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Biomedical subjects

S M Hecht

Publications and source records attributed to S M Hecht.

At least 91 records · Page 5Linked to original sources
PubMed1985

DNA damage and growth inhibition in cultured human cells by bleomycin congeners.

Bleomycin is hypothesized to cause cell growth inhibition and cell death via DNA cleavage. We have attempted to determine if net DNA cleavage is directly related to growth inhibition by measuring whether both parameters vary in parallel. Of primary importance to these studies was use of several bleomycin congeners. We have shown that these congeners vary in their abilities both to inhibit cell growth and to cause DNA damage. Bleomycin B2, tallysomycin, and phleomycin were the most potent growth inhibitors, and bleomycin B2 caused the most DNA damage. N-Acetylbleomycin A2 was inactive in both assays. The net amount of DNA damage measured at two levels of growth inhibition was compared for each congener and was found to vary widely among the congeners. Similarly, the degree of growth inhibition at a given level of submaximal DNA damage was found to vary widely when individual congeners were compared to each other. Hence, growth inhibition and net DNA damage due to bleomycin are not directly correlated with each other when individual congeners are compared to each other.

Bleomycin↗
PubMed1985

DNA damage induced by bleomycin in the presence of dibucaine is not predictive of cell growth inhibition.

Growth inhibition and cell killing by bleomycin are believed to be related to the ability of this antibiotic to cleave chromosomal DNA. Because bleomycin has an intracellular site of action, its ability to cross biological membranes must be critical to its overall effectiveness as an antitumor agent. The local anesthetic dibucaine acts to enhance membrane fluidity; therefore, the reported ability of this local anesthetic to modulate bleomycin effects on KB cells was investigated. Cells were treated with various bleomycin congeners in the presence or absence of dibucaine for 24 h. Dibucaine enhanced the inhibition of cell growth mediated by bleomycin A2, demethylbleomycin A2, bleomycin B2, and isobleomycin A2. N-Acetylbleomycin A2 did not inhibit cell growth in the absence of dibucaine, but it was inhibitory in the presence of dibucaine. Cells treated simultaneously for analysis of DNA breakage on alkaline sucrose gradients revealed that breakage was also enhanced in the presence of dibucaine. The degree of enhancement varied with dose and bleomycin congener. N-Acetylbleomycin A2 did not induce DNA breakage in either the absence or the presence of dibucaine. While growth inhibition and net DNA breakage correlated reasonably well in the absence of dibucaine for each bleomycin analogue tested, proportionality was lost in the presence of dibucaine, and very little DNA breakage was present when growth inhibition was complete. These observations imply that, at least in the presence of dibucaine, bleomycin may mediate growth inhibition at some locus in addition to chromosomal DNA and, also, that a given net amount of bleomycin analogue induced DNA damage per se does not produce a specific degree of growth inhibition.

Bleomycin↗
PubMed1985

Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission.

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.

Bleomycin↗
PubMed1984

Bleomycin may be activated for DNA cleavage by NADPH-cytochrome P-450 reductase.

In the presence of NADPH and O2, NADPH-cytochrome P-450 reductase was found to activate Fe(III)-bleomycin A2 for DNA strand scission. Consistent with observations made previously when cccDNA was incubated in the presence of bleomycin and Fe(II) + O2 or Fe(III) + C6H5IO, degradation of DNA by NADPH-cytochrome P-450 reductase activated Fe(III)-bleomycin A2 produced both single- and double-strand nicks with concomitant formation of malondialdehyde (precursors). Cu(II)-bleomycin A2 also produced nicks in SV40 DNA following activation with NADPH-cytochrome P-450 reductase, but these were not accompanied by the formation of malondialdehyde (precursors). These findings confirm the activity of copper bleomycin in DNA strand scission and indicate that it degrades DNA in a fashion that differs mechanistically from that of iron bleomycin. The present findings also-establish the most facile pathways for enzymatic activation of Fe(III)-bleomycin and Cu(II)-bleomycin, provide data concerning the nature of the activated metallobleomycins, and extend the analogy between the chemistry of cytochrome P-450 and bleomycin.

Bleomycin↗
PubMed1984

T4 RNA ligase mediated preparation of novel "chemically misacylated" tRNAPheS.

