Biomedical subjects

E A Thompson

Publications and source records attributed to E A Thompson.

At least 19 recordsLinked to original sources

PubMed1999

Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Animals↗

PubMed1999

Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote.

In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.

Animals↗

PubMed1998

The cyclin E promoter is activated by human cytomegalovirus 86-kDa immediate early protein.

Human cytomegalovirus (HCMV) activates cyclin E/Cdk2, which regulates cell cycle progression in G1 and S phase of the cell cycle. HCMV activation of cyclin E/Cdk2 can be demonstrated in cells that are refractory to normal mitotic stimuli. This observation suggests that the virus has some means to overcome the stringent control on expression of cell cycle progression factors that is characteristic of cells in the G0 state. One of the mechanisms involved in activation of cyclin E/Cdk2 is the induction of cyclin E expression. We report here that HCMV induces cyclin E expression through a transcriptional mechanism. The cyclin E gene is activated by the HCMV 86-kDa immediate early gene product (IE86), which directly binds to nucleotide sequences within the cyclin E promoter. An IE86 DNA-binding mutant neither binds nor activates the cyclin E promoter. IE86-binding sites within the cyclin E promoter are required for IE86-mediated activation, and deletion of the IE86-binding site inhibits IE86 activation of the cyclin E promoter. We also demonstrate that mutation of the known E2F-binding sites in the cyclin E promoter does not block activation by HCMV or IE86. These data provide a molecular mechanism for HCMV induction of cyclin E and represent the first report of IE86 directly binding to a cellular promoter.

Base Sequence↗

PubMed1998

TGF-beta1 effects on proliferation of rat intestinal epithelial cells are due to inhibition of cyclin D1 expression.

Transforming growth factor-beta 1 (TGF-beta1) arrests intestinal epithelial cells (RIE-1 and IEC-6) in the G1 phase of the cell cycle and inhibits cyclin D1 expression. This report describes experiments designed to elucidate the mechanism of cyclin D1 inhibition and to determine whether inhibition of cyclin D1 expression is the cause, rather than the result, of TGF-beta1-mediated cell cycle arrest. TGF-beta1 inhibition of IEC-6 cell proliferation was associated with a decrease in the abundance of cyclin D1/Cdk4 complexes and a corresponding decrease in Cdk4-dependent phosphorylation of the retinoblastoma protein. Metabolic labeling studies indicated that TGF-beta1 inhibited cyclin D1 synthesis without altering the rate of cyclin D1 protein degradation. Cyclin D1 antisense oligonucleotides blocked serum-stimulated induction of cyclin D1 and DNA synthesis, whereas cyclin D1 sense oligonucleotides had no effect. RIE-1 cells were engineered to overexpress human cyclin D1 under the control of a tetracycline-repressible promoter. These cells entered S phase in the presence of TGF-beta1 only when human cyclin D1 was derepressed by the withdrawal of tetracycline. These data indicate that TGF-beta1 inhibits the synthesis of cyclin D1 in gut epithelial cells and that this inhibition is the cause, rather than the result, of TGF-beta1-mediated arrest of intestinal epithelial cell proliferation.

Animals↗

PubMed1998

Ictal cognitive assessment of partial seizures and pseudoseizures.

BACKGROUND: Previous studies suggest that responsiveness is impaired during complex partial seizures (CPS) and pseudoseizures (PS); however, to our knowledge, there has been no systematic comparison using both response and memory testing. OBJECTIVE: To compare CPS with PS using ictal cognitive assessment (ICA) of responsiveness and memory. PATIENTS AND METHODS: We used a nonautomated method of ICA by bedside observers, consisting of family members and staff, during video electroencephalographic monitoring to test responsiveness and memory during the ictal phase in 245 events. We assessed the adequacy of testing and compared the testing results in 31 patients during CPS and 13 patients during PS. RESULTS: The ictal presentation of a command was successful in 58% of the events. The ictal presentation of at least 2 memory items with testing for recall after orientation was adequate in 57% of events. Impaired responsiveness was shown during both CPS and PS. However, some response was detected during 48% of PS compared with 18% of CPS (P<.01). Memory items were recalled during 63% of PS but during only 4% of CPS (P<.001). The International Classification of Epileptic Seizures remained useful, but in 11 events (8%), distinguishing complex from simple partial seizures was difficult. Recall of various types of stimuli (aural-verbal vs visual-pictorial) during ICA did not correlate with the side or location of the seizure focus, but this may have been confounded by the rarity of any memory recall during CPS. CONCLUSIONS: Ictal cognitive assessment by bedside observers is practical and provides the interaction necessary for properly classifying seizures; ICA, especially memory, may help to distinguish CPS from PS.

