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Biomedical subjects

D W Mullins

Publications and source records attributed to D W Mullins.

11 recordsLinked to original sources
PubMed1988

Effect of hyperthermia and doxorubicin on nucleoid sedimentation and poly (ADP-ribose) polymerase activity in L1210 cells.

We report on the individual and combined effects of doxorubicin (DOX) and hyperthermia (HYP) on nucleoid sedimentation and poly (ADP-ribose) polymerase (PARP) activity of L1210 cells. The effects of HYP and DOX on nucleoid sedimentation (increased sedimentation) were similar and correlated with cell viability. No correlation of PARP activity with cell toxicity was evident; the activity of PARP was inhibited by HYP (42 degrees C; 1-3 h) and stimulated by DOX (1-10 microM; 30 min). The HYP-induced inhibition of PARP was actually ameliorated by simultaneous exposure to DOX. Although separate studies have previously suggested that chromatin alterations or the inhibition of PARP might play a role in the effect of HYP, the correlation of nucleoid changes (rather than PARP activity) with cell viability emphasizes the contribution of the former. Furthermore, the results suggest that the nucleoid technique may prove useful in screening potential treatment modalities.

Animals↗
PubMed1986

Comparative rates of esterification of 5'-AMP with hydrophobic amino acids: relevance to the genetic-code assignments.

We have continued our program aimed at understanding the origin and evolution of the genetic code and the process of protein synthesis by comparing the rates of esterification of 5'-AMP by a series of hydrophobic N-acetylamino acids. The reaction clearly shows differences in reaction rate (AcPhe greater than AcLeu greater than AcVal greater than AcIle) among the amino acids having A as middle letter of their anticodons. However, there were no significant differences in reaction rate between AcLeu, AcNorleu, and Ac-alpha-aminobutyric acid, and AcGly reacted faster than all of these and AcPhe. Consequently, this simple reaction with AMP can distinguish only among those amino acids that actually have A as the middle anticodonic nucleotide. The relevance of these studies to the origins of the process of protein synthesis and of the genetic code is discussed in conjunction with results from other studies of a similar nature.

Acetylation↗
PubMed1986

Aliphatic amino acid side chains associate with the "face" of the adenine ring.

We have synthesized the free amino acid adenylate anhydrides of phenylalanine, leucine, isoleucine and valine. These activated compounds are very labile at high pH, but at low pH they become more stable. Proton NMR spectra of these adenylates show that in every case, the hydrophobic side chains, even in these small molecules at low pH and low concentration, are associated with the "face" of the adenine ring. Although aromatic rings are known to associate with adenine in this fashion, to our knowledge this is the first report of an intercalative-type interaction of aliphatic side chains with nucleic acid bases. Since adenine is the most hydrophobic base, these interactions are of a hydrophobic character, and occur in spite of the fact that the adenine ring is protonated. These results may have implications regarding recognition processes in DNA-protein and RNA-protein interactions.

Adenine↗
PubMed1985

Rationalization of some genetic anticodonic assignments.

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.

Amino Acids↗
PubMed1985

Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride.

All earthly creatures use only L-amino acids in template directed protein synthesis. The reason for this exclusive use of the L-isomer is not yet apparent, although recent experiments by Usher and his colleagues have shown some stereoselectivity in the aminoacylation of di- and polynucleotides. We have separately reported on intramolecular interactions between hydrophobic amino acid side chains and the adenine ring in aminoacyl adenylates. There was a preferential association of Phe greater than Leu = Ile greater than Val with the adenine in these studies, but we made no attempts to address the question of D, L selectivity. Recently, in 1H NMR studies of N-acetylphenylalanyl adenylate anhydride, we noticed evidence that both D- and L-isomers of the amino acid were present and, furthermore, that one isomer seemed to be associating with the adenine ring more strongly than the other. Using HPLC, we have separated the two diastereoisomers and have enzymatically determined that the isomer which associates more strongly is the biologically important one, the L-isomer. We present those studies here and discuss the evolutionary significance of this finding.

Adenosine Monophosphate↗
PubMed1977

Poly(adenosine diphosphate-ribose) polymerase: the distribution of a chromosome-associated enzyme within the chromatin substructure.