T4 RNA ligase was employed for the condensation of Escherichia coli tRNAPhe missing cytidine-75 and adenosine-76 (tRNAPhe-COH; the acceptor "oligomer") with each of several chemically acylated derivatives of pCpA (the donor "oligomer"). The resulting "chemically misacylated " tRNAPheS were obtained in 20-65% yields following chromatographic workup on DEAE-cellulose and benzoylated DEAE-cellulose. Characterization of the chemically misacylated tRNAs was accomplished by (i) enzymatic reaminoacylation of chemically misacylated tRNAPhe with phenylalanine by E. coli phenylalanyl-tRNA synthetase following chemical deacylation of the "incorrect" amino acid, (ii) comparison of the hydrolytic effects of Cu2+ solutions on chemically and enzymatically prepared samples of N-acetyl-L-phenylalanyl- tRNAPheS , and (iii) measurement of the chromatographic behavior of the tRNA species derived from chemical misacylation .

Escherichia coli↗
PubMed1983

Loss of positional specificity in the aminoacylation of Escherichia coli tRNAGly.

The positional specificity in the aminoacylation of Escherichia coli tRNAGly by its cognate aminoacyl-tRNA synthetase has been studied using tRNAGlys terminating in 2'- or 3'-deoxyadenosine under conditions believed to alter tRNA conformation. Although E. coli tRNAGly terminating in 3'-deoxyadenosine has been reported not to be a good substrate for activation by the homologous glycyl-tRNA synthetase, by systematic variation of the conditions employed for aminoacylation it was possible to activate this tRNA to essentially the same extent as unmodified tRNAGly. Activation of tRNAGly terminating in 3'-deoxyadenosine was carried out optimally at 45 degrees C in an incubation mixture containing 0.3-0.4 M NaCl; 10% methanol, ethanol, and dimethyl sulfoxide were found to facilitate activation of the modified tRNA. Interestingly, the conditions employed to enhance activation of this modified tRNAGly had no effect on the activation of unmodified tRNAGly or tRNAGly terminating in 2'-deoxyadenosine. These experiments afford insight into the activation of tRNAGly by glycyl-tRNA synthetase and provide facile access to positionally defined, isomeric glycl-tRNAGlys.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Transfer RNA pyrophosphorolysis with CTP(ATP):tRNA nucleotidyltransferase. A direct route to tRNAs modified at the 3' terminus.

The pyrophosphorolysis of tRNA by yeast CTP-(ATP):tRNA nucleotidyltransferase has been studied in an effort to define the behavior of the enzyme and the experimental parameters that lead to net loss of the 3'-terminal nucleotide or to nucleotide exchange. It was found that removal of AMP from the terminus of tRNA proceeded optimally at 1.0 mM PPi; incorporation of 2'- or 3'-dAMP was also studied and shown to proceed optimally at a 6.0 mM concentration of deoxynucleoside triphosphate. CTP was shown to inhibit the pyrophosphorolysis and nucleotide exchange observed when starting from intact tRNA, but apparently not by inhibiting removal of CMP from tRNA missing the 3'-terminal adenosine moiety. The optimized conditions for nucleotide exchange were used for the preparative conversion of tRNAs to species terminating in 2'- and 3'-deoxyadenosine.

Kinetics↗
PubMed1983

Dipeptide formation with misacylated tRNAPhes.

Several misacylated P-site tRNAs have been prepared by the T4 RNA ligase-mediated coupling of Escherichia coli tRNAPhe missing the 3'-terminal cytidine and adenosine moieties and N-acetylaminoacyl-pCpA derivatives. The reaction proceeded in reasonable yield in each case and the tRNA products were purified conveniently by successive chromatographies on DEAE-cellulose and benzoylated DEAE-cellulose. The misacylated tRNAPhes were assayed for participation in the peptidyltransferase reaction, using E. coli ribosomes and poly(U) as message. When phenylalanyl-tRNAPhe was employed as the A-site tRNA, "chemically aminoacylated" N-acetyl-L-phenylalanyl-tRNAPhe produced dipeptide to virtually the same extent as authentic N-acetyl-L-phenylalanyl-tRNAPhe, which was prepared by enzymatic activation of tRNAPhe followed by chemical acetylation. Significant dipeptide formation was also obtained with N-acetyl-L-tyrosyl-tRNAPhe, but much less dipeptide was obtained when the D-isomers of these two N-acetylaminoacyl-tRNAs were employed in the same assay system. Interestingly, N-acetyl-beta-phenylalanyl-tRNAPhe formed dipeptide at least to the same extent as authentic N-acetyl-L-phenylalanyl-tRNAPhe.

