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PubMed1976

Molecular weights, poly(A)--content, and partial separation of chick globin mRNAs.

Poly(A)-containing 9S RNA from chick reticulocytes was electrophoresed on formamide-polyacrylamide gels. The molecular weight was determined to be 211 000 +/- 10 000 daltons. The RNA was separated into three different fractions with respect to molecular weight. These RNAs were translated in a wheat germ cell-free system. The lower molecular weight RNA directed up to 95% alpha-chain synthesis, compared to 60% for the higher molecular weight RNA. This was accompanied by a relative increase for beta-chain synthesis with increasing molecular weight. It could also be shown by hybridization with labelled poly(U) that the average poly(A) length decreased from about 83 nucleotides for fraction I to 36 nucleotides for fraction III. Our results suggest that fractionation of avian 9 S globin mRNA by electrophoresis on formamide-polyacrylamide gels is dependent upon two parameters, namely differences in the lengths of the non-poly(A)-containing portion of the alpha and beta mRNAs and differences in the poly(A) lengths.

Animals↗

PubMed1988

In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localisation of mRNA in neuronal and endocrine tissue. An analysis of procedural variables.

Methodological variables for in situ hybridization using 32P labelled oligodeoxyribonucleotides (oligomers) have been examined. Four different oligomers directed against proglucagon messenger RNA (mRNA) and two different oligomers against prosomatostatin mRNA have been used. Specific hybridization was obtained in adult rat brain, stomach and pancreas and in neonatal rat ileum. Tissue was perfusion fixed with 4% paraformaldehyde 0.2% glutaraldehyde and hybridization was carried out in 50% formamide for 72 h at 42 degrees C. Using hybridization conditions of lower stringency (33% formamide) labelling was also obtained in guinea pig tissue. Other variables which affected hybridization signal intensity were the inclusion of a prehybridization dehydration stage, the probe concentration, the inclusion of ammonium acetate in the posthybridization dehydrating ethanols and in the autoradiographic emulsion, and the exposure time. The localisation of proglucagon mRNA in rat pancreas using a 20mer was used as a model tissue for testing these methodological variables and the results were found generally also to apply to the other probes and tissues tested. The methods described provide single cell resolution and show that 32P labelled oligomers may be used to localise neuropeptide and endocrine mRNAs in different types of tissue and in different mammalian species.

Animals↗

PubMed1994

Antagonism of cadmium cytotoxicity by differentiation inducers.

Studies on the antagonism of toxicity can provide information about toxic mechanisms and suggest chemotherapeutic strategies. A rapid cell growth assay that measures the effects of test agents on the accumulation of cell protein (Shopsis and Eng, Toxicol. Lett. 1985;26:1) has been applied to studies of the antagonism of the cytotoxicity of cadmium. Exposure of Balb/c mouse 3T3 cells to 15 mumol/L Cd2+ for 24 h or 7 mumol/L Cd2+ for 48 h caused a 50% decrease in total cell protein. Zn2+ and selenite ion, antagonists of Cd toxicity in vivo, antagonized Cd2+ cytotoxicity when added in micromolar concentrations at the initiation of exposure to Cd2+. A diverse group of chemicals that can induce differentiation in vitro in cultured erythroleukemia and other cells were also found to antagonize the cytotoxic effects of Cd2+ to 3T3 cells. Dimethyl sulfoxide (DMSO), hexamethylene bisacetamide, N,N-dimethyl formamide, N-methyl formamide, dimethyl acetamide, hypoxanthine, hemin, ouabain, and sodium butyrate, when added to cultures simultaneously with Cd2+, each antagonized Cd2+ toxicity. These agents were used at concentrations equal to or lower than the concentrations at which they induce cellular differentiation. Other cytotoxicity assays and morphological studies confirmed these observations. DMSO added as much as 6 h after the initiation of a 24-h exposure to Cd2+ still protected cells; conversely, pretreatment of cultures with butyrate or DMSO for 24 h followed by their removal did not confer protection against subsequent Cd2+ challenge. Ethanol and methanol (noninducers of differentiation) did not antagonize Cd2+ cytotoxicity, and differentiation-inducing agents did not protect the cells from Zn(2+)- or Hg(2+)-induced cytotoxicity. DMSO treatment does not induce an increase in the concentrations of metallothionein or glutathione in these cells.

3T3 Cells↗

PubMed2001

Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments.

Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide.

Base Sequence↗

PubMed2000

Characterisation of a cyanide hydratase gene in the phytopathogenic fungus Leptosphaeria maculans.

