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Biomedical subjects

D A Thompson

Publications and source records attributed to D A Thompson.

At least 37 records · Page 2Linked to original sources
PubMed2000

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5.

HIV-1 entry into CD4(+) cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

Amides↗
PubMed2000

Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa.

Mutation of a receptor tyrosine kinase gene, Mertk, in the Royal College of Surgeons (RCS) rat results in defective phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) and retinal degeneration. We screened the human orthologue, MERTK, located at 2q14.1 (ref. 10), in 328 DNA samples from individuals with various retinal dystrophies and found three mutations in three individuals with retinitis pigmentosa (RP). Our findings are the first conclusive evidence implicating the RPE phagocytosis pathway in human retinal disease.

Adult↗
PubMed2000

Sexually transmitted diseases in a family planning and an antenatal clinic in Peru: limitations of current practices and analysis of the use of potential markers, pH testing, and Whiff testing.

BACKGROUND: Little data exist on sexually transmitted diseases (STDs), including HIV, in family planning and antenatal clinics in Peru. GOAL: To evaluate (1) the prevalence of infection, (2) associated factors, (3) current clinical practices, and (4) the sensitivity of whiff and pH testing for STDs. STUDY DESIGN: A study of 363 women from an antenatal (n = 259) and a family planning (n = 104) clinic in central Lima, Peru that included oral histories and physical examinations. Samples were collected for laboratory diagnosis of common STDs, including HIV. RESULTS: Overall, 10.8% of antenatal clinic patients and 15.4% of family planning clinic patients had an STD, but no HIV infection was found. The diagnostic sensitivity of health providers was low, especially for cervical infections (sensitivity, 0%). In addition, few factors were associated with cervical infections or trichomoniasis. In the family planning clinic, pH testing was 100% sensitive for trichomoniasis and bacterial vaginosis. In both clinics, whiff testing was 84% sensitive and 47% specific for trichomoniasis and 88% sensitive and 53% specific for bacterial vaginosis. CONCLUSION: The burden of STDs was high and the sensitivity of current diagnostic practices was low. These results point to the need for simple diagnostic tools. Whiff and pH testing was found potentially useful for the diagnosis of vaginal infections. These simple tests should be implemented as screening tools.

Adult↗
PubMed2000

Setting-based practice variation in the management of simple febrile seizure.

OBJECTIVE: To identify provider-based differences in the ED assessment and management of children presenting with uncomplicated, first-time febrile seizures. METHODS: Multicenter, retrospective cohort study of seven EDs in-the Chicago area: two tertiary academic pediatric EDs (PEDs) and five community-based general EDs (GEDs). The visits of all patients with a discharge diagnosis including the term "seizure" were identified from a 30-month period. Records of patients who met criteria for simple, first-time febrile seizure were reviewed (age 6-60 months; temperature > or =38.0 degrees C; single, generalized, tonic-clonic seizure <20 minutes; "alert" or "arousable" on presentation; absence of known neurologic disease). RESULTS: Four hundred fifty-five records were included: 330 and 125 patients presenting to GEDs and PEDs, respectively. The two groups did not differ in mean age, vital signs, reported duration of seizure, or prior antibiotic use. Lumbar puncture (LP) was performed more often in the GED group (33% vs 22%). No patients were found to have bacterial meningitis. The patients in the GED group were more likely to receive parenteral antibiotics in the ED (56% vs 22%) and to be admitted or transferred (18% vs 4%). In a logistic regression model incorporating age, temperature, seizure duration, seizure in the ED, prior antibiotic use, primary care, and insurance status, the GED patients remained more likely to have an LP (OR 1.5), receive parenteral antibiotics (OR 2.5), and be admitted or transferred (OR 2.5). CONCLUSIONS: There were significant setting-based differences in the evaluation and management of children with simple febrile seizures presenting to GEDs and PEDs.

Adult↗
PubMed2000

Predictors of pain control in patients undergoing flexible bronchoscopy.

