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E W Gerner

Publications and source records attributed to E W Gerner.

At least 55 records · Page 3Linked to original sources
PubMed1992

Delta-type DNA polymerase characterized from Drosophila melanogaster embryos.

Genetic and biochemical evidence suggests there are at least three DNA polymerases required for replication in eukaryotic cells. However, Drosophila embryonic cells have a very short duration S phase which is regulated differently. To address the question of whether embryos utilize different DNA polymerases, we employed Mono Q anion exchange chromatography to resolve the DNA polymerase activities. Two types of DNA polymerase, DNA polymerase delta and DNA polymerase alpha, were distinguished by: 1. copurification of DNA primase or 3'-5'exonuclease activities; 2. immunoblot analysis with alpha-specific polyclonal antisera; 3. sensitivity to aphidicolin and BuPdGTP; and 4. processivity measurements with and without Proliferating Cell Nuclear Antigen. These observations suggest that Drosophila embryos, similar to nonembryonic cells, have both alpha- and delta-type DNA polymerases.

Animals↗
PubMed1992

Purtscher's retinopathy preceding acute pancreatitis.

A patient with a long history of alcohol abuse had diminished vision. The clinical and angiographic findings were consistent with Purtscher's retinopathy. Subsequently, the patient was hospitalized for acute pancreatitis. To our knowledge, this is the first reported case of pancreatic retinopathy preceding the systemic findings of acute pancreatitis.

Acute Disease↗
PubMed1991

Polyamine catabolism in rodent and human cells in culture.

N1-Acetylspermidine (N1AcSpd) accumulates in late exponential phase, or after certain stresses such as heat shock, in both human tumour (A549) and rodent (HTC, CHO) cells, grown in medium containing an inhibitor of the FAD-dependent polyamine oxidase (PAO). Inhibition of PAO has little effect on cell growth or on the cellular content of the major polyamines, putrescine, spermidine or spermine, found in proliferating cells in culture, but decreases cellular putrescine content in heat shocked cells. Putrescine and spermidine are generated when N1AcSpd or N1-acetylspermine (N1AcSpm) respectively is added to either human or rodent cells depleted of the former amines by alpha-difluoromethylornithine. N1AcSpm is formed in polyamine-depleted human A549 cells when N1AcSpd is added to cultures treated with the PAO inhibitor. This reaction does not occur in either rodent line, suggesting that N1AcSpd can be converted directly into N1AcSpm in human, but not rodent, cells under specific conditions. The data presented demonstrate that a variety of human and rodent cells express PAO activity and catabolize polyamines by a mechanism which includes PAO. PAO activity is of little consequence to proliferating A549, HTC or CHO cells in culture, but does produce new putrescine in both late-exponential-phase and heat-shocked cells. These findings suggest that polyamine catabolism is part of a general response of both rodent and human cells to a variety of environmental and physiological stresses.

Animals↗
PubMed1991

Spermidine acetylation in response to a variety of stresses in Escherichia coli.

Heat shock, cold shock, ethanol, and alkaline shift, but not hydrogen peroxide, stimulate the accumulation of monoacetylspermidine in Escherichia coli. Acetylation occurs with nearly equal frequencies at both the N1 and N8 positions of this ubiquitous polycation. Spermidine acetylation does not appear to be associated with known stress regulons, such as htpR, oxyR, and SOS. E. coli, capable of acetylating spermidine, constitutively express a spermidine acetyltransferase activity during all phases of growth, and this activity is unaffected by cold shock. A mutant strain, incapable of acetylating spermidine, does not express this enzyme activity but grows at an identical rate as the parent strain at 37 degrees C. These results demonstrate that the monoacetylation of spermidine in E. coli is regulated by some mechanism other than a stress-inducible acetyltransferase and is not essential for growth of these cells. They suggest that polyamine acetylation is involved in the responses of these organisms to a variety of chemical and physical stresses.

Acetylation↗
PubMed1990

Polyamine regulation of heat-shock-induced spermidine N1-acetyltransferase activity.