Acylation↗
PubMed1983

gamma Ray induced deoxyribonucleic acid strand breaks. 3' Glycolate termini.

Gamma irradiation of DNA in aqueous solution produces single strand breaks with two types of 3'-termini. These termini have been identified as a simple phosphoryl group and a glycolate moiety attached to the 3'-phosphoryl group via the 2"-OH of glycolic acid. Irradiation of 5'-end labeled poly(dG) produces compounds that comigrate with authentic 3',5'-dGDP and 2'-deoxyguanosine-3'-(phospho-2"-O-glycolic acid-5'-monophosphate by thin layer chromatography and polyacrylamide gel electrophoresis. Irradiation of poly(dGdC) followed by digestion to 3'-nucleotides produces 2'-deoxyguanosine-3'-(phospho-2"-O-glycolic acid). The presence of glycolate termini at some strand breaks may affect intracellular repair of these lesions.

Chemical Phenomena↗
PubMed1982

A structurally modified yeast tRNAPhe with six nucleotides in the anticodon loop lacks significant phenylalanine acceptance.

The deletion of nucleoside 37 from yeast tRNAPhe was accomplished in a three-step procedure that involved (i) specific depurination of this purine under acidic conditions and removal of the carbohydrate moiety with aniline-2-aminopyridine, (ii) removal of phosphate monoesters from the 5'-half-molecule with alkaline phosphatase, and (iii) resealing of the anticodon loop with T4 RNA ligase. The course of the resealing reaction was monitored by polyacrylamide gel electrophoresis and found to be complete within a few hours when incubated at 37 degrees C in the presence of 215 units/ml of RNA ligase. Although the half-molecules used to prepare the modified tRNA had substantial phenylalanine acceptance when assayed at low ionic strength, the modified species itself was essentially devoid of phenylalanine acceptance. We conclude that the anticodon loop of tRNAPhe is involved in the activation of this tRNA and that both the presence of specific nucleotides in this loop and their ability to assume an appropriate spatial or conformational arrangement may be important for enzyme recognition.

Anticodon↗
PubMed1982

Specificity of deoxyribonucleic acid cleavage by bleomycin, phleomycin, and tallysomycin.

The sites of cleavage of DNA by bleomycin A2, bleomycin B2, phleomycin, tallysomycin A, and Blenoxane (Bristol-Meyers) in reactions containing equimolar Fe2+ and atmospheric oxygen were analyzed by gel electrophoresis of 32P end labeled DNA fragments. Bleomycin A2 and bleomycin B2 reactions cleaved DNA at all sites with a frequency equal to that of Blenoxane. At high concentrations of bleomycin the site specificity of cleavage was unchanged. Bleomycin cleavage sites and phleomycin cleavage sites are a subset of sites cleaved in reactions containing tallysomycin A. The nature of 5' and 3' termini induced by bleomycin cleavage was investigated. Electrophoresis of bleomycin-induced fragments after alkaline phosphatase or polynucleotide kinase treatment indicated that 5' termini are phosphoryl groups but 3' termini are not simple phosphoryl groups. Analysis of bleomycin cleavage of single-stranded DNA substrate showed that cleavage occurs only in regions of potentially double-stranded looped-back sequences. Possible mechanisms for determination of bleomycin cleavage sequence specificity are discussed.

Base Sequence↗
PubMed1982

Oxygen transfer from bleomycin-metal complexes.

Both Fe(III) and Cu(II) complexes of bleomycin (BLM), but not N-acetyl BLM . Fe(III), mediated the transfer of oxygen from iodosobenzene to organic substrates. In analogy with results obtained using certain cytochrome P-450 analogs, cis-stilbene was converted cleanly to the respective oxide, while no more than traces of trans-stilbene oxide were formed from trans-stilbene under identical conditions. The possible relevance of these observations to the degradation of DNA by bleomycin was also studied. In both the presence and absence of O2, BLM . Cu(II) . C6H5IO effected DNA degradation, as judged by the release of [3H]thymine from radiolabeled Escherichia coli DNA. These findings provide a valuable new assay system for the study of bleomycin analogs and suggest the possibility that bleomycin may function as an "oxygen transferase" in its degradation of DNA in situ.