A gene encoding a cyanide hydratase was cloned from an aggressive isolate of Leptosphaeria maculans, the fungus which causes blackleg disease of oilseed Brassica spp. This enzyme catalyses the breakdown of hydrogen cyanide to a less toxic compound, formamide. The predicted amino acid sequence of cyanide hydratase in L. maculans is 77% and 82% identical to cyanide hydratases from two other ascomycetes, Gloeocercospora sorghi and Fusarium lateritium, respectively. The gene is present as a single copy in the L. maculans genome, in both aggressive and non-aggressive isolates, although there is a restriction fragment length polymorphism between these two isolate groups for this gene. The cyanide hydratase promoter contains four putative target sites for GATA transcription factors, proteins that regulate nitrogen metabolism and other processes. Transcription of cyanide hydratase in an aggressive L. maculans isolate is induced strongly by potassium cyanide. Transcription of the gene is detectable in cotyledons of Brassica juncea and B. napus during infection. L. maculans can utilise the reaction product, formamide, as a sole source of nitrogen.

Amino Acid Sequence↗

PubMed2006

Molecular forms of coumarin-307 in sol-gel glasses.

Coumarin-307(C-307) impregnated sol-gel glass samples were prepared by two methods - (I) predoping using HNO3 as catalyst, and (II) postdoping using HNO3 as catalyst and formamide as drying control chemical additive (DCCA). The absorption/fluorescence properties including fluorescence lifetime of the dye in these sol-gel host matrices were studied and results were compared with dye in methanol (MeOH). In addition to usual non-polar form of C-307 that exists in MeOH, amino-protonated form and dimer of dye have also been found to co-exist in the dried state of samples prepared by predoping method. This behaviour of dye was uncommon to sol and gel states of materials. The fluorescence quantum yield (Qf) of C-307 doped solid was found to be 0.77 and it was almost constant in the concentration range 5 x 10(16) to 8.8 x 10(17) cm(-3). On the other hand, only non-polar form of C-307 has been observed in the samples prepared by postdoping method. The Qf value of dye was 0.96 in the postdoped samples. To explain such behaviour of C-307 in the two types of sol-gel matrices: dye concentration, role of HNO3, water, formamide and matrix effect have been taken into account.

Journal Article↗

PubMed1988

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes.

We present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G + C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. Optimal conditions for washing were 0.5X standard sodium citrate (SSC) at 42 degrees C for 2 X 15 min. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11 degrees C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5X SSC and found to be 43, 58, and 60 degrees C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.

Base Sequence↗

PubMed1990

High-performance liquid chromatographic separation of hyaluronan and four proteoglycans produced by human bone cell cultures.

Four proteoglycans and hyaluronan synthesized by cultured human bone cells were isolated using a two-step high-performance liquid chromatography system involving desalting and buffer exchange with a TSK-GEL HW 40(S) column followed by ion-exchange separation on a Nucleogen 4000-10 DEAE column. The desalting of 4 M guanidinium HCl extracts by a TSK-GEL HW 40(S) column equilibrated in a formamide:KH2PO4 buffer produces greater than 95% recoveries, enables quantitation of label incorporation and requires only 40 min to complete. The Nucleogen 4000-10 DEAE column utilizes the same buffer system and requires only 100 min for the resolution of four distinct types of proteoglycans. The formamide:KH2PO4 buffer system is compatible with a previously developed polyacrylamide gel system for the electrophoretic profiling of proteoglycans. After separation by charge density, proteoglycans were further resolved by size distribution using a calibrated TSK-GEL HW 75(F) column which also enabled the estimation of the apparent Mr of hyaluronan produced by the bone cells. The same TSK-GEL HW 40(S) resin is used to exchange pooled proteoglycans into buffers for analyzing enzyme digests of glycosaminoglycan chains and core proteins. The technique has been applied to the analysis of biosynthetically labeled proteoglycans produced in culture by fetal and adult human bone cells. A distinct pattern of proteoglycan size and secretion for both cell types could be shown using this method. The method of analysis is useful for high yield and rapid screening of various cell types for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.

Age Factors↗

PubMed1977

Nuclear steady-state RNA from chicken immature red blood cells. Distribution of globin-coding and poly (A) sequences.