The enzyme spermidine/spermine N1-acetyltransferase (N1-SAT) is rapidly induced by heat shock in CHO and A549 cells, with activity declining by 24 h. Depletion of intracellular polyamines by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, blocks this induction. Re-addition of putrescine to these cultures restores the response to heat shock, with a concomitant increase in intracellular N1-acetylspermidine. Diaminopropane is more than twice as effective as the naturally occurring diamine putrescine, suggesting that the propylamine moiety of spermidine is involved in the regulation of N1-SAT induction. Inhibitor studies indicate transcriptional activation and that the enzyme has an apparent half-life of 30-60 min. A second heat shock rapidly inhibits induced N1-SAT activity, which decays with a half-life of 2-3 min. Despite its induction by heat, N1-SAT is not a stable enzyme, suggesting that the activity observed is not due to a modification of an existing peptide, but is due to a transcriptional event, which may justify the inclusion of this enzyme in the family of heat-shock proteins.

Acetyltransferases↗
PubMed1990

Alteration of cellular adhesion by heat shock.

Chinese hamster ovary (CHO) cells were analyzed for their ability to reassemble microfilament bundles, to remain attached to a tissue culture surface, or to initiate and complete attachment onto a substrate after heat shock (45 degrees C/10 min). The cells remained attached to the tissue culture surface during and after the heat shock while the actin microfilament bundles were reversibly disrupted. Heat shock inhibited the ability of the cells to initiate and complete attachment onto a new tissue culture surface or onto a plastic surface coated with vitronectin. An inspection of the proteins present in substrate-attached material (SAM) revealed 11 major proteins containing glucosamine whose apparent Mr values were 250,000, 200,000, 150,000, 140,000, 90,000, 86,000, 82,000, 68,000, 54,000, 47,000, and 46,000. Three of the proteins (p200, p150, and p46) bound to wheat germ agglutinin while p150 and p140 bound to concanavalin A. The composition of the 11 proteins of the SAM fraction synthesized previous to the heat shock was not altered during heat shock. However, the appearance of the newly synthesized proteins in the SAM fraction was delayed by heat shock (0.5 h for p150 and 6 h for p82). The ability of heat-shocked cells to reattach onto a vitronectin-coated surface correlated with the appearance of newly synthesized p150 and p82 in the SAM fraction. Our results suggest that in addition to the microfilament bundles, heat shock may reversibly disrupt the cellular adhesion site. Further, p150 and p82, proteins whose appearance in the SAM fraction is delayed by heat shock, may be involved in the cellular attachment onto substrates, including vitronectin.

Actin Cytoskeleton↗
PubMed1990

Polyamine biosynthesis inhibitors combined with systemic hyperthermia in cancer therapy.

A Phase I clinical trial has been initiated at the University of Arizona Cancer Center which combines escalating oral doses of the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO), with systemic hyperthermia (approximately 41.5 degrees C) in the treatment of metastatic melanoma. The rationale for the combination of hyperthermia and polyamine biosynthesis inhibitors in the treatment of human cancers includes studies which show that depletion of endogenous polyamines, as a result of treatment with DFMO, sensitizes both rodent and human tumor cells to the cytotoxic effects of hyperthermia. Heat shock induces the first enzyme in polyamine catabolism, spermidine/spermine N1-acetyltransferase (N1-SAT). The consequently acetylated forms of spermidine and spermine are then constitutively oxidized by the enzyme polyamine oxidase (PAO). Both CHO and human A549 lung cancer cells exhibit heat-inducible polyamine acetylation, display potent heat sensitization after polyamine depletion, and ultimately reveal prolonged expression of thermotolerance. Conversely, HeLa cells do not demonstrate heat-inducible polyamine catabolism, are not sensitized to heat with DFMO, and display more rapid kinetics of thermotolerance decay. These laboratory studies suggest that enhancement of the cytotoxic action of hyperthermia by DFMO occurs as a consequence of the inhibition of polyamine catabolism, a heat-inducible process that affords some form of protection to cells undergoing heat stress. Human melanoma cultures demonstrate heat-inducible polyamine catabolism and are sensitized to hyperthermic cytotoxicity by DFMO. To date, 24 systemic hyperthermia treatments have been delivered to nine patients with metastatic melanoma in conjunction with oral DFMO under this Phase I clinical trial.