The purpose of this study was to assess the extent to which patients undergoing flexible bronchoscopy (FOB) experience pain and to identify patient factors and process of care factors that are associated with pain. We conducted a prospective cohort study on 481 patients undergoing FOB. Overall control of pain during FOB was the primary outcome. The mean age of the patients was 48 yr, 50% were male, and 32% required supplemental oxygen prior to FOB. Pain control was excellent in 36% of patients, but 10% considered it to be fair or poor. Patient factors associated with excellent pain control were excellent health (versus poor health, OR = 6.25 [95% CI, 2.28-16.67]), more education (college education versus high school education, OR = 1.72 [95% CI, 1.05-2.86]), and not having asthma (OR = 2.86 [95% CI, 1.09-7.14]). Process of care factors associated with excellent pain control were not being bothered by scope insertion (versus bothered, OR = 3.65 [95% CI, 1.99-6.98]), no memory of FOB (versus some memory, OR = 2.33 [95% CI, 1.24-4.44]), and higher ratings of information about the procedure (per 1-point increase on a 12-point scale, OR = 1.57 [95% CI, 1.41-1.78]). This is the first large-scale, prospective study to evaluate patient and process of care factors that influence pain control during FOB. It demonstrated that there are patient characteristics and process of care factors that need to be considered when evaluating pain during bronchoscopy. Improved preparation of patients with lower education, inferior health status, and asthma may lead to decreased pain during FOB. Bronchoscopists may be able to reduce pain during FOB by identifying methods to decrease pain on scope insertion, by improving the information provided to patients, and by achieving greater levels of amnesia during FOB.

Adolescent↗
PubMed2000

Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration.

PURPOSE: To characterize the spectrum of RPE65 mutations present in 453 patients with retinal dystrophy with an interest in understanding the range of functional deficits attributable to sequence variants in this gene. METHODS: The 14 exons of RPE65 were amplified by polymerase chain reaction (PCR) from patients' DNA and analyzed for sequence changes by single-strand conformation polymorphism (SSCP) and direct sequencing. Haplotype analysis was performed using RPE65 intragenic polymorphisms. Patients were examined clinically and with visual function tests. RESULTS: Twenty-one different disease-associated DNA sequence changes predicting missense or nonsense point mutations, insertions, deletions, and splice site defects in RPE65 were identified in 20 patients in homozygous or compound heterozygous form. In one patient, paternal uniparental isodisomy (UPD) of chromosome 1 resulted in homozygosity for a probable functional null allele. Eight of the disease-associated mutations (Y79H, E95Q, E102X, D167Y, 669delCA, IVS7+4a-->g, G436V, and G528V) and one mutation likely to be associated with disease (IVS6+5g-->a) have not been reported previously. The most commonly occurring sequence variant identified in the patients studied was the IVS1+5g-->a mutation, accounting for 9 of 40 (22.5%) total disease alleles. This splice site mutation, as well as R91W, the most common missense mutation, exists on at least two different genetic backgrounds. The phenotype resulting from RPE65 mutations appears to be relatively uniform and independent of mutation class, suggesting that most missense mutations (15 of 40 disease alleles [37.5%]) result in loss of function. At young ages, this group of patients has somewhat better subjective visual capacity than is typically associated with Leber congenital amaurosis (LCA) type I, with a number of patients retaining some useful visual function beyond the second decade of life. CONCLUSIONS: RPE65 mutations account for a significant percentage (11.4%) of disease alleles in patients with early-onset retinal degeneration. The identification and characterization of patients with RPE65 mutations is likely to represent an important resource for future trials of rational therapies for retinal degeneration.

Adolescent↗
PubMed1999

Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines.

