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E J Morris

Publications and source records attributed to E J Morris.

At least 19 recordsLinked to original sources
PubMed2001

Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage.

DNA damage has been implicated as one important initiator of cell death in neuropathological conditions such as stroke. Accordingly, it is important to understand the signaling processes that control neuronal death induced by this stimulus. Previous evidence has shown that the death of embryonic cortical neurons treated with the DNA-damaging agent camptothecin is dependent on the tumor suppressor p53 and cyclin-dependent kinase (CDK) activity and that the inhibition of either pathway alone leads to enhanced and prolonged survival. We presently show that p53 and CDKs are activated independently on parallel pathways. An increase in p53 protein levels, nuclear localization, and DNA binding that result from DNA damage are not affected by the inhibition of CDK activity. Conversely, no decrease in retinoblastoma protein (pRb) phosphorylation was observed in p53-deficient neurons that were treated with camptothecin. However, either p53 deficiency or the inhibition of CDK activity alone inhibited Bax translocation, cytochrome c release, and caspase-3-like activation. Taken together, our results indicate that p53 and CDK are activated independently and then act in concert to control Bax-mediated apoptosis.

Active Transport, Cell Nucleus↗
PubMed2001

Retinoblastoma protein partners.

Studies of the retinoblastoma gene (Rb) have shown that its protein product (pRb) acts to restrict cell proliferation, inhibit apoptosis, and promote cell differentiation. The frequent mutation of the Rb gene, and the functional inactivation of pRb in tumor cells, have spurred interest in the mechanism of pRb action. Recently, much attention has focused on pRb's role in the regulation of the E2F transcription factor. However, biochemical studies have suggested that E2F is only one of many pRb-targets and, to date, at least 110 cellular proteins have been reported to associate with pRb. The plethora of pRb-binding proteins raises several important questions. How many functions does pRb possess, which of these functions are important for development, and which contribute to tumor suppression? The goal of this review is to summarize the current literature of pRb-associated proteins.

Carrier Proteins↗
PubMed2001

Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside.

Cytosine arabinoside (AraC) is a nucleoside analog that produces significant neurotoxicity in cancer patients. The mechanism by which AraC causes neuronal death is a matter of some debate because the conventional understanding of AraC toxicity requires incorporation into newly synthesized DNA. Here we demonstrate that AraC-induced apoptosis of cultured cerebral cortical neurons is mediated by oxidative stress. AraC-induced cell death was reduced by treatment with several different free-radical scavengers (N-acetyl-L-cysteine, dipyridamole, uric acid, and vitamin E) and was increased following depletion of cellular glutathione stores. AraC induced the formation of reactive oxygen species in neurons as measured by an increase in the fluorescence of the dye 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. AraC produced DNA single-strand breaks as measured by single-cell gel electrophoresis and the level of DNA strand breakage was reduced by treatment with the free radical scavengers. These data support a model in which AraC induces neuronal apoptosis by provoking the generation of reactive oxygen species, causing oxidative DNA damage and initiating the p53-dependent apoptotic program. These observations suggest the use of antioxidant therapies to reduce neurotoxicity in AraC chemotherapeutic regimens.

Acetylcysteine↗
PubMed2001

The role of infertility nurses in ovulation induction programmes.

The role of the infertility nurse is continually expanding and changing to meet the demands of couples undergoing assisted reproduction. This article examines the responsibilities infertility nurses have in ovulation induction programmes in fertility clinics today. A simple questionnaire was sent to all fertility units listed in The Patients' Guide to DI and IVF Clinics. There was a response rate of 71%. Specialist infertility units had a higher number of nurses available to carry out various tasks within the fertility programme compared with other types of staff. Members of the nursing staff were involved in initial consultation, transvaginal ultrasound scanning, intrauterine inseminations, administration of medication, sperm preparation and pregnancy tests. Infertility nurses played a major role in ovulation induction programmes. In 39% of units, nurses performed intrauterine inseminations alone, in 23% of units they made decisions as to the requirement for human chorionic gonadotrophin administration, and in over 77% of units transvaginal scans were done by nurses. This role could be even broader, assuming adequate training is provided and undertaken. The extended role of infertility nurse practitioners allows more continuity of care and better understanding of patients' needs and results in the involvement of fewer people in the overall care.

Female↗
PubMed2000

Induction and modulation of cerebellar granule neuron death by E2F-1.