Acetylation↗
PubMed1989

Effects of diethyldithiocarbamate and endogenous polyamine content on cellular responses to hydrogen peroxide cytotoxicity.

In exponential-phase Chinese-hamster cells, 0.1 mM-diethyldithiocarbamate (DDC) afforded greater than 1 log survival protection to cultures treated before and during exposure to 1 mM-H2O2. Both DDC and H2O2 treatment stimulated the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, within 4 h of exposure. DDC, and to a lesser degree H2O2, also stimulated the activity of spermidine N1-acetyltransferase (SAT), the rate-limiting enzyme in polyamine catabolism. The increase in SAT activity, after exposure to DDC or another stress (heat shock), was inhibited in cells depleted of putrescine and spermidine by alpha-difluoromethylornithine (DFMO), the enzyme-activated suicide inhibitor of ODC. Pretreatment with DFMO or heat shock also induced resistance to H2O2 cytotoxicity. Since SAT activity is low in resting cells, yet stimulation of enzyme activity depends on endogenous spermidine pools, these results suggest that the expression of SAT activity occurs by a mechanism involving a stress-dependent displacement of spermidine into a new intracellular compartment. The stimulation of ODC and SAT activities does not appear to be a necessary component of the mechanism by which DDC protects cells from H2O2 cytotoxicity, although spermidine displacement may be a common facet of the cellular response to stress.

Acetyltransferases↗
PubMed1989

Heat shock stimulates polyamine oxidation by two distinct mechanisms in mammalian cell cultures.

Heat shock stimulates both exogenous and endogenous polyamine oxidation in mammalian cells, but by distinct biochemical mechanisms. Exogenously added polyamines are oxidized in serum via the temperature-dependent activation of a single class of enzymes, the copper-dependent amine oxidases. Endogenous polyamines undergo a two-step reaction sequence involving acetylation by a heat-inducible acetyltransferase and subsequent oxidation by a constitutively expressed, flavin-dependent polyamine oxidase. In both instances, polyamine oxidation generates hydrogen peroxide and reactive aldehydes which influence cell viability as demonstrated by inhibitor studies. Aminoguanidine, an inhibitor of the copper-dependent amine oxidases, confers protection to cells during either a severe 43 degrees C heat shock or a relatively nontoxic heat stress followed by incubation at 37 degrees C, all in the presence of exogenous spermidine. Specific inhibition of the endogenous polyamine oxidase will also confer partial survival protection after heat shock, but only in cultures that have been previously depleted of cellular glutathione. These data confirm that hyperthermic stress can generate an oxidative stress in mammalian cells via induction of polyamine oxidation. Further, through distinct extracellular and intracellular mechanisms, these temperature-dependent polyamine oxidation reactions can modulate cell viability.

Acetyltransferases↗
PubMed1989

Ocular toxicity of tamoxifen.

Tamoxifen is a preferred agent for the treatment of breast cancer. While efficacious, it is not without serious side effects. Ocular toxicity to the cornea, retina, and optic nerve have been reported. We present a case of tamoxifen retinopathy and emphasize the need for periodic ophthalmologic examinations to prevent loss of vision.

Adult↗
PubMed1988

Inhibition of ionizing radiation recovery processes in polyamine-depleted Chinese hamster cells.