We measured the insulin-stimulated amount of Akt1, Akt2, and Akt3 enzymatic activities in four breast cancer cell lines and three prostate cancer cell lines. In the estrogen receptor-deficient breast cancer cells and the androgen-insensitive prostate cells, the amount of Akt3 enzymatic activity was approximately 20-60-fold higher than in the cells that were estrogen- or androgen-responsive. In contrast, the levels of Akt1 and -2 were not increased in these cells. The increase in Akt3 enzyme activity correlated with an increase in both Akt3 mRNA and protein. In a prostate cancer cell line lacking the tumor suppressor PTEN (a lipid and protein phosphatase), the basal enzymatic activity of Akt3 was constitutively elevated and represented the major active Akt in these cells. Finally, reverse transcription-PCR was used to examine the Akt3 expression in 27 primary breast carcinomas. The expression levels of Akt3 were significantly higher in the estrogen receptor-negative tumors in comparison to the estrogen receptor-positive tumors. To see if the increase in Akt3 could be due to chromosomal abnormalities, the Akt3 gene was assigned to human chromosome 1q44 by fluorescence in situ hybridization and radiation hybrid cell panel analyses. These results indicate that Akt3 may contribute to the more aggressive clinical phenotype of the estrogen receptor-negative breast cancers and androgen-insensitive prostate carcinomas.

Breast Neoplasms↗
PubMed1999

Aminooxypentane-RANTES, an inhibitor of R5 human immunodeficiency virus type 1, increases the interferon gamma to interleukin 10 ratio without impairing cellular proliferation.

Studies have demonstrated that the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta suppress human immunodeficiency type 1 (HIV-1) replication in vitro. Infection with HIV-1 requires expression of CD4 antigen and the chemokine receptor CXCR4 (X4) or CCR5 (R5) on the surface of target cells. The engagement of these receptors with the viral surface proteins is essential for the membrane fusion process. This study investigated the anti-HIV-1 activity of a derivative of RANTES, the CCR5 antagonist aminooxypentane (AOP)-RANTES, on R5 HIV-1 isolates in peripheral blood mononuclear cells. In drug exposure experiments, AOP-RANTES efficiently inhibited viral replication of HIV-1 R5 strains, with a viral breakthrough observed after the withdrawal of the compound. The HIV-1-specific proliferative capacity was maintained under all conditions when compared with controls. An increase in IFN-gamma production accompanied by a parallel decrease in the generation of IL-10 was observed following the in vitro exposure of cells to AOP-RANTES in the presence of three of four HIV-1 R5 isolates. These experiments confirmed that the chemokine receptor antagonist AOP-RANTES was effective as an inhibitor of HIV-1 R5 strain infectivity in peripheral blood mononuclear cells. The capacity of this compound to maintain HIV-1-specific proliferative activity with a shift toward a type 1 cytokine profile makes this compound a unique molecule, one adopting an immunological pathway to limit HIV-1 infection.

Anti-HIV Agents↗
PubMed1999

Expression of the HPV E7 oncoprotein mimics but does not evoke a p53-dependent cellular DNA damage response pathway.

Acute expression of the human papillomavirus E7 oncoprotein in preimmortal human fibroblasts induces changes in the abundances of multiple cellular regulatory proteins. These alterations include a destabilization of the retinoblastoma tumor suppressor protein pRB, stabilization of the tumor suppressor protein p53, and increases in the level of the cyclin-dependent kinase inhibitor p21(cip1). Since the HPV E7 oncoproteins can interfere with several cell cycle checkpoints and similar alterations in the levels of pRB, p53, and p21(cip1) are also observed in a p53-dependent response to DNA damage, we investigated whether E7 expression triggers this signal transduction pathway. The results demonstrate that E7-mediated destabilization of pRB does not require p53 activity and is independent of the ability of E7 to induce apoptosis. Moreover, E7-mediated increases in p21(cip1) levels are largely p53-independent and involve stabilization of the p21(cip1) protein. In contrast the decreases in pRB expression in response to DNA damage involve transcriptional downregulation of RB gene expression.

