Biomedical subjects
Publications and source records attributed to M A Thompson.
Surrogate markers such as the CD4 lymphocyte count and viral p24 antigen level have now been joined by assays of HIV nucleic acid in patients' plasma as a measure of viral burden. The findings are already assisting in the development and clinical testing of antiretroviral agents. They also may facilitate therapeutic decision making for the individual patient.
Owing to its sparingly soluble properties, the potential anticancer drug pancratistatin (1) resisted conventional drug formulation procedures and the synthesis of a water-soluble prodrug became necessary. That important objective for further pre-clinical development was met by devising a route to a disodium phosphate derivative (5). The key step in the synthesis of the phenolic phosphate was phosphorylation of 1,2,3,4-tetraacetoxy-pancratistatin (2) with dibenzyloxy(N,N-diisopropylamido)-phosphine. Subsequent oxidation with m-chloroperbenzoic acid afforded phosphate 4a. Hydrogenolysis of the benzyl esters followed by base-catalysed hydrolysis of the acetate groups led to the water-soluble prodrug 5 in high yield.
Nature has provided potent insecticidal toxins as fermentation products of many Bacillus thuringiensis strains. Elucidation of structure-function relationships for this class of natural toxins is in its early stages. Both direct experimentation and application of theoretical structure-function principles emerging from the rapidly growing field of protein structure are accelerating understanding of these toxins. Coupled with the increasing demand for biologically sound pesticides, the benefits from engineering nature's toxins for improved performance set the stage for exciting, fast growth in discovery, characterization and commercialization of new active ingredients for biopesticides and transgenic plants.
OBJECTIVES: The purpose of this study was to determine whether early qualitative or quantitative angiographic features can predict reocclusion after initially successful coronary thrombolysis. BACKGROUND: Although both the benefits of early reperfusion and the consequences of subsequent reocclusion after thrombolysis for acute myocardial infarction have been well described, efforts to describe angiographic markers of lesions at high risk for reocclusion have produced conflicting results. The Global Utilization of Streptokinase and t-PA for Occluded Coronary Arteries (GUSTO) angiographic trial provides the opportunity to examine these relations in the largest single, prospective patient cohort studied to date. METHODS: We studied 559 patients undergoing follow-up angiography at 90 min and 5 to 7 days after thrombolysis in the GUSTO trial. Patients received one of four thrombolytic regimens: 1) streptokinase with intravenous heparin; 2) streptokinase with subcutaneous heparin; 3) accelerated-dose recombinant tissue-type plasminogen activator (rt-PA) with intravenous heparin; or 4) a combination of streptokinase and conventionally dosed rt-PA with intravenous heparin. Qualitative variables examined at 90-min angiography included Thrombolysis in Myocardial Infarction (TIMI) flow grade, visible thrombus and lesion morphology. Quantitative variables included percent diameter stenosis, percent area stenosis, minimal lumen diameter and lesion length. The study contained a power > 0.85 to detect clinically important differences in percent diameter stenosis, percent area stenosis and minimal lumen diameter between the groups with subsequent reocclusion and sustained patency at the p = 0.05 level. RESULTS: At follow-up, 33 patients (5.9%) had reocclusion. The reocclusion rate for patients with early TIMI grade 2 flow was 6.3% versus 5.6% for TIMI grade 3 flow (p = NS). When the group with reocclusion was compared with the group with continued patency, there were no differences in presence of early visible thrombus, complex lesion morphology, percent diameter stenosis, percent area stenosis, minimal lumen diameter or lesion length. CONCLUSIONS: Our findings demonstrate that neither qualitative nor quantitative angiographic variables at 90 min after initiation of thrombolytic therapy can be used to predict subsequent coronary reocclusion.
Kindling is a form of experimental epilepsy in which periodic electrical stimulation of a brain pathway induces a permanently hyperexcitable state. A previous study demonstrated that kindling enhances the sensitivity of hippocampal CA3 pyramidal cells to NMDA (N-methyl-D-aspartate), consistent with a greater expression of NMDA receptors. We have tested the possibility that kindling also changes the sensitivity of these neurons to competitive NMDA receptor antagonists. When depolarizing responses to NMDA were studied with a grease-gap preparation 1-5 months after the last evoked seizure, higher concentrations of competitive antagonist were required to reduce response amplitudes. Schild analysis yielded higher KD values for all three antagonists tested. This finding suggests that kindling provokes the expression by CA3 pyramidal cells of NMDA receptors with reduced affinity for competitive antagonists.