Polyamines are involved in many cellular processes, including DNA structure and function. Since DNA, or some DNA-containing structure, is known to be the target for cell killing induced by ionizing radiation and a number of chemotherapeutic agents, we investigated the effects of polyamine depletion on cytotoxic responses of Chinese hamster cells to X-irradiation. Colony forming ability after single, acute radiation exposures of cells growing under oxic conditions was minimally affected by endogenous putrescine and spermidine depletion, achieved after treatment with alpha-difluoromethylornithine. Survival of cells rendered hypoxic and then irradiated was unaffected by alpha-difluoromethylornithine treatment. However, cellular recovery processes were nearly completely suppressed in polyamine-depleted cells, including sublethal damage recovery, as evidenced by split-dose irradiations in log phase cultures, and potentially lethal damage recovery, observed when growth-inhibited cultures were allowed time to repair radiation damage prior to being plated for colony formation. Both these recovery processes were restored by exogenous putrescine treatment. Reaccumulation of intracellular spermidine content closely correlated with restoration of potentially lethal damage recovery. Depletion of putrescine and spermidine pools had little effect on either single or double strand DNA break production or rejoining. These data demonstrate that both sublethal and potentially lethal damage recovery are polyamine-dependent processes in Chinese hamster cells, and imply that the mechanisms by which hamster cells recovery from these types of radiation damage are unrelated to their ability to rejoin DNA strand breaks, at least during the first hour after irradiation. Finally, these results suggest that the depletion of tumor polyamine content may be an effective method of enhancing the sensitivity of human tumors to fractionated radiotherapy.

Animals↗
PubMed1988

Ornithine decarboxylase and polyamine levels in columnar upper gastrointestinal mucosae in patients with Barrett's esophagus.

Ornithine decarboxylase (ODC) activity was elevated in the premalignant metaplastic columnar epithelium (mean activity, 0.13 unit/mg protein, N = 18 individual samples from 18 patients), compared to either adjacent gastric (mean activity, 0.02 unit/mg protein, N = 9) or small intestinal (mean activity, 0.02 unit/mg protein, N = 9) epithelium in patients with Barrett's esophagus. Enzyme activity ranged from 0 (less than detectable) to more than 0.5 unit/mg protein in the metaplastic tissue. However, neither putrescine, spermidine, spermine (as individual parameters), nor total polyamine contents were related to ODC activity in the individual patient biopsies. Spermidine/spermine ratios ranged from 0.38 to 2.18 and were also not related to enzyme activity in any apparent manner. Nevertheless, cell strains derived from the metaplastic tissue were growth inhibited by alpha-difluoromethylornithine, an enzyme-activated, suicide inhibitor of ODC. In two different cell strains derived from Barrett's epithelium, growth was affected with drug concentrations as low as 0.05 mM. While the mechanism responsible for the elevation in enzyme activity is unknown, the regulation of polyamine metabolism appears to be altered in this premalignant tissue. The growth inhibition of Barrett's epithelium-derived cell lines by ODC inhibitors suggests a potential role for these compounds in the treatment of this disease.

Barrett Esophagus↗
PubMed1987

Hypusine formation in protein by a two-step process in cell lysates.

The putative protein synthesis initiation factor eukaryotic initiation factor 4D (eIF-4D) is post-translationally modified by the polyamine spermidine, forming the rare amino acid hypusine from a lysine residue. The hypusine precursor, deoxyhypusine, was formed in crude cell lysates at pH 9.5 and converted to hypusine at pH 7.1. The modification occurred in eIF-4D, since the isoelectric points and molecular weights of the proteins modified in intact cells and lysates were indistinguishable. Only lysates from cells treated with alpha-difluoromethylornithine, to deplete endogenous polyamine pools, supported the formation of deoxyhypusine, suggesting that unmodified eIF-4D accumulated in spermidine deficient cells. Guazatine, an inhibitor of enzymes which form delta 1-pyrroline from spermidine, blocked deoxyhypusine formation in lysates by nearly 70% at 100 microM and completely at 1 mM. Other mammalian amine oxidase inhibitors had little or no effect on this reaction. Thus, deoxyhypusine formation in eIF-4D is catalyzed by a guazatine-sensitive enzyme with a basic pH optimum.

Animals↗
PubMed1987

Heat shock proteins in thermotolerance and other cellular processes.

