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Biomedical subjects

David A Davis

Publications and source records attributed to David A Davis.

At least 19 recordsLinked to original sources
PubMed2025

The elevated expression of ORF75, a KSHV lytic gene, in Kaposi sarcoma lesions is driven by a GC-rich DNA cis element in its promoter region.

The spindle cells of Kaposi sarcoma (KS) lesions primarily express Kaposi sarcoma herpesvirus (KSHV) latent genes with minimal expression of lytic genes. However, recent transcriptome analyses of KS lesions have shown high expression of KSHV open reading frame (ORF) 75, which is considered a late lytic gene based on analyses in primary effusion lymphoma (PEL) lines. ORF75 encodes a pseudo-amidotransferase that is part of the viral tegument, acts as a suppressor of innate immunity, and is essential for viral lytic replication. We assessed a representative KS lesion by RNAscope and found that ORF75 RNA was expressed in the majority of latency-associated nuclear antigen (LANA)-expressing cells. Luciferase fusion reporter constructs of the ORF75 promoter were analyzed for factors potentially driving its expression in KS. The ORF75 promoter construct showed high basal transcriptional activity in vitro in endothelial cells, mediated by a proximal consensus specificity protein 1 (Sp1) (GGGGCGGGGC) element along with two distal CCAAT boxes. Sp proteins formed complexes with the proximal consensus Sp1 element to activate ORF75 promoter transcription. We also found evidence that a repressive factor or factors in B cells, but not endothelial or epithelial cells, interacted with more distal elements in the ORF75 promoter region to repress constitutive ORF75 expression in B cells. Alternate forms of Sp1 were found to accumulate during latency and showed substantial enrichment during viral lytic replication in PEL cells and infected endothelial cells, but their functional significance is unclear. We also found that ORF75 can in turn upregulate its own expression and that of other KSHV genes. Thus, while ORF75 acts primarily as a lytic gene in PEL cell lines, Sp proteins induce substantial constitutive ORF75 transcription in infected endothelial cells and this can account for its high expression in KS lesions.

Herpesvirus 8, Human↗
PubMed2006

Accuracy of physician self-assessment compared with observed measures of competence: a systematic review.

CONTEXT: Core physician activities of lifelong learning, continuing medical education credit, relicensure, specialty recertification, and clinical competence are linked to the abilities of physicians to assess their own learning needs and choose educational activities that meet these needs. OBJECTIVE: To determine how accurately physicians self-assess compared with external observations of their competence. DATA SOURCES: The electronic databases MEDLINE (1966-July 2006), EMBASE (1980-July 2006), CINAHL (1982-July 2006), PsycINFO (1967-July 2006), the Research and Development Resource Base in CME (1978-July 2006), and proprietary search engines were searched using terms related to self-directed learning, self-assessment, and self-reflection. STUDY SELECTION: Studies were included if they compared physicians' self-rated assessments with external observations, used quantifiable and replicable measures, included a study population of at least 50% practicing physicians, residents, or similar health professionals, and were conducted in the United Kingdom, Canada, United States, Australia, or New Zealand. Studies were excluded if they were comparisons of self-reports, studies of medical students, assessed physician beliefs about patient status, described the development of self-assessment measures, or were self-assessment programs of specialty societies. Studies conducted in the context of an educational or quality improvement intervention were included only if comparative data were obtained before the intervention. DATA EXTRACTION: Study population, content area and self-assessment domain of the study, methods used to measure the self-assessment of study participants and those used to measure their competence or performance, existence and use of statistical tests, study outcomes, and explanatory comparative data were extracted. DATA SYNTHESIS: The search yielded 725 articles, of which 17 met all inclusion criteria. The studies included a wide range of domains, comparisons, measures, and methodological rigor. Of the 20 comparisons between self- and external assessment, 13 demonstrated little, no, or an inverse relationship and 7 demonstrated positive associations. A number of studies found the worst accuracy in self-assessment among physicians who were the least skilled and those who were the most confident. These results are consistent with those found in other professions. CONCLUSIONS: While suboptimal in quality, the preponderance of evidence suggests that physicians have a limited ability to accurately self-assess. The processes currently used to undertake professional development and evaluate competence may need to focus more on external assessment.

Clinical Competence↗
PubMed2006

Inhibition of HIV-1 replication by a peptide dimerization inhibitor of HIV-1 protease.

Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus type-1 (HIV-1) protease and on the C-terminus of p6* can inhibit HIV-1 protease activity by preventing dimerization. We developed a peptide dimerization inhibitor, P27, that included these domains and a cell permeable domain derived from HIV-1 Tat. P27 inhibited wild type (WT) and protease inhibitor (PI)-resistant HIV-1 protease (IC50: 0.23-0.32 microM). Kinetic and biochemical assays confirmed that P27 inhibits protease dimerization. Fluorescein-labeled peptide accumulated in MT-2 cells and protected acutely infected MT-2 cells from HIV-1-induced cytotoxicity (IC50: 5.1 microM). P27 also inhibited p24 accumulation from H9 and U937 cells chronically infected with WT or PI-resistant HIV-1. Immunoblot analysis on the supernatants and infected cells revealed a block in virus release by P27 rather than an inhibition of polyprotein processing. However, inhibition of p55 Gag processing by active-site inhibitors was enhanced when combined with P27, suggesting that P27 can affect protease function in maturing virions. Although P27 was rationally designed to block dimerization of the mature HIV-1 protease, the effects of P27 on HIV-1 replication may be related to partial inhibition of Gag-Pol processing leading to a disruption in virus release.

Cell Line↗
PubMed2006

Increased therapeutic potential of an experimental anti-mitotic inhibitor SB715992 by genistein in PC-3 human prostate cancer cell line.

BACKGROUND: Kinesin spindle proteins (KSP) are motor proteins that play an essential role in mitotic spindle formation. HsEg5, a KSP, is responsible for the formation of the bipolar spindle, which is critical for proper cell division during mitosis. The function of HsEg5 provides a novel target for the manipulation of the cell cycle and the induction of apoptosis. SB715992, an experimental KSP inhibitor, has been shown to perturb bipolar spindle formation, thus making it an excellent candidate for anti-cancer agent. Our major objective was a) to investigate the cell growth inhibitory effects of SB715992 on PC-3 human prostate cancer cell line, b) to investigate whether the growth inhibitory effects of SB715992 could be enhanced when combined with genistein, a naturally occurring isoflavone and, c) to determine gene expression profile to establish molecular mechanism of action of SB715992. METHODS: PC-3 cells were treated with varying concentration of SB715992, 30 microM of genistein, and SB715992 plus 30 microM of genistein. After treatments, PC-3 cells were assayed for cell proliferation, induction of apoptosis, and alteration in gene and protein expression using cell inhibition assay, apoptosis assay, microarray analysis, real-time RT-PCR, and Western Blot analysis. RESULTS: SB715992 inhibited cell proliferation and induced apoptosis in PC-3 cells. SB715992 was found to regulate the expression of genes related to the control of cell proliferation, cell cycle, cell signaling pathways, and apoptosis. In addition, our results showed that combination treatment with SB715992 and genistein caused significantly greater cell growth inhibition and induction of apoptosis compared to the effects of either agent alone. CONCLUSION: Our results clearly show that SB715992 is a potent anti-tumor agent whose therapeutic effects could be enhanced by genistein. Hence, we believe that SB715992 could be a novel agent for the treatment of prostate cancer with greater success when combined with a non-toxic natural agent like genistein.

Antimitotic Agents↗
PubMed2006

Preferential inhibition of the magnesium-dependent strand transfer reaction of HIV-1 integrase by alpha-hydroxytropolones.

Integration is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1); therefore, inhibitors of HIV-1 integrase are candidates for antiretroviral therapy. Two 7-hydroxytropolone derivatives (alpha-hydroxytropolones) were found to inhibit HIV-1 integrase. A structure-activity relationship investigation with several tropolone derivatives from The National Cancer Institute compound repository demonstrated that the 7-hydroxy group is essential for integrase inhibition. alpha-Hydroxytropolones preferentially inhibit strand transfer and are inhibitory both in the presence of magnesium or manganese. Lack of inhibition of disintegration in the presence of magnesium coupled with results from different cross-linking assays suggests alpha-hydroxytropolones as interfacial inhibitors. We propose that alpha-hydroxytropolones chelate the divalent metal (Mg2+ or Mn2+) in the enzyme active site. The most active compound against HIV-1 integrase in biochemical assays [2,4,6-cycloheptatrien-1-one, 2,7-dihydroxy-4-isopropyl (NSC 18806) IC50 = 4.8 +/- 2.5 microM] exhibits weak cytoprotective activity against HIV-1(IIIB) in a cell-based assay. alpha-Hydroxytropolones represent a new family of inhibitors for the development of novel drugs against HIV infection.