Growing evidence suggests that certain cell cycle regulators also mediate neuronal death. Of relevance, cyclin D1-associated kinase activity is increased and the retinoblastoma protein (Rb), a substrate of the cyclin D1-Cdk4/6 complex, is phosphorylated during K(+) deprivation-evoked death of cerebellar granule neurons (CGNs). Cyclin-dependent kinase (CDK) inhibitors block this death, suggesting a requirement for the cyclin D1/Cdk4/6-Rb pathway. However, the downstream target(s) of this pathway are not well defined. The transcription factor E2F-1 is regulated by Rb and is reported to evoke death in proliferating cells when overexpressed. Accordingly, we examined whether E2F-1 was sufficient to evoke death of CGNs and whether it was required for death evoked by low K(+). We show that adenovirus-mediated expression of E2F-1 in CGNs results in apoptotic death, which is independent of p53, dependent upon Bax, and associated with caspase 3-like activity. In addition, we demonstrate that levels of E2F-1 mRNA and protein increase during K(+) deprivation-evoked death. The increase in E2F-1 protein is blocked by the CDK inhibitor flavopiridol. Finally, E2F-1-deficient neurons are modestly resistant to death induced by low K(+). These results indicate that E2F-1 expression is sufficient to promote neuronal apoptosis and that endogenous E2F-1 modulates the death of CGNs evoked by low K(+).

Adenoviridae↗
PubMed2000

Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage.

Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.

Animals↗
PubMed2000

E2F1 mediates death of B-amyloid-treated cortical neurons in a manner independent of p53 and dependent on Bax and caspase 3.

Although B-amyloid (AB) is suggested to play an important role in Alzheimer's disease, the mechanisms that control AB-evoked toxicity are unclear. We demonstrated previously that the cell cycle-related cyclin-dependent kinase 4/6/retinoblastoma protein pathway is required for AB-mediated death. However, the downstream target(s) of this pathway are unknown. We show here that neurons lacking E2F1, a transcription factor regulated by the retinoblastoma protein, are significantly protected from death evoked by AB. Moreover, p53 deficiency does not protect neurons from death, indicating that E2F1-mediated death occurs independently of p53. Neurons protected by E2F1 deficiency have reduced Bax-dependent caspase 3-like activity. However, protection afforded by E2F1, Bax, or caspase 3 deficiency is transient. In the case of E2F1, but not with Bax or caspase 3 deficiency, delayed death is accompanied by DEVD-AFC cleavage activity. Taken together, these results demonstrate the required role of E2F1, Bax, and caspase 3 in AB evoked death, but also suggest the participation of elements independent of these apoptosis regulators.

Amyloid beta-Peptides↗
PubMed2000

Misdirected vimentin messenger RNA alters cell morphology and motility.

Localized messenger RNAs were first observed as embryonic determinants that altered development when mislocalized. In recent years localized mRNAs have been found for several cytoskeletal proteins, including actin, vimentin and several microtubule associated proteins. We sought to determine whether redirecting mRNA for a cytoskeletal protein to an inappropriate address would alter cellular phenotypes. To do so we generated vimentin mRNAs with a myc epitope tag and the (beta)-actin 3' untranslated region (3' UTR) as a localization signal. When misdirected vimentin mRNAs are expressed in either fibroblasts or SW13 cells, cells develop numerous, extremely long processes; these cells also move more slowly to enter a wound of the monolayer. In situ hybridization revealed that the misdirected mRNA was often localized in the processes, in contrast to endogenous vimentin mRNA. The processes usually contained actin distal to the transgenic vimentin and microtubules proximal to it. SW13 cells lacking vimentin produced fewer and shorter processes, suggesting a dominant negative effect that involves recruitment of endogenous vimentin. Control experiments that transfected in constructs expressing tagged, correctly localized vimentin, or (beta)-galactosidase that localized through the (beta)-actin 3' UTR, indicate that neither the shape nor the motility changes are solely due to the level of vimentin expression in the cell. This is direct evidence that the site of expression for at least one cytoskeletal mRNA alters the phenotype of the cell in which it is expressed. Messenger RNA localization is proving to be as essential for the normal maintenance of somatic cell phenotypes as embryonic determinants are for embryogenesis.

Animals↗
PubMed1999

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

Animals↗
PubMed1999

Optimization of single-cell gel electrophoresis (SCGE) for quantitative analysis of neuronal DNA damage.

