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At least 973 records · Page 54Linked to original sources
PubMed1981

Electron microscopic evidence of circular molecules in 9-S globin mRNA from rabbit reticulocytes.

9S globin mRNA prepared by the proteinase K method from polysomes of rabbit reticulocytes consists of 40% circular molecules as revealed by electron microscopy, if spreading of the molecules is performed from a solution of 50% formamide, 0.5 M NaCl, 25 mM Tris, 10 mM EDTA, pH 8, after 16 h incubation at 42 degrees C. We assume a noncovalent nature of the circularization because of the fact that a total transformation into the well known linear form occurs if strong denaturing conditions for spreading were used. The biological significance of the circular globin mRNA molecules is unknown.

Animals↗
PubMed1981

Identification of ceruloplasmin messenger RNA sequences in heterogeneous nuclear RNA from rat liver.

The distribution of the sequences of ceruloplasmin mRNA in different fractions of heterogeneous nuclear RNA from rat liver was studied using cDNA transcripts of highly purified mRNA as hybridization probe. The content of ceruloplasmin mRNA sequences in poly(A)-containing and poly(A)-free subfractions of heterogeneous nuclear RNA is respectively 1 and 27 molecules per a hepatocyte. Heterogeneous nuclear RNA carrying the sequences of ceruloplasmin mRNA sedimented in sucrose gradients containing formamide, as a broad zone around the 56S peak. Denaturing electrophoresis followed by the transfer of RNA onto diabenzyloxymethyl paper and hybridization with [32P]-cDNA revealed multiple high molecular weight fractions of ceruloplasmin pre = mRNA (9.0, 6.6, 2.2 and 1.6 megadaltons) in the non-adenylated fraction of nuclear RNA and a single 1.1-1.2 megadalton zone in poly(A)-containing nuclear RNA, the latter being equal in size to the mature ceruloplasmin mRNA from liver polysomes.

Animals↗
PubMed1981

Activation of the alternative pathway of complement in human serum by Propionibacterium acnes (Corynebacterium parvum) cell fractions.

Activation of the alternative pathway of complement is known to be initiated by bacterial structures. We have fractionated Propionibacterium acnes cells, purified various cell fractions, and tested their complement-activating ability in human serum chelated with ethyleneglycol bis-(beta-aminoethylether)-N,N1-tetraacetic acid. The majority of complement-activating activity was localized in the wall fraction. This activity was resistant to lipid extraction, protease, RNAse, DNAse and lysozyme treatment. NaIO4, formamide, and hot (but not cold) trichloroacetic acid (TCA) extraction ablated the complement-activating capacity of cell walls. Compounds removed by extraction failed to consume significant hemolytic activity against antibody-coated sheep erythrocytes (EA). Addition of TCA-extracted soluble material to cell wall suspensions resulted in an inhibition of hemolytic consumption by the cell wall. These results indicate that, in P. acnes, complement-activating molecules are located in the cell wall and are carbohydrate in nature. Peptidoglycan, lipid, protein, and nucleic acid do not appear to contribute to the cell wall's ability to activate complement.

Carbohydrates↗
PubMed1985

Improved in situ hybridization and G-banding by pretreatment with Denhardt's solution and gelatin-chrome alum.

Various pretreatments of metaphase spreads were examined to obtain optimal DNA labelling patterns while maintaining chromosome integrity during in situ hybridization procedures. Preparations of African green monkey (AGM) chromosomes fixed in methanol-acetic acid (CV-1 cell line) were treated by coating with Denhardt's solution, dilute gelatin-chrome alum, nonfat instant dry milk dissolved in saline-citrate solution (SSC) and/or acetylation prior to denaturation of chromosomal DNA in 70% formamide-2 X SSC for 2 min at 70 degrees C. A 3H-labelled, cloned DNA fragment of the highly repetitive AGM component alpha DNA was hybridized to the chromosomes by incubation at 45 degrees C for 16 h. Treatment with gelatin-chrome alum prior to denaturation greatly improved chromosome morphology and decreased background, but reduced pericentromeric labelling. Sequential treatment with 5 X Denhardt's solution followed by gelatin-chrome alum resulted in enhanced specificity of labelling and excellent chromosome morphology, as well as reduced levels of background. Acetylation had little effect after pretreatment with gelatin-chrome alum, but reduced background levels after pretreatment with Denhardt's solution. Chromosomes treated with Denhardt's solution plus gelatin-chrome alum can be routinely G-banded using trypsin after in situ hybridization.

Animals↗
PubMed1981

Propidium iodide as a probe for the study of chromatin thermal denaturation in situ.