Apoptosis↗
PubMed1999

NMR structure of a minimized human agouti related protein prepared by total chemical synthesis.

The structure of the chemically synthesized C-terminal region of the human agouti related protein (AGRP) was determined by 2D 1H NMR. Referred to as minimized agouti related protein, MARP is a 46 residue polypeptide containing 10 Cys residues involved in five disulfide bonds that retains the biological activity of full length AGRP. AGRP is a mammalian signaling molecule, involved in weight homeostasis, that causes adult onset obesity when overexpressed in mice. AGRP was originally identified by homology to the agouti protein, another potent signaling molecule involved in obesity disorders in mice. While AGRP's exact mechanism of action is unknown, it has been identified as a competitive antagonist of melanocortin receptors 3 and 4 (MC3r, MC4r), and MC4r in particular is implicated in the hypothalamic control of feeding behavior. Full length agouti and AGRP are only 25% homologous, however, their active C-terminal regions are approximately 40% homologous, with nine out of the 10 Cys residues spatially conserved. Until now, 3D structures have not been available for either agouti, AGRP or their C-terminal regions. The NMR structure of MARP reported here can be characterized as three major loops, with four of the five disulfide bridges at the base of the structure. Though its fold is well defined, no canonical secondary structure is identified. While previously reported structural models of the C-terminal region of AGRP were attempted based on Cys homology between AGRP and certain toxin proteins, we find that Cys spacing is not sufficient to correctly determine the 3D fold of the molecule.

Agouti Signaling Protein↗
PubMed1999

Contribution of melanocortin receptor exoloops to Agouti-related protein binding.

Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the alpha-melanocyte stimulating hormone analogue [Nle4,D-Phe7] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.

Agouti-Related Protein↗
PubMed1999

Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site.

The family of protein kinases called Akt, protein kinase B (PKB), or related to A and C kinase (RAC) have been implicated in numerous biological processes including adipocyte and muscle differentiation, glycogen synthesis, glucose uptake, apoptosis and cellular proliferation. There are 3 known isoforms of this enzyme in mammalian cells (1/alpha, 2/beta and 3/gamma). Akt1 and 2 contain a key regulatory serine phosphorylation site in the carboxy-terminal region of the protein. However, the reported sequence of the rat Akt3 protein differed significantly from this in that it lacked 25 amino acids in the C-terminal region, including this key regulatory serine phosphorylation site (Biochem. Biophys. Res. Commun. 216, 526-534). In the present studies we show that the deduced sequence of human Akt3 contains this serine and that it is phosphorylated in response to insulin. These results indicate that human Akt3 is regulated similarly to Akt1 and Akt2.

Amino Acid Sequence↗
PubMed1999

GATA-3 is expressed in association with estrogen receptor in breast cancer.

To better understand the molecular basis for the hormone-responsive phenotype in breast cancer, we have used a human cDNA array to compare patterns of gene expression between breast carcinoma cell lines discordant for estrogen receptor (ER) expression. These experiments indicated abundant expression of the transcription factor GATA-3 in the ER-positive cell lines MCF7 and T-47D, with minimal or no expression in the ER-negative cells lines MDA-MB-231 and HBL-100. Northern blot analysis of a panel of human breast carcinoma cell lines demonstrated a correlation between ER and GATA-3 expression. Studies of MCF7 cells grown in the absence or presence beta-estradiol indicated that GATA-3 expression was not responsive to estradiol. Protein immunoprecipitation and gel shift analysis confirmed the presence of functional GATA-3 protein in MCF7 but not in HBL-100 nuclear extracts. A panel of 47 primary breast cancers was characterized for expression of ER and GATA-3 using immunoperoxidase assay. In primary tumors, a statistically significant correlation between ER and GATA-3 expression was established (p < 0.0001, chi2). Our results indicate that GATA-3, in association with ER, is likely to regulate genes critical to the hormone-responsive breast cancer phenotype.

Blotting, Northern↗