Neuronal death can be induced by DNA-damaging agents and occurs by apoptosis involving a specific signal-transduction pathway. However, to our knowledge, methods for the quantitative determination of DNA damage in individual neurons have not yet been described. Here we optimize the single-cell gel electrophoresis (SCGE) or "comet"-assay to measure DNA damage within individual neurons growing in dissociated cell culture. In addition, we have written a macro for the NIH Image program to determine the tail moment of individual comets. We have calibrated this method using gamma-irradiated (0-16 Gy) cerebral cortical neurons from the rat central nervous system. Neuronal DNA damage (in the form of DNA strand breaks) occurs in a linear, dose-dependent manner, which can be quantitatively determined in vitro using the SCGE assay. These data demonstrate that the SCGE assay is an effective method to measure DNA damage in individual neurons and may be highly useful for the study of neuronal DNA damage formation, repair and apoptosis.

Animals↗
PubMed1998

Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus-induced expression of CKIs p16(ink4), p21(waf/cip1), and p27(kip1), as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation- and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1-associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.

Animals↗
PubMed1998

Multiple pathways of neuronal death induced by DNA-damaging agents, NGF deprivation, and oxidative stress.

Here, we compare the pathways by which DNA-damaging agents, NGF deprivation, and superoxide dismutase 1 (SOD1) depletion evoke apoptosis of sympathetic neurons. Previous work raised the hypothesis that cell cycle signaling plays a required role in neuronal apoptosis elicited by NGF deprivation and the DNA-damaging agent camptothecin. To test this hypothesis, we extended our investigation of DNA-damaging agents to cytosine arabinoside (AraC) and UV irradiation. As with NGF deprivation and camptothecin treatment, the cyclin-dependent kinase inhibitors flavopiridol and olomoucine protected neurons from apoptosis induced by AraC and UV treatment. These observations support the model that camptothecin, AraC, and UV treatment cause DNA damage, which leads to apoptosis by a mechanism that, as in the case of NGF deprivation, includes activation of cell cycle components. Flavopiridol and olomoucine, however, had no effect on death induced by SOD1 depletion, suggesting that CDKs do not play a role in this paradigm of neuronal death. To compare further the mechanisms of death evoked by NGF withdrawal, SOD1 depletion, and DNA-damaging agents, we investigated their responses to inhibitors of cysteine aspartases, elements of apoptotic pathways. The V-ICEinh and BAF, two peptide inhibitors of cysteine aspartases, protected neurons in all three death paradigms. In contrast, the cysteine aspartase inhibitory peptide zVAD-fmk conferred protection from NGF withdrawal and SOD1 depletion, but not DNA-damaging agents, whereas acYVAD-cmk protected only from SOD1 depletion. Taken together, these findings indicate that three different apoptotic stimuli activate separate pathways of death in the same neuron type.

Animals↗
PubMed1997

G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis.

Previous studies have demonstrated that G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases (CDKs) prevent the death of nerve growth factor (NGF)-deprived PC12 cells and sympathetic neurons, suggesting that proteins normally involved in the cell cycle may also serve to regulate neuronal apoptosis. Past findings additionally demonstrate that DNA-damaging agents, such as the DNA topoisomerase (topo-I) inhibitor camptothecin, also induce neuronal apoptosis. In the present study, we show that camptothecin-induced apoptosis of PC12 cells, sympathetic neurons, and cerebral cortical neurons is suppressed by the G1/S blockers deferoxamine and mimosine, as well as by the CDK-inhibitors flavopiridol and olomoucine. In each case, the IC50 values were similar to those reported for inhibition of death induced by NGF-deprivation. In contrast, other agents that arrest DNA synthesis, such as aphidicolin and N-acetylcysteine, failed to block death. This suggests that the inhibition of DNA synthesis per se is insufficient to provide protection from camptothecin. We find additionally that the cysteine aspartase family protease inhibitor zVAD-fmk inhibits apoptosis evoked by NGF-deprivation but not camptothecin treatment. Thus, despite their shared sensitivity to G1/S blockers and CDK inhibitors, the apoptotic pathways triggered by these two causes of death diverge at the level of the cysteine aspartase. In summary, neuronal apoptosis induced by the DNA-damaging agent camptothecin appears to involve signaling pathways that normally control the cell cycle. The consequent death signals of such deregulation, however, are different from those that result from trophic factor deprivation.

Animals↗
PubMed1996

Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity.