Bleomycin↗
PubMed1982

Structural basis for the deoxyribonucleic acid affinity of bleomycins.

The role of the bithiazole moiety of bleomycin in the interaction of the antibiotic with DNA has been studied by the use of synthetic bithiazole derivatives. The DNA affinity of individual C-terminal (bithiazole) analogues of bleomycin was measured in terms of the ability of these species to block the binding of bleomycin to DNA, as judged by diminution of the DNA degradation that attends bleomycin binding. DNA degradation was monitored both by release of [3H]thymine from radiolabeled PM-2 DNA and by alteration of bleomycin-treated DNA oligomers of defined sequence derived from Escherichia coli plasmid pLJ3. It was found that the affinity of the bithiazole derivatives for DNA depended on the presence of the bithiazole moiety itself but more importantly on the number and spacing of positively charged groups; 2'-(2-aminoethyl)-2,4'-bithiazole-4-[3-[(4-aminobutyl) amino]propyl]carboxamide (14), having three positively charged groups at neutral pH, was a reasonably effective inhibitor of DNA degradation by bleomycin. Consistent with the importance of the spacing of the positively charged groups, tetrapeptide S (12) was found to be significantly less inhibitory toward DNA degradation by bleomycin than tripeptide S, in spite of their equal number of positively charged groups and the greater structural similarity of the former to bleomycin A2. Bleomycin is known to cleave DNA perferentially at certain sequences. It was shown that the inhibitors employed in this study diminished DNA cleavage proportionately at each cleavage site; no alteration was observed in the specificity of cleavage. A number of the bithiazole analogues employed as inhibitors of bleomycin-mediated DNA degradation were also utilized in fluorescence quenching experiments with calf thymus DNA. Consistent with the belief that these species inhibit bleomycin degradation by competitive binding to the DNA substrate, the best inhibitors exhibited the greatest fluorescence quenching upon admixture of DNA.

Bacteriophages↗
PubMed1982

Deglyco-bleomycin. Degradation of DNA and formation of a structurally unique Fe(II) . CO complex.

In analogy with bleomycin, deglyco-bleomycin B2 has been found to form a stable, diamagnetic complex with Fe(II) and CO. Although the stoichiometry of this complex appeared to be the same as that formed with bleomycin, the geometry of the deglyco-bleomycin complex was fundamentally different, especially as regards orientation of the beta-aminoalanine moiety. In the presence of Fe(II) and O2, deglyco-bleomycin A2 and deglyco-bleomycin B2 were found to release [3H]thymine from radiolabeled PM-2 DNA; when employed at limiting concentrations, deglyco-bleomycin A2 and B2 gave about half as much [3H]thymine release as the respective bleomycins. In view of the spectral evidence (Burger, R. M., Horwitz, S. B., Peisach, J., and Wittenberg, J. B. (1979) J. Biol. Chem. 254, 12299-12302) that Fe(II) . bleomycin . CO has the same geometry as the complex formed by initial association of bleomycin, Fe(II), and O2, the accumulated data suggest strongly that all metal complexes of bleomycin (derivatives) capable of DNA degradation need not have the same geometry.

Bleomycin↗
PubMed1981

Design and preparation of affinity columns for the purification of eukaryotic messenger ribonucleic acid cap binding protein.

2',3'-O-[1-(2-Carboxyethyl) ethylidene]-7-methylguanosine 5'-diphosphate (5) and 7-(5-carboxypentyl) guanosine 5'-diphosphate (13) have been synthesized and immobilized on AH-Sepharose 4B to the extent of 17.4 and 36.6 mumol of ligand/g of gel, respectively. The affinity resins thus derives were employed in columns for the purificaton of 24K cap binding protein (CBP) from rabbit reticulocytes. Each resin was found to retain the protein of interest; elution of 24K CBP could then be effected by washing with 70 microM m7GDP. The 24K CBPs released from both columns were found to be active, both as judged by a cross-linking assay that utilized 10(4)-oxidized methyl-3H-labeled reovirus mRNA as a substrate for the protein and also by the ability of the isolated 24K CBP to stimulate the translocation of capped Sindbis virus mRNA in HeLa cell extracts.

Carrier Proteins↗