Riluzole is believed to exert its anticonvulsant and neuroprotective actions by reducing glutamate release. This study demonstrated that 10-30 microM riluzole reduces the K(+)-evoked release of glutamate and aspartate from slices of hippocampal area CA1. Only higher concentrations reduced gamma-aminobutyrate (GABA) release. These actions of riluzole were not occluded by tetrodotoxin. Riluzole did not diminish the ability of glutamate analogues to depolarize CA1 pyramidal cells, as determined from grease-gap recordings. Therefore the anticonvulsant and neuroprotective actions of riluzole in the hippocampus may be at least partly explained by its ability to inhibit glutamate/aspartate release from synaptic terminals.
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Mammalian circadian rhythms are regulated by a pacemaker within the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanisms controlling the synchronization of the circadian pacemaker are unknown; however, immediate early gene (IEG) expression in the SCN is tightly correlated with entrainment of SCN-regulated rhythms. Antibodies were isolated that recognize the activated, phosphorylated form of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB). Within minutes after exposure of hamsters to light, CREB in the SCN became phosphorylated on the transcriptional regulatory site, Ser133. CREB phosphorylation was dependent on circadian time: CREB became phosphorylated only at times during the circadian cycle when light induced IEG expression and caused phase shifts of circadian rhythms. These results implicate CREB in neuronal signaling in the hypothalamus and suggest that circadian clock gating of light-regulated molecular responses in the SCN occurs upstream of phosphorylation of CREB.
Increased intracellular cAMP and Ca2+ levels can activate transcription of a number of eukaryotic genes through a common promoter element, the cAMP/Ca2+ response element. This element is the binding site for activating transcription factor (ATF)/CREB proteins, an extensive transcription factor family. Here we report that one member of this family, ATF-1, can mediate both Ca2+ and cAMP transcriptional responses, but that the responses to the two pathways differ in magnitude. In contrast, another family member, CREB, has been shown to mediate Ca2+ and cAMP responses to similar levels. Taken together, these results suggest a mechanism that allows cells to integrate and differentiate gene regulation by cAMP and Ca2+.
There are three types of ovarian neoplasms: (1) Those which arise from the surface epithelium covering the ovary. (2) Those which are derived from the cortical mesenchymal stroma. (3) Those which develop from germ cells. Our laboratory has concentrated its effort on solid tumors in women of the first type, epithelial, which has the highest incidence and is the most lethal. Development of these tumors is correlated with aging in the ovary. They form primarily during the perimenopause. Among women, 64% of the total ovarian cancer cases are diagnosed between ages 41 and 60 years. Our approach has been to establish stable tumor cell lines from patient specimens for use as in vitro models. We have investigated the response of these cells to steroid hormones because we hypothesized that these tumors retain some metabolic characteristics which are specific to the ovary. Our data demonstrate that testosterone and androstenedione, but not cortisol, inhibited proliferation of ovarian tumor cells in vitro by a mechanism which was independent of steroid receptors. These androgens are routinely synthesized and secreted by human ovary, and in the menopausal ovary the primary source of androgen is the stromal cell compartment. Because a relatively high local concentration of ovarian androgen exists in vivo, it is possible that androgen may suppress ovarian epithelial carcinoma in women as well. If it does, then development of this carcinoma may be facilitated when the postmenopausal ovary fails to produce adequate androgen during postreproductive years.
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The snRNP E protein is one of several proteins associated with the U family of small nuclear RNAs that are involved in RNA processing. Isolation and characterization of the snRNP E protein cDNA sequences from mouse and chicken revealed a 100% conservation of the predicted amino acid sequence when compared to that of the human homologue. Further characterization of a genomic clone for the mouse snRNP E protein gene revealed that the 5' untranslated region and the immediate 5' upstream region have also been highly conserved: 72 and 70%, respectively. Conserved 5' regions include multiple copies of the CTTCCG hexamer sequence which are involved in regulating transcription of the human snRNP E protein gene. Mobility shift assays using corresponding DNA fragments from both human and mouse reveal that both fragments can compete for binding of at least one common transcription factor. These studies demonstrate that along with the amino acid sequence conservation between human and mouse, the snRNP E protein gene has also maintained a high DNA sequence conservation within its basal promoter structure.
