-germ RNA polymerase II was relatively inefficient at transcribing chromatin of elongated spermatids. Such behaviour can be predicted if unmasked double-stranded DNA is present in elongated spermatids. PMID:346018

Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells.

PubMed Central

Hendrick, J P; Wolin, S L; Rinke, J; Lerner, M R; Steitz, J A

1981-01-01

Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed. Images PMID:6180298

Epstein-Barr virus associated T-cell lymphoproliferative disease misdiagnosed as ulcerative colitis: a case report.

PubMed

Zheng, Xiaodan; Xie, Jianlan; Zhou, Xiaoge

2015-01-01

Epstein-Barr virus (EBV)-associated T-cell lymphoproliferative disease (LPD) is not uncommon in China, but gastrointestinal involvement is very rare. We report on an immunocompetent patient with EBV-associated T-cell LPD of the colon. The 26-year-old man was initially misdiagnosed with ulcerative colitis (UC). A colon biopsy revealed the presence of small to medium-sized lymphoid cells infiltrating the intestinal wall. The neoplastic cells expressed CD3, CD5, and granzyme B, not CD56. EBV-encoded small ribonucleic acid was detected in the tumor cells of the colon as well as the lymph node, and the T-cell receptor gene rearrangement result displayed δ gene monoclonal rearrangement. The patient died 2 moths after the diagnosis. The clinical course of EBV-associated T-cell LPD is aggressive and the prognosis is poor, the wrong diagnosis may delay treatment. Therefore, we should be very careful to prevent misdiagnosis. When patients have multiple intestinal ulcers that are not typical of UC and the clinical course is unusual, although morphology looks like inflammatory change, pathologist should consider the possibility of EBV-associated LPD. The treatment strategy and prognosis of these two diseases are different.

Energy parasites trigger oncogene mutation.

PubMed

Pokorný, Jiří; Pokorný, Jan; Jandová, Anna; Kobilková, Jitka; Vrba, Jan; Vrba, Jan

2016-10-01

Cancer initialization can be explained as a result of parasitic virus energy consumption leading to randomized genome chemical bonding. Analysis of experimental data on cell-mediated immunity (CMI) containing about 12,000 cases of healthy humans, cancer patients and patients with precancerous cervical lesions disclosed that the specific cancer and the non-specific lactate dehydrogenase-elevating (LDH) virus antigen elicit similar responses. The specific antigen is effective only in cancer type of its origin but the non-specific antigen in all examined cancers. CMI results of CIN patients display both healthy and cancer state. The ribonucleic acid (RNA) of the LDH virus parasitizing on energy reduces the ratio of coherent/random oscillations. Decreased effect of coherent cellular electromagnetic field on bonding electrons in biological macromolecules leads to elevating probability of random genome reactions. Overlapping of wave functions in biological macromolecules depends on energy of the cellular electromagnetic field which supplies energy to bonding electrons for selective chemical bonds. CMI responses of cancer and LDH virus antigens in all examined healthy, precancerous and cancer cases point to energy mechanism in cancer initiation. Dependence of the rate of biochemical reactions on biological electromagnetic field explains yet unknown mechanism of genome mutation.

A novel knowledge-based potential for RNA 3D structure evaluation

NASA Astrophysics Data System (ADS)

Yang, Yi; Gu, Qi; Zhang, Ben-Gong; Shi, Ya-Zhou; Shao, Zhi-Gang

2018-03-01

Ribonucleic acids (RNAs) play a vital role in biology, and knowledge of their three-dimensional (3D) structure is required to understand their biological functions. Recently structural prediction methods have been developed to address this issue, but a series of RNA 3D structures are generally predicted by most existing methods. Therefore, the evaluation of the predicted structures is generally indispensable. Although several methods have been proposed to assess RNA 3D structures, the existing methods are not precise enough. In this work, a new all-atom knowledge-based potential is developed for more accurately evaluating RNA 3D structures. The potential not only includes local and nonlocal interactions but also fully considers the specificity of each RNA by introducing a retraining mechanism. Based on extensive test sets generated from independent methods, the proposed potential correctly distinguished the native state and ranked near-native conformations to effectively select the best. Furthermore, the proposed potential precisely captured RNA structural features such as base-stacking and base-pairing. Comparisons with existing potential methods show that the proposed potential is very reliable and accurate in RNA 3D structure evaluation. Project supported by the National Science Foundation of China (Grants Nos. 11605125, 11105054, 11274124, and 11401448).

Panax ginseng induces the expression of CatSper genes and sperm hyperactivation

PubMed Central

Park, Eun Hwa; Kim, Do Rim; Kim, Ha Young; Park, Seong Kyu; Chang, Mun Seog

2014-01-01

The cation channel of sperm (CatSper) protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca2+ influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca2+ levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression. PMID:24969054

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

PubMed Central

Fout, G. Shay; Cashdollar, Jennifer L.; Griffin, Shannon M.; Brinkman, Nichole E.; Varughese, Eunice A.; Parshionikar, Sandhya U.

2016-01-01

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water. PMID:26862985

Sequential Formation and Accumulation of Primary and Secondary Shunt Metabolic Products in Claviceps purpurea1

PubMed Central

Taber, W. A.

1964-01-01

The fungus Claviceps purpurea was grown on a rich and a limited nutrient medium such that alkaloid was produced after 8 days on the former medium and after 3 days on the latter medium. Cultures grown on both were assayed for the primary shunt metabolic products, polyols, trehalose, lipids, ribonucleic acid, and polyphosphate, and the secondary metabolic product, ergot alkaloid. Although differing considerably in composition, the two media nevertheless allowed formation of both primary and secondary shunt products. In both instances, however, the secondary product, ergot alkaloid, did not form until formation and accumulation of the primary products had ceased and the mycelial content of these products was actually decreasing. In both instances, alkaloid formation took place after the total dry weight of the mycelium had begun to decrease but while the dry weight of the residual, or structural portion of the mycelium, was either constant or increasing. The dilution of labeling in mannitol isolated from mycelia grown on rich medium containing 1,6-C14-labeled mannitol was 2.2. Thus, about half of the mycelial mannitol was actually mannitol which had been taken up directly from the medium. PMID:14199021

Structural determinants of an internal ribosome entry site that direct translational reading frame selection

PubMed Central

Ren, Qian; Au, Hilda H.T.; Wang, Qing S.; Lee, Seonghoon; Jan, Eric

2014-01-01

The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome. PMID:25038250

Anti-inflammatory effect of linear polarized infrared irradiation on interleukin-1beta-induced chemokine production in MH7A rheumatoid synovial cells.

PubMed

Shibata, Yasuko; Ogura, Naomi; Yamashiro, Keisuke; Takashiba, Shogo; Kondoh, Toshirou; Miyazawa, Keiji; Matsui, Masaru; Abiko, Yoshimitsu

2005-12-01

We examined the anti-inflammatory effect of infrared linear polarized light irradiation on the MH7A rheumatoid fibroblast-like synoviocytes (FLS) stimulated with the proinflammatory cytokine interleukin (IL)-1beta. Expression of messenger ribonucleic acids (mRNAs) encoding IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted), growth-related gene alpha (GROalpha), and macrophage inflammatory protein-1alpha (MIP1alpha) was measured using real-time reverse transcription polymerase chain reaction, and the secreted proteins were measured in the conditioned media using enzyme-linked immunosorbent assays. We found that irradiation with linear polarized infrared light suppressed IL-1beta-induced expression of IL-8 mRNA and, correspondingly, the synthesis and release of IL-8 protein in MH7A cells. This anti-inflammatory effect was equivalent to that obtained with the glucocorticoid dexamethasone. Likewise, irradiation suppressed the IL-1beta-induced expression of RANTES and GROalpha mRNA. These results suggest that the irradiation of the areas around the articular surfaces of joints affected by rheumatoid arthritis (RA) using linear polarized light may represent a useful new approach to treatment.

Novel Compound Heterozygous Mutations Expand the Recognized Phenotypes of FARS2-Linked Disease.

PubMed

Walker, Melissa A; Mohler, Kyle P; Hopkins, Kyle W; Oakley, Derek H; Sweetser, David A; Ibba, Michael; Frosch, Matthew P; Thibert, Ronald L

2016-08-01

Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic. FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygous FARS2 mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance imaging (MRI), serum and cerebrospinal fluid analyses, skeletal muscle biopsy histology, and autopsy gross and histologic findings, which include features shared with Alpers-Huttenlocher syndrome, Leigh syndrome, and a previously published case of FARS2 mutation associated infantile onset disease. The authors also present structure-guided analysis of the relevant mutations based on published mitochondrial phenylalanyl transfer RNA synthetase and related protein crystal structures as well as biochemical analysis of the corresponding recombinant mutant proteins. © The Author(s) 2016.

Cell death in response to antimetabolites directed at ribonucleotide reductase and thymidylate synthase

PubMed Central

Asuncion Valenzuela, Malyn M; Castro, Imilce; Gonda, Amber; Diaz Osterman, Carlos J; Jutzy, Jessica M; Aspe, Jonathan R; Khan, Salma; Neidigh, Jonathan W; Wall, Nathan R

2015-01-01

New agent development, mechanistic understanding, and combinatorial partnerships with known and novel modalities continue to be important in the study of pancreatic cancer and its improved treatment. In this study, known antimetabolite drugs such as gemcitabine (ribonucleotide reductase inhibitor) and 5-fluorouracil (thymidylate synthase inhibitor) were compared with novel members of these two drug families in the treatment of a chemoresistant pancreatic cancer cell line PANC-1. Cellular survival data, along with protein and messenger ribonucleic acid expression for survivin, XIAP, cIAP1, and cIAP2, were compared from both the cell cytoplasm and from exosomes after single modality treatment. While all antimetabolite drugs killed PANC-1 cells in a time- and dose-dependent manner, neither family significantly altered the cytosolic protein level of the four inhibitors of apoptosis (IAPs) investigated. Survivin, XIAP, cIAP1, and cIAP2 were found localized to exosomes where no significant difference in expression was recorded. This inability for significant and long-lasting expression may be a reason why pancreatic cancer lacks responsiveness to these and other cancer-killing agents. Continued investigation is required to determine the responsibilities of these IAPs in their role in chemoresistance in pancreatic adenocarcinoma. PMID:25767396

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak

2014-04-20

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from themore » GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.« less

The extraction and purification of a cysteine transfer ribonucleic acid from baker's yeast.

PubMed Central

Holness, N J; Atfield, G

1976-01-01

1. A modification of the RPC 1 system of A.D. Kelmers, G.D. Novelli & M.P. Stulberg (1965) (J. Biol. Chem. 240, 3979-3983) is described in which the support medium is a Celite of narrow range particle size treated with dichlorodimethylsilane. 2. By using this system an apparently pure preparation of tRNA Cys was isolated from baker's yeast tRNA. 3. This preparation accepted at least 60% of the theoretical quantity of [3-14C]cysteine in a conventional assay and failed to accept isoleucine, phenylalanine, proline, serine or tyrosine. 4. A theoretical countercurrent-distribution curve calculated by assuming a distribution coefficient K of 2.03 was in excellent agreement with the profiles of E260 and cysteine-acceptor ability after 537 transfers in the 1.85 M-phosphate/formamide/propan-2-ol system of C.M. Connelly & B.P. Doctor (1965) (J. Biol. Chem. 241, 715-719). 5. Chromatography of tRNA Cys on Bio-Gel P100 polyacrylamide beads afforded two components one of which was far less efficient than the other in accepting cysteine. The base compositions of the two were similar. PMID:776175

DOE Office of Scientific and Technical Information (OSTI.GOV)

Awata, T.; Matsumoto, C.; Iwamoto, Y.

We studied a woman with acanthosis nigricans and insulin resistance. The patient`s Epstein-Barr virus-transformed lymphocytes revealed slightly decreased insulin binding and markedly decreased insulin-stimulated autophosphorylation of the insulin receptor. The nucleotide sequence analysis of the patient`s genomic DNA revealed a 3-basepair in-frame deletion of one allele, resulting in the loss of leucine at position 999 of the insulin receptor ({Delta}Leu{sup 999}). The messenger ribonucleic acid transcripts from the mutant allele in the patient`s lymphocytes were not decreased. Insulin-stimulated autophosphorylation of the insulin receptor from cells expressing {Delta}Leu{sup 999} mutant insulin receptor complementary DNA was markedly decreased. The proband, her mother,more » elder brother, and younger brother, who were heterozygous for this mutation, showed moderate or marked hyperinsulinemia during oral glucose tolerance tests. Although fasting glucose levels were normal and fasting insulin values were preserved in all subjects with the mutation for the 8-yr period of observation, a tendancy of progressive increase in postload glucose levels were observed. These results suggest that the {Delta}Leu{sup 999} mutation, which reduces tyrosine kinase activity, was responsible for insulin resistance and contributed to postload hyperglycemia. 27 refs., 3 figs., 1 tab.« less

Accumulation of 70S Monoribosomes in Escherichia coli After Energy Source Shift-Down

PubMed Central

Ruscetti, Francis W.; Jacobson, Lewis A.

1972-01-01

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as “complexed” rather than “free” particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of 3H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down. PMID:4591472

Simulated physiological stretch increases expression of extracellular matrix proteins in human bladder smooth muscle cells via integrin α4/αv-FAK-ERK1/2 signaling pathway.

PubMed

Chen, Shulian; Peng, Chuandu; Wei, Xin; Luo, Deyi; Lin, Yifei; Yang, Tongxin; Jin, Xi; Gong, Lina; Li, Hong; Wang, Kunjie

2017-08-01

To investigate the effect of simulated physiological stretch on the expression of extracellular matrix (ECM) proteins and the role of integrin α4/αv, focal adhesion kinase (FAK), extracellular regulated protein kinases 1/2 (ERK1/2) in the stretch-induced ECM protein expression of human bladder smooth muscle cells (HBSMCs). HBSMCs were seeded onto silicone membrane and subjected to simulated physiological stretch at the range of 5, 10, and 15% elongation. Expression of primary ECM proteins in HBSMCs was analyzed by real-time polymerase chain reaction and Western blot. Specificity of the FAK and ERK1/2 was determined by Western blot with FAK inhibitor and ERK1/2 inhibitor (PD98059). Specificity of integrin α4 and integrin αv was determined with small interfering ribonucleic acid (siRNA) transfection. The expression of collagen I (Col1), collagen III (Col3), and fibronectin (Fn) was increased significantly under the simulated physiological stretch of 10 and 15%. Integrin α4 and αv, FAK, ERK1/2 were activated by 10% simulated physiological stretch compared with the static condition. Pretreatment of ERK1/2 inhibitor, FAK inhibitor, integrin α4 siRNA, or integrin αv siRNA reduced the stretch-induced expression of ECM proteins. And FAK inhibitor decreased the stretch-induced ERK1/2 activity and ECM protein expression. Integrin α4 siRNA or integrin αv siRNA inhibited the stretch-induced activity of FAK. Simulated physiological stretch increases the expression of ECM proteins in HBSMCs, and integrin α4/αv-FAK-ERK1/2 signaling pathway partly modulates the mechano-transducing process.

High-resolution mass spectrometry of skin mucus for monitoring physiological impacts and contaminant biotransformation products in fathead minnows exposed to wastewater effluent.

PubMed

Mosley, Jonathan D; Ekman, Drew R; Cavallin, Jenna E; Villeneuve, Daniel L; Ankley, Gerald T; Collette, Timothy W

2018-03-01

High-resolution mass spectrometry is advantageous for monitoring physiological impacts and contaminant biotransformation products in fish exposed to complex wastewater effluent. We evaluated this technique using skin mucus from male and female fathead minnows (Pimephales promelas) exposed to control water or treated wastewater effluent at 5, 20, and 100% levels for 21 d, using an on-site, flow-through system providing real-time exposure. Both sex-specific and non-sex-specific responses were observed in the mucus metabolome, the latter suggesting the induction of general compensatory pathways for xenobiotic exposures. Altogether, 85 statistically significant treatment-dependent metabolite changes were observed out of the 310 total endogenous metabolites that were detected (156 of the 310 were annotated). Partial least squares-regression models revealed strong covariances between the mucus metabolomes and up-regulated hepatic messenger ribonucleic acid (mRNA) transcripts reported previously for these same fish. These regression models suggest that mucus metabolomic changes reflected, in part, processes by which the fish biotransformed xenobiotics in the effluent. In keeping with this observation, we detected a phase II transformation product of bisphenol A in the skin mucus of male fish. Collectively, these findings demonstrate the utility of mucus as a minimally invasive matrix for simultaneously assessing exposures and effects of environmentally relevant mixtures of contaminants. Environ Toxicol Chem 2018;37:788-796. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Investigation of RNA Hairpin Loop Folding with Time-Resolved Infrared Spectroscopy

NASA Astrophysics Data System (ADS)

Stancik, Aaron Lee

Ribonucleic acids (RNAs) are a group of functional biopolymers central to the molecular underpinnings of life. To complete the many processes they mediate, RNAs must fold into precise three-dimensional structures. Hairpin loops are the most ubiquitous and basic structural elements present in all folded RNAs, and are the foundation upon which all complex tertiary structures are built. A hairpin loop forms when a single stranded RNA molecule folds back on itself creating a helical stem of paired bases capped by a loop. This work investigates the formation of UNCG hairpin loops with the sequence 5'-GC(UNCG)GC-3' (N = A, U, G, or C) using both equilibrium infrared (IR) and time-resolved IR spectroscopy. Equilibrium IR melting data were used to determine thermodynamic parameters. Melting temperatures ranged from 50 to 60°C, and enthalpies of unfolding were on the order of 100 kJ/mol. In the time-resolved work, temperature jumps of up to 20°C at 2.5°C increments were obtained with transient relaxation kinetics spanning nanoseconds to hundreds of microseconds. The relaxation kinetics for all of the oligomers studied were fit to first or second order exponentials. Multiple vibrational transitions were probed on each oligomer for fully folded and partially denatured structures. In the time-resolved limit, in contrast to equilibrium melting, RNA does not fold according to two-state behavior. These results are some of the first to show that RNA hairpins fold according to a rugged energy landscape, which contradicts their relatively simple nature. In addition, this work has proven that time-resolved IR spectroscopy is a powerful and novel tool for investigating the earliest events of RNA folding, the formation of the hairpin loop.

Electrochemical DNA biosensors based on long-range electron transfer: investigating the efficiency of a fluidic channel microelectrode compared to an ultramicroelectrode in a two-electrode setup.

PubMed

Horny, M-C; Lazerges, M; Siaugue, J-M; Pallandre, A; Rose, D; Bedioui, F; Deslouis, C; Haghiri-Gosnet, A-M; Gamby, J

2016-11-01

Here, we describe the transposition of an ultramicroelectrode (UME) setup into a microfluidic chip configuration for DNA biosensors. The hydrodynamic properties of the fluidic channel microelectrode were screened with an [Fe(iii)(CN) 6 ] 3- /[Fe(ii)(CN) 6 ] 4- redox couple by cyclic voltammetry to provide a basis for further biological processes. A 23-base DNA probe was self-assembled into a monolayer on gold microelectrodes both in classical configuration and integrated in a microfluidic setup. Special interest was focused on the DNA target mimicking the liver-specific micro-ribonucleic acid 122 (miRNA122). Long-range electron transfer was chosen for transducing the hybridization. This direct transduction was indeed significantly enhanced after hybridization due to DNA-duplex π-stacking and the use of redox methylene blue as a DNA intercalator. Quantification of the target was deduced from the resulting electrical signal characterized by cyclic voltammetry. The limit of detection for DNA hybridization was 0.1 fM in stopped flow experiments, where it can reach 1 aM over a 0.5 μL s -1 flow rate, a value 10 4 -fold lower than the one measured with a conventional UME dipped into an electrolyte droplet under the same analytical conditions. An explanation was that forced convection drives more biomolecules to the area of detection even if a balance between the speed of collection and the number of biomolecules collected has been found. The latter point is discussed here along with an attempt to explain why the sensor has reached such an unexpected value for the limit of detection.

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland

PubMed Central

2010-01-01

Introduction Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ)/Smad pathway as an example. Methods We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ. Results IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium. PMID:20942910

Dynamic and geometric analyses of Nudaurelia capensis ω virus maturation reveal the energy landscape of particle transitions.

PubMed

Tang, Jinghua; Kearney, Bradley M; Wang, Qiu; Doerschuk, Peter C; Baker, Timothy S; Johnson, John E

2014-04-01

Quasi-equivalent viruses that infect animals and bacteria require a maturation process in which particles transition from initially assembled procapsids to infectious virions. Nudaurelia capensis ω virus (NωV) is a T = 4, eukaryotic, single-stranded ribonucleic acid virus that has proved to be an excellent model system for studying the mechanisms of viral maturation. Structures of NωV procapsids (diameter = 480 Å), a maturation intermediate (410 Å), and the mature virion (410 Å) were determined by electron cryo-microscopy and three-dimensional image reconstruction (cryoEM). The cryoEM density for each particle type was analyzed with a recently developed maximum likelihood variance (MLV) method for characterizing microstates occupied in the ensemble of particles used for the reconstructions. The procapsid and the mature capsid had overall low variance (i.e., uniform particle populations) while the maturation intermediate (that had not undergone post-assembly autocatalytic cleavage) had roughly two to four times the variance of the first two particles. Without maturation cleavage, the particles assume a variety of microstates, as the frustrated subunits cannot reach a minimum energy configuration. Geometric analyses of subunit coordinates provided a quantitative description of the particle reorganization during maturation. Superposition of the four quasi-equivalent subunits in the procapsid had an average root mean square deviation (RMSD) of 3 Å while the mature particle had an RMSD of 11 Å, showing that the subunits differentiate from near equivalent environments in the procapsid to strikingly non-equivalent environments during maturation. Autocatalytic cleavage is clearly required for the reorganized mature particle to reach the minimum energy state required for stability and infectivity. Copyright © 2014 John Wiley & Sons, Ltd.

Mutants of Escherichia coli defective in membrane phospholipid synthesis: macromolecular synthesis in an sn-glycerol 3-phosphate acyltransferase Km mutant.

PubMed

Bell, R M

1974-03-01

sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane preparations from the mutants than those of the parent. All extracts prepared from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase activity, but most contained less than 10% of the wild-type level. Membrane preparations from revertants of the acyltransferase mutants had apparent K(m)'s for G3P similar to that of the parent. Strains have been derived in which the G3P requirement can be satisfied with glycerol in the presence of glucose, presumably because the glycerol kinase was desensitized to inhibition by fructose 1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately; growth continued for about one doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein nearly doubled paralleling the growth curve; the rate of phospholipid synthesis assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the cells grew and then decreased as the cells stopped growing; the rate of protein synthesis showed no increase and declined more slowly than the rates of RNA and DNA synthesis when the cells stopped growing. The cells retained and gained in the capacity to synthesize phospholipids upon glycerol deprivation. These data indicate that net phospholipid synthesis is not required for continued macromolecular synthesis for about one doubling, and that the rates of these processes are not coupled during this time period.

Regucalcin and cell regulation: role as a suppressor protein in signal transduction.

PubMed

Yamaguchi, Masayoshi

2011-07-01

Regucalcin was discovered in 1978 as a calcium-binding protein that does not contain EF-hand motif of calcium-binding domain (Yamaguchi and Yamamoto Chem Pharm Bull 26:1915-1918, 1978). The name regucalcin was proposed for this calcium-binding protein, which can regulate various Ca(2+)-dependent enzyme activations in liver cells. The regucalcin gene is localized on the chromosome X, and the organization of the regucalcin gene consists of seven exons and six introns. AP-1, NF1-A1, and RGPR-p117 bind to the promoter region of the rat regucalcin gene and enhance transcription activity of regucalcin gene expression that is mediated through calcium signaling. Regucalcin plays a pivotal role in the keep of intracellular calcium ion (Ca(2+)) homeostasis due to activating Ca(2+) pump enzymes in the plasma membrane (basolateral membrane), microsomes (endoplasmic reticulum), mitochondria, and nuclei of many cell types. Regucalcin has a suppressive effect on calcium signaling from the cytoplasm to the nucleus in the proliferative cells. Regucalcin has also been demonstrated to transport to the nucleus, and it can inhibit Ca(2+)-dependent protein kinase and protein phosphatase activities, Ca(2+)-activated deoxyribonucleic acid (DNA) fragmentation, and DNA and ribonucleic acid (RNA) synthesis in the nucleus. Overexpression of regucalcin suppresses cell death and apoptosis in the cloned rat hepatoma cells induced by various signaling factors. Regucalcin can inhibit the enhancement of cell proliferation due to hormonal stimulation. Regucalcin plays an important role as a regulatory protein in cell signaling system, and it is proposed to play a pivotal role in keep of cell homeostasis and function.

Cellular distribution and regulation of ghrelin messenger ribonucleic acid in the rat pituitary gland.

PubMed

Caminos, J E; Nogueiras, R; Blanco, M; Seoane, L M; Bravo, S; Alvarez, C V; García-Caballero, T; Casanueva, F F; Diéguez, C

2003-11-01

Ghrelin, a 28-amino-acid acylated peptide, strongly stimulates GH release and food intake. In the present study, we found that ghrelin is expressed in somatotrophs, lactotrophs, and thyrotrophs but not in corticotrophs or gonadotrophs of rat pituitary. Persistent expression of the ghrelin gene is found during postnatal development in male and female rats, although the levels significantly decrease in both cases from pituitaries of 20-d-old rats onward, but at 60 d old, the levels were higher in male than female rats. This sexually dimorphic pattern appears to be mediated by estrogens because ovariectomy, but not orchidectomy, increases pituitary ghrelin mRNA levels. Taking into account that somatotroph cell function is markedly influenced by thyroid hormones, glucocorticoids, GH, and metabolic status, we also assessed such influence. We found that ghrelin mRNA levels decrease in hypothyroid- and glucocorticoid-treated rats, increase in GH-deficient rats (dwarf rats), and remain unaffected by food deprivation. In conclusion, we have defined the specific cell types that express ghrelin in the rat anterior pituitary gland. These data provide direct morphological evidence that ghrelin may well be acting in a paracrine-like fashion in the regulation of anterior pituitary cell function. In addition, we clearly demonstrate that pituitary ghrelin mRNA levels are age and gender dependent. Finally, we show that pituitary ghrelin mRNA levels are influenced by alteration on thyroid hormone, glucocorticoids, and GH levels but not by fasting, which indicates that the regulation of ghrelin gene expression is tissue specific.

[Special application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in clinical microbiological diagnostics].

PubMed

Nagy, Erzsébet; Abrók, Marianna; Bartha, Noémi; Bereczki, László; Juhász, Emese; Kardos, Gábor; Kristóf, Katalin; Miszti, Cecilia; Urbán, Edit

2014-09-21

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a new possibility for rapid identification of bacteria and fungi revolutionized the clinical microbiological diagnostics. It has an extreme importance in the routine microbiological laboratories, as identification of the pathogenic species rapidly will influence antibiotic selection before the final determination of antibiotic resistance of the isolate. The classical methods for identification of bacteria or fungi, based on biochemical tests, are influenced by many environmental factors. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a rapid method which is able to identify a great variety of the isolated bacteria and fungi based on the composition of conserved ribosomal proteins. Recently several other applications of the method have also been investigated such as direct identification of pathogens from the positive blood cultures. There are possibilities to identify bacteria from the urine samples in urinary tract infection or from other sterile body fluids. Using selective enrichment broth Salmonella sp from the stool samples can be identified more rapidly, too. The extended spectrum beta-lactamase or carbapenemase production of the isolated bacteria can be also detected by this method helping the antibiotic selection in some cases. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry based methods are suitable to investigate changes in deoxyribonucleic acid or ribonucleic acid, to carry out rapid antibiotic resistance determination or other proteomic analysis. The aim of this paper is to give an overview about present possibilities of using this technique in the clinical microbiological routine procedures.

BIO-ORGANIC CHEMISTRY QUARTERLY REPORT. December 1961, January and February 1962

DOE Office of Scientific and Technical Information (OSTI.GOV)

Various,

1962-04-03

Progress is reported in investigations on the polymerization of formaldehyde, ultraviolet irradiation of aqueous HC/sup 14/N, radiation chemistry of nucleic acid constituents, oxidation of free sugars and aldonic acid derivatives by Acetobacter suboxydans, preparation and isolation of C/sup 14/O/ sub 2/~ enzyme, metabolism of C/sup 14/-ribulose diphosphate by Nitrobacter agilis, C/sup 14/O/sub 2/ metabolism of Hordeum valgare seedlings during the development of the photosynthetic apparatus, location and chemical characterization of RNA in the chloroplasts of Spinacea oleracea, inhibition of dark bleaching by stroma extracts and by inert gases, ESR studies on chromatophores from Rhodospirillium rubrum and on quantasomes from spinachmore » chloroplasts, and phthalocyanine manganese and etioporphyrin manganese complexes. (J.R.D.) It has been known for a hundred years that formaldehyde polymerizes to carbohydrate substances in alkaline media. Although the reaction has long attracted much attention, only recently has a detailed qualitative analysis of the products been carried out by chromatographic methods. We have started to re-examine this reaction by combining chromatography with radioactive tracer techniques in the hope of refining the quantitative aspects of the analysis. Our particular interest has been to develop methods for determining the relative proportions of ribose and ribulose in the mixtures of sugars formed in basic media, as well as under other polymerizing conditions. The finding of large amounts of these sugars might help to explain the occurrence of ribose as the only basic sugar in the fundamental replicating molecules--the nucleic acids. Formaldehyde is thought to have been present in the primitive reducing atmosphere which existed before life first appeared. The ribonucleic acids must have appeared in the constitution of reproducing systems at a very early stage in the development of living organisms. In this study, the polymerizations of formaldehyde were carried out in calcium hydroxide suspensions at 40{sup o}. Aliquots of the reaction mixtures were withdrawn at after various time intervals and the alkali was neutralized with sulfuric acid or, in later experiments, with carbon dioxide. The hydrolysis with sulfuric acid that was used initially to break down any polymers was shown to be unnecessary, as identical products were obtained with this treatment and with simple carbon dioxide neutralization.« less

Solution Preserves Nucleic Acids in Body-Fluid Specimens

NASA Technical Reports Server (NTRS)

Pierson, Duane L.; Stowe, Raymond P.

2004-01-01

A solution has been formulated to preserve deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in specimens of blood, saliva, and other bodily fluids. Specimens of this type are collected for diagnostic molecular pathology, which is becoming the method of choice for diagnosis of many diseases. The solution makes it possible to store such specimens at room temperature, without risk of decomposition, for subsequent analysis in a laboratory that could be remote from the sampling location. Thus, the solution could be a means to bring the benefits of diagnostic molecular pathology to geographic regions where refrigeration equipment and diagnostic laboratories are not available. The table lists the ingredients of the solution. The functions of the ingredients are the following: EDTA chelates divalent cations that are necessary cofactors for nuclease activity. In so doing, it functionally removes these cations and thereby retards the action of nucleases. EDTA also stabilizes the DNA helix. Tris serves as a buffering agent, which is needed because minor contaminants in an unbuffered solution can exert pronounced effects on pH and thereby cause spontaneous degradation of DNA. SDS is an ionic detergent that inhibits ribonuclease activity. SDS has been used in some lysis buffers and as a storage buffer for RNA after purification. The use of the solution is straightforward. For example, a sample of saliva is collected by placing a cotton roll around in the subject's mouth until it becomes saturated, then the cotton is placed in a collection tube. Next, 1.5 mL of the solution are injected directly into the cotton and the tube is capped for storage at room temperature. The effectiveness of the solution has been demonstrated in tests on specimens of saliva containing herpes simplex virus. In the tests, the viral DNA, as amplified by polymerase chain reaction, was detected even after storage for 120 days.

227 Views of RNA: Is RNA Unique in Its Chemical Isomer Space?

PubMed Central

Meringer, Markus; Goodwin, Jay

2015-01-01

Abstract Ribonucleic acid (RNA) is one of the two nucleic acids used by extant biochemistry and plays a central role as the intermediary carrier of genetic information in transcription and translation. If RNA was involved in the origin of life, it should have a facile prebiotic synthesis. A wide variety of such syntheses have been explored. However, to date no one-pot reaction has been shown capable of yielding RNA monomers from likely prebiotically abundant starting materials, though this does not rule out the possibility that simpler, more easily prebiotically accessible nucleic acids may have preceded RNA. Given structural constraints, such as the ability to form complementary base pairs and a linear covalent polymer, a variety of structural isomers of RNA could potentially function as genetic platforms. By using structure-generation software, all the potential structural isomers of the ribosides (BC5H9O4, where B is nucleobase), as well as a set of simpler minimal analogues derived from them, that can potentially serve as monomeric building blocks of nucleic acid–like molecules are enumerated. Molecules are selected based on their likely stability under biochemically relevant conditions (e.g., moderate pH and temperature) and the presence of at least two functional groups allowing the monomers to be incorporated into linear polymers. The resulting structures are then evaluated by using molecular descriptors typically applied in quantitative structure–property relationship (QSPR) studies and predicted physicochemical properties. Several databases have been queried to determine whether any of the computed isomers had been synthesized previously. Very few of the molecules that emerge from this structure set have been previously described. We conclude that ribonucleosides may have competed with a multitude of alternative structures whose potential proto-biochemical roles and abiotic syntheses remain to be explored. Key Words: Evolution—Chemical evolution—Exobiology—Prebiotic chemistry—RNA world. Astrobiology 15, 538–558. PMID:26200431

Turnip mosaic virus Moves Systemically through Both Phloem and Xylem as Membrane-Associated Complexes1

PubMed Central

Zheng, Huanquan

2015-01-01

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K2, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K2-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem. PMID:25717035

RNA interference targeting cytosolic NADP(+)-dependent isocitrate dehydrogenase exerts anti-obesity effect in vitro and in vivo.

PubMed

Nam, Woo Suk; Park, Kwon Moo; Park, Jeen-Woo

2012-08-01

A metabolic abnormality in lipid biosynthesis is frequently associated with obesity and hyperlipidemia. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) is an essential reducing equivalent for numerous enzymes required in fat and cholesterol biosynthesis. Cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) has been proposed as a key enzyme for supplying cytosolic NADPH. We report here that knockdown of IDPc expression by Ribonucleic acid (RNA) interference (RNAi) inhibited adipocyte differentiation and lipogenesis in 3T3-L1 preadipocytes and mice. Attenuated IDPc expression by IDPc small interfering RNA (siRNA) resulted in a reduction of differentiation and triglyceride level and adipogenic protein expression as well as suppression of glucose uptake in cultured adipocytes. In addition, the attenuation of Nox activity and Reactive oxygen species (ROS) generation accompanied with knockdown of IDPc was associated with inhibition of adipogenesis and lipogenesis. The loss of body weight and the reduction of triglyceride level were also observed in diet-induced obese mice transduced with IDPc short-hairpin (shRNA). Taken together, the inhibiting effect of RNAi targeting IDPc on adipogenesis and lipid biosynthesis is considered to be of therapeutic value in the treatment and prevention of obesity and obesity-associated metabolic syndrome. © 2012 Elsevier B.V. All rights reserved.

Dual RNA regulatory control of a Staphylococcus aureus virulence factor.

PubMed

Chabelskaya, Svetlana; Bordeau, Valérie; Felden, Brice

2014-04-01

In pathogens, the accurate programming of virulence gene expression is essential for infection. It is achieved by sophisticated arrays of regulatory proteins and ribonucleic acids (sRNAs), but in many cases their contributions and connections are not yet known. Based on genetic, biochemical and structural evidence, we report that the expression pattern of a Staphylococcus aureus host immune evasion protein is enabled by the collaborative actions of RNAIII and small pathogenicity island RNA D (SprD). Their combined expression profiles during bacterial growth permit early and transient synthesis of Sbi to avoid host immune responses. Together, these two sRNAs use antisense mechanisms to monitor Sbi expression at the translational level. Deletion analysis combined with structural analysis of RNAIII in complex with its novel messenger RNA (mRNA) target indicate that three distant RNAIII domains interact with distinct sites of the sbi mRNA and that two locations are deep in the sbi coding region. Through distinct domains, RNAIII lowers production of two proteins required for avoiding innate host immunity, staphylococcal protein A and Sbi. Toeprints and in vivo mutational analysis reveal a novel regulatory module within RNAIII essential for attenuation of Sbi translation. The sophisticated translational control of mRNA by two differentially expressed sRNAs ensures supervision of host immune escape by a major pathogen.

Structural studies of RNA-protein complexes: A hybrid approach involving hydrodynamics, scattering, and computational methods.

PubMed

Patel, Trushar R; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A; Bujnicki, Janusz M

2017-04-15

The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Tumor-specific RNA interference targeting Pokemon suppresses tumor growth and induces apoptosis in prostate cancer.

PubMed

Li, Yining; Xu, Shuxiong; Wang, Xiangwei; Shi, Hua; Sun, Zhaolin; Yang, Zhao

2013-02-01

To explore the exact mechanism of Pokemon in prostate cancer. Pokemon is a member of the POK family of transcriptional repressors. Its main function is suppression of the p14ARF (alternate reading frame) tumor suppressor gene. Although Pokemon expression has been found to be increased in various types of lymphoma, the exact mechanism of the gene in prostate cancer is not clear. In the present study, prostate cancer cells were transfected with the specific short hairpin ribonucleic acid (RNA) expression vector targeting Pokemon. The expression of Pokemon messenger RNA and its protein was detected by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cell growth and cell apoptosis were also examined using the methyl thiazolyl tetrazolium assay and flow cytometry. The results demonstrated that specific RNA interference (RNAi) could decrease the expression levels of Pokemon gene messenger RNA and protein in prostate cancer cells. In addition, that specific RNAi significantly inhibited the cell proliferation and increased the apoptotic rate. In vivo experiments showed that specific RNAi inhibited the tumorigenicity of prostate cancer cells and significantly suppressed tumor growth. Therefore, an RNAi-targeted Pokemon gene strategy could be a potential approach to prostate cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

Vasopressin synthesis by the magnocellular neurons is different in the supraoptic nucleus and in the paraventricular nucleus in human and experimental septic shock.

PubMed

Sonneville, Romain; Guidoux, Céline; Barrett, Lucinda; Viltart, Odile; Mattot, Virginie; Polito, Andrea; Siami, Shidasp; de la Grandmaison, Geoffroy Lorin; Blanchard, Anne; Singer, Mervyn; Annane, Djillali; Gray, Françoise; Brouland, Jean-Philippe; Sharshar, Tarek

2010-05-01

Impaired arginine vasopressin (AVP) synthesis and release by the neurohypophyseal system, which includes the neurohypophysis and magnocellular neurons of the paraventricular and supraoptic nuclei, have been postulated in septic shock, but changes in this system have never been assessed in human septic shock, and only partially experimentally. We investigated AVP synthesis and release by the neurohypophyseal system in 9 patients who died from septic shock and 10 controls, and in 20 rats with fecal peritonitis-induced sepsis and 8 sham-operation controls. Ten rats died spontaneously from septic shock, and the others were sacrificed. In patients with septic shock, as in rats that died spontaneously following sepsis induction, AVP immunohistochemical expression was decreased in the neurohypophysis and supraoptic magnocellular neurons, whereas it was increased in the paraventricular magnocellular neurons. No significant change was observed in AVP messenger RiboNucleic Acid (mRNA) expression assessed by in situ hybridization in either paraventricular or supraoptic magnocellular cells. This study shows that both in human and experimental septic shock, AVP posttranscriptional synthesis and transport are differently modified in the magnocellular neurons of the supraoptic and paraventricular nuclei. This may account for the inappropriate AVP release in septic shock and suggests that distinct pathogenic mechanisms operate in these nuclei.

Gene Transfer of Brain-derived Neurotrophic Factor (BDNF) Prevents Neurodegeneration Triggered by FXN Deficiency.

PubMed

Katsu-Jiménez, Yurika; Loría, Frida; Corona, Juan Carlos; Díaz-Nido, Javier

2016-05-01

Friedreich's ataxia is a predominantly neurodegenerative disease caused by recessive mutations that produce a deficiency of frataxin (FXN). Here, we have used a herpesviral amplicon vector carrying a gene encoding for brain-derived neurotrophic factor (BDNF) to drive its overexpression in neuronal cells and test for its effect on FXN-deficient neurons both in culture and in the mouse cerebellum in vivo. Gene transfer of BDNF to primary cultures of mouse neurons prevents the apoptosis which is triggered by the knockdown of FXN gene expression. This neuroprotective effect of BDNF is also observed in vivo in a viral vector-based knockdown mouse cerebellar model. The injection of a lentiviral vector carrying a minigene encoding for a FXN-specific short hairpin ribonucleic acid (shRNA) into the mouse cerebellar cortex triggers a FXN deficit which is accompanied by significant apoptosis of granule neurons as well as loss of calbindin in Purkinje cells. These pathological changes are accompanied by a loss of motor coordination of mice as assayed by the rota-rod test. Coinjection of a herpesviral vector encoding for BDNF efficiently prevents both the development of cerebellar neuropathology and the ataxic phenotype. These data demonstrate the potential therapeutic usefulness of neurotrophins like BDNF to protect FXN-deficient neurons from degeneration.

Quantification and determinants of the amount of respiratory syncytial virus (RSV) shed using real time PCR data from a longitudinal household study.

PubMed

Wathuo, Miriam; Medley, Graham F; Nokes, D James; Munywoki, Patrick K

2016-12-14

Background A better understanding of respiratory syncytial virus (RSV) epidemiology requires realistic estimates of RSV shedding patterns, quantities shed, and identification of the related underlying factors. Methods RSV infection data arise from a cohort study of 47 households with 493 occupants, in coastal Kenya, during the 2009/2010 RSV season. Nasopharyngeal swabs were taken every 3 to 4 days and screened for RSV using a real time polymerase chain reaction (PCR) assay. The amount of virus shed was quantified by calculating the 'area under the curve' using the trapezoidal rule applied to rescaled PCR cycle threshold output. Multivariable linear regression was used to identify correlates of amount of virus shed. Results The median quantity of virus shed per infection episode was 29.4 (95% CI: 15.2, 54.2) log 10 ribonucleic acid (RNA) copies. Young age (<1 year), presence of upper respiratory symptoms, intra-household acquisition of infection, an individual's first infection episode in the RSV season, and having a co-infection of RSV group A and B were associated with increased amount of virus shed. Conclusions The findings provide insight into which groups of individuals have higher potential for transmission, information which may be useful in designing RSV prevention strategies.

miR-186 inhibits cell proliferation in multiple myeloma by repressing Jagged1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Zengyan; Department of Hematology, Hospital Affiliated to Binzhou Medical University, 661 Second Huanghe Street, Binzhou 256603; Zhang, Guoqiang

2016-01-15

MicroRNAs (miRNAs) are small, noncoding ribonucleic acids that regulate gene expression by targeting mRNAs for translational repression and degradation. Accumulating experimental evidence supports a causal role of miRNAs in hematology tumorigenesis. However, the specific functions of miRNAs in the pathogenesis of multiple myeloma (MM) remain to be established. In this study, we demonstrated that miR-186 is commonly downregulated in MM cell lines and patient MM cells. Ectopic expression of miR-186 significantly inhibited cell growth, both in vitro and in vivo, and induced cell cycle G{sub 0}/G{sub 1} arrest. Furthermore, miR-186 induced downregulation of Jagged1 protein expression by directly targeting its 3′-untranslated regionmore » (3′-UTR). Conversely, overexpression of Jagged1 rescued cells from miR-186-induced growth inhibition. Our collective results clearly indicate that miR-186 functions as a tumor suppressor in MM, supporting its potential as a therapeutic target for the disease. - Highlights: • miR-186 expression is decreased in MM. • miR-186 inhibits MM cell proliferation in vitro and in vivo. • Jagged1 is regulated by miR-186. • Overexpression of Jagged1 reverses the effects of miR-186.« less

Aging of Escherichia coli

PubMed Central

Clifton, C. E.

1966-01-01

Clifton, C. E. (Stanford University, Stanford, Calif.). Aging of Escherichia coli. J. Bacteriol. 92:905–912. 1966.—The rates of endogenous and exogenous (glucose) respiration decreased much more rapidly than did the viable count during the first 24 hr of aging of washed, C14-labeled cells of Escherichia coli K-12 suspended in a basal salt medium devoid of ammonium salts. The rates of decrease of respiration and of death approached each other as the age of the cells increased, but death was not the only factor involved in decreased respiratory activity of the suspensions. The greatest decrease in cellular contents with aging was noted in the ribonucleic acid fraction, of which the ribose appeared to be oxidized, while uracil accumulated in the suspension medium. The viable count and respiratory activities remained higher in glucose-fed than in nonfed suspensions. Proline-labeled cells fed glucose tended to lose more of their proline and to convert more proline into C14O2 than in unfed controls. On the other hand, uracil-labeled cells fed glucose retained more of the uracil than did nonfed cells, but glucose elicited some oxidation of uracil. An exogenous energy source such as glucose aided in the maintenance of a population, but it was not the only factor needed for such maintenance. PMID:5332874

[Development of a diagnostic test system for early non-invasive detection of prostate cancer based on PCA3 mRNA levels in urine sediment using quantitative reverse tanscription polymerase chain reaction (qRT-PCR)].

PubMed

Pavlov, K A; Shkoporov, A N; Khokhlova, E V; Korchagina, A A; Sidorenkov, A V; Grigor'ev, M É; Pushkar', D Iu; Chekhonin, V P

2013-01-01

The wide introduction of prostatic specific antigen (PSA) determination into clinical practice has resulted in a larger number of prostate biopsies, while the lower age threshold for PSA has led to a larger number of unnecessary prostate biopsies. Hence, there is a need for new biomarkers that can detect prostate cancer. PCA3 is a noncoding messenger ribonucleic acid (mRNA) that is expressed exclusively in prostate cells. The aim of the study has been to develop a diagnostic test system for early non-invasive detection of prostate cancer based on PCA3 mRNA levels in urine sediment using quantitative reverse transcription polymerase chain reaction (qRT-PCR). As part of the study, a laboratory diagnostic test system prototype has been designed, an application methodology has been developed and specificity and sensitivity data of the method has been assessed. The diagnostic system has demonstrated its ability to detect significantly elevated levels of PCA 3/KLK 3 in samples from prostate cancer (PCa) patients compared with those from healthy men. The findings have shown relatively high diagnostic sensitivity, specificity and negative-predictive values for an early non-invasive screening of prostate cancer

Milk’s Role as an Epigenetic Regulator in Health and Disease

PubMed Central

Melnik, Bodo C.; Schmitz, Gerd

2017-01-01

It is the intention of this review to characterize milk’s role as an epigenetic regulator in health and disease. Based on translational research, we identify milk as a major epigenetic modulator of gene expression of the milk recipient. Milk is presented as an epigenetic “doping system” of mammalian development. Milk exosome-derived micro-ribonucleic acids (miRNAs) that target DNA methyltransferases are implicated to play the key role in the upregulation of developmental genes such as FTO, INS, and IGF1. In contrast to miRNA-deficient infant formula, breastfeeding via physiological miRNA transfer provides the appropriate signals for adequate epigenetic programming of the newborn infant. Whereas breastfeeding is restricted to the lactation period, continued consumption of cow’s milk results in persistent epigenetic upregulation of genes critically involved in the development of diseases of civilization such as diabesity, neurodegeneration, and cancer. We hypothesize that the same miRNAs that epigenetically increase lactation, upregulate gene expression of the milk recipient via milk-derived miRNAs. It is of critical concern that persistent consumption of pasteurized cow’s milk contaminates the human food chain with bovine miRNAs, that are identical to their human analogs. Commercial interest to enhance dairy lactation performance may further increase the epigenetic miRNA burden for the milk consumer. PMID:28933365

Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes.

PubMed

Ito, Akira; Aoyama, Tomoki; Iijima, Hirotaka; Tajino, Junichi; Nagai, Momoko; Yamaguchi, Shoki; Zhang, Xiangkai; Kuroki, Hiroshi

2015-05-01

To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41°C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37°C were higher than the corresponding values at 32°C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37°C compared to those at 32°C, whereas they were considerably inhibited at 41°C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Symbiont-driven sulfur crystal formation in a thiotrophic symbiosis from deep-sea hydrocarbon seeps

PubMed Central

Eichinger, Irmgard; Schmitz-Esser, Stephan; Schmid, Markus; Fisher, Charles R; Bright, Monika

2014-01-01

The siboglinid tubeworm Sclerolinum contortum symbiosis inhabits sulfidic sediments at deep-sea hydrocarbon seeps in the Gulf of Mexico. A single symbiont phylotype in the symbiont-housing organ is inferred from phylogenetic analyses of the 16S ribosomal ribonucleic acid (16S rRNA) gene and fluorescent in situ hybridization. The phylotype we studied here, and a previous study from an arctic hydrocarbon seep population, reveal identical 16S rRNA symbiont gene sequences. While sulfide is apparently the energy source for the symbionts (and ultimately the gutless host), both partners also have to cope with its toxicity. This study demonstrates abundant large sulfur crystals restricted to the trophosome area. Based on Raman microspectroscopy and energy dispersive X-ray analysis, these crystals have the same S8 sulfur configuration as the recently described small sulfur vesicles formed in the symbionts. The crystals reside adjacent to the symbionts in the trophosome. This suggests that their formation is either extra- or intracellular in symbionts. We propose that formation of these crystals provides both energy-storage compounds for the symbionts and serves the symbiosis by removing excess toxic sulfide from host tissues. This symbiont-mediated sulfide detoxification may have been crucial for the establishment of thiotrophic symbiosis and continues to remain an important function of the symbionts. PMID:24992535

Molecular and cellular profiling of acute responses to total body radiation exposure in ovariectomized female cynomolgus macaques.

PubMed

DeBo, Ryne J; Register, Thomas C; Caudell, David L; Sempowski, Gregory D; Dugan, Gregory; Gray, Shauna; Owzar, Kouros; Jiang, Chen; Bourland, J Daniel; Chao, Nelson J; Cline, J Mark

2015-06-01

The threat of radiation exposure requires a mechanistic understanding of radiation-induced immune injury and recovery. The study objective was to evaluate responses to ionizing radiation in ovariectomized (surgically post-menopausal) female cynomolgus macaques. Animals received a single total-body irradiation (TBI) exposure at doses of 0, 2 or 5 Gy with scheduled necropsies at 5 days, 8 weeks and 24 weeks post-exposure. Blood and lymphoid tissues were evaluated for morphologic, cellular, and molecular responses. Irradiated animals developed symptoms of acute hematopoietic syndrome, and reductions in thymus weight, thymopoiesis, and bone marrow cellularity. Acute, transient increases in plasma monocyte chemoattractant protein 1 (MCP-1) were observed in 5 Gy animals along with dose-dependent alterations in messenger ribonucleic acid (mRNA) signatures in thymus, spleen, and lymph node. Expression of T cell markers was lower in thymus and spleen, while expression of macrophage marker CD68 (cluster of differentiation 68) was relatively elevated in lymphoid tissues from irradiated animals. Ovariectomized female macaques exposed to moderate doses of radiation experienced increased morbidity, including acute, dose-dependent alterations in systemic and tissue-specific biomarkers, and increased macrophage/T cell ratios. The effects on mortality exceeded expectations based on previous studies in males, warranting further investigation.

Aqueous solutions of didecyldimethylammonium chloride and octaethylene glycol monododecyl ether: Toward synergistic formulations against enveloped viruses.

PubMed

Nardello-Rataj, Véronique; Leclercq, Loïc

2016-09-10

Micellization of di-n-decyldimethylammonium chloride, [DiC10][Cl], and octaethylene glycol monododecyl ether, C12E8, mixtures have been investigated by surface tension and conductivity measurements. From these results, various physicochemical and thermodynamic key parameters (e.g. micellar mole fraction of [DiC10][Cl], interaction parameter, free energy of micellization, etc.) have been evaluated and discussed in detail. The results prove high synergistic effect between the two surfactants. Based on these results, the virucidal activity of an equimolar mixture of [DiC10][Cl] and C12E8 has been investigated. A marked synergism was observed on lipid-containing deoxyribonucleic and ribonucleic acid viruses, such as herpes virus, respiratory syncytial virus, and vaccinia viruses. In contrast, Coxsackievirus (non-enveloped virus) was not inactivated. These results support that the mechanism is based on the extraction of lipids and/or proteins from the envelope inside the mixed micelles. This extraction creates "holes" the size of which increases with concentration up to a specific value which triggers the virus inactivation. Such a mixture could be used to extend the spectrum of virucidal activity of the amphiphiles virucides commonly employed in numerous disinfectant solutions. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Differentially Expressed miRNAs in Colorado Potato Beetles (Leptinotarsa decemlineata (Say)) Exposed to Imidacloprid.

PubMed

Morin, Mathieu D; Lyons, Pierre J; Crapoulet, Nicolas; Boquel, Sébastien; Morin, Pier Jr

2017-12-16

The Colorado potato beetle ( Leptinotarsa decemlineata (Say)) is a significant pest of potato plants that has been controlled for more than two decades by neonicotinoid imidacloprid. L. decemlineata can develop resistance to this agent even though the molecular mechanisms underlying this resistance are not well characterized. MicroRNAs (miRNAs) are short ribonucleic acids that have been linked to response to various insecticides in several insect models. Unfortunately, the information is lacking regarding differentially expressed miRNAs following imidacloprid treatment in L. decemlineata . In this study, next-generation sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were used to identify modulated miRNAs in imidacloprid-treated versus untreated L. decemlineata . This approach identified 33 differentially expressed miRNAs between the two experimental conditions. Of interest, miR-282 and miR-989, miRNAs previously shown to be modulated by imidacloprid in other insects, and miR-100, a miRNA associated with regulation of cytochrome P450 expression, were significantly modulated in imidacloprid-treated beetles. Overall, this work presents the first report of a miRNA signature associated with imidacloprid exposure in L. decemlineata using a high-throughput approach. It also reveals interesting miRNA candidates that potentially underly imidacloprid response in this insect pest.

Synthesis of thiol-containing analogues of puromycin and a study of their interaction with N-acetylphenylalanyl-transfer ribonucleic acid on ribosomes to form thioesters

PubMed Central

Gooch, John; Hawtrey, Arthur O.

1975-01-01

1. The thiol-containing analogue of puromycin, 6-dimethylamino-9-{1′-[3′-(2″-mercapto-3″-phenylpropionamido)-3′-deoxy-β-d-ribofuranosyl]}purine (XVII) in which the primary amino group of the antibiotic is replaced with a thiol grouping, was synthesized chemically (compound XVII is abbreviated to thiopuromycin). 2. Thiopuromycin (XVII) was found to be active in releasing N-[3H]acetylphenylalanine from its tRNA carrier as the thioester, N-acetylphenylalanylthiopuromycin (XIX) in the Escherichia coli ribosomal system. The reaction product (XIX) was synthesized chemically from thiopuromycin and N-acetylphenylalanine and found to be stable to hydrolysis in the standard incubation medium at pH7.6. dl-Phenyl-lactylpuromycin (XXI), the hydroxy analogue of puromycin, was also synthesized chemically and shown to release N-acetylphenylalanine from its tRNA carrier in the E. coli ribosomal system, thus confirming the previous results of Fahnestock et al. [Biochemistry (1970) 9, 2477–2483]. 3. In marked contrast with the results obtained in the E. coli system, both thiopuromycin (XVII) and hydroxypuromycin (XXI) were found to be inactive in releasing N-acetylphenylalanine from its tRNA carrier in the rat liver ribosomal system. PMID:1103886

Interaction of elongation factor Tu from Escherichia coli with aminoacyl-tRNA carrying a fluorescent reporter group on the 3' terminus.

PubMed

Ott, G; Faulhammer, H G; Sprinzl, M

1989-09-15

Transfer ribonucleic acids containing 2-thiocytidine in position 75 ([s2C]tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3'-shortened tRNA using ATP(CTP):tRNA nucleotidyltransferase. [s2C]tRNA was selectively alkylated with fluorescent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) on the 2-thiocytidine residue. The product [AEDANS-s2C]aminoacyl-tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore. From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast [AEDANS-s2C]Tyr-tRNATyr, yeast Tyr-tRNATyr, and the homologous E. coli Phe-tRNAPhe, respectively, interacting with E. coli elongation factor Tu.GTP. The measurement of the association and dissociation rates of the interaction of [AEDANS-s2C]Tyr-tRNATyr with EF-Tu.GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol-1 K-1 for delta S degrees' and -34.7 kJ mol-1 for delta H degrees' of this reaction.

CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation

PubMed Central

Puustinen, Pietri; Rytter, Anna; Mortensen, Monika; Kohonen, Pekka; Moreira, José M.

2014-01-01

mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A’s reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation. PMID:24590173

Influence of Na+ and Mg2+ ions on RNA structures studied with molecular dynamics simulations.

PubMed

Fischer, Nina M; Polêto, Marcelo D; Steuer, Jakob; van der Spoel, David

2018-06-01

The structure of ribonucleic acid (RNA) polymers is strongly dependent on the presence of, in particular Mg2+ cations to stabilize structural features. Only in high-resolution X-ray crystallography structures can ions be identified reliably. Here, we perform molecular dynamics simulations of 24 RNA structures with varying ion concentrations. Twelve of the structures were helical and the others complex folded. The aim of the study is to predict ion positions but also to evaluate the impact of different types of ions (Na+ or Mg2+) and the ionic strength on structural stability and variations of RNA. As a general conclusion Mg2+ is found to conserve the experimental structure better than Na+ and, where experimental ion positions are available, they can be reproduced with reasonable accuracy. If a large surplus of ions is present the added electrostatic screening makes prediction of binding-sites less reproducible. Distinct differences in ion-binding between helical and complex folded structures are found. The strength of binding (ΔG‡ for breaking RNA atom-ion interactions) is found to differ between roughly 10 and 26 kJ/mol for the different RNA atoms. Differences in stability between helical and complex folded structures and of the influence of metal ions on either are discussed.

Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

PubMed Central

Whitfeld, P R; Leaver, C J; Bottomley, W; Atchison, B

1978-01-01

A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves. Images Fig. 8. PMID:743229

Short Hairpin Ribonucleic Acid Constructs Targeting Insulin-like Growth Factor Binding Protein-3 Reversed Decreased Testosterone Concentrations in Diabetic Rats

PubMed Central

Zhou, Zhang-Yan; Fei-Li; Cheng, Shao-Ping; Huang, Hui; Peng, Bi-Wen; Wang, Jing; Liu, Chang-Mao; Xing, Cheng; Sun, Ya-Ling; Bsoul, Najeeb; Pan, Hui; Yi, Cun-Jian; Liu, Rong-Hua; Zhong, Guang-Jun

2015-01-01

Background The aim of this study was to determine if shRNA constructs targeting insulin-like growth factor binding protein-3 can rehabilitate decreased serum testosterone concentrations in streptozotocin-induced diabetic rats. Material/Methods After 12 weeks of intracavernous administration of IGFBP-3 shRNA, intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated. The expression of IGFBP-3 at mRNA and protein levels was detected by quantitative real-time PCR analysis and Western blot, respectively. The concentrations of serum testosterone and cavernous cyclic guanosine monophosphate were detected by enzyme-linked immunosorbent assay. Results After 12 weeks of intracavernous administration of IGFBP-3 shRNA, the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic control group (p<0.01). Cavernous IGFBP-3 expression at both mRNA and protein levels was significantly inhibited. Both serum testosterone and cavernous cyclic guanosine monophosphate concentrations were significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic control group (p<0.01). Conclusions These results suggest that IGFBP-3 shRNA may rehabilitate erectile function via increases of concentrations of serum testosterone and cavernous cyclic guanosine monophosphate in streptozotocin-induced diabetic rats. PMID:25582342

Dysregulation of pulmonary endothelial protein C receptor and thrombomodulin in severe falciparum malaria-associated ARDS relevant to hemozoin

PubMed Central

Maknitikul, Sitang; Luplertlop, Natthanej; Grau, Georges E. R.

2017-01-01

To investigate the role of the protein C system, endothelial protein C receptor (EPCR) and thrombomodulin (TM) in the pathogenesis of malaria-associated acute respiratory distress syndrome (ARDS) in relation to hemozoin and proinflammatory cytokines-induced type II pneumocyte injury and -aggravated pulmonary resolution. A total of 29 left-over lung specimens that were obtained from patients who died from severe falciparum malaria were examined. Histopathological, immunohistochemical and electron microscopic analyses revealed that ARDS coexisted with pulmonary edema and systemic bleeding; the severity was dependent on the level of hemozoin deposition in the lung and internal alveolar hemorrhaging. The loss of EPCR and TM was primarily identified in ARDS patients and was related to the level of hemozoin, parasitized red blood cell (PRBC) and white blood cell accumulation in the lung. Moreover, an in vitro analysis demonstrated that interleukin-13 and -31 and hemozoin induced pneumocytic cell injury and apoptosis, as assessed by EB/AO staining, electron microscopy and the up-regulation of CARD-9 mRNA (caspase recruitment domain-9 messenger-ribonucleic acid). The dysregulation of EPCR and TM in the lung, especially in those with increased levels of hemozoin, may play an important role in the pathogenesis of malaria-associated ARDS through an apoptotic pathway. PMID:28732053

The DcpS inhibitor RG3039 improves motor function in SMA mice

PubMed Central

Van Meerbeke, James P.; Gibbs, Rebecca M.; Plasterer, Heather L.; Miao, Wenyan; Feng, Zhihua; Lin, Ming-Yi; Rucki, Agnieszka A.; Wee, Claribel D.; Xia, Bing; Sharma, Shefali; Jacques, Vincent; Li, Darrick K.; Pellizzoni, Livio; Rusche, James R.; Ko, Chien-Ping; Sumner, Charlotte J.

2013-01-01

Spinal muscular atrophy (SMA) is caused by mutations of the survival motor neuron 1 (SMN1) gene, retention of the survival motor neuron 2 (SMN2) gene and insufficient expression of full-length survival motor neuron (SMN) protein. Quinazolines increase SMN2 promoter activity and inhibit the ribonucleic acid scavenger enzyme DcpS. The quinazoline derivative RG3039 has advanced to early phase clinical trials. In preparation for efficacy studies in SMA patients, we investigated the effects of RG3039 in severe SMA mice. Here, we show that RG3039 distributed to central nervous system tissues where it robustly inhibited DcpS enzyme activity, but minimally activated SMN expression or the assembly of small nuclear ribonucleoproteins. Nonetheless, treated SMA mice showed a dose-dependent increase in survival, weight and motor function. This was associated with improved motor neuron somal and neuromuscular junction synaptic innervation and function and increased muscle size. RG3039 also enhanced survival of conditional SMA mice in which SMN had been genetically restored to motor neurons. As this systemically delivered drug may have therapeutic benefits that extend beyond motor neurons, it could act additively with SMN-restoring therapies delivered directly to the central nervous system such as antisense oligonucleotides or gene therapy. PMID:23727836

The effect of ethionine on ribonucleic acid synthesis in rat liver.

PubMed Central

Swann, P F

1975-01-01

1. By 1h after administration of ethionine to the female rat the appearance of newly synthesized 18SrRNA in the cytoplasm is completely inhibited. This is not caused by inhibition of RNA synthesis, for the synthesis of the large ribosomal precursor RNA (45S) and of tRNA continues. Cleavage of 45S RNA to 32S RNA also occurs, but there was no evidence for the accumulation of mature or immature rRNA in the nucleus. 2. The effect of ethionine on the maturation of rRNA was not mimicked by an inhibitor of protein synthesis (cycloheximide) or an inhibitor of polyamine synthesis [methylglyoxal bis(guanylhydrazone)]. 3. Unlike the ethionine-induced inhibition of protein synthesis, this effect was not prevented by concurrent administration of inosine. A similar effect could be induced in HeLa cells by incubation for 1h in a medium lacking methionine. The ATP concentration in these cells was normal. From these two observations it was concluded that the effect of etionine on rRNA maturation is not caused by an ethionine-induced lack of ATP. It is suggested that ethionine, by lowering the hepatic concentration of S-adenosylmethionine, prevents methylation of the ribosomal precursor. The methylation is essential for the correct maturation of the molecule; without methylation complete degradation occurs. PMID:1212195

The genome editing revolution: A CRISPR-Cas TALE off-target story.

PubMed

Stella, Stefano; Montoya, Guillermo

2016-07-01

In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Nucleosome regulatory dynamics in response to TGFβ

PubMed Central

Enroth, Stefan; Andersson, Robin; Bysani, Madhusudhan; Wallerman, Ola; Termén, Stefan; Tuch, Brian B.; De La Vega, Francisco M.; Heldin, Carl-Henrik; Moustakas, Aristidis; Komorowski, Jan; Wadelius, Claes

2014-01-01

Nucleosomes play important roles in a cell beyond their basal functionality in chromatin compaction. Their placement affects all steps in transcriptional regulation, from transcription factor (TF) binding to messenger ribonucleic acid (mRNA) synthesis. Careful profiling of their locations and dynamics in response to stimuli is important to further our understanding of transcriptional regulation by the state of chromatin. We measured nucleosome occupancy in human hepatic cells before and after treatment with transforming growth factor beta 1 (TGFβ1), using massively parallel sequencing. With a newly developed method, SuMMIt, for precise positioning of nucleosomes we inferred dynamics of the nucleosomal landscape. Distinct nucleosome positioning has previously been described at transcription start site and flanking TF binding sites. We found that the average pattern is present at very few sites and, in case of TF binding, the double peak surrounding the sites is just an artifact of averaging over many loci. We systematically searched for depleted nucleosomes in stimulated cells compared to unstimulated cells and identified 24 318 loci. Depending on genomic annotation, 44–78% of them were over-represented in binding motifs for TFs. Changes in binding affinity were verified for HNF4α by qPCR. Strikingly many of these loci were associated with expression changes, as measured by RNA sequencing. PMID:24771338

An investigation into the effects of silver nanoparticles on natural microbial communities in two freshwater sediments.

PubMed

Bao, Shaopan; Wang, Han; Zhang, Weicheng; Xie, Zhicai; Fang, Tao

2016-12-01

The expanding production and usage of commercial silver nanoparticles (AgNPs) will inevitably increase their environmental release, with sediments as a substantial sink. However, little knowledge is available about the potential impacts of AgNPs on freshwater sediment microbial communities, as well as the interactions between microbial communities and biogeochemical factors in AgNPs polluted sediment. To address these issues, two different sediments: a eutrophic freshwater sediment and an oligotrophic freshwater sediment, were exposed to 1 mg/g of either AgNO 3 , uncoated AgNPs (35-nm and 75-nm), or polyvinylpyrrolidone coated AgNPs (PVP-AgNPs) (30-50 nm) for 45 days. High-throughput sequencing of 16S ribosomal ribonucleic acid (16S rRNA) genes using the Illumina MiSeq platform was conducted to evaluate the effects of Ag addition on bacterial community composition. Moreover, sediment microbial biomass and activity were assessed by counting cultivable bacterial number and determining enzyme activities. During the 45-day exposure, compared with no amendment control, some treatments had resulted in significant changes and alterations of sediment biomass or bacterial enzyme activities shortly. While the microbial components at phylum level were rarely affected by AgNPs addition, and as confirmed by the statistical analysis with two-factor analysis of similarities (ANOSIM), there were no significant differences on bacterial community structure across the amended treatments. Redundancy analysis further demonstrated that chemical parameters acid-volatile sulfide (AVS) and simultaneously extracted silver (SE-Ag) in sediment significantly structured the overall bacterial community in sediments spiked with various silver species. In summary, these findings suggested that the ecotoxicity of AgNPs may be attenuated by the transformation under complex environmental conditions and the self-adaption of sediment microbial communities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Degradation of Adenine on the Martian Surface in the Presence of Perchlorates and Ionizing Radiation: A Reflectron Time-of-flight Mass Spectrometric Study

NASA Astrophysics Data System (ADS)

Góbi, Sándor; Bergantini, Alexandre; Kaiser, Ralf I.

2017-04-01

The aim of the present work is to unravel the radiolytic decomposition of adenine (C5H5N5) under conditions relevant to the Martian surface. Being the fundamental building block of (deoxy)ribonucleic acids, the possibility of survival of this biomolecule on the Martian surface is of primary importance to the astrobiology community. Here, neat adenine and adenine-magnesium perchlorate mixtures were prepared and irradiated with energetic electrons that simulate the secondary electrons originating from the interaction of the galactic cosmic rays with the Martian surface. Perchlorates were added to the samples since they are abundant—and therefore relevant oxidizers on the surface of Mars—and they have been previously shown to facilitate the radiolysis of organics such as glycine. The degradation of the samples were monitored in situ via Fourier transformation infrared spectroscopy and the electron ionization quadruple mass spectrometric method; temperature-programmed desorption profiles were then collected by means of the state-of-the-art single photon photoionization reflectron time-of-flight mass spectrometry (PI-ReTOF-MS), allowing for the detection of the species subliming from the sample. The results showed that perchlorates do increase the destruction rate of adenine by opening alternative reaction channels, including the concurrent radiolysis/oxidation of the sample. This new pathway provides a plethora of different radiolysis products that were identified for the first time. These are carbon dioxide (CO2), isocyanic acid (HNCO), isocyanate (OCN-), carbon monoxide (CO), and nitrogen monoxide (NO); an oxidation product containing carbonyl groups (R1R2-C=O) with a constrained five-membered cyclic structure could also be observed. Cyanamide (H2N-C≡N) was detected in both irradiated samples as well.

Comparison of gene delivery techniques for therapeutic angiogenesis ultrasound-mediated destruction of carrier microbubbles versus direct intramuscular injection.

PubMed

Kobulnik, Jeremy; Kuliszewski, Michael A; Stewart, Duncan J; Lindner, Jonathan R; Leong-Poi, Howard

2009-10-27

This study was designed to compare the efficacy of angiogenic gene delivery by ultrasound-mediated (UM) destruction of intravenous carrier microbubbles to direct intramuscular (IM) injections. Current trials of gene therapy for angiogenesis remain limited by suboptimal, invasive delivery techniques. Hind-limb ischemia was produced by iliac artery ligation in 99 rats. In 32 rats, UM delivery of green fluorescent protein (GFP)/vascular endothelial growth factor-165 (VEGF(165)) plasmid deoxyribonucleic acid was performed. Thirty-five animals received IM injections of VEGF(165)/GFP plasmid. Remaining rats received no treatment. Before delivery (day 14 after ligation) and at days 17, 21, and 28 and week 8 after ligation, microvascular blood volume and microvascular blood flow to the proximal hind limbs were assessed by contrast-enhanced ultrasound (n = 8 per group). Total transfection was assessed by reverse transcriptase-polymerase chain reaction, and localization of transfection was determined by immunohistochemistry. By day 28, both IM and UM delivery of VEGF(165) produced significant increases in microvascular blood volume and microvascular blood flow. Whereas increases in microvascular blood volume were similar between treatment groups, microvascular blood flow was greater (p < 0.005) in UM-treated animals as compared with IM-treated animals, persisting to week 8. The VEGF(165)/GFP messenger ribonucleic acid expression was greater (p < 0.05) for IM-treated animals. A strong GFP signal was detected for both groups and was localized to focal perivascular regions and myocytes around injection sites for IM and to the vascular endothelium of arterioles/capillaries in a wider distribution for UM delivery. Despite lower transfection levels, UM delivery of VEGF(165) is as effective as IM injections. The UM delivery results in directed vascular transfection over a wider distribution, which may account for the more efficient angiogenesis.

Specific telomere dysfunction induced by GRN163L increases radiation sensitivity in breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gomez-Millan, Jaime; Goldblatt, Erin M.; Gryaznov, Sergei M.

Purpose: Telomerase is expressed in 80-90% of tumor cells, but is absent in most somatic cells. The absence of telomerase activity results in progressive telomere shortening, leading to cellular senescence or death through deoxyribonucleic acid (DNA) damage signals. In addition, a role for telomerase in DNA damage repair has also been suggested. A specific telomerase inhibitor, GRN163L that is complementary to the template region of the telomerase ribonucleic acid component (hTR). We hypothesized that exposure to GRN163L, either through immediate inhibition of telomerase activity or through eventual telomere shortening and dysfunction, may enhance radiation sensitivity. Our goal was to testmore » whether the treatment with GRN163L enhances sensitivity to irradiation (IR) in MDA-MB-231 breast cancer cells. Methods and Materials: The MDA-MB-231 breast cancer cells were treated with or without GRN163L for 2-42 days. Inhibition of telomerase activity and shortening of telomeres were confirmed. Cells were then irradiated and clonogenic assays were performed to show cell survival differences. In vivo studies using MDA-MB-231 xenografts were performed to corroborate the in vitro results. Results: We show that cells with shortened telomeres due to GRN163L enhance the effect on IR reducing survival by an additional 30% (p < 0.01). These results are confirmed in vivo, with a significant decrease in tumor growth in mice exposed to GRN163L. Conclusions: We found that GRN163L is a promising adjuvant treatment in combination with radiation therapy that may improve the therapeutic index by enhancing the radiation sensitivity. These studies prompt further investigation as to whether this combination can be applied to other cancers and the clinic.« less

Surface-Active Agents for Isolation of the Core Component of Avian Myeloblastosis Virus 1

PubMed Central

Stromberg, Kurt

1972-01-01

Sixty-one surface-active agents were evaluated in a procedure designed to assess their ability to remove the envelope from the core component of avian myeloblastosis virus (AMV). The procedure consisted of centrifugation of intact AMV through a series of sucrose gradients each containing an upper layer of agent at one of eight concentrations between 0.01 and 10%. The effectiveness of an agent in producing AMV cores was indicated by (i) the appearance of light-scattering bands in the region of core buoyant density in gradient tubes; (ii) the range of surfactant concentration over which these bands appeared; and (iii) an electron microscopy assessment by the negative-staining technique of the relative proportion of core to non-core material in each of these bands. Six nonionic surfactants were selected by this screening method for comparison in regard to recovery of core protein and endogenous ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, as well as further morphologic evaluation by electron microscopy. The nonionic surfactants of the polyoxyethylene alcohol class (particularly, Sterox SL) were most effective. Nonionic surfactants of the polyoxyethylene alkylphenol class (particularly, Nonidet P-40) were also effective. Sterox SL and Nonidet P-40 each gave a more than fivefold increase in specific activity of endogenous RNA-dependent DNA polymerase, and each gave a low recovery of core protein. Sterox SL did not interfere to the extent that Nonidet P-40 did in procedures which involved spectrophotometric assay at 260 nm. The use of Sterox SL resulted in the least envelope contamination of core preparations by electron microscopy examination, the most recovery of protein and endogenous RNA-dependent DNA polymerase activity, and a core buoyant density in sucrose of 1.27 g/ml. Images PMID:4112071

Mentha piperita as a pivotal neuro-protective agent against gamma irradiation induced DNA fragmentation and apoptosis : Mentha extract as a neuroprotective against gamma irradiation.

PubMed

Hassan, Hanaa A; Hafez, Hani S; Goda, Mona S

2013-01-01

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells, in particular to DNA, is due to oxidative stress enhancing apoptotic cell death. Our present study aimed to characterize and semi-quantify the radiation-induced apoptosis in CNS and the activity of Mentha extracts as neuron-protective agent. Our results through flow cytometry exhibited the significant disturbance and arrest in cell cycle in % of M1: SubG1 phase, M2: G0/1 phase of diploid cycle, M3: S phase and M4: G2/M phase of cell cycle in brain tissue (p < 0.05). Significant increase in % of apoptosis and P53 protein expression as apoptotic biomarkers were coincided with significant decrease in Bcl(2) as an anti-apoptotic marker. The biochemical analysis recorded a significant decrease in the levels of reduced glutathione, superoxide dismutase, deoxyribonucleic acid (DNA) and ribonucleic acid contents. Moreover, numerous histopathological alterations were detected in brain tissues of gamma irradiated mice such as signs of chromatolysis in pyramidal cells of cortex, nuclear vacuolation, numerous apoptotic cell, and neural degeneration. On the other hand, gamma irradiated mice pretreated with Mentha extract showed largely an improvement in all the above tested parameters through a homeostatic state for the content of brain apoptosis and stabilization of DNA cycle with a distinct improvement in cell cycle analysis and antioxidant defense system. Furthermore, the aforementioned effects of Mentha extracts through down-regulation of P53 expression and up-regulation of Bcl(2) domain protected brain structure from extensive damage. Therefore, Mentha extract seems to have a significant role to ameliorate the neuronal injury induced by gamma irradiation.

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

PubMed

Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

2015-05-01

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Small interfering RNA-mediated suppression of serum response factor, E2-promotor binding factor and survivin in non-small cell lung cancer cell lines by non-viral transfection.

PubMed

Walker, Tobias; Nolte, Andrea; Steger, Volker; Makowiecki, Christina; Mustafi, Migdat; Friedel, Godehard; Schlensak, Christian; Wendel, Hans-Peter

2013-03-01

Serum response factor (SRF), E2F1 and survivin are well-known factors involved in a multitude of cancer-related regulation processes. However, to date, no suitable means has been found to apply their potential in the therapy of non-small cell lung cancer (NSCLC). This study deals with questions of small interfering ribonucleic acid (siRNA) transfection efficiency by a non-viral transfection of NSCLC cell-lines and the power of siRNA to transiently influence cell division by specific silencing. Different NSCLC cell lines were cultured under standard conditions and transfected, with specific siRNA targeting SRF, E2F1 and survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as controls. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for messenger RNA (mRNA) expression levels. Additionally, transfection efficiency was evaluated by flow cytometry. The analysis of cell proliferation was determined with a CASY cell counter 3 days after transfection with SRF or SCR-siRNA. Transfection of the NSCLC cell lines with specific siRNAs against SRF, E2F1 and survivin resulted in a very considerable reduction of the intracellular mRNA concentration. CASY confirmation of cell viability demonstrated an excellent survival of the cell lines treated with non-specific siRNA, in contrast to with application of specific siRNA. This study reports a reliable transfectability of NSCLC-cell lines by siRNA, initially in a non-viral manner, and a reproducible knockdown of the focussed targets, consequently leading to the death of the tumour cells. This constitutes a strong candidate for a new assessment strategy in the therapy of non-small cell lung cancer.

Complex extreme learning machine applications in terahertz pulsed signals feature sets.

PubMed

Yin, X-X; Hadjiloucas, S; Zhang, Y

2014-11-01

This paper presents a novel approach to the automatic classification of very large data sets composed of terahertz pulse transient signals, highlighting their potential use in biochemical, biomedical, pharmaceutical and security applications. Two different types of THz spectra are considered in the classification process. Firstly a binary classification study of poly-A and poly-C ribonucleic acid samples is performed. This is then contrasted with a difficult multi-class classification problem of spectra from six different powder samples that although have fairly indistinguishable features in the optical spectrum, they also possess a few discernable spectral features in the terahertz part of the spectrum. Classification is performed using a complex-valued extreme learning machine algorithm that takes into account features in both the amplitude as well as the phase of the recorded spectra. Classification speed and accuracy are contrasted with that achieved using a support vector machine classifier. The study systematically compares the classifier performance achieved after adopting different Gaussian kernels when separating amplitude and phase signatures. The two signatures are presented as feature vectors for both training and testing purposes. The study confirms the utility of complex-valued extreme learning machine algorithms for classification of the very large data sets generated with current terahertz imaging spectrometers. The classifier can take into consideration heterogeneous layers within an object as would be required within a tomographic setting and is sufficiently robust to detect patterns hidden inside noisy terahertz data sets. The proposed study opens up the opportunity for the establishment of complex-valued extreme learning machine algorithms as new chemometric tools that will assist the wider proliferation of terahertz sensing technology for chemical sensing, quality control, security screening and clinic diagnosis. Furthermore, the proposed algorithm should also be very useful in other applications requiring the classification of very large datasets. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Microbial aerosol liberation from soiled textiles isolated during routine residuals handling in a modern health care setting.

PubMed

Handorean, Alina; Robertson, Charles E; Harris, J Kirk; Frank, Daniel; Hull, Natalie; Kotter, Cassandra; Stevens, Mark J; Baumgardner, Darrel; Pace, Norman R; Hernandez, Mark

2015-12-09

A wide variety of specialty textiles are used in health care settings for bedding, clothing, and privacy. The ability of textiles to host or otherwise sequester microbes has been well documented; however, their reciprocal potential for liberating airborne bacteria remains poorly characterized. In response, a multi-season survey of bacterial bioaerosols was conducted in the origin and terminus of residual paths which are specifically designed to isolate soiled hospital textiles as they are moved to laundering. This survey used conventional optical particle counting which incorporated multi-channel fluorescence in conjunction with molecular phylogenetic analyses to characterize the bioaerosols liberated during soiled textile storage--immediately before and after the occupation of a modern hospital. Although outfitted with a HEPA filtration system, the number of airborne particles presenting fluorescing optical signatures consistent with airborne bacteria and fungi significantly increased in textile holding rooms soon after the hospital's commissioning, even though these isolated residual areas rarely host personnel. The bioaerosol liberated during textile storage was characterized using Illumina MiSeq sequencing of bacterial 16S ribosomal ribonucleic acid (rRNA) genes. Gene copies recovered by quantitative PCR from aerosol collected in co-located impingers were consistent with fluorescence gated optical particle counting. The relative abundance patterns of proximal bacterial bioaerosol were such that the air in the origin and terminus of textile storage rooms could not be differentiated once the hospital began processing soiled linens. Genes from microbes typically associating with human skin, feces, and hair--Staphylococcus, Propionibacteria, Corynebacteria, Lactobacillus, and Streptococcus spp.--dominated the aerosol abundance profiles in textile holding rooms, which were generally far less diverse than communities recovered from surfaces in patient rooms. These results suggest that aerosol partitioning from the routine handling of soiled textiles can contribute to airborne exposures in the health care environment.

RNA-FISH to Study Regulatory RNA at the Site of Transcription.

PubMed

Soler, Marta; Boque-Sastre, Raquel; Guil, Sonia

2017-01-01

The increasing role of all types of regulatory RNAs in the orchestration of cellular programs has enhanced the development of a variety of techniques that allow its precise detection, quantification, and functional scrutiny. Recent advances in imaging and fluoresecent in situ hybridization (FISH) methods have enabled the utilization of user-friendly protocols that provide highly sensitive and accurate detection of ribonucleic acid molecules at both the single cell and subcellular levels. We herein describe the approach originally developed by Stellaris ® , in which the target RNA molecule is fluoresecently labeled with multiple tiled complementary probes each carrying a fluorophore, thus improving sensitivity and reducing the chance of false positives. We have applied this method to the detection of nascent RNAs that partake of special regulatory structures called R loops. Their growing role in active gene expression regulation (Aguilera and Garcia-Muse, Mol Cell 46:115-124, 2012; Ginno et al., Mol Cell 45:814-825, 2012; Sun et al., Science 340:619-621, 2013; Bhatia et al., Nature 511:362-365, 2014) imposes the use of a combination of in vivo and in vitro techniques for the detailed analysis of the transcripts involved. Therefore, their study is a good example to illustrate how RNA FISH, combined with transcriptional arrest and/or cell synchronization, permits localization and temporal characterization of potentially regulatory RNA sequences.

Distinct Effects of the Cervicovaginal Microbiota and Herpes Simplex Type 2 Infection on Female Genital Tract Immunology

PubMed Central

Gajer, P.; Yi, T. J.; Ma, B.; Humphrys, M. S.; Thomas-Pavanel, J.; Chieza, L.; Janakiram, P.; Saunders, M.; Tharao, W.; Huibner, S.; Shahabi, K.; Ravel, J.; Kaul, R.

2017-01-01

Abstract Background. Genital inflammation is a key determinant of human immunodeficiency virus (HIV) transmission, and may increase HIV-susceptible target cells and alter epithelial integrity. Several genital conditions that increase HIV risk are more prevalent in African, Caribbean, and other black (ACB) women, including bacterial vaginosis and herpes simplex virus type-2 (HSV-2) infection. Therefore, we assessed the impact of the genital microbiota on mucosal immunology in ACB women and microbiome-HSV-2 interactions. Methods. Cervicovaginal secretions and endocervical cells were collected by cytobrush and Instead Softcup, respectively. T cells and dendritic cells were assessed by flow cytometry, cytokines by multiplex enzyme-linked immunosorbent assay (ELISA), and the microbiota by 16S ribosomal ribonucleic acid gene sequencing. Results. The cervicovaginal microbiota of 51 participants were composed of community state types (CSTs) showing diversity (20/51; 39%) or predominated by Lactobacillus iners (22/51; 42%), L. crispatus (7/51; 14%), or L. gasseri (2/51; 4%). High-diversity CSTs and specific bacterial phyla (Gardnerella vaginalis and Prevotella bivia) were strongly associated with cervicovaginal inflammatory cytokines, but not with altered endocervical immune cells. However, cervical CD4+ T-cell number was associated with HSV-2 infection and a distinct cytokine profile. Conclusions. This suggests that the genital microbiota and HSV-2 infection may influence HIV susceptibility through independent biological mechanisms. PMID:28201724

Mapping Optimal Charge Density and Length of ROMP-Based PTDMs for siRNA Internalization.

PubMed

Caffrey, Leah M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

2016-10-10

A fundamental understanding of how polymer structure impacts internalization and delivery of biologically relevant cargoes, particularly small interfering ribonucleic acid (siRNA), is of critical importance to the successful design of improved delivery reagents. Herein we report the use of ring-opening metathesis polymerization (ROMP) methods to synthesize two series of guanidinium-rich protein transduction domain mimics (PTDMs): one based on an imide scaffold that contains one guanidinium moiety per repeat unit, and another based on a diester scaffold that contains two guanidinium moieties per repeat unit. By varying both the degree of polymerization and, in effect, the relative number of cationic charges in each PTDM, the performances of the two ROMP backbones for siRNA internalization were evaluated and compared. Internalization of fluorescently labeled siRNA into Jurkat T cells demonstrated that fluorescein isothiocyanate (FITC)-siRNA internalization had a charge content dependence, with PTDMs containing approximately 40 to 60 cationic charges facilitating the most internalization. Despite this charge content dependence, the imide scaffold yielded much lower viabilities in Jurkat T cells than the corresponding diester PTDMs with similar numbers of cationic charges, suggesting that the diester scaffold is preferred for siRNA internalization and delivery applications. These developments will not only improve our understanding of the structural factors necessary for optimal siRNA internalization, but will also guide the future development of optimized PTDMs for siRNA internalization and delivery.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs.

PubMed

Moreno, Renata; Hernández-Arranz, Sofía; La Rosa, Ruggero; Yuste, Luis; Madhushani, Anjana; Shingler, Victoria; Rojo, Fernando

2015-01-01

The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putida Hfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

The presence of Epstein-Barr virus (EBV) in diffuse large B-cell lymphomas (DLBCLs) in Turkey: special emphasis on 'EBV-positive DLBCL of the elderly'.

PubMed

Uner, Aysegul; Akyurek, Nalan; Saglam, Arzu; Abdullazade, Samir; Uzum, Nuket; Onder, Sevgen; Barista, Ibrahim; Benekli, Mustafa

2011-04-01

Accumulated evidence has shown the importance of Epstein-Barr virus in the pathogenesis of various lymphomas. This study aimed to determine the prevalence of Epstein-Barr virus expression and its effect on survival amongst diffuse large B-cell lymphoma (DLBCL) cases from two large tertiary care centres in Turkey with a particular interest in identifying cases of 'Epstein-Barr virus-positive DLBCL of the elderly'. Diffuse large B-cell lymphoma cases diagnosed between 1999 and 2009 were retrieved and 340 cases were used to construct tissue microarrays. The presence of Epstein-Barr virus small ribonucleic acids was examined by in situ hybridization using Epstein-Barr virus (EBV)-encoded small RNA (EBER) oligonucleotides. A total of 18 cases (5.3%) showed Epstein-Barr virus expression. Twelve cases were classified as Epstein-Barr virus-positive DLBCL of the elderly. Epstein-Barr virus-positive DLBCL cases showed a significantly inferior overall survival as compared with Epstein-Barr virus-negative cases (p < 0.001). In our study group Epstein-Barr virus expression is not prevalent in DLBCLs. Epstein-Barr virus-positive DLBCL of the elderly is also rare in the Turkish population. The presence of Epstein-Barr virus, however, is associated with poor prognosis. © 2011 The Authors. APMIS © 2011 APMIS.

Apoptosis and Inflammation Associated Gene Expressions in Monocrotaline-Induced Pulmonary Hypertensive Rats after Bosentan Treatment

PubMed Central

Hong, Young Mi; Kwon, Jung Hyun; Choi, Shinkyu

2014-01-01

Background and Objectives Vascular wall remodeling in pulmonary hypertension can be caused by an aberration in the normal balance between proliferation and apoptosis of endothelial cell in the pulmonary artery. The objective of this study was to evaluate the effect of bosentan on apoptosis in monocrotaline (MCT)-induced pulmonary hypertension. Materials and Methods Sprague-Dawley rats were divided into three groups: control (C) group, M group (MCT 60 mg/kg) and B group (MCT 60 mg/kg plus bosentan 20 mg/day orally). Gene expressions of Bcl (B cell leukemia/lymphoma)-2, caspase-3, complement component (C)-6, vascular endothelial growth factor (VEGF), interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were analyzed by real time polymerase chain reaction and western blot analysis. Results The messenger ribonucleic acid (mRNA) expressions of caspase-3 and VEGF were significantly increased in the M group compared with the C group, and significantly decreased in the B group compared with the M group in week 4. mRNA expression of IL-6 was significantly decreased in weeks 1, 2, and 4 in the B group compared with the M group. mRNA expression of TNF-α was significantly decreased on day 5 and in weeks 1 and 2 in the B group compared with the M group. Conclusion Bosentan may have potential for preventing apoptosis and inflammation. PMID:24653739

Neurotransmitter Systems in a Mild Blast Traumatic Brain Injury Model: Catecholamines and Serotonin.

PubMed

Kawa, Lizan; Arborelius, Ulf P; Yoshitake, Takashi; Kehr, Jan; Hökfelt, Tomas; Risling, Mårten; Agoston, Denes

2015-08-15

Exposure to improvised explosive devices can result in a unique form of traumatic brain injury--blast-induced traumatic brain injury (bTBI). At the mild end of the spectrum (mild bTBI [mbTBI]), there are cognitive and mood disturbances. Similar symptoms have been observed in post-traumatic stress disorder caused by exposure to extreme psychological stress without physical injury. A role of the monoaminergic system in mood regulation and stress is well established but its involvement in mbTBI is not well understood. To address this gap, we used a rodent model of mbTBI and detected a decrease in immobility behavior in the forced swim test at 1 d post-exposure, coupled with an increase in climbing behavior, but not after 14 d or later, possibly indicating a transient increase in anxiety-like behavior. Using in situ hybridization, we found elevated messenger ribonucleic acid levels of both tyrosine hydroxylase and tryptophan hydroxylase 2 in the locus coeruleus and the dorsal raphe nucleus, respectively, as early as 2 h post-exposure. High-performance liquid chromatography analysis 1 d post-exposure primarily showed elevated noradrenaline levels in several forebrain regions. Taken together, we report that exposure to mild blast results in transient changes in both anxiety-like behavior and brain region-specific molecular changes, implicating the monoaminergic system in the pathobiology of mbTBI.

Quantification of therapeutic miRNA mimics in whole blood from nonhuman primates.

PubMed

Kelnar, Kevin; Peltier, Heidi J; Leatherbury, Neil; Stoudemire, Jay; Bader, Andreas G

2014-02-04

MRX34, a microRNA (miRNA)-based therapy for cancer, has recently entered clinical trials as the first clinical candidate in its class. It is a liposomal nanoparticle loaded with a synthetic mimic of the tumor suppressor miRNA miR-34a as the active pharmaceutical ingredient. To understand the pharmacokinetic properties of the drug and to rationalize an optimal dosing regimen in the clinic, a method is needed to quantitatively detect the miRNA mimic. Here, we report the development and qualification of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay in support of pharmacokinetic and toxicokinetic assessments in the nonhuman primate. Detection and quantification were performed on total ribonucleic acid (RNA) isolated from whole blood. The qualified range of the standard curve spans 6 orders of magnitude from 2.5 × 10(-7) to 2.5 × 10(-1) ng per reverse transcription (RT) reaction, corresponding to an estimated blood concentration from 6.2 × 10(-5) to 6.2 × 10(1) ng/mL. Our results demonstrate that endogenous as well as the exogenous miR-34a can be accurately and precisely quantified. The assay was used to establish the pharmacokinetic profile of MRX34, showing a favorable residence time and exposure of the miRNA mimic in whole blood from nonhuman primates.

Involvement of TRPV1 and AQP2 in hypertonic stress by xylitol in odontoblast cells.

PubMed

Tokuda, M; Fujisawa, M; Miyashita, K; Kawakami, Y; Morimoto-Yamashita, Y; Torii, M

2015-02-01

To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress. OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay. Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation. OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.

All-atom four-body knowledge-based statistical potential to distinguish native tertiary RNA structures from nonnative folds.

PubMed

Masso, Majid

2018-09-14

Scientific breakthroughs in recent decades have uncovered the capability of RNA molecules to fulfill a wide array of structural, functional, and regulatory roles in living cells, leading to a concomitantly significant increase in both the number and diversity of experimentally determined RNA three-dimensional (3D) structures. Atomic coordinates from a representative training set of solved RNA structures, displaying low sequence and structure similarity, facilitate derivation of knowledge-based energy functions. Here we develop an all-atom four-body statistical potential and evaluate its capacity to distinguish native RNA 3D structures from nonnative folds based on calculated free energy scores. Atomic four-body nearest-neighbors are objectively identified by their occurrence as tetrahedral vertices in the Delaunay tessellations of RNA structures, and rates of atomic quadruplet interactions expected by chance are obtained from a multinomial reference distribution. Our four-body energy function, referred to as RAMP (ribonucleic acids multibody potential), is subsequently derived by applying the inverted Boltzmann principle to the frequency data, yielding an energy score for each type of atomic quadruplet interaction. Several well-known benchmark datasets reveal that RAMP is comparable with, and often outperforms, existing knowledge- and physics-based energy functions. To the best of our knowledge, this is the first study detailing an RNA tertiary structure-based multibody statistical potential and its comparative evaluation. Copyright © 2018 Elsevier Ltd. All rights reserved.

A pharmacokinetic evaluation of 14C-labeled afovirsen sodium in patients with genital warts.

PubMed

Crooke, S T; Grillone, L R; Tendolkar, A; Garrett, A; Fratkin, M J; Leeds, J; Barr, W H

1994-12-01

Afovirsen sodium is a 20-mer phosphorothioate oligonucleotide designed to be complementary to the messenger ribonucleic acid sequence for the translation initiation codon of the E2 protein vital to replication of human papillomaviruses types 6 and 11. 14C-Labeled afovirsen was given as a single-dose intradermal injection in each of four warts of five patients to determine the time-dependent changes in concentration of intact afovirsen in genital warts and to determine the systemic absorption and elimination of radiolabeled compound. Intact afovirsen in genital warts was determined by high pressure liquid chromatography analysis of protease K digested extracts. Intact afovirsen was present in wart tissue for at least 72 hours at concentrations several times in excess of the estimated minimal inhibitory concentration of 1 mumol/L. Absorption of radiolabeled afovirsen from the injection site was rapid, with a peak plasma concentration achieved within 1 hour. Clearance of afovirsen was primarily attributable to slow metabolism, with about 30% of the radiolabel eliminated as 14C-CO2 in expired air over a 6-day period after dosing. Radioactivity eliminated in urine represented metabolites of afovirsen. From the clinical pharmacokinetic data presented here and from previously published pharmacokinetic data in rats, the disposition of afovirsen in humans appears to be relatively similar to that in rats. These data suggest that once or twice weekly dosing regimen in the clinic may be appropriate.

Neuropeptide metabolism on intact, regional brain slices: effect of dopaminergic agents on substance P, cholecystokinin and Met-enkephalin degradation.

PubMed

Waters, S M; Konkoy, C S; Davis, T P

1995-08-01

Neuroleptic drugs have been shown to affect the level and messenger ribonucleic acid of specific neuropeptides. The effect of subchronically administered neuroleptics on neuropeptide metabolism, however, has not been systematically characterized. In the present study, the effect of neuroleptics and other dopaminergic compounds on substance P (SP), cholecystokinin and met-enkephalin degradation was determined on intact, regional, rat brain slices. After 7-day administration of haloperidol (1 mg/kg) or chlorpromazine (20 mg/kg), SP degradation was decreased in caudate-putamen and nucleus accumbens. After administration of the dopaminergic agonist apomorphine (5 mg/kg, b.i.d.), SP degradation was increased in the nucleus accumbens. The dopamine D2-receptor antagonist sulpiride (100 mg/kg, b.i.d.) produced no effect on SP degradation. Met-enkephalin degradation was decreased after haloperidol administration in both frontal cortex and caudate-putamen and unaffected by apomorphine administration. The metabolism of cholecystokinin was not affected by neuroleptic treatment. Studies performed with specific peptidase inhibitors suggested that neutral endopeptidase 24.11, metalloendopeptidase 24.15 and aminopeptidases degrade SP on caudate-putamen and nucleus accumbens slices. Therefore, alterations in these peptidases may be responsible for the change noted in SP degradation after dopaminergic compound administration. These metabolic changes noted after neuroleptic administration may therefore contribute to neuroleptic-induced alterations in regional peptide levels.

Isolation and characterization of porcine adipose tissue-derived adult stem cells.

PubMed

Williams, Kellie J; Picou, Alicia A; Kish, Sharon L; Giraldo, Angelica M; Godke, Robert A; Bondioli, Kenneth R

2008-01-01

Stem cell characteristics such as self-renewal, differentiation and expression of CD34 and CD44 stem cell markers have not been identified in porcine adipose tissue-derived adult stem (ADAS) cells. The objective of this study was to develop a protocol for the isolation and culture of porcine adipose tissue-derived cells and to determine stem cell-like characteristics. Primary cultures were established and cell cultures were maintained. Cloning capacity was determined using a ring cloning procedure. Primary cultures and clones were differentiated and stained for multiple differentiated phenotypes. CD34 and CD44 messenger ribonucleic acid (mRNA) was isolated and reverse transcriptase polymerase chain reaction was used to compare expression profiles. An average of 2,700,000 nucleated cells/ml was isolated; 26% were adherent, and cells completed a cell cycle approximately every 3.3 days. Ring cloning identified 19 colonies. Primary cultures and clones were determined to differentiate along osteogenic, adipogenic and chondrogenic tissue lineages. The mRNA expression profiles showed CD34 expression was higher for undifferentiated ADAS cells versus differentiated cell types and the CD34 expression level was lower than that of CD44 among differentiated cells. Improved culture conditions and defined cellular characteristics of these porcine ADAS cells have been identified. Porcine ADAS can self-renew, can differentiate into multiple tissue lineages and they express CD34. Copyright 2008 S. Karger AG, Basel.