Adult↗

PubMed1998

Semiparametric estimation of major gene effects for age of onset.

Analysis of age of onset is a key factor in the segregation and linkage analysis of some complex genetic traits. Previous work in the genetics literature has used parametric distributional assumptions on age of onset. In this paper, a Cox model with latent major gene effects is used: a semiparametric model with unspecified baseline hazard. A Monte Carlo EM procedure is used to obtain maximum likelihood estimates. Markov chain Monte Carlo is used to realize genotypic configurations from the posterior distribution given the current model and the observed data, and these genotypic configurations are used to estimate the expectations in the EM algorithm. Simulated data sets indicate that the parameters can be estimated well, and one real data set shows the practical applicability of the proposed method.

Age Distribution↗

PubMed1998

Disequilibrium likelihoods for fine-scale mapping of a rare allele.

Genetic linkage studies based on pedigree data have limited resolution, because of the relatively small number of segregations. Disequilibrium mapping, which uses population associations to infer the location of a disease mutation, provides one possible strategy for narrowing the candidate region. The coalescent process provides a model for the ancestry of a sample of disease alleles, and recombination events between disease locus and marker may be placed on this ancestral phylogeny. These events define the recombinant classes, the sets of sampled disease copies descending from the meiosis at which a given recombination occurred. We show how Monte Carlo generation of the recombinant classes leads to a linkage likelihood for fine-scale mapping from disease haplotypes. We compare single-marker disequilibrium mapping with interval-disequilibrium mapping and discuss how the approach may be extended to multipoint-disequilibrium mapping. The method and its properties are illustrated with an example of simulated data, constructed to be typical of fine-scale mapping of a rare disease in the Japanese population. The method can take into account known features of population history, such as changing patterns of population growth.

Algorithms↗

PubMed1997

Inhibition of cellular Cdk2 activity blocks human cytomegalovirus replication.

Human cytomegalovirus is a herpesvirus that induces numerous cellular processes upon infection. Among these are activation of cyclin-dependent kinase 2, which regulates cell cycle progression in G1 and S phase. We report here that inhibition of cellular Cdk2 activity blocks HCMV replication. Inhibition of Cdk2 activity by roscovitine inhibits HCMV DNA synthesis, production of infectious progeny, and late antigen expression in infected cells in a dose-dependent manner. HCMV replication is also inhibited by the expression of a Cdk2 dominant negative mutant, whereas expression of wild-type Cdk2 has no effect on viral replication. These data indicate that activation of cellular Cdk2 is necessary for HCMV replication.

Antigens, Viral↗

PubMed1997

MCMC segregation and linkage analysis.

Our objective was to infer the genetic model for the quantitative traits using a variety of methods developed in our group. Only a single data set was analyzed in any one analysis, although some comparison between data sets was made. In addition, the simulated model was not known during the course of the analysis. Basic modeling and segregation analyses for the five quantitative traits was followed by several simple genome scans to indicate areas of interest. A Markov chain Monte Carlo (MCMC) multipoint quantitative trait locus (QTL) mapping approach was then used to estimate the posterior probabilities of linkage of QTL to each chromosome simultaneously with trait model parameters, and to further localize the genes. Comparisons between the nuclear family and pedigree data sets indicated a greater power for QTL detection and mapping with the pedigree data sets. Even with the pedigree data, however, precise localization of the QTL did not appear to be possible using single replicate data sets. Two of the three genes with effects on trait Q1 were detected by the MCMC method.

