Inhibition of mammalian deoxyribonucleic acid synthesis by neocarzinostatin: selective effect on replicon initiation in CHO cells and resistant synthesis in ataxia telangiectasia fibroblasts

Treatment of CHO cells with low doses of the protein antibiotic neocarzinostatin severely inhibited DNA replicon initiation but had no effect on chain elongation. The selectivity of the effect on initiation, which was greater than that seen with other chemical agents and comparable to that seen with X-rays, explains the biphasic dose response seen for DNA synthesis inhibition by this drug. Parallel experiments employing the nucleoid sedimentation technique indicated that half-maximal relaxation of domains of DNA supercoiling and half-maximal inhibition of replicon initiation required the same dose of neocarzinostatin, approximately 0.03 micrograms/mL. These results, similar results obtained with the protein antibiotic auromomycin, and previous results obtained with X-rays suggest a quantitative correlation between inhibition of replicon initiation and induction of sufficient strand breakage to relax domains of supercoiling in DNA of mammalian cells. Results in human ataxia telangiectasia fibroblasts indicated that neocarzinostatin, like X-rays, is much less effective in inhibiting DNA synthesis in these cells than in normal human fibroblasts. This finding is consistent with the hypothesis that the genetic defect in ataxia telangiectasia involves a failure to recognize the presence of strand breaks in cellular DNA.     

Properties of recombinant cells capable of growing on serine without NhaB Na+/H+ antiporter in Escherichia coli.

Escherichia coli HIT-1 has a mutation in the Na+/H+ antiporter gene, nhaB (P. Thelen, T. Tsuchiya, and E. B. Goldberg, J. Bacteriol. 173:6553-6557, 1991). This strain is not able to utilize serine as a carbon source (T. Ishikawa, H. Hama, M. Tsuda, and T. Tsuchiya, J. Biol. Chem. 262:7443-7446, 1987), because an active NhaB is required to maintain the electrochemical potential of Na+, which drives serine transport via the Na+/serine carrier, the major transport system for serine. We isolated recombinant cells from a cross between strains HIT-1 and Hfr, and these cells were able to grow on serine even though the NhaB Na+/H+ antiporter of the recombinant cells was still defective. We found that the activity of the H+/serine cotransport system, one of the minor serine transport systems in E. coli, was elevated in the recombinant cells. H+/serine cotransport activity was induced by leucine in the recombinant cells more strongly than in strain HIT-1. A kinetic analysis showed that the Vmax, but not the Km, of the transport system was much higher in the recombinant cells than in strain HIT-1 cells.     

Quantification and excretion profiles of pteridines in primate urine.

Biopterin, 6-hydroxymethyl-pterin, isoxanthopterin, neopterin and, pterin were quantified in stress-free collected spontaneous morning urine samples from Callithrix jacchus, Saguinus fuscicollis, Saguinus labiatus, Saimiri sciureus, Presbytis entellus, Cercopithecus albogularis, Cercocebus torquatus, Macaca fascicularis, Hylobates concolor, Pongo pygmaeus, and Gorilla gorilla. In most species, biopterin was the most frequent urinary pteridine followed by neopterin. Sex differences in biopterin and neopterin excretion were observed in Gorilla gorilla and Pongo pygmaeus. Pterin and isoxanthopterin were only present in minor concentrations. 6-hydroxymethyl-pterin was barely detectable and not present in the urine of Saguinus labiatus, Saimiri sciureus, and both male Gorilla gorilla and Pongo pygmaeus.     

Chromosomal deletion 4p15.32----p14 in a Treacher Collins syndrome patient: exclusion of the disease locus from and mapping of anonymous DNA sequences to this region.