Heat shock proteins appear to be causatively involved in the acquisition of thermotolerance in prokaryotes but not in eukaryotes. Further, the enhanced synthesis of hsps may be necessary for some cellular responses to stress but not others. In prokaryotic cells the development of thermotolerance, as measured by cell survival, is dependent upon protein synthesis. However, in eukaryotes, enhanced hsp synthesis following an inducing stress and prior to a subsequent heat shock is neither necessary nor sufficient for the development of thermotolerance as measured by colony-forming assays. The enhanced expression of hsps may be required for some mammalian cellular stress responses, such as the ability to reform both actin microfilament bundles and nucleolar morphology. These latter two thermotolerant responses have not been correlated with colony-forming ability. Future work should address the relationships between these various physiological responses to stress and determine if hsps function in some repair mode with regard to colony formation responses. Evidence is accumulating that hsps or their cognates may function in growth and differentiation in some manner as yet to be fully explained. Recent studies indicate that genes controlling cell division in E. coli may be linked to those of several stress regulons, and it would not be surprising to find a similar relationship in eukaryotes. At this time, it is important that studies investigating the role of hsps in stress and other cellular responses such as growth and differentiation define the specific gene (including its regulatory sequences) that encodes the protein being investigated, in order to avoid apparently contradictory and confusing reports of hsps expression.

Animals↗
PubMed1987

Ornithine decarboxylase production in vitro by using mouse cDNA.

Microgram quantities of ornithine decarboxylase (ODC, EC 4.1.1.17)-specific mRNA were synthesized by transcription techniques in vitro, by using a plasmid containing mouse cDNA coding for this enzyme. The homogeneous RNA preparation was then used for cell-free synthesis of ODC protein, in rabbit reticulocyte lysates. Analysis of products translated in vitro by polyacrylamide-gel electrophoresis revealed predominantly one protein produced, with Mr approx. 54,000, which was immunoprecipitable by anti-ODC serum. Two-dimensional gel-electrophoretic analysis showed that the protein ODC synthesized in vitro had a pI of approx. 5.4, similar to the native enzyme isolated from mouse tissues. In addition, quantification of activity and protein amount showed that the enzyme synthesized in vitro had a specific activity of approx. 63,000 units (nmol/min)/mg, consistent with the purified mouse kidney enzyme's specific activity of approx. 47,000 units/mg. An average of nearly 200 pg of ODC protein was produced in vitro from various RNA preparations. These data demonstrate that ODC-specific mRNA and active ODC protein can be produced by 'in vitro' technology, which should prove useful in studying functional and structural characteristics of these molecules.

Cell-Free System↗
PubMed1987

Sensitization of Chinese hamster ovary cells to heat shock by alpha-difluoromethylornithine.

When exposed to alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, Chinese hamster ovary cells become increasingly sensitive to the cytotoxic effects of elevated temperatures (D.J.M. Fuller and E.W. Gerner, Cancer Res., 42:5046-5049, 1982). This sensitization becomes marked at times greater than 24 h after drug removal, and by 48 h, polyamine-depleted cells that have been exposed to 43 degrees C for 90 min have clonogenic survival values more than two orders of magnitude lower than control populations. Dose response studies demonstrate that, when measured 36 h after removal of the drug, hyperthermic cytotoxicity is maximally potentiated by exposure to DFMO for times as short as 2 to 4 h. A drug concentration of 1 mM for 8 h also elicits maximal response. An additional 8-h drug treatment 24 h after the first fails to further reduce survival in response to heat shock, suggesting the effects of the first exposure are persistent. Intracellular putrescine pools are depleted by the drug within 8 h, and spermidine levels continue to decline for up to 50 h. Consistent with these observations, ornithine decarboxylase (EC 4.1.1.17) activity is found to be reduced for up to 48 h after drug removal. The concomitant depression of spermidine is reflected in the elevation of S-adenosylmethionine decarboxylase (EC 4.1.1.50), which is substrate limited. Putrescine and spermidine show no sign of reaccumulation until approximately 4 days after DFMO exposure. Exposure to exogenous putrescine reversed the sensitization to heat shock induced by DFMO. This effect is quite specific for putrescine (1.4-diaminobutane) and is not replicated by other diamine homologues ranging from 1.3-diaminopropane to 1.8-diaminooctane. Polyamine-depleted cells express thermotolerance with kinetics similar to control cells although overall survival levels are lower. These results suggest that the mechanism of induction and expression of thermotolerance is independent of the role of acid-soluble polyamine pools in cellular responses to heat shock.

Animals↗