The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturation in situ has been examined. Smears of lymphocytes and hepatocyte nuclei from 15-day-old rats were fixed in acetic acid--ethanol (1:3 v/v), treated with RNAse and submitted to different protein extraction procedures, namely, incubation with pepsin, trypsin and sodium chloride. Denaturation experiments were performed in Sörensen buffer at pH 7.4 containing 10% formamide at temperatures between 27 and 95 degrees C. The samples were stained with propidium iodide and mounted in buffer or glycerol. Measurements were performed with a microfluorometer at a wavelength of 446 nm. The results indicate a higher thermostability of lymphocytes as compared to hepatocytes. The denaturation pattern suggests a certain organization complexity of chromatin, better emphasized by the derivative curves which show the presence of at least three fractions with different melting points. After protein extraction, the denaturation curves exhibit a somewhat simplified pattern, with the disappearance of the most stable peak in the derivative curves. The samples mounted in glycerine exhibit a better stability of staining with time, and an increased quantum efficiency of the fluorochrome with regard to those mounted in buffer. These data confirm the importance of protein--DNA interactions in the organization of chromatin and point to some differences, depending on the cell type and on functional activity.

Animals↗
PubMed1975

Histochemical conditions influencing metachromatic staining. A comparative study by means of a model system of polyacrylamide films.

The influence of different histochemical conditions on some metachromatic staining reactions has been studied using polyacrylamide films containing pure glycostaminoglycans. The films were incubated in fixatives without staining, and in glycerol, diethylene glycol and other glycols, formamide, N,N-dimethylformamide, dimethyl sulphoxide and ethanol (of several concentrations) after staining and their absorption (metachromatic) spectra recorded. In the case of heparin and heparan sulphate the metachromasy was disturbed when the films were immersed before staining in some fixative solutions containing formaldehyde and acid. After equilibration of stained films in organic solvents, changes in the absorption peaks were found to depend on the type and concentration of solvent, the type of glycosaminoglycan and the type of dye. Films containing glycosaminoglycan plus protein were used to investigate the blocking of the metachromatic reaction as the result of ionic interactions with proteins. The parameters that influence this phenomenon (e.g type of protein, glycosaminoglycan and dye, pH of staining) are discussed and a three-dimensional picture is introduced which can explain some of the results obtained in these experiments.

Acrylamides↗
PubMed1986

Solvation properties of ubiquinone-10 in solvents of different polarity.

The solvation properties of ubiquinone-10 and ubiquinol-10 in a wide variety of solvents of polarity varying from alkanes to water are reported. Greatest solubility is observed in solvents of intermediate polarity and particularly where low polarity is combined with a pronounced tendency to interact with the benzoquinone substituent of the ubiquinone molecule. This includes solvents like chloroform and benzene. Ubiquinone-10 is somewhat less polar than ubiquinol-10 as judged by comparative solubilities of the two molecules. Proton-NMR chemical shift measurements and aggregation studies in selected solvents indicate that in ubiquinone-10 in the liquid phase and in solution in hydrocarbons like dodecane the molecules have a preferred association possibly involving stacking of the benzoquinone rings. Surface balance studies indicated that the surface-active character of ubiquinone-10 is relatively weak and only in a comparatively polar and highly structured solvent, formamide, was there evidence of an effect on surface tension of the solvent. The critical micelle concentration in this solvent was estimated to be about 5 microM on the basis of surface tension measurements. Ubiquinone-10 is well known to form virtually insoluble monolayers at the air/water interface. Studies of the partition of ubiquinone-10 in binary mixtures of solvents suggest that the interaction of the benzoquinone ring substituent with structured polar solvents is considerably weaker than the internal cohesion between molecules of the solvent. No evidence on the basis of wide-angle X-ray diffraction measurements was obtained to indicate that solvent molecules were a component of the crystal lattice of ubiquinone-10 that had precipitated from solvent mixtures.

Alkanes↗
PubMed1987

On the specificity of antisense RNA to arrest in vitro translation of mRNA coding for Drosophila hsp 23.

The specificity of action of antisense RNA for one of Drosophila low molecular weight heat shock proteins (hsp 23) was tested at the translational level using the rabbit reticulocyte lysate cell-free system. T7 polymerase-driven transcripts of hsp 23 in the antisense orientation were mixed with mRNA from heat-shocked cells under various stringency conditions prior to translation in vitro. Although the four small hsps show considerable sequence homology in their coding sequences, antisense hsp 23 RNA was shown to specifically inhibit hsp 23 mRNA translation under both high (formamide, 45 degrees C) and low stringency (37 degrees C) conditions. This suggests that the 5' leader and the ribosome binding region of mRNA are of prime importance in the specificity of action of antisense RNA at the translational level.