Chromosome Mapping↗

PubMed1997

Intestinal cell cycle regulation.

The intestinal epithelium is maintained by a balance between proliferation, differentiation and death that occurs as cells migrate up the crypt-villus axis. Cell cycle regulators such as cyclins, cyclin-dependent kinases (Cdks) and Cdk inhibitory proteins are expressed in a distinct pattern along the crypt-villus structure, suggesting their role in controlling intestinal cells. This is supported by observations that these cell cycle proteins are regulated by growth factors, nutrients and cell-cell contact in cultured intestinal epithelial cells. One of the key regulators of intestinal cell proliferation and differentiation is transforming growth factor-beta, which is expressed in the gut epithelium.

Animals↗

PubMed1997

Cell cycle-mediated regulation of hepatic regeneration.

BACKGROUND: Hepatic regeneration after partial hepatectomy (PH) is characterized by a synchronous induction of normally quiescent hepatocytes to reenter the cell cycle, leading to a complete restoration of hepatic mass. Cell cycle progression requires activation of cyclin-dependent kinases (Cdks) that are regulated by cyclins and Cdk inhibitors. METHODS: Protein expression of the cyclins (D-type and E), Cdks (Cdk2 and 4), and Cdk inhibitors (p21 and p27) was measured by Western blot after SHAM operation or PH in F344 rats. In addition, Cdk2-associated kinase activity was measured. RESULTS: Rapid induction of D-type and E cyclins, as well as their catalytic partners, Cdk2 and Cdk4, occurred after PH in rats. Complexes containing cyclin E and Cdk2 assembled in the regenerating liver, leading to increased Cdk2-associated kinase activity. The regenerating liver returned to preresection weight by day 7, at which time the Cdk2 activity also returned to SHAM levels. Biphasic induction of the Cdk inhibitor p21 was observed; the first peak occurred as early as 6 hours after PH, with a subsequent peak in expression occurring at 24 to 72 hours after PH. CONCLUSIONS: Taken together, these data support the concept that cyclins, Cdks, and Cdk inhibitors regulate cell cycle progression in the regenerating liver. In addition, the induction of p21 at two time points suggests that this protein may regulate both early proliferation and subsequent inhibition of hepatocyte regeneration.

Animals↗

PubMed1997

Human cytomegalovirus infection results in altered Cdk2 subcellular localization.

Human cytomegalovirus (HCMV) stimulates numerous cellular pathways upon infection. One of these pathways involves activation of cyclin E/Cdk2. Recent reports have demonstrated that Cdk2 is retained in the cytoplasm of cells arrested in GO by serum deprivation, sequestered from its regulatory subunit cyclin E which is located within the nucleus. Cdk2 rapidly enters the nucleus and becomes active upon stimulation of these cells with serum growth factors. The ability of HCMV to activate cyclin E/Cdk2 in both serum-arrested cells and contact-inhibited cells suggests that HCMV infection may also result in the translocation of Cdk2 into the nucleus. In this report, we demonstrate that Cdk2 is sequestered in the cytoplasm of cells arrested in GO by contact inhibition, as well as those arrested by serum deprivation. HCMV infection results in translocation of Cdk2 from the cytoplasm into the nucleus within 24 h of infection, both in serum-arrested and contact-inhibited cells.

CDC2-CDC28 Kinases↗

PubMed1997

Glucocorticoid inhibition of fibroblast proliferation and regulation of the cyclin kinase inhibitor p21Cip1.