Nuclear steady-state RNA and polysomal RNA of chicken immature red blood cells were isolated and separated on formamide sucrose gradients. For comparison the distribution of 9 S globin mRNA was investigated by gradient centrifugation of 125I-labelled mRNA. The material was either pooled into two fractions (less than 20 S; greater than 20 S) and translated in an Ehrlich ascites cell-free system or each gradient fraction was analyzed by hybridization with [3H]-poly (U) or [3H]-labelled DNA complementary to purified 9 S globin mRNA (globin cDNA). In neither case could evidence be obtained for the existence of a high molecular weight RNA as a probable globin mRNA precursor. Further analysis was performed by electrophoresis of RNA on exponential polyacrylamide gels in formamide and subsequent hybridization with cDNA. The results are consistent with those of gradient centrifugation and demonstrate that the distribution of globin-coding sequences in nuclear steady state RNA corresponds to that of cytoplasmic 9 S globin mRNA.

Animals↗

PubMed1978

Composition and properties of messenger ribonucleoprotein fragments containing and lacking polyadenylate.

Ribonucleoprotein fragments were isolated from Ehrlich ascites cell messenger ribonucleoprotein (mRNP), immobilized on oligo(dT)-cellulose, by successive RNAase and formamide treatments and their RNA and protein moieties were characterized. A 8--10 S formamide-eluted (F) RNP primarily contained the poly(A) segment as an RNA moiety, as evidenced by its characteristic electrophoretic mobility, resistance to RNAase digestion, affinity for nitrocellulose filters and poly(U), and base composition. In contrast, the RNAase-eluted (R) fraction was composed of a 2--3 S RNP carrying a much shorter polyribonucleotide fragment of heterogeneous base composition and properties unlike poly(A). Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the protein moieties of the R fraction were composed primarily of 56 000, 67 000, and 71 000 molecular weight polypeptides whereas those of the F fraction contained the latter three polypeptides as well as a major species exhibiting a molecular weight of 81 000. The data demonstrate that RNA sequences other than poly(A) interact with protein in mRNP, that three major mRNP polypeptides are associated with both poly(A) and non-poly(A) RNA binding sites, and that one major polypeptide is specifically bound to the poly-(A) region of mRNP.

Animals↗

PubMed1976

Some properties of erythrocuprein treated by organic solvents.

The effect of various types of organic solvents on the properties of bovine erythrocuprein was studied. Three organic solvents were found in which the protein is soluble, these were: dimethyl sulfoxide, formamide and N-methylformamide. It was shown that in formamide and dimethyl sulfoxide media the protein possees superoxide dismutase activity, but in N-methylformamide the protein has negligible activity. In organic solvents the substrate (superoxide radical) and solvated electron result in reduction of the protein copper. At high concentrations of superoxide radical or solvated electrons an inactivation of protein and stabilization of superoxide radicals was noted. The stabilization is most pronounced in N-methylformamide. The protein that is reduced by the radical or the solvated electron may be reoxidized by molecular oxygen, the latter being reduced to the superoxide radical.

Animals↗

PubMed1990

Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecules which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for association with D. However, we find that the interaction between D and lysine in the presence of water is actually thermochemically repulsive, and that the presence of formamide does not affect the interaction between D and lysine, implying no useful complex formation between formamide and D. We were also unable to demonstrate that the blocking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seems that present concepts of cryoprotectant toxicity are in need of serious revision.

Amides↗

PubMed1983

Tests of spool models for DNA packaging in phage lambda.

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine. In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map. In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with 32P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found. These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on lambda DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982). A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into lambda phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophage lambda↗

PubMed1991

Detection of human papillomavirus in head and neck tumors with DNA hybridization and immunohistochemical analysis.

The presence of human papillomavirus (HPV) DNA in oral, sinus, pharynx, and larynx lesions of Japanese patients was studied by Southern blot hybridization under less stringent (25% formamide, 42 degrees C) and stringent (50% formamide, 42 degrees C) conditions. Three samples from 10 benign tumors, and 3 of 30 malignant tumors, contained HPV DNA or HPV-related sequences. The HPV DNAs harbored in three laryngeal papillomas were HPV-11, -6, and -6 or -11, respectively. The HPV DNA and viral capsid antigens were easily detected by in situ hybridization, Western blotting, and peroxidase-antiperoxidase staining. However, neither the typical restriction pattern of HPV DNA nor viral antigen was identified in the malignant tumors, suggesting that subgenomic fragments remained integrated in the host cell DNA.

Antigens, Viral↗

PubMed1991

Analysis of the gene encoding the outer capsid glycoprotein (VP7) of group C rotaviruses by northern and dot blot hybridization.

