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Biomedical subjects

D R Morris

Publications and source records attributed to D R Morris.

At least 55 records · Page 3Linked to original sources
PubMed1988

Mitogenic signaling pathways regulating expression of c-myc and ornithine decarboxylase genes in bovine T-lymphocytes.

Expression of the c-myc and ornithine decarboxylase (ODC) genes is elevated early after mitogenic activation of T-lymphocytes, and regulation of the two genes seems to be coupled to transmembrane signaling pathways that are in part different. The evidence is consistent with protein kinase C (PKC) being both necessary and sufficient to induce expression of the ODC gene in response to treatment of T-cells with either the mitogen concanavalin A (Con A) or biologically active phorbol esters. Furthermore, there seems to be no involvement of events dependent on calmodulin (CaM) in the regulation of ODC in these cells. The situation with c-myc is more complex. In contrast to ODC, transcription of this gene is not stimulated by treatment of resting T-cells with phorbol esters alone, but the cells respond to phorbol esters in combination with the calcium ionophore ionomycin. Induction of the c-myc gene by Con A is inhibited by CaM antagonists. These results are consistent with a model in which transcriptional activation of the c-myc gene in resting T-lymphocytes requires two signals, one from PKC and the other involving CaM.

Animals↗
PubMed1987

Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen-activated lymphocytes.

Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15-min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.

Actins↗
PubMed1987

Aggregation of DNA by analogs of spermidine; enzymatic and structural studies.

A homologous series of spermidine analogs, with defined abilities to replace the natural polyamine in supporting cell growth, was examined for its influence on the structure of supercoiled, aggregated DNA and on the ability of the DNA aggregates to act as substrates for various enzymes. The concentration of amine necessary to aggregate negatively supercoiled Col E1 DNA was progressively increased as the diaminobutane moiety of spermidine was extended beyond 5 methylene groups. 1H- and 31P-NMR spectroscopy suggested that less rigid DNA aggregates were formed by spermidine analogs than by spermidine itself. Spermidine and its analogs differentially modulated the activities of bacterial and mammalian type I topoisomerases and EcoRI restriction endonuclease on aggregated DNA in a manner reminiscent of the abilities of the amines to stimulate cell growth. When DNA was not aggregated, the influence of the various amines on these reactions was almost identical. These results are discussed in relation to the structures of the DNA aggregates in the presence of the various triamines.

Animals↗
PubMed1987

Messenger RNAs coding for enzymes of polyamine biosynthesis are induced during the G0-G1 transition but not during traverse of the normal G1 phase.

The events occurring during emergence of cells from quiescence ("G0") are not necessarily identical to those in the G1 phase of continuously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c-myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum-induced levels rather than the reduced values found in serum-starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c-myc, are growth controlled, but not regulated during traverse of a normal cell cycle.

Adenosylmethionine Decarboxylase↗
PubMed1987

Different early-signaling pathways coupled to transcriptional and posttranscriptional regulation of gene expression during mitogenic activation of T lymphocytes.

Two categories of mitogen-induced mRNAs were defined in T lymphocytes. The type 1 messages (represented by c-myc) were regulated transcriptionally, and their expression seemed to be calmodulin dependent. The type 2 messages (ornithine decarboxylase, actin, and alpha-tubulin) were regulated posttranscriptionally through activation of protein kinase C.

Animals↗
PubMed1986

Isolation of a cDNA clone encoding S-adenosylmethionine decarboxylase. Expression of the gene in mitogen-activated lymphocytes.

S-Adenosylmethionine decarboxylase was purified from bovine liver and digested with endopeptidase Lys-C; the resulting peptides were chromatographically separated. Peptides containing either methionine or tryptophan were subjected to sequence analysis. An oligonucleotide mixture of 48 sequences, which was 17 nucleotides in length, was synthesized based on one of these peptide sequences. This synthetic oligonucleotide mixture was labeled and used to screen a bovine cDNA library in phage lambda gt11. A clone was identified which contained a 1350-nucleotide insert. This insert contained nucleotide sequences coding for amino acid sequences of two of the peptides that were analyzed, thus proving that this cDNA clone codes for S-adenosylmethionine decarboxylase. A subcloned fragment from the coding region of the cDNA was used as a probe to analyze the expression of this gene in mitogen-activated lymphocytes. Northern blots revealed two message species of 2.4 and 3.6 kilobases in length. Both mRNAs were coordinately expressed and were present in polysomes. The levels of these mRNAs increased approximately 4-fold by 9 h after activation of the cells. The magnitude of the increase in these messages is to be compared with an 8- to 10-fold increase in the rate of synthesis of the protein. The apparent increase in translational efficiency of this message upon lymphocyte activation was confirmed by analyzing polysomes from these cells. In resting lymphocytes, the average size of polysomes containing mRNA coding for S-adenosylmethionine decarboxylase was 1.4 ribosomes per mRNA, and this value increased to 2.7 in stimulated cells. Thus, it appears that the increase in translational efficiency of this mRNA arises from an elevated rate of translational initiation, leading to more ribosomes per polysome encoding this particular message. This is not a general effect on the expression of all proteins, since there is no change in the translational efficiency of cytoplasmic actin upon activation of lymphocytes.