Animals↗
PubMed1975

Asymmetric adsorption by quartz: a model for the prebiotic origin of optical activity.

One mechanism previously proposed for the abiotic accumulation of molecules of one chirality in nature is asymmetric adsorption on the chiral surfaces of optically active quartz crystals. Earlier literature in this field is reviewed, with the conclusion that previous investigations of this phenomenon, using optical rotation criteria, have afforded ambiguous results. We now have studied the adsorption of radioactive D- and L-alanine on powdered d- and l-quartz, using change in radioactivity level as a criterion for both gross and differential adsorption. d-Quartz preferentially adsorbed D-alanine from anhydrous dimethyl-formamide solution, and l-quartz L-alanine. The differential adsorption varied between 1.0 and 1.8%. The implications of these observations are discussed from the viewpoint of early chemical evolution and the origin of optically active organic compounds in nature.

Alanine↗
PubMed1978

Size and secondary structure of avian myeloblastosis virus associated ribosomal RNA: comparison with cellular and precursor ribosomal RNA.

Ribosomal RNA isolated from ribosomes present inside avian myeloblastosis virus (AMV) was characterized by electron microscopy using the formamide-urea spreading technique. The molecular weight and the secondary structures were compared with those of r-RNA and precursor r-NA isolated from host cells, the leukemic myeloblasts. The molecular weight of viral r-RNA (1.62 +/- 0.18 X 10(6) and 0.69 +/- 0.10 X 10(6)) and the molecular weight of cellular r-RNA (1.63 +/- 0.18 X 10(6) and 0.67 +/- 0.09 X 10(6)), the latter obtained from avian myeloblasts, were found to be identical and comparable with the molecular weight of chicken liver r-RNA. Likewise, the secondary structures of viral r-RNA were identical to those of cellular r-RNA. The postulated possible precursor character of viral r-RNA was excluded, since the molecules of viral r-RNA do not show any similarity to those of precursor r-RNA. Previously observed differences in behavior of viral and cellular (myeloblastic) r-RNA in sedimentation and electrophoretic mobility are discussed.

Animals↗
PubMed1994

Chemiluminescent detection of Fiji disease virus with biotinylated DNA probes.

Biotinylated Fiji disease fijivirus specific cDNA probes detected the presence of the virus in total nucleic acid extracts from infected sugarcane plants. Hybridised biotinylated probes were detected with streptavidin-alkaline phosphatase conjugate and the light generating substrate AMPPD. Samples were either blotted manually, or by alkaline capillary transfer using 100 mM NaOH. Transfer of nucleic acids to charge modified nylon with sodium hydroxide was superior to denaturation with glyoxal or formamide and salt-citrate buffer transfer as the bands were clearly resolved and no degradation of the FDV dsRNA was observed. Transfer of either total nucleic acid extracts or purified dsRNA in manifold blots generated false positive signals with the non-radio-active chemiluminescent detection systems tested. Manual or northern blots had a limit of detection for purified target double-stranded RNA of approximately 10 pg and 0.5 pg respectively. Manual blots were tested for practical application to screen germplasma for FDV infection. The virus was detected in leaf samples from FDV-infected plants, in some instances prior to development of the characteristic gall symptom.

Biotin↗
PubMed1989

A theoretical study of Zn++ interacting with models of ligands present at the thermolysin active site.

The binding energy and the geometrical arrangements of the complexes formed by the zinc dication with OH-, one, four, five or six water molecules, SH-, H2S, formic acid, the formate anion, imidazole, its anion and formamide are calculated using the MNDO method. The comparison of the results obtained with those of ab initio computations on the same complexes induced us to propose for Zn++ a set of parameters different from the one determined by Dewar for the neutral metal atom. Using the two MNDO parametrizations, similar calculations are carried out for Zn++ interacting with two molecules of 2-aminoethanethiol and with models of the four ligands which are present at the thermolysin active site, in order to evaluate the possibilities and limitations of this semiempirical method for theoretical studies concerning zinc metalloenzymes. In the last case, the results obtained suggest that, in the crystal state, the water molecule could be deprotonated. This finding is discussed in relation with the mechanism of action of the enzyme which has been proposed.

Binding Sites↗
PubMed1990

Human papillomavirus type 11DNA in papillary squamous cell lung carcinoma.