Camptothecin is an S-phase-specific anticancer agent that inhibits the activity of the enzyme DNA topoisomerase-I (topo-I). Irreversible DNA double-strand breaks are produced during DNA synthesis in the presence of camptothecin, suggesting that this agent should not be toxic to nondividing cells, such as neurons. Unexpectedly, camptothecin induced significant, dose-dependent cell death of postmitotic rat cortical neurons in vitro; astrocytes were more resistant. Aphidicolin, an inhibitor of DNA polymerase alpha, did not prevent camptothecin-induced neuronal death, while death was prevented by actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole as well as cycloheximide and anisomycin, inhibitors of RNA and protein synthesis, respectively. Camptothecin-induced neuronal death was apoptotic, as characterized by chromatin condensation, cytoplasmic shrinking, plasma membrane blebbing, and fragmentation of neurites. DNA fragmentation was also confirmed by the use of the in situ DNA end labeling assay. In addition, aurintricarboxylic acid, an inhibitor of the apoptotic endonuclease, partially protected against camptothecin-induced neuronal death. The toxicity of stereoisomers of a camptothecin analogue was stereospecific, demonstrating that toxicity was a result of inhibition of topo-I. The difference in sensitivity to camptothecin between neurons and astrocytes correlated with their transcriptional activity and level of topo-I protein expression. These data indicate important roles for topo-I in postmitotic neurons and suggest that topo-I inhibitors can induce apoptosis independent of DNA synthesis. We suggest a model based on transcriptionally mediated DNA damage, a novel mechanism of action of topo-I poisons.

Animals↗
PubMed1995

Transesophageal echocardiography in the diagnosis of traumatic rupture of the aorta.

BACKGROUND: Rupture of the aorta is a major cause of death after motor vehicle accidents. Survival depends on early diagnosis, and emergency aortography is the standard imaging method. Although transesophageal echocardiography is noninvasive and can provide high-resolution images of the aorta, information about its value in patients with trauma is limited. We conducted this study to assess prospectively the value of transesophageal echocardiography in the emergency evaluation of patients at risk for aortic injury. METHODS: Transesophageal echocardiography of the aorta was attempted in 101 patients admitted to the emergency room with a diagnosis of possible traumatic rupture of the aorta. Echocardiography and aortography personnel were notified simultaneously of the arrival of the patient, and the two tests were performed sequentially by operators who were blinded to the results of the other test. The sensitivity and specificity of transesophageal echocardiography were calculated on the basis of the results of aortography of the arch, surgery, or autopsy. RESULTS: Transesophageal echocardiography was attempted in 101 patients. The study was successfully performed in 93 patients but could not be completed in 8 because of lack of cooperation on the part of the patient (7 patients) or maxillofacial trauma (1 patient). Despite a high injury-severity score (mean, 29.6), transesophageal echocardiography was performed without complications, and within a mean (+/- SD) of 29 +/- 12 minutes. Eleven of the 93 studies (12 percent) demonstrated rupture of the aorta near the isthmus. The findings were confirmed in 10 of the 11 patients by aortography (9 patients), surgery (9 patients), or autopsy (1 patient), yielding a sensitivity of 100 percent and specificity of 98 percent for the detection of injury to the aorta. There was one false positive echocardiogram. CONCLUSIONS: Transesophageal echocardiography is a highly sensitive and specific method of detecting injury to the thoracic aorta. This technique can be used safely and quickly in critically injured patients with suspected traumatic rupture of the aorta and compares favorably with arch aortography.

Acute Disease↗
PubMed1995

L-dopa cytotoxicity to PC12 cells in culture is via its autoxidation.

The mechanism of cytotoxicity of L-DOPA was studied in the rat pheochromocytoma PC12 cell line. The cytotoxicity of L-DOPA to PC12 cells was time and concentration dependent. Carbidopa, which inhibited the conversion of L-DOPA to dopamine, did not protect against L-DOPA cytotoxicity in PC12 cells. Furthermore, clorgyline, a selective inhibitor of monoamine oxidase type A, and pargyline, an inhibitor of both monoamine oxidase types A and B, both did not have an effect on L-DOPA toxicity. These findings suggest that cytotoxicity was not due to dopamine formed from L-DOPA. Catalase or superoxide dismutase each partially protected against L-DOPA toxicity in PC12 cells. In combination, the effects were synergistic and provided almost total protection against cytotoxicity. 6-Cyano-7-nitroquinoxaline-2,3-dione, an antagonist of non-NMDA receptors, did not protect against L-DOPA toxicity. These data suggest that toxicity of L-DOPA is most likely due to the action of free radicals formed as a result of its autoxidation. Furthermore, these findings suggest that patients on long-term L-DOPA therapy are potentially at risk from the toxic intermediates formed as a result of its autoxidation.

Animals↗