The genetic diversity of gene 8 (encoding the outer capsid glycoprotein VP7) among group (Gp) C rotaviruses was examined by Northern and dot blot hybridization. A cDNA clone of the porcine Gp C Cowden strain gene 8 was labeled with 32P by nick translation and used as a probe. The gene 8 probe hybridized with the corresponding gene of one human (88-196) and four porcine (Cowden, NB, WH, and Wi) strains of Gp C rotaviruses under both moderate (50% formamide, 5X SSC, and 42 degrees) and high (50% formamide, 5X SSC, and 52 degrees) stringency conditions. However, under high stringency conditions little or no hybridization was detected with the corresponding gene of one bovine (Shintoku) and three other porcine (Ah, HF, and KH) strains of Gp C rotaviruses. In control experiments, the Cowden gene 8 probe did not hybridize with Gp A (Gottfried strain) or Gp B (Ohio strain) rotaviruses. These data demonstrate that the Cowden gene 8 probe is Gp C rotavirus-specific and that genetic diversity exists among Gp C rotaviruses in the gene encoding the outer capsid glycoprotein VP7. Our gene 8 probe may be useful in hybridization assays for serotyping Gp C rotaviruses, analogous to the use of VP7 probes for serotyping Gp A rotaviruses. However, final confirmation of our genetic approach to serotype Gp C rotaviruses awaits the serologic analysis of these viruses.

Animals↗

PubMed1991

New findings in the metabolism of N,N-dimethylformamide--consequences for evaluation of occupational risk.

Using a novel gas chromatographic method, specific mercapturic acids produced in the biotransformation of several formamide analogues have been quantified. Thus, N-acetyl-S-(N-methylcarbamoyl)cysteine, derived from an important industrial solvent N,N-dimethylformamide, was found to be a minor metabolite in rodents but an important one in humans. Because manifestations of hepatotoxicity of formamide analogues were always linked with the production of mercapturic acids, the risk from exposure to DMF in humans appears to be higher than that estimated from toxicological experiments on laboratory animals.

Air Pollutants, Occupational↗

PubMed1990

Influence of the oversulfation method and the degree of sulfation on the anticoagulant properties of dermatan sulfate derivatives.

Six oversulfated dermatan sulfate (DS) derivatives, differing in their tissue origin (porcine skin, bovine and porcine intestinal mucosa), and the oversulfation method of preparation, have been tested for their anticoagulant properties. In the first method, the SO3-trimethylamine complex is added to a DS sodium salt dissolved in formamide while it is added to a DS-benzethonium salt dissolved in dimethyl formamide in the second method. The rate of sulfation of these compounds ranged from 7.8 to 11.5 percent of sulfur on a weight basis, whereas it is 6% and 12% for natural DS and for heparin respectively. The anticoagulant potency was assessed by determining the catalytic effect of each glycosaminoglycan on the inhibition of thrombin added to (i) plasma (ii) purified heparin cofactor II(HC II) or (iii) purified antithrombin III(AT III). The catalytic effect on Factor Xa inhibition in the presence of AT III has also been investigated. The increased sulfation is found to enhance the antithrombin activity of the native dermatan sulfate whatever the method used, while the Factor Xa inhibition by AT III could be catalysed only by the most sulfated derivative obtained by the second method. The two derivatives which were less oversulfated, by the first oversulfation method, exhibit equal or even higher catalytic effects on thrombin inhibition when compared to the four other derivatives. The use of the first oversulfation method provides slightly oversulfated derivatives which exhibit strong anticoagulant properties and may constitute effective antithrombotic drugs with no bleeding tendency, a side effect perhaps related to a high rate of sulfation.

Animals↗

PubMed1981

O6-alkylguanine and structural modifications in liver DNA of dialkylnitrosamine-treated rats.

Isolated rat liver DNA was fractionated on benzoylated-naphthoylated-DEAE-cellulose (BND-cellulose) columns. In experiments with dimethyl-nitrosamine (DMN)-treated (1-30 mg/kg) rats killed 24 h after injection, the amount of DNA eluting in the formamide fraction (i.e., containing single-stranded or partially denatured regions) was increased in a dose-dependent way. The absence of an effect at 6 days after DMN indicated that above regions disappear much more rapidly from rat liver DNA than do those DMN-induced lesions that give rise to more persistent DNA fragmentation in alkaline sucrose gradients. This indicates that 2 different lesions are involved. When the extents of alkylation at the O6- and 7-position of guanine in the NaCl-eluted and formamide-eluted DNA were compared, no significant differences were observed. Our results indicate that, at least in the present system, neither the presence nor the repair of O6-alkylguanine is related to the formation of single-stranded regions in rat liver DNA.

Animals↗