Mercurated nucleic acid probes, a new principle for non-radioactive in situ hybridization

This report describes the localization of specific nucleic acid sequences in interphase nuclei and metaphase chromosomes by a new hybridocytochemical method based on the use of mercurated nucleic acid probes. After the hybridization a sulfhydryl-hapten compound is reacted with the hybrids formed. A number of such ligands were synthesized and tested. A fluorescyl ligand could be used for the direct visualization of highly repetitive sequences. For indirect immunocytochemical visualization trinitrophenyl ligands were found to be more sensitive than biotinyl analogues. These ligands were applied for the detection of target sequences in metaphase chromosomes and interphase nuclei of somatic cell hybrids, human lymphoid cell lines and blood cell cultures. The sequences were in the range of high to low copy numbers. The lower limit of sensitivity is indicated by the visualization of two human unique DNA fragments (40 and 15.6 kb) in human metaphases. The method is rapid, gives consistent results and can be used for both RNA and DNA probes. Other potentials of the new principle are discussed.     

Histochemical demonstration of creatine kinase activity using polyvinyl alcohol and auxiliary enzymes

Creatine kinase activity (EC 2.7.3.2.) has been demonstrated in myocardium and skeletal muscle from rats by a method based on the incubation of cryostat sections with a polyvinyl alcohol-containing medium and the use of auxiliary enzymes. Hexokinase and glucose-6-phosphate dehydrogenase were spread on object glasses before mounting the sections to be incubated. In this way, the auxiliary enzymes were interposed between glass slide and section thus preventing loss of formazan generated within the sections. Creatine kinase activity was found to be localized in finely dispersed form along the myofibrils and as large granules in the sarcoplasm of myocardium and skeletal muscle. The formazan produced specifically by creatine kinase (test minus control), as measured cytophotometrically at 585 nm, was completely inhibited by 2 mM 2,4-dinitrofluorobenzene, a specific inhibitor of creatine kinase activity. The control reaction was unaffected by the inhibitor. The results obtained with the present method are similar to results obtained with the far more complicated semipermeable membrane technique. The introduction of auxiliary enzymes in the polyvinyl alcohol method enables the development of histochemical methods for many enzymes by linking the reactions to a dehydrogenase reaction.     

Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction

Summary Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction.Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60–120mm. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section.p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (K _m ) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of thea form only in periportal areas and of thea andb forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4–16 min). A linear relationship was also found with section thickness (4–10 µm). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity.It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.     

Repair of N-methyl-N''-nitro-N-nitrosoguanidine-induced DNA damage by ABC excinuclease.

Escherichia coli has several overlapping DNA repair pathways which act in concert to eliminate the DNA damage caused by a diverse array of physical and chemical agents. The ABC excinuclease which is encoded by the uvrA, uvrB, and uvrC genes mediates both the incision and excision steps of nucleotide excision repair. Traditionally, this repair pathway has been assumed to be active against DNA adducts that cause major helical distortions. To determine the level of helical deformity required for recognition and repair by ABC excinuclease, we have evaluated the substrate specificity of this enzyme by using DNA damaged by N-methyl-N'-nitro-N-nitrosoguanidine. ABC excinuclease incised methylated DNA in vitro in a dose-dependent manner in a reaction that was ATP dependent and specific for the fully reconstituted enzyme. In vivo studies with various alkylation repair-deficient mutants indicated that the excinuclease participated in the repair of DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.     

Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Heparan sulfate proteoglycans of human lung fibroblasts. Structural heterogeneity of the core proteins of the hydrophobic cell-associated forms

Heparan sulfate proteoglycans (HSPG) were solubilized from human lung fibroblast monolayers with detergent. Presumptive membrane-associated forms displaying hydrophobic properties were purified by gel filtration on Sepharose CL-4B, by ion-exchange chromatography on Mono Q and by incorporation in lipid vesicles. The HSPG preparations were 125I-iodinated and treated with heparitinase before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five radiolabeled proteins with apparent molecular weights of 125,000, 90,000, 64,000, 48,000, and 35,000 were visualized by autoradiography. A sixth protein, identified in nonreduced 125I-HSPG preparations, appeared as a non-HS chain-bearing Mr 35,000 peptide which was disulfide-linked to an HS chain-bearing peptide of similar size. This multiplicity of core proteins did not seem to result from proteolysis during the heparitinase treatment itself, since some of the core proteins migrated independently during gel filtration before heparitinase digestion. Moreover, heparitinase digestion of 125I-HSPG purified by affinity chromatography on an immobilized monoclonal antibody yielded only the Mr 64,000 protein. Alternative depolymerizations of the HS chains by heparinase or HNO2 also yielded multiple protein bands. These results imply that heterogeneity of the core protein moiety may be a genuine property of the hydrophobic HSPG of human lung fibroblasts. The occurrence of multiple integral membrane HSPG forms may be relevant for the multiple functions that have been ascribed to cell-surface HSPG.     

Differential effects of temperature on cAMP-induced excitation, adaptation, and deadaptation of adenylate and guanylate cyclase in Dictyostelium discoideum

Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of approximately 1 s; excitation of adenylate cyclase is much slower with a lag time of 30 s. Excitation of both enzyme activities is less than twofold slower at 0 degrees C than at 20 degrees C. Adaptation of guanylate cyclase is very fast (t1/2 = 2.4 s at 20 degrees C), and virtually absent at 0 degrees C. Adaptation of adenylate cyclase is much slower (t1/2 = 110 s at 20 degrees C) but not very temperature sensitive (t1/2 = 290 s at 0 degrees C). At 20 degrees C, deadaptation of adenylate cyclase is about twofold slower than deadaptation of guanylate cyclase (t1/2 = 190 and 95 s, respectively). Deadaptation of adenylate cyclase is absent at 0 degrees C, while that of guanylate cyclase proceeds slowly (t1/2 = 975 s). The results show that excitation, adaptation, and deadaptation of guanylate cyclase have different kinetics and temperature sensitivities than those of adenylate cyclase, and therefore are probably independent processes.