Cellular immediate early genes (IEGs) are a class of genes whose transcription is transiently activated within minutes of exposure of cells to a wide range of extracellular stimuli. In mature neurons IEG expression can be triggered by a variety of neutrotransmitters and neurotrophic factors. The IEGs, many of which encode transcription factors, are believed to control the physiological response of the cells to the initial stimulation event by activating secondary programs of gene expression. The mechanism by which membrane depolarization/Ca2+ influx trigger the activation of one IEG, c-fos, has been characterized in PC12 cells. In these cells, the cAMP response element-binding protein (CREB) functions as a Ca2+ regulated transcription factor. In addition, CREB is an in vitro substrate for several Ca2+ calmodulin-dependent protein kinases (CaM kinases). These results suggest a model whereby activation of voltage sensitive Ca2+ channels stimulates CaM kinase activation leading to CREB phosphorylation and c-fos transcriptional activation.
Previous studies have implicated adaptations in the cyclic AMP system in mechanisms of opiate tolerance, dependence, and withdrawal in the rat locus coeruleus. It has been speculated that such adaptations may occur at the level of gene expression. To understand better the mechanism by which opiates produce these intracellular adaptations, we studied morphine regulation of the state of phosphorylation of cyclic AMP response element-binding protein (CREB), a transcription factor that mediates some of the effects of the cyclic AMP system on gene expression. We show here, by use of a back phosphorylation and immunoprecipitation procedure, that acute morphine decreases the state of phosphorylation of CREB, an effect that becomes completely attenuated after chronic morphine administration. In contrast, acute precipitation of opiate withdrawal, via administration of an opiate receptor antagonist, increases the phosphorylation state of CREB. Such regulation of CREB phosphorylation could be part of the molecular pathway by which opiates produce changes in gene expression that lead to addiction.
The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor (NGF) stimulates PC12 cells to differentiate into neurons. We have studied its transcriptional regulation in order to better understand the neuronal-specific end steps of the signal transduction pathway of NGF. By 5' deletion mapping of the peripherin promoter, we have localized two positive regulatory elements necessary for full induction by NGF: a distal positive element and a proximal constitutive element within 111 bp of the transcriptional start site. In addition, there is a negative regulatory element (NRE; -179 to -111), the deletion of which results in elevated basal expression of the gene. Methylation interference footprinting of the NRE defined a unique sequence, GGCAGGGCGCC, as the binding site for proteins present in nuclear extracts from both undifferentiated and differentiated PC12 cells. However, DNA mobility shift assays using an oligonucleotide probe containing the footprinted sequence demonstrate a prominent retarded complex in extracts from undifferentiated PC12 cells which migrates with slower mobility than do the complexes produced by using differentiated PC12 cell extract. Transfection experiments using peripherin-chloramphenicol acetyltransferase constructs in which the footprinted sequence has been mutated confirm that the NRE has a functional, though not exclusive, role in repressing peripherin expression in undifferentiated and nonneuronal cells. We propose a two-step model of activation of peripherin by NGF in which dissociation of a repressor from the protein complex at the NRE, coupled with a positive signal from the distal positive element, results in depression of the gene.
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Expression of the protooncogene c-Myb protein was assessed in normal mucosa and in tumor samples resected from six patients. We found that the tumor samples always expressed higher levels of full length Myb protein than the normal tissue. This contrasts with the situation in c-myb-associated hemopoietic malignancies of the mouse and chicken, in which Myb proteins are generally amino or carboxyl truncated. Tissues from five patients with colonic adenomatous polyps were also examined and found to express levels of Myb that were, in general, intermediate between those found in normal tissues and tumors. Of particular interest is that the more dysplastic polyps displayed higher Myb levels. In one patient with carcinoma and multiple colonic polyps, some polyps had intermediate levels of Myb, whereas one polyp with carcinoma in situ expressed tumor-like levels of Myb. To directly test the hypothesis that Myb expression may be important in determining the rate of colonic cell proliferation, we examined three colonic carcinoma cell lines and one polyp cell line. We found that the cell lines with the most rapid doubling times exhibited the highest Myb levels. In addition, we show that antisense myb oligonucleotides retard the proliferation of one of these colonic cell lines which expresses the highest level of Myb.
The mechanism by which Ca2+ mediates gene induction in response to membrane depolarization was investigated. The adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) was shown to function as a Ca(2+)-regulated transcription factor and as a substrate for depolarization-activated Ca(2+)-calmodulin-dependent protein kinases (CaM kinases) I and II. CREB residue Ser133 was the major site of phosphorylation by the CaM kinases in vitro and of phosphorylation after membrane depolarization in vivo. Mutation of Ser133 impaired the ability of CREB to respond to Ca2+. These results suggest that CaM kinases may transduce electrical signals to the nucleus and that CREB functions to integrate Ca2+ and cAMP signals.