The distribution of a chromatin-bound, nuclear protein modifying enzyme, poly (adenosine diphosphate-ribose) polymerase, and its product, poly(ADP-ribose), among various fractions of sheared and nuclease-digested HeLa cell chromatin has been examined. Epichlorohydrin-tris(hydroxymethyl)aminomethane-cellulose and glycerol gradient fractionation of solubilized chromatin indicated that poly(ADP-ribose)polymerase activity was associated primarily with the template active regions (euchromatin), whereas the transcriptionally inert chromatin fractions were found to contain relatively low levels of ADP-ribosylating activity. When isolated HeLa cell nuclei were digested in situ with micrococcal nuclease and the resultant chromatin was fractionated into nucleosome monomers (v bodies) and oligomers by sucrose gradient centrifugation, only material sedimenting faster than the 11S monomers was found to contain appreciable poly(ADP-ribose) polymerase activity. If, on the other hand, isolated HeLa cell nuclei were first incubated with labeled NAD, the substrate for poly(ADP-ribose) polymerase, prior to the preparation and fractionation of nuclease-digested chromatin, it was found that those chromatin fractions which possess significant poly(ADP-ribose) polymerase activity (nucleosome oligomers) are relatively deficient in the labeled product of this enzyme, and that a considerable portion of the homopolymeric product is ultimately associated with the 11S v bodies. Additional evidence is presented which indicates that the absence of nucleosome monomer-associated poly(ADP-ribose) polymerase activity is not due to the absence of a suitable acceptor on these structures, and that the activity of this enzyme within the chromatin is most probably dependent upon the physical integrity of the oligomeric structures themselves.

Cell Nucleus↗
PubMed1977

A putative role for nicotinamide adenine dinucleotide-promoted nuclear protein modification in the antitumor activity of N-methyl-N-nitrosourea.

Incubation of HeLa cells with the anticancer agent N-methyl-N-nitrosourea (MNU) results in: (a) depression of intracellular nicotinamide adenine dinucleotide levels; (b) stimulation of the chromatin-associated, chromosomal protein-modifying enzyme polyadenosine diphosphoribose [poly(ADP-ribose)] polymerase, which uses nicotinamide adenine dinucleotide as substrate; and (c) some fragmentation of cellular DNA. DNase treatment of HeLa nuclei in vitro also stimulates poly(ADP-ribose) polymerase activity, but not in nuclei derived from MNU-treated cells unless they have been subsequently incubated to allow for recovery from MNU damage. DNA polymerase activity is stimulated in vitro by poly(ADP) ribosylation of nuclear proteins. By using intact nuclei derived from MNU-treated HeLa cells, the repair via elongation of single-strand DNA breaks is demonstrated in vitro. This repair is dependent on DNA polymerase activity and is enhanced by adenosine diphosphate ribosylation of histones. Inhibition of poly(ADP-ribose) polymerase with nicotinamide results in extensive degradation of MNU-damaged DNA. Taken as a whole, these results suggest that poly(ADP-ribose) polymerase may play a role in the repair of alkylation damage to cellular DNA and that the inhibition of this enzyme in vivo might be exploited to potentiate the antitumor and carcinogenic activities of MNU.

Alkylation↗
PubMedDate not supplied

Restoration of hyperthermia-associated increased protein to DNA ratio of nucleoids.

The sedimentation of L1210 nucleoids has been used to demonstrate a hyperthermia-associated increased protein to DNA ratio and an apparent inhibition of processes involved in the restoration of the protein to DNA ratio. The distance of nucleoid sedimentation increased as a function of exposure temperature and exposure time, and was proportional to an increased protein to DNA ratio in the nucleoids. Studies in which control and hyperthermia-treated cells were mixed prior to nucleoid preparation indicated that the association of protein with the nucleoid occurred during hyperthermia treatment and not during nucleoid preparation. However, double-labelling studies suggested an interaction between control and hyperthermia-treated cells since the presence of control cells during lysis resulted in near normalization of nucleoid sedimentation. Treatment with proteinase K also restored the nucleoid sedimentation. Incubation at 37 degrees C following hyperthermia revealed a rapid restoration of nucleoid sedimentation and a slower restoration of the protein to DNA ratio. Ethidium bromide-induced changes in nucleoid sedimentation were altered by the hyperthermia-associated increased protein content of nucleoids and the alterations were overcome by enzymatic digestion of the protein prior to the ethidium bromide exposure. Thus, hyperthermia caused, and inhibited the repair of, an increased protein content of nucleoids. The restoration of the increased protein possibly occurs by a heat-sensitive proteolytic enzyme. The temporal use of an appropriate chemotherapy agent to inhibit the restoration of hyperthermia-associated changes may be a useful treatment option.

Animals↗