Glucocorticoids inhibit the proliferation of fibroblastic cells in vivo and in culture; however, the molecular mechanism that accounts for this effect has remained obscure. We have undertaken to elucidate the mechanism whereby glucocorticoids decrease the rate of proliferation of mouse L929 fibroblastic cells. Addition of dexamethasone to mid-log phase fibroblasts prolongs G1 phase. This increase in the G1 interval is associated with, and probably due to, inhibition of phosphorylation of the product of the Rb-1 tumor suppressor gene, pRb. Inhibition of pRb phosphorylation by cyclin D-dependent kinases can be demonstrated in vitro. Nevertheless, there is no detectable change in the expression of cyclin D1, cyclin D2, or cyclin D3. Cyclin-dependent kinase-4 (Cdk4) and Cdk6 are not down-regulated in L929 cells after addition of glucocorticoids, and the abundance of cyclin D/Cdk4 complexes does not change. Inhibition of pRb kinase activity is associated with an increase in the abundance of one of the Cdk inhibitors, p21Cip1. The abundance of another cyclin kinase inhibitor, p27Kip1, remains constant. The amount of Cdk4 that is bound to p21Cip1 increases rapidly after addition of dexamethasone, and the activity of Cdk4-pRb kinase decreases in parallel. These results indicate that glucocorticoid inhibition of fibroblast proliferation is due to induction of p21Cip1, which binds to and inactivates cyclinD/Cdk4 complexes. The abundance of p21 mRNA increases about 5-fold within 2 h after addition of dexamethasone. This effect does not obtain in L929 mutants that are null for the glucocorticoid receptor, and a variant that expresses the glucocorticoid receptor from a tetracycline-repressible expression vector demonstrates induction of p21 mRNA only in the absence of tetracycline. Cycloheximide does not block induction of p21 mRNA, and dexamethasone has no detectable effect on the apparent rate of degradation of p21 mRNA. Nuclear run-on transcription of the Cip1 gene increases within 2 h after addition of dexamethasone. This effect can be blocked by tetracycline-mediated repression of the glucocorticoid receptor.

Animals↗

PubMed1997

The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry.

Exit from mitosis in animal cells is substantially delayed when spindle assembly is inhibited, spindle bipolarity is disrupted, or when a monopolar spindle is formed. These observations have led to the proposal that animal cells have a 'spindle assembly' checkpoint for the metaphase-anaphase transition that monitors bipolar spindle organization. However, the existence of such a checkpoint is uncertain because perturbations in spindle organization can produce unattached kinetochores, which by themselves are known to delay anaphase onset. In this study we have tested if cells monitor bipolar spindle organization, independent of kinetochore attachment, by analyzing the duration of mitosis in sea urchin zygotes and vertebrate somatic cells containing multipolar spindles in which all kinetochores are attached to spindle poles. We found that sea urchin zygotes containing tripolar or tetrapolar spindles progressed from nuclear envelope breakdown to anaphase onset with normal timing. We also found that the presence of supernumerary, unpaired spindle poles did not greatly prolong mitosis. Observation of untreated PtK1 cells that formed tripolar or tetrapolar spindles revealed that they progressed through mitosis, on average, at the normal rate. More importantly, the interval between the bipolar attachment of the last monooriented chromosome and anaphase onset was normal. Thus, neither of these cell types can detect the presence of gross aberrations in spindle architecture that inevitably lead to aneuploidy. We conclude that animal cells do not have a checkpoint for the metaphase-anaphase transition that monitors defects in spindle architecture independent of the checkpoint that monitors kinetochore attachment to the spindle. For dividing cells in which spindle microtubule assembly is not experimentally compromised, we propose that the completion of kinetochore attachment is the event which limits the time of the metaphase-anaphase transition.

Anaphase↗

PubMed1997

Enhancing outcomes in an indicated drug prevention program for high-risk youth.

