Localization of a gene involved in complementation of the defect in xeroderma pigmentosum group A cells on human chromosome 1

Human, Chinese hamster or Chinese hamster/human hybrid cytoplasts were fused with UV-irradiated xeroderma pigmentosum group A (XP-A) cells. Unscheduled DNA synthesis (UDS) of the XP-A nucleus was measured 0-2 and 2-4 h after seeding of the fused population. Human cytoplasts did correct the defect in the XP-A nucleus immediately after fusion, whereas the chinese hamster cytoplasts did not show this rapid increase in excision repair. The results obtained after fusion of cytoplasts isolated from a panel of 26 Chinese hamster-human hybrids showed that chromosome 1 bears genetic information that is necessary for the rapid correction of the XP-A defect. Furthermore, this genetic information was regionally assigned to 1q42-qter by analysing hybrid cell lines having retained various segments of chromosome 1. Cytoplasts from a Chinese hamster/XP-A hybrid containing chromosome 1 of XP-A origin corrected also the defect with fast kinetics. This result indicate that the correcting factor consists of human and Chinese hamster components. As a consequence, the gene mapped on chromosome 1 may not be the gene which is mutated in XP-A cells.     

Ultrastructural localization of the circulating anodic antigen and the circulating cathodic antigen in the liver of mice infected with Schistosoma mansoni: a sequential study

The present study describes the ultrastructural localization of two important circulating schistosome antigens--the circulating anodic antigen (CAA) and the circulating cathodic antigen (CCA)--in livers of mice at various time intervals after infection with Schistosoma mansoni. For the demonstration of these antigens at the electron microscope level use was made of a direct, double immunogold labeling procedure, in which CAA-specific monoclonal antibodies, labeled with 5-nm gold particles, and CCA-specific monoclonal antibodies, labeled with 15-nm gold particles, were used. Both antigens were localized in granules and in inclusion bodies of Kupffer cells and granuloma macrophages and it was found that in these compartments the degree of 5- and 15-nm gold labeling increased with the duration of the infection. Sometimes gold particles were also encountered on the cell surface and in endocytotic vesicles of these cells, in endothelial cells, and in the space of Disse. From these data it was concluded that in the liver CAA and CCA were primarily accumulated in granules and inclusion bodies of Kupffer cells and granuloma macrophages. It is discussed whether at these locations both antigens are degraded by lysosomal enzymes and whether these antigens are complexed with antibodies.     

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.     

Effect of artemisinin (qinghaosu) and chloroquine on drug-sensitive and drug-resistant strains of Plasmodium falciparum malaria: use of [2,8-3H]adenosine as an alternative to [G-3H]hypoxanthine in the assessment of in vitro antimalarial activity

Using [G-3H]hypoxanthine uptake as a radioactive indicator for the growth of malarial parasites, we measured the antimalarial activity of artemisinin (Qinghaosu, QHS) against FCMSU1/Sudan strain (chloroquine-sensitive strain) and FCB K+ strain (chloroquine-resistant strain) of Plasmodium falciparum in continuous culture in vitro. The 50% inhibitory concentrations (IC50) for QHS against FCMSU1/Sudan strain and FCB K+ strain were 2.8 X 10(-8) and 3.0 X 10(-8) M, respectively. On the contrary, the response of the two strains to chloroquine was quite different. The IC50 for chloroquine against FCMSU1/Sudan strain was 5.6 ng/ml, whereas that for the FCB K+ strain was 65.6 ng/ml. Therefore, QHS did not appear to exhibit any cross-resistance with chloroquine. If [2,8-3H]adenosine was used as a radioactive precursor instead of [G-3H]hypoxanthine for the determination of antimalarial activity, virtually identical results were obtained. Therefore, [2,8-3H]adenosine can be used as an alternative to [G-3H]hypoxanthine for the assessment of antimalarial action.     

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.     

Characterization of dolichol kinase from bovine thyroid microsomes

Bovine thyroid microsomes are able to phosphorylate exogenous [1-3H]dolichol as well as endogenous dolichol. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [1-3H]dolichol to [1-3H]DMP as well as the formation of [32P]DMP from endogenous dolichol and [gamma-32P]CTP. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca2+- and Mn2+-ions. In accordance, EDTA was strongly inhibitory. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. The apparent Km-value for exogenous dolichol amounted to 4 microM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1-3H]dolichol depending on the source of CTP. With endogenous dolichol Km-values for CTP of 27.8 and 6.1 microM were calculated in respectively the absence and presence of 5 mM VO4(3-). Triton X-100 (0.15%) was necessary in the [1-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [gamma-32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). The levels of the enzymatic activity could be doubled by the inclusion of 18-21 mM NaF [( 1-3H]dolichol kinase) as phosphatase inhibitor: VO4(3-) had practically no effect. In contrast with [gamma-32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO4(3-) while F- resulted into no appreciable effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.