Catalytic Domain↗
PubMed2006

Genetic organization and hypoxic activation of the Kaposi's sarcoma-associated herpesvirus ORF34-37 gene cluster.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We previously reported that hypoxia activates KSHV lytic replication and that the promoter for open reading frame 34 (ORF34) contains a functional hypoxia-responsive element (HRE). ORF34 is part of a cluster of lytic genes (ORF34-37) that includes ORF36, a phosphotransferase, and ORF37, a shutoff exonuclease. Rapid amplification of cDNA ends analysis revealed that they share a common polyadenylation signal but have two start sites. Two transcripts were identified, one 3.4 kb encoding ORF35-37, and the other 4.2 kb encoding ORF34 and also having coding potential for ORF35-37. Exposure of PEL cell lines to hypoxia induced messages of lengths consistent with those of these transcripts. Reporter assays with Hep3B cells showed activation of both transcripts by hypoxia. The ORF34-37 promoter region has six consensus HREs. Sequential deletion, site-directed mutagenesis experiments, and Northern blot analysis of RNA produced by constructs indicated that the second HRE (HRE-2) plays a critical role in the hypoxic activation of both RNA transcripts. The ORF35-37 transcript was upregulated by cotransfected hypoxia-inducible factor (HIF). Electrophoretic mobility shift assays demonstrated that HRE-2 and ancillary sequences bind and compete for HIF with hypoxic Hep3B nuclear extract. The activation of this gene cluster by hypoxia may have implications for the pathogenesis of PEL and KS. Moreover, the activation of ORF36 by hypoxia might be exploited to develop targeted therapy for PEL, which arises in a hypoxic environment (pleural effusions).

Base Sequence↗
PubMed2005

Differential gene up-regulation by hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha in HEK293T cells.

Cells exposed to hypoxia respond by increasing the level of hypoxia-inducible factor-1 (HIF-1). This factor then activates a number of genes by binding to hypoxia response elements in their promoter regions. A second hypoxia-responsive factor, HIF-2, can activate many of the same genes as HIF-1. Overexpression of HIFs accompanies the pathogenesis of many tumors. It is unclear, however, as to the respective role of these factors in responsiveness to hypoxia and other stresses. To address this issue, we used microarray technology to study the genes activated in HEK293T cells by hypoxia or transfection with the alpha chain of HIF-1 (or mutant HIF-1 resistant to degradation) or HIF-2. Fifty-six genes were found to be up-regulated at least 3-fold by either hypoxia or transfection. Of these, 21 were elevated both by transfection with HIF-1alpha and with HIF-2alpha, and 14 were preferentially activated by HIF-1alpha including several involved in glycolysis. Ten genes were preferentially activated by HIF-2alpha, including two (CACNA1A and PTPRZ1) implicated in neurologic diseases. Interestingly, most HIF-2alpha-responsive genes were not substantially activated by hypoxia. An additional 10 genes were up-regulated by hypoxia but minimally activated by HIF-1alpha or HIF-2alpha transfection. Ten of the genes were studied by quantitative real-time PCR and/or by Northern blot and the results paralleled those found with microarray technology. Although confirmation in other systems will be necessary, these results indicate that whereas some genes are robustly activated by both HIF-1 and HIF-2, others can be preferentially activated by one or the other factor.

Basic Helix-Loop-Helix Proteins↗
PubMed2005

Identification of carboxypeptidase N as an enzyme responsible for C-terminal cleavage of stromal cell-derived factor-1alpha in the circulation.

The chemokine stromal-derived factor-1alpha (SDF-1alpha) is an essential regulator of hematopoiesis, lymphocyte homing, pre-B-cell growth, and angiogenesis. As SDF-1alpha is constitutively expressed in many tissues, chemokine function is mostly regulated by proteolytic degradation. Human serum cleaves the 68-amino acid chemokine, SDF-1alpha, at both termini. The enzyme or enzymes responsible for the removal of the carboxy-terminal lysine from SDF-1alpha, leading to significant reduction in biologic activity, have not been identified. Using a new biochemical assay for measuring the carboxy-terminal cleavage activity, we purified from serum and plasma a peptidase that specifically removes the carboxy-terminal lysine from SDF-1alpha and identified it as carboxypeptidase N (CPN, also known as kininase I, arginine carboxypeptