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PubMed1979

Low molecular weight RNAs as components of nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA.

70-130 S polyparticles as well as 38 S monoparticles were isolated from rat liver nuclei and analyzed in respect to their RNA components by microgel polyacrylamide electrophoresis in formamide. In addition to the high molecular weight polydisperse hnRNA of polyparticles several low molecular weight RNAs (snRNA) were detected. There are at least six distinct snRNA species in polyparticles. Except for one species, which is missing, 38 S monoparticles showed a similar snRNA pattern. From densitometer tracings of microgels the snRNAs were estimated to represent about 11% of the total polyparticle RNA. The number of nucleotides for the various snRNAs were determined from a plot of relative electrophoretic mobility versus log number of nucleotides. The possibility that the snRNAs are degradation products of the hnRNA was excluded on the basis of the following findings. (1) The snRNA pattern was similar in mono- and polyparticles. (2) Whereas the hnRNA of polyparticles incubated at 37 degrees C was extensively degraded, the snRNA did not show a corresponding increase. (3) After a 30 min pulse with [3H]orotate the hn RNA was readily labeled; none of the snRNAs, however, incorporated radioactivity. The snRNAs were still found after treatment of polyparticles with 2 M NaCl excluding contamination by nucleoplasm.

Animals↗
PubMed1981

The effect of essential amino acid deprivation on poly(A) metabolism in Neurospora crassa.

Poly(A) messenger RNa was isolated from a methionine auxotroph of Neurospora crassa grown to exponential and early stationary phases, and from mycelia deprived of the essential amino aid. The polyadenylate regions were isolated from the mRNA preparations by enzymatic digestion and affinity chromatography, and their lengths determined by electrophoresis on 10% acrylamide/90% formamide gels. There was no significant difference in the lengths of the poly(A) regions between exponential and early stationary phase cultures. However, the poly(A) regions of mRNA isolated from cells undergoing amino acid deprivation were larger and more heterogeneous than the poly(A) regions isolated from cells grown in amino acid-supplemented medium. Whole cell extracts prepared from both amino acid-supplemented and amino acid-deprived cultures were assayed for their ability to synthesize and degrade poly(A). No significant difference between the two extracts was detected in either poly(A) polymerase or poly(A)-degrading activities. It was concluded that continuing translational activity in required for the shortening of poly(A) tracts in N. crassa.

Amino Acids↗
PubMed1981

Poly(A)- and oligo(A)-adjacent sequences in RNA from the large hnRNP complexes of rat liver.

The present report describes the isolation of hnRNP complexes with sedimentation coefficients greater than 80 S from rat liver nuclei without the use of ribonuclease inhibitors. The RNA moiety from these complexes is heterogeneous in size, with a mean sedimentation coefficient of 16 S when assayed with polyacrylamide-formamide gel electrophoresis. About 3% of this RNA binds to oligo(dT)-cellulose, the size of the bound fraction being somewhat smaller than the bulk of the hnRNP-RNA. This poly(A) RNA contains two adenylate tracts, one with about 30 and the second with about 200 adenylate residues. Reverse transcription of the poly(A)-containing RNA and hybridization of the cDNA with their respective templates shows two well-defined frequency populations, the first one separated from the second by almost 2.5 logs in the R0t curve. The same distribution was found, whether the hybrids were analysed with S1 nuclease or hydroxyapatite. Both separated frequency classes hybridize to total genomic DNA, the abundant one at C0t values typical for repetitive and unique sequences, the scarce one only at C0t values typical for unique sequences. The two populations are also able to hybridize with polysomal polyadenylated mRNA, the dilution of both frequencies being very similar in the cytoplasm.

Adenine Nucleotides↗
PubMed1976

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds.

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT greater than nitrofurantoin greater than 5-nitro-2-furamidoxime greater than 5-nitrofurfurylidene diacetate greater than 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with beta-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.

Animals↗
PubMed1977

Cytotoxicity and DNA damage to mammalian cells by nitrofurans.

Nitrofurazone, nitrofurantoin, furazolidone, furaltadone and N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) were toxic to cultured mouse L cells. The extent of toxocity and the rate of reduction of nitrofurazone increased markedly as the oxygen content of the incubation medium was lowered. The toxic effect of nitrofurans was decreased by addition of serum and was much greater in phosphate-buffered saline containing glucose (PSG) than in medium. Damage to L cell DNA by nitrofurans increased as the oxygen concentration decreased from 21% to 0%. The concentration of nitrofurazone and duration of exposure also determined the number of DNA single-strand breaks. It is suggested that toxicity and DNA damage may result from the actions of toxic intermediates in the metabolic reduction of nitrofurans.

Cell Survival↗
PubMed1983

The mode of action of cytotoxic and antitumor 1-nitroacridines. III. In vivo interstrand cross-linking of DNA of mammalian or bacterial cells by 1-nitroacridines.

To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin [Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine], its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system. Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx. 20 X 10(3) (B. subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs. The first cross-links were introduced in B. subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C. Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system. Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B. subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C. Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines.

Aminoacridines↗
PubMed1985

Alkali lability and rapid initiation of excision repair following photoaffinity damage by ethidium azide.

DNA damage and repair provoked by ethidium azide (EA) photoaffinity labeling in mouse leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on ice, but restoration of DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated viral DNA with EA failed to introduce DNA strand breaks. However, it was discovered that photoaffinity labeling of DNA with EA resulted in alkali labile sites shown by single strand breaks produced on alkaline sucrose sedimentation or by alkali exposure followed by sedimentation on neutral formamide gradients. These results suggest that the drug attachment sites should be identifiable by the location of such single strand breaks.

Affinity Labels↗
PubMed1987

Elevation of adenylate cyclase activity during leukemic cell differentiation.

Exposure of the various human myeloid leukemic cell lines (HL60 and RDFD) to various compounds results in marked differentiation of the cells. This differentiation is associated with a marked increase in both basal and NaF-stimulated adenylate cyclase (AC) activity. The increase in AC activity occurs regardless of the differentiation inducer one has utilized (retinoic acid (RA), dimethyl formamide (DMF), hypoxanthine (HPX) or actinomycin D (actD) and is correlated with this process, as a variant of the HL60 cell (HL60-Blast) that does not differentiate upon exposure to the various inducers does not demonstrate this increase in AC activity. In addition, the differentiation process is associated with a rapid increase in intracellular cAMP within hours of adding the inducer, followed by a gradual decrease.

Adenylyl Cyclases↗
PubMed1991

Low-cost, high-sensitivity laser-induced fluorescence detection for DNA sequencing by capillary gel electrophoresis.

A low cost, 0.75-mW helium neon laser, operating in the green region at 534.5 nm, is used to excite fluorescence from tetramethylrhodamine isothiocyanate-labelled DNA fragments that have been separated by capillary gel electrophoresis. The detection limit (3 sigma) for the dye is 500 ymol [1 yoctomole (1 ymol) = 10(-24) mol] or 300 analyte molecules in capillary zone electrophoresis; the detection limit for labeled primer separated by capillary gel electrophoresis is 2 zmol [1 zeptomole (1 zmol) = 10(-21) mol]. The Richardson-Tabor peak-height encoded sequencing technique is used to prepare DNA sequencing samples. In 6% T, 5% C acrylamide, 7 M urea gels, sequencing rates of 300 bases/hour are produced at an electric field strength of 200 V/cm; unfortunately, the data are plagued by compressions. These compressions are eliminated with addition of 20% formamide to the sequencing gel; the gel runs slowly and sequencing data are generated at a rate of about 70 bases/hour.

Base Sequence↗
PubMed1992

Reversed-phase high-performance liquid chromatography-thermospray mass spectrometry of radiation-induced decomposition products of thymine and thymidine.

High-performance liquid chromatography-thermospray mass spectrometry was applied to the analysis of various radiation-induced decomposition products of thymidine including N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide and the various diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine, 5-hydroxy-5,6-dihydrothymidine and 5,6-dihydrothymidine. This method combines high sensitivity and product resolution, rendering it particularly useful for monitoring the formation of radiation-induced base damage within DNA.

Chromatography, High Pressure Liquid↗
PubMed1994

Chromatographic studies of the enantiomeric composition of some therapeutic compositions applied in the treatment of liver and kidney diseases.

A gas-liquid chromatographic system with alpha-cyclodextrin in formamide medium (coated on Chromosorb) was used for the separation of enantiomers of alpha-pinene, beta-pinene, limonene and camphene in medicines applied in the therapy of liver and kidney diseases. The drugs under investigation were produced in Poland (Terpichol and Terpinex), in Germany (Rowachol and Rowatinex) and in Slovenia (Uroterp). It was found that, depending on the manufacturer, medicines possessing similar chemical compositions differ considerably from one another regarding the content of enantiomers, mainly those of alpha-pinene.

Bicyclic Monoterpenes↗
PubMed1988

Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification.

Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 X 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45,000 X g, 4 degrees C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4 degrees C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70,000 molecule which upon reduction electrophoresed with an apparent Mr of 85,000-90,000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.

Avidin↗
PubMed1984

Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo.

U1 small nuclear RNA is thought to be involved in messenger RNA splicing by binding to complementary sequences in pre-mRNA. We have investigated intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa cells were pulse-labeled with [3H]uridine under conditions in which hnRNA is preferentially labeled. Isolated nuclei were treated with aminomethyltrioxsalen , which produces interstrand crosslinks at sites of base-pairing between hnRNA and U1 RNA. hnRNA-ribonucleoprotein (hnRNP) particles were isolated in sucrose gradients containing 50% formamide, to dissociate non-crosslinked U1 RNA, and then analyzed by immunoaffinity chromatography using a human autoantibody that is specific for the ribonucleoprotein form of U1 RNA (anti-U1 RNP). After psoralen crosslinking, pulse-labeled hnRNA in hnRNP particles reproducibly bound to anti-U1 RNP. The amount of hnRNA bound to anti-U1 RNP was reduced 80 to 85% when psoralen crosslinking of nuclei was omitted, or if the crosslinks between U1 RNA and hnRNA were photo-reversed prior to immunoaffinity chromatography. Analysis of the proteins bound to anti-U1 RNP after crosslink reversal revealed polypeptides having molecular weights similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human immunoglobulin G. These results indicate that the subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is a ribonucleoprotein. This raises the possibility that these proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.

Chromatography, Affinity↗
PubMed1984

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs.

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Identification by proton NMR of N-(hydroxymethyl)-N-methylformamide as the major urinary metabolite of N,N-dimethylformamide in mice.

Urine samples from mice which had received N,N-dimethylformamide were investigated by high field 1H-NMR spectroscopy. The most prominent signals in the N-CH3 region had chemical shifts identical with those of N,N-dimethylformamide (delta 2.85, 3.01) and N-(hydroxymethyl)-N-methylformamide (delta 2.91, 3.05). Resonances downfield of delta 7.5 (from formyl protons) also coincided with those of the reference formamides. When [14C]methyl-labelled N,N-dimethylformamide was injected and urine samples investigated by radio thin layer chromatography, the major area of radioactivity corresponded to the Rf of N-(hydroxymethyl)-N-methylformamide. Dimethylamine and methylamine were found to be minor metabolites of N,N-dimethylformamide.

Animals↗
PubMed1975

The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.

Benz(a)Anthracenes↗
PubMedDate not supplied

The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae.

MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Käfer, 1988). However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with gamma-valerolactone and propionitrile. In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M. An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and gamma-valerolactone in yeast strain D7.

Acetates↗
PubMed1994

Serotypes and putative virulence markers of Streptococcus suis isolates from cats and dogs.

Thirteen isolates of Streptococcus suis from cats and dogs were characterised. Formamide extracts of 10 of the 13 cultures reacted with group D specific antiserum, and serotyping of the cultures with specific antisera against capsular types 1 to 28 revealed the serotypes 9, 20, 1/2, 22, 26 and 4; the remaining cultures were untypable. In addition one of the cultures reacted with monoclonal antibodies against muramidase-released protein and in parallel with monoclonal antibodies against the extracellular factor (EF) of S suis. The latter reacted with proteins with a molecular weight of more than 150,000 indicating that the strain exhibited EF-related proteins. One of the 13 cultures haemagglutinated erythrocytes from human beings and various animal species and adhered in large numbers to HeLa cells.

Animals↗