We report a case of papillary squamous cell carcinoma of the lung developing in relation to a condylomatous papilloma and related to human papillomavirus (HPV) infection. The viral origin of the bronchial papillomatous lesion is strongly suggested by cytological and histological features with marked condylomatous changes. No viral capsid antigen was detected by immunohistochemistry. Transmission electron microscopy failed to reveal intranuclear viral-like particles in the papillary part of the carcinoma, but typical ultrastructural koilocytotic cells with irregular nucleus and coarse chromatin were observed. HPV DNA type 11 was detected by in situ hybridization using biotinylated probes on paraffin-embedded specimens, under stringent conditions (Tm-12 degrees, 50% formamide). Papillary squamous cell carcinoma may result from the malignant conversion of benign squamous papilloma of the bronchus. HPV type 11 may be associated with malignant conversion of benign papilloma of the pulmonary tract, as in the upper respiratory tract. In situ hybridization with biotinylated probes is a relatively simple and appropriate method for retrospective analysis of HPV DNA sequences in surgical specimens.

Bronchial Neoplasms↗
PubMed1986

Permeability of small nonelectrolytes through lipid bilayer membranes.

Diffusion of small nonelectrolytes through planar lipid bilayer membranes (egg phosphatidylcholine-decane) was examined by correlating the permeability coefficients of 22 solutes with their partition coefficients between water and four organic solvents. High correlations were observed with hexadecane and olive oil (r = 0.95 and 0.93), but not octanol and ether (r = 0.75 and 0.74). Permeabilities of the seven smallest molecules (mol wt less than 50) (water, hydrofluoric acid, hydrochloric acid, ammonia, methylamine, formic acid and formamide) were 2- to 15-fold higher than the values predicted by the permeabilities of the larger molecules (50 less than mol wt less than 300). The "extra" permeabilities of the seven smallest molecules were not correlated with partition coefficients but were inversely correlated with molecular volumes. The larger solute permeabilities also decreased with increasing molecular volume, but the relationship was neither steep nor significant. The permeability pattern cannot be explained by the molecular volume dependence of partitioning into the bilayer or by the existence of transient aqueous pores. The molecular volume dependence of solute permeability suggests that the membrane barrier behaves more like a polymer than a liquid hydrocarbon. All the data are consistent with the "solubility-diffusion" model, which can explain both the hydrophobicity dependence and the molecular volume dependence of nonelectrolyte permeability.

Carbon Radioisotopes↗
PubMed1981

Nitroblue-tetrazolium test for the functional evaluation of phagocytic cells: a critical analysis of the methodology.

The reduction of NBT to formazan has been suggested as an indicator of the reduction potential of biological systems. An increase in the amount of reduced formazan reflects the activation of the hexose monophosphate shunt of phagocytes cultivated in vitro, as a result of cellular stimulation by chemical or biological factors, or during phagocytosis. This phenomenon has been widely used for the determination of activated phagocytes by different methods. However, the technical limitations of these methods have not been evaluated carefully. In the investigations presented here three solvents for formazan, pyridine, dioxane and dimethyl-formamide, have been tested for their suitability as extraction agents. For each solvent the optimal wavelength for photometric evaluation has been determined and dose relation curves between dissolved formazan and OD have been established. Several factors (time, temperature, pH, contamination with water or acid) affecting the dissolving properties and stability of formazan in different solvents have been investigated. With the solvents tested, dioxane proved to be the most suitable agent for extracting NBF. Thus a methodology for the quantitative evaluation of NBT has been established. This method can be used for the identification of activators as well as of inhibitors of the phagocyte system.

Chemical Phenomena↗
PubMed1995

Adrenoceptor-mediated modulation of Evans blue dye permeation of rat small intestine.

In the present study, we addressed the problem whether sympathoadrenal mechanisms could influence the paracellular permeation of macromolecules from the lumen to the lamina propria of the small intestine. Experiments were conducted with rats that were anesthetized with ether for 10-20 min, during which time laparotomy was performed and six consecutive loops (each of 5 cm length) of the jejunum were prepared. A 3% solution of the azo dye, Evans blue (EB; MW 960.83) in phosphate-buffered saline, was instilled into each loop at a volume of 0.3 ml, this compound serving as a marker for tight junctional permeability. Thereafter, the abdomen was closed and the rats were allowed to wake up, but were killed after 60 min. The loops were dissected, opened, and rinsed with acetylcysteine in order to remove the adherent mucus layer. Each loop was weighed and incubated with formamide for 24 hr to elute the amount of EB absorbed, which was quantitated spectrophotometrically. In the control situation, the uptake was homogenous along the loops. beta-Adrenoceptor-blocking, or -stimulating agents could influence the uptake considerably. The data obtained could indicate that noradrenergic nerves, via an activation of beta 2-adrenoceptors, may cause an increase of tight junction permeability for macromolecules, but circulatory mechanisms also must be taken into account in order to explain the observed effects.