Adenosylmethionine Decarboxylase↗
PubMed1986

Modulation of the relaxing activity of Escherichia coli topoisomerase I by single-stranded DNA binding proteins.

Removal of negative superhelical turns in ColE1 plasmid DNA by Escherichia coli topoisomerase I was markedly enhanced by the presence of single-stranded DNA binding protein from E. coli. A lack of species specificity makes unlikely the possibility of physical association between topoisomerase I and single-stranded DNA binding proteins. Stabilization of single-stranded regions in supercoiled DNA by single-stranded DNA binding protein would appear to be the basis of the enhancement of topoisomerase activity.

DNA Topoisomerases, Type I↗
PubMed1986

Prosthodontics. Urgent care.

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Dental Abutments↗
PubMed1985

Nuclear magnetic resonance studies of polyamine binding to a defined DNA sequence.

The binding of spermine to the self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been studied by nuclear magnetic resonance spectroscopy. Free spermine gives narrow resonance lines and positive nuclear overhauser effects are observed between the spermine protons, as expected for a small molecule rotating freely in solution. In the spermine-DNA complex, there was no broadening of the spermine spectrum and very weak positive nuclear overhauser effects were observed, indicating that the spermine still has a remarkably short rotational correlation time. Spermine induced no changes in the DNA spectrum beyond those found upon addition of other salts. Although spermine interacts with DNA with a binding constant of approximately 10(6) at the low ionic strength under which these experiments were performed, it appears that the nature of the complex and the lifetime of the ligand on the DNA are such that the mobility of the spermine molecule is effectively independent of that of the DNA molecule.

Base Sequence↗
PubMed1985

Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases.

In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly. Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+. On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine. Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme. In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA. These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness.

Animals↗
PubMed1985

Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization.

pBR322 DNA, linearized by lysis of an oxolinic acid-treated culture of Escherichia coli strain DK6recA- (pBR322) with sodium dodecyl sulfate, was purified, treated with DNA polymerase in the presence of the four deoxynucleoside triphosphates, and ligated to DNA linkers containing the XhoI recognition sequence. Most of the drug-resistant colonies resulting from transformation of E. coli with this material bore plasmids that appeared by restriction enzyme analysis to differ from pBR322 only by the introduction of an XhoI site. The XhoI sites in plasmids from 93 transformants were distributed unevenly around the pBR322 map. Maxam-Gilbert DNA sequence analysis of 36 of these plasmids, labeled at the 5' termini of the XhoI sites, revealed that 29 of them contained, in addition to the XhoI linker, a duplication of four base-pairs of the pBR322 sequence surrounding the linker. Therefore, oxolinic acid-induced linearization must have resulted in 5'-terminal extensions of four bases, the configuration known to result from oxolinic acid-induced DNA cleavage by DNA gyrase in vitro. The sequence data thus allowed the determination of the precise point at which linearization occurred, apparently by the abortion of a gyrase-DNA covalent intermediate that existed in vivo. When the 19 different sites of the 29 plasmids were compared, the following set of rules could be derived: (formula; see text) where N is any nucleotide, R is a purine, and Y is a pyrimidine. Cleavage occurred at the line between the eighth and ninth positions from the left. The parenthetical G and T were preferred secondarily to T and G, respectively, whereas T and G in the 13th position from the left were equally preferred. Several of these rules are similar to those proposed previously based on several in vitro gyrase cleavage sites. Some of our rules show dyad symmetry around the axis midway between the cleavage points in the two strands, while others are distinctly asymmetric.

Base Sequence↗
PubMed1984

Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme.

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.

Chromatography, Affinity↗
PubMed1984

Inhibition of S-adenosylmethionine decarboxylase and diamine oxidase activities by analogues of methylglyoxal bis(guanylhydrazone) and their cellular uptake during lymphocyte activation.

Several congeners of methylglyoxal bis(guanylhydrazone) were tested for their ability to inhibit eukaryotic putrescine-activated S-adenosylmethionine decarboxylase (EC 4.1.1.50) and intestinal diamine oxidase (EC 1.4.3.6). All the compounds tested, namely methylglyoxal bis(guanylhydrazone), ethylglyoxal bis(guanylhydrazone), dimethylglyoxal bis(guanylhydrazone) and the di-N"-methyl derivative of methylglyoxal bis(guanylhydrazone), were strong inhibitors of both yeast and mouse liver adenosylmethionine decarboxylase activity in vitro. The enzyme from both sources was most powerfully inhibited by ethylglyoxal bis(guanylhydrazone). All the diguanidines likewise inhibited diamine oxidase activity in vitro. The maximum intracellular concentrations of the ethyl and dimethylated analogues achieved in activated lymphocytes were only about one-fifth of that of the parent compound. However, both derivatives appeared to utilize the polyamine-carrier system, as indicated by competition experiments with spermidine.