This study examined the net effects of refining a high school-based indicated drug prevention program. The Personal Growth Class (PGC), tailored to meet the needs of high-risk youth, was designed to increase control of drug use, school performance, and emotional well-being. The program integrates social support and life-skills training. Process evaluation revealed the need for program enhancements to address underestimated levels of depression, anger, and suicidal behaviors prevalent among high-risk youth and to ensure that core content was being emphasized consistently. Youth participating in Late cohorts received the refined PGC with enhanced skills training. Study participants (N = 280) were youth, primarily ages fifteen to-seventeen, at high-risk for school failure or dropout. Indicators of emotional well-being (e.g., depression, stress, anger, self-esteem, personal control), drug involvement, and school performance were compared for Late versus Early cohorts. Regression analyses revealed the Late versus Early cohorts showed significantly greater decreases in hard drug use, depression, perceived stress, and anger, and greater increases in self-esteem. The results support arguments that effective indicated prevention programs should target specific high-risk youth employing strategies to counteract the multifaceted risk factors they experience and enhance needed protective factors.

Adolescent↗

PubMed1997

N-(4-hydroxyphenyl)retinamide induces apoptosis in T lymphoma and T lymphoblastoid leukemia cells.

We demonstrate that N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), a synthetic retinoic acid (RA) derivative, is a potent and selective inducer of apoptosis in malignant T lymphoid cells, but has little effect on normal lymphoid cells of the thymus or spleen. 4-HPR and its stereoisomer, 9-cis-4-HPR, are 50 to > 150 times more potent than 7 other retinoids in killing CEM-C7 human T lymphoblastoid leukemia cells and P1798-C7 murine T lymphoma cells. 4-HPR's apoptotic action requires the intact molecule bearing both the retinoid moiety and the hydroxyphenol ring; 4-HPR remains unmetabolized after uptake into CEM-C7 and P1798-C7 cells for up to 24 hours. We also show that glucocorticoid (GC)-resistant variants are equally susceptible to 4-HPR as are GC-sensitive cells. Thus, 4-HPR may be potentially important as a new chemotherapeutic drug for use as alternative to, or in combination with, conventional drugs for treating lymphoid malignancies.

Aminophenols↗

PubMed1997

Allelic disequilibrium and allele frequency distribution as a function of social and demographic history.

Allelic disequilibrium between closely linked genes is a common observation in human populations and often gives rise to speculation concerning the role of selective forces. In a previous treatment, we have developed a population model of the expected distribution of rare variants (including private polymorphisms) in Amerindians and have argued that, because of the great expansion of Amerindian numbers with the advent of agriculture, most of these rare variants are of relatively recent origin. Many other populations have similar histories of striking recent expansions. In this treatment, we demonstrate that, in consequence of this fact, a high degree of linkage disequilibrium between two nonhomologous alleles <0.5 cM apart is the "normal" expectation, even in the absence of selection. This expectation is enhanced by the previous subdivision of human populations into relatively isolated tribes characterized by a high level of endogamy and inbreeding. We also demonstrate that the alleles associated with a recessive disease phenotype are expected to exist in a population in very variable frequencies: there is no need to postulate positive selection with respect to the more common disease-associated alleles for such entities as phenylketonuria or cystic fibrosis.

Alleles↗

PubMed1996

Human cytomegalovirus inhibits cellular DNA synthesis and arrests productively infected cells in late G1.

Human embryonic lung fibroblasts (LU) can be productively infected with human cytomegalovirus (HCMV). During the course of productive infection, the virus elicits a number of responses that resemble certain aspects of G1 cell cycle progression. The virus activates cyclin E/Cdk2 kinase in both subconfluent, serum-arrested, and density-arrested cultures. Activation of cyclin E-dependent kinase is due, in part, to induction of cyclin E and, in part, to inhibition of the cyclin kinase inhibitors, Cip1 and Kip1. However, G1 progression is incomplete in HCMV-infected cells. Neither cyclin A nor cyclin D is induced, and cellular DNA synthesis does not occur if one takes care to avoid addition of fresh serum to serum-starved cultures. The data indicate that the virus induces a state of late G1 arrest, in which cyclin E/Cdk2 activates nucleotide metabolism and other biosynthetic processes that are necessary for viral replication. Failure to activate host cell DNA synthesis ensures that the virus will have uncompleted access to such precursors.

CDC2-CDC28 Kinases↗