Animals↗
PubMed1990

Triple combination of retinoic acid plus actinomycin D plus dimethylformamide induces differentiation of human acute myeloid leukaemic blasts in primary culture.

Differentiation induction therapy provides an alternative for treatment of acute myeloid leukaemia (AML) patients who are either unsuitable for or unresponsive to conventional cytotoxic chemotherapy. The effect of a triple combination of retinoic acid (RA) + actinomycin D (Act-D) + dimethylformamide (DMF) on differentiation of blasts from 24 AML patients was studied. Non-adherent mononuclear cells were seeded at a concentration of 5 x 10(5) cells/ml in 24-well tissue-culture plates containing RPMI 1640 culture medium with 20% fetal calf serum, 10% autologous serum and 10% 5637-conditioned medium and incubated with 10(-6) M retinoic acid, 5 nM actinomycin D and/or 100 mM dimethylformamide alone and in combination with each other for 6 days at 37 degrees C in a humidified incubator and an atmosphere containing 5% CO2. The triple combination of 10(-6) M retinoic acid + 5 nM actinomycin D + 100 mM dimethylformamide induced 90% of the blasts from 22 of the 24 AML patients to differentiate. The combination of N-methylformamide (a compound similar to dimethylformamide) with cyclophosphamide significantly increased the in vivo activity with no concomitant increase in its reversible hepatotoxicity. Since several polar compounds related to dimethyl-formamide, e.g. hexamethylene bisacetamide and N-methylformamide, are currently undergoing phase II clinical trials, it may be feasible to combine one of these with retinoic acid and/or actinomycin D in the treatment of AML patients.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Sensitized photooxygenation of the fungicide furalaxyl.

BACKGROUND: The photolysis of pesticides is of high current interest since light is one of the most important abiotic factors which are responsible for the environmental fate of these substances and may induce their conversion into noxious products. The action of light can also be mediated by oxygen and synthetic or naturally occurring substances which act as sensitizers. Our objective in this study was to investigate the photochemical behaviour of the systemic fungicide furalaxyl in the presence of oxygen and various sensitizers, and to compare the toxicity of the main photoproduct(s) to that of the parent compound. Previous reports on the direct photolysis of the pesticide demonstrated a very slow degradation and the only identified photoproducts were N-2,6-xylyl-D,L-alaninare and 2,6-dimethylaniline. METHODS: Solutions of furalaxyl in CH3CN were photooxygenate using a 500W high-pressure mercury lamp (through a Pyrex glass filter, lambda>300 nm) or a 650W halogen lamp or sunlight and the proper sensitizer. When sunlight was used, aqueous solutions were employed. The photodegradation was checked by NMR and/or GC-MS. The photoproducts were spectroscopically evidenced and, when possible, isolated chromatographically. Acute toxicity tests were performed on the rotifer Brachionus calyciflorus, the crustacean cladoceran Daphnia magna and the anostracan Thamnocephalus platyurus, while chronic toxicity tests (sublethal endpoints) comprised a producer, the alga Pseudokirchneriella subcapitata and the crustacean Ceriodaphnia dubia, as a consumer. RESULTS AND DISCUSSION: In the presence of both oxygen and sensitizer, furalaxyl underwent rapid photochemical transformations mainly to N-disubstituted formamide, maleic anhydride and a 2(5H)-furanone derivative. The formation of these products was rationalized in terms of a furan endoperoxide intermediate derived from the reaction of furalaxyl with active dioxygenated species (singlet oxygen, superoxide anion or ground state oxygen). The 2(5H)-furanone exhibited a higher toxicity than the parent compound. CONCLUSION: This work reports the first data on the photosensitized oxygenation of furalaxyl with evidence of the high tendency of the pesticide to undergo photodegradation under these conditions leading, among other things, to a 2(5H)-furanone, which is more toxic than the starting furalaxyl towards aquatic organisms. RECOMMENDATIONS AND OUTLOOK: Investigation highlights that the photolytic fate of a pesticide, although quite stable to direct photoreaction due to its low absorption of solar radiation at ground level, can be significantly influenced in the environment by the presence of substances with energy or electron-transfer properties as natural dyes, e.g. chlorophyll, or synthetic pollutants, e.g. polycyclic aromatic hydrocarbons (PAH).

Animals↗