Adenosylmethionine Decarboxylase↗
PubMed1984

Physiological effects in bovine lymphocytes of inhibiting polyamine synthesis with ethylglyoxal bis(guanylhydrazone).

Previous results have suggested that ethylglyoxal bis(guanylhydrazone) is a more specific inhibitor of polyamine biosynthesis than the widely used methylglyoxal bis(guanylhydrazone). The physiological effects on mitogenically activated lymphocytes of polyamine depletion with ethylglyoxal bis(guanylhydrazone) were examined. In the presence of ethylglyoxal bis(guanylhydrazone) and the ornithine decarboxylase inhibitor alpha-difluoromethylornithine, the cellular contents of putrescine, spermidine, and spermine were decreased by 75 to 90, 65 to 80, and 40 to 60%, respectively, compared with control cultures. Inhibition of DNA synthesis in these polyamine-deficient cells was always greater than that of protein synthesis. Upon addition of spermidine to the deficient cells, the cellular spermidine content was restored within 4 hr, but the complete recovery of macromolecular synthesis took 10 to 20 hr. Thymidine kinase and DNA polymerase alpha activities in polyamine-deficient cells were lower than those in normal cells, whereas RNA polymerase II and leucyl transfer RNA synthase activities were nearly equal to those in normal cells. These results and studies with 2-dimensional gel electrophoresis raise the possibility that polyamines may regulate the synthesis of specific proteins. Decreased synthesis of replication proteins in polyamine-deficient cells may be one reason for the reduced synthesis of DNA.

Animals↗
PubMed1984

Comparison of specificity of inhibition of polyamine synthesis in bovine lymphocytes by ethylglyoxal bis(guanylhydrazone) and methylglyoxal bis(guanylhydrazone).

Ethylglyoxal bis(guanylhydrazone) (EGBG) was compared as an inhibitor of polyamine biosynthesis with methylglyoxal bis(guanylhydrazone) (MGBG) in bovine small lymphocytes stimulated by concanavalin A. EGBG brought about a decrease in spermidine and spermine levels equal to that found with MGBG, but at a 5-fold lower intracellular drug concentration. Despite identical polyamine levels, the degree of inhibition of DNA and protein synthesis by EGBG was smaller than that observed with MGBG, in either the presence or absence of the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. It was found that in vitro protein synthesis and in vivo mitochondrial function were inhibited by concentrations of MGBG necessary to inhibit polyamine synthesis in cells (1 to 3 mM), but not by efficacious levels of EGBG (0.2 to 0.6 mM). These results suggest that EGBG is more suitable as a specific inhibitor of polyamine biosynthesis and that use of this drug, rather than MGBG, in combination with alpha-difluoromethylornithine may be useful for studying the physiological functions of polyamines in animal cells.

Animals↗
PubMed1983

Structural specificity of the triamines sym-homospermidine and aminopropylcadaverine in stimulating growth of spermidine auxotrophs of Escherichia coli.

sym-Homospermidine (HSpe) was compared with its structural isomer, aminopropylcadaverine (AP5), and the naturally-occurring triamine, spermidine (Spe), in its ability to stimulate the growth of Spe auxotrophs of Escherichia coli. HSpe was taken up by the cells, but was less effective than Spe in stimulating growth. In at least one E. coli auxotroph, HSpe was also less effective than AP5.

Cadaverine↗
PubMed1983

Regulation of protein synthesis in mitogen-activated bovine lymphocytes. Analysis of actin-specific and total mRNA accumulation and utilization.

In response to the mitogenic lectin concanavalin A, the rate of protein synthesis in bovine small lymphocytes increased about 3-fold in 10 h and about 6-fold in 24 h. The rate of synthesis of the cytoskeletal protein actin, which comprises about 10% of total protein synthesis, increased in parallel with total protein synthesis. The cellular levels of actin mRNA were determined at various times after mitogen addition using a cDNA probe derived from a recombinant plasmid carrying a fragment coding for bovine actin. Actin mRNA sequences were found to increase significantly within 3 h and to increase 3.6 +/- 1.2-fold within 10 h after mitogen addition. Therefore, the increase in actin synthesis after mitogenic activation seems to be accounted for by accumulation of actin mRNA sequences. Total translatable mRNA and poly(A) sequences also accumulated in parallel with total protein synthesis, suggesting that regulation of actin synthesis was not unique. Two direct studies of mRNA utilization indicated that there is little, if any, translational regulation of actin synthesis. A detailed examination of actin mRNA-containing polysomes and total polysomes revealed no significant change in the average size of polysomes after cell activation. Therefore, given that the rates of polypeptide elongation and termination are constant after mitogen addition (Kay, J.E., Ahern, T., Lindsay V.J., and Sampson, J. (1975) Biochim. Biophys. Acta 378, 241-250), the rate of translational initiation per active mRNA must be unchanged. In addition, no significant amount of unutilized, nonpolysomal actin mRNA was detected in total cell extracts from either unstimulated or activated cells. Together these data suggest that elevated protein synthesis in activated lymphocytes is regulated by mRNA level.

Actins↗