Treacher Collins syndrome is an autosomal dominant condition of bilateral craniofacial abnormalities of structures derived from the first and second branchial arches. A patient with severe manifestations of Treacher Collins syndrome and a de novo chromosomal deletion in region 4p15.32----p14 was identified. Anonymous DNA sequences of loci D4S18, D4S19, D4S20, D4S22, and D4S23 were mapped to the deleted region. DNA probes previously mapped to loci on chromosome 4p (D4S10, D4S15, D4S16, D4S26, D4S35, D4S95, D4S144, RAF1P1, QDPR, and HOX7) were not deleted in this patient. Linkage analysis between the D4S18, D4S23, and QDPR loci and Treacher Collins syndrome in eight families excluded the Treacher Collins syndrome locus from the region of the deletion.     

Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers.

Polycationic polymers have been noted for their effects in promoting cell adhesion to various surfaces, but previous studies have failed to describe a mechanism dealing with this type of adhesion. In the present study, three polycationic polymers (chitosan, poly-L-lysine, and lysozyme) were tested for their effects on microbial hydrophobicity, as determined by adhesion to hydrocarbon and polystyrene. Test strains (Escherichia coli, Candida albicans, and a nonhydrophobic mutant, MR-481, derived from Acinetobacter calcoaceticus RAG-1) were vortexed with hexadecane in the presence of the various polycations, and the extent of adhesion was measured turbidimetrically. Adhesion of all three test strains rose from near zero values to over 90% in the presence of low concentrations of chitosan (125 to 250 micrograms/ml). Adhesion occurred by adsorption of chitosan directly to the cell surface, since E. coli cells preincubated in the presence of the polymer were highly adherent, whereas hexadecane droplets pretreated with chitosan were subsequently unable to bind untreated cells. Inorganic cations (Na+, Mg2+) inhibited the chitosan-mediated adhesion of E. coli to hexadecane, presumably by interfering with the electrostatic interactions responsible for adsorption of the polymer to the bacterial surface. Chitosan similarly promoted E. coli adhesion to polystyrene at concentrations slightly higher than those which mediated adhesion to hexadecane. Poly-L-lysine also promoted microbial adhesion to hexadecane, although at concentrations somewhat higher than those observed for chitosan. In order to study the effect of the cationic protein lysozyme, adhesion was studied at 0 degree C (to prevent enzymatic activity), using n-octane as the test hydrocarbon. Adhesion of E. coli increased by 70% in the presence of 80 micrograms of lysozyme per ml. When the negatively charged carboxylate residues on the E. coli cell surface were substituted for positively charged ammonium groups, the resulting cells became highly hydrophobic, even in the absence of polycations. The observed "hydrophobicity" of the microbial cells in the presence of polycations is thus probably due to a loss of surface electronegativity. The data suggest that enhancement of hydrophobicity by polycationic polymers is a general phenomenon.     

Kinetic characterization of early immunoreactive intermediates during the refolding of guanidine-unfolded Escherichia coli tryptophan synthase beta 2 subunits.

This paper deals with stopped-flow studies on the kinetics of the regain of immunoreactivity toward five distinct monoclonal antibodies during the folding of the guanidine-unfolded beta 2 subunit of Escherichia coli tryptophan synthase and of two complementary proteolytic fragments of beta, F1 (N-terminal; Mw = 29,000) and F2 (C-terminal; Mw = 12,000). It is shown that, while selected as being "specific" for the native protein, these antibodies are all able to recognize early folding intermediates. The two antigenic determinants carried by the F2 domain and the antigenic site carried by the hinge peptide linking F1 and F2 are present so early during the folding process that their kinetics of appearance could not be followed. On the contrary, the rate constants of appearance of two "native-like" epitopes, carried by F1, could be determined during the folding of beta chains. The rate constant of appearance of the epitope to antibody 19 was found to be k = 0.065 s-1 at 12 degrees C. This value is very similar to that we reported previously for the appearance of an early epitope to the same antibody during the folding of acid-denatured beta chains. Thus, in spite of the important structural differences between guanidine-unfolded and acid-denatured beta chains, the same early folding events seem to be involved in the appearance of this epitope. The rate constant was found to be significantly smaller (k = 0.02 s-1 at 12 degrees C) for the appearance of the epitope to antibody 9. This shows that the regain of immunoreactivity is not concerted within the F1 domain.(ABSTRACT TRUNCATED AT 250 WORDS)