Isolation and characterization of the diastereoisomers of a series of phosphate-ethylated dinucleoside monophosphates.

Internucleotide phosphate esterification is a common reaction of many potent carcinogenic alkylating agents. It can give rise to two stereochemically distinct molecules about a triesterified phosphorus atom. The eight individual diastereoisomers derived from phosphate ethylation of d-ApT, d-CpT, d-GpT, and d-TpT were prepared from o-chlorophenyl phosphotriester intermediates and isolated by reverse-phase HPLC. Each pair of isomers, together with its parent analog, was examined by variable temperature circular dichroism. The results are interpreted in terms of secondary structure changes from which the absolute configurations of the ethylated phosphate groups can be inferred. These configurational assignments were confirmed by 31P NMR.

Synthesis and properties of oligodeoxyribonucleotides containing an ethylated internucleotide phosphate.

Internucleotide phosphotriesters comprise an important class of DNA lesions produced by carcinogenic alkylating agents. To avoid confusion resulting from the presence of other DNA lesions, synthetically prepared oligonucleotides containing ethylated internucleotide phosphates as the sole form of damage were employed to investigate several chemical and biochemical properties of DNA alkyl phosphotriesters. A total of four oligonucleotides were synthesised for this study, the dimers Tp(Et)T and pTp(Et)T and the decamer d-TpTpTp(Et)TpCpTpApTpTpT together with its unmodified analogue. The dimers were characterized by UV and phosphorus NMR spectroscopy and the decamers by two-dimensional homochromatography, alkali hydrolysis, and variable-temperature circular dichroism (CD). Alkali hydrolysis of the ethylated decamer produced strand breaks in approximately 75% of the molecules. This is in close agreement with data previously obtained for dinucleoside ethyl phosphotriesters and triesters in alkylated cellular DNA. Results from the CD study suggest that the ethyl substituent does not disrupt base stacking within the oligomer. The interactions of two enzymes with the alkylated oligonucleotides were examined. First, it was found that ethylation of the internucleotide phosphate renders TpT inactive as a substrate for T4 polynucleotide kinase, implying that a negative charge is required on the 3'-phosphate group of the nucleotide to be phosphorylated. Hence, postlabeling assays of DNA damage that depend upon enzymatic phosphorylation of modified 3'-nucleotides cannot be applied to dinucleoside alkyl phosphotriesters. Second, both decamers, when annealed to a single-stranded plasmid template, were able to prime DNA synthesis, catalyzed by Escherichia coli DNA polymerase I, with equal effectiveness. The use of this reaction as a means of site-specifically incorporating phosphotriesters into viral vectors is recognized.

The cytotoxicity of kaolin towards macrophages in vitro.

The inhalation of china clay dust by man can cause pneumoconiosis. In an attempt to identify the factors responsible the cytotoxicity in vitro of china clay dust towards mouse peritoneal macrophages was examined. Respirable dusts collected at china clay drying plants were cytotoxic towards the cells. This activity was caused by kaolinite (the major mineral in china clay) and was not due to the presence of ancillary minerals. The cytotoxicity of kaolinite was not due to particle morphology and the positively charged edges of the mineral contributed only slightly to cytotoxicity. An electron microscope study showed that macrophages phagocytosed PVPNO-coated kaolinite particles indicating that the low cytotoxicity of these particles was not due to poor phagocytosis. Residence of china clay in rat lungs appeared to reduce its cytotoxicity. It was concluded that the cytotoxicity of kaolinite was probably related to the proposed amorphous silica-rich gel coating on the particles. The relevance of the findings in vitro to the effects in vivo of china clay is discussed.

Colloidal gold markers and probes for routine application in microscopy.

Colloidal gold can be used as an electron-dense cytochemical probe for direct or indirect labelling techniques. Gold markers can be prepared in a size range of 5-150 nm, and show size-dependent shape characteristics and absorption spectra. Size and shape distribution increases with mean particle diameter, with appreciable overlapping between populations of different mean size. Using radio isotope-binding assays, spectrophotometric analysis and an innovative rapid microtitration technique, the effect of pH, ionic strength and protein concentration on gold-protein interaction has been studied. Efficient adsorption of protein to gold occurs at, or near, the pI of the protein. The amount of protein needed to effect stabilization is both a function of pH and of ionic strength, but does not reflect the amount of protein binding for all proteins. There is evidence for multilamellar adsorption of proteins to gold, which is discussed in context of the bioactivity, and stability of the probe. A working protocol for the routine reproducible manufacture of protein-gold probes is given, making use of the microtitration assay.

Cell surface distribution of fibronectin in cultures of fibroblasts and bladder derived epithelium: SEM-immunogold localization compared to immunoperoxidase and immunofluorescence..

Expression of cell surface fibronectin in cultures of untransformed fibroblasts is well documented, but little is known of its presence and distribution in cultured epithelial cells. Using species monospecific anti-fibronectin antibodies, the distribution of fibronectin in untransformed fibroblasts and in normal and neoplastic bladder epithelial cells was characterized by indirect labelling experiments using immunogold scanning electron microscopy (SEM). The surface matrix of fibronectin expressed in rodent and human fibroblast cell lines was demonstrated with ease by SEM of gold-tagged second antibodies. However, no fibronectin could be detected on any of the mouse and human bladder epithelium-derived cells studied in single or in mixed epithelial-fibroblast cultures. These SEM-immunogold observations were compared to and confirmed by immunofluorescence and immunoperoxidase microscopy. Immunofluorescence and SEM localization of the fibronectin in the extracellular matrix presented similar distribution patterns but the higher resolution of the SEM provided a more detailed analysis.

Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.

The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.

Mass spectral characterisation of the major DNA--carcinogen adduct formed from the metabolically activated carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one.

E. coli DNA, labelled with [14C]adenine and [14C]-guanine, was allowed to react with the [3H]-labelled carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one in the presence of a microsomal metabolising system. Enzymatic hydrolysis of the DNA followed by Sephadex LH20 chromatography of its constituent nucleosides established that the major DNA - carcinogen adduct involved guanine, and not adenine. This was confirmed by submitting calf thymus DNA, which had been allowed to react with the unlabelled carcinogen, to pyrolysis electron impact mass spectrometry without further derivatisation. Analysis of a selected ion product (m/z 368) by means of mass-analysed kinetic energy spectrometry, a technique which allows study of the further fragmentation of the single, selected ion, revealed that the guanine moiety was attached via the nitrogen atom of its exocyclic amino group to C-1 of a 1,2,3,4-tetrahydro-2,3,4-trihydroxy derivative of the original carcinogen.

A review of the colloidal gold marker system.

Colloidal gold can be used as a particulate marker for the detection and localization of target molecules by various modes of microscopy (light and fluorescent microscopy, scanning and transmission electron microscopy) using both direct and indirect labeling approaches. Several techniques are available for the preparation of gold markers in a size range of 5nm to 150nm, their mean size and shape characteristics and absorption spectra varying with particle size. Under appropriate conditions, colloidal gold will bind macromolecules by non-covalent electrostatic adsorption with little change in the specific activity of the bound macromolecule. This interaction is influenced by a number of factors including ionic concentration, pH conditions (in correlation with the protein pI values) and protein stabilizing levels. Presence of reactive protein on probes can be demonstrated and quantitated by direct and indirect radioactive binding assays and agglutination assays. These assays provide convenient procedures for characterizing stability, and behaviour in storage, of gold probes. Stability of gold probes under conditons where competing proteins are present, under freeze-thaw cycles and under SEM preparation conditions have been evaluated in this paper. Some of the basic procedures in the application of gold probes to cell labeling are briefly discussed together with certain limitations of the colloidal gold marker system. A bibliography of gold probe cell labeling studies is included.

Colloidal gold probes--a further evaluation.

Colloidal gold provides an immuno- and cytochemical marker suitable both for TEM and SEM. Various parameters involved in the preparation and stability of gold markers of various sizes and of probes have been reviewed and modifications in methodology suggested based on a study of factors affecting the performance of the gold marker system. In particular, dependence of protein adsorption on pH conditions correlating with protein pI is further confirmed. Presence of bioactive protein in gold probes is demonstrated and quantitated by radioassay (125I) and agglutination procedures.

The synthesis and enzymatic polymerization of 5-thio- and 5-methylmercurithio-pyrimidine nucleotides.

The 5-thio and 5-methylmercurithio derivatives of UTP, dUTP and dCTP have been synthesized and tested as substrates for nucleic acid polymerases. The 5-thio-nucleotides were polymerized inefficiently by both RNA polymerase and DNA polymerase I of Escherichia coli. The 5-methylmercurithio derivatives of dUTP and dCTP were, however, utilized by DNA polymerase I, an enzyme insensitive to mercurial compounds, although they were potent inhibitors of all other polymerases tested. While polymers containing the 5-thio substituent possess structural abnormalities, most likely interstrand disulfide bridges, polymers containing 5-methylmercurithio groups appear normal. The latter polynucleotides are readily separated from non-sulfated polymers by chromatography on mercuriagarose.

Conversion of covalently mercurated nucleic acids to tritiated and halogenated derivatives.

Mercurated nucleic acids are converted to the corresponding tritiated, brominated, and iodinated derivatives by treatment with sodium borotritiide, N-bromosuccinimide, and elemental iodine, respectively. All three reactions occur under mild conditions in neutral aqueous solutions. Mercury-halogen conversions are essentially quantitative at both the mono- and polynucleotide levels. Tritiation reactions also proceed efficiently with mononucleotides, although polymers undergo incomplete demercuration. In spite of the latter limitation , these reactions provide novel and efficient synthetic routes to radiolabeled nucleic acid derivatives.

Direct covalent mercuration of nucleotides and polynucleotides.

Nucleotides of cytosine and uracil are readily mercurated by heating at 37-50 degrees in buffered aqueous solutions (pH 5.0-8.0) containing mercuric acetate. Proton magnetic resonance, elemental, electrophoretic, and chromatographic analyses have shown the products to be 5-mercuricytosine and 5-mercuriuracil derivatives, where the mercury atom is covalently bonded. Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA while only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of polymer mercuration is, unlike that of mononucleotides, markedly influenced by the ionic strength of the reaction mixture: the lower the ionic strength the faster the reaction rate. Pyrimidine residues in single- and double-stranded polymers react at essentially the same rate. Although most polynucleotides can be extensively mercurated at pH 7.0 in sodium or Trisacetate buffers, tRNA undergoes only limited substitution in Tris buffers. The mild reaction conditions give minimal single-strand breakage and, unlike direct iodination procedures, do not produce pyrimidine hydrates. Mercurated polynucleotides can be exploited in a variety of ways, particularly by crystallographic and electron microscopic techniques, as tools for studying polynucleotide structure.

A histochemical study of acid phosphatase in normal and virus-transformed cultured fibroblasts.

The distribution of acid phosphatase has been investigated in normal and virus-transformed cultured hamster and mouse fibroblasts. The enzyme was found to be present in lysosomes, autophagic vacuoles and elements of the Golgi apparatus. It was also found to be associated with a surface coat in some virus-transformed mouse cells and in the cytoplasm of both normal and transformed hamster cells.

The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis.

A simple acetoxymercuration reaction for introducing covalently bound mercury atoms into nucleotides is described. The 5-mercuriacetate derivatives of UTP, CTP, dUTP, and dCTP, as well as the 7-mercuriacetate derivative of 7-deazaATP, have been prepared by this procedure and tested as substrates for nucleic acid polymerases. These nucleotides, in the absence of added mercaptan, are not polymerized and in most instances are potent enzyme inhibitors. However, conversion of these mercuriacetates to mercurithio compounds in situ by the addition of one of various mercaptans, yields nucleoside triphosphates that are excellent substrates for all polymerases tested: Escherichia coli and T7 RNA polymerases, DNA polymerase I of E. coli, DNA polymerase of avian myeloblastosis virus, and calf-thymus terminal deoxynucleotidyl transferase. By varying the mercaptan used to promote syntheses it is possible to access certain structural limitations in the enzyme's nucleoside triphosphate binding site. These mercurinucleotides appear to have a diversity of potential applications: (1) as heavy-atom reagents for crystallographic and microscopic studies; (2) as affinity probes for enzymes sensitive to sulfhydryl modification; (3) as steric probes of substrate-binding sites on enzymes; and (4) as reagents for forming covalent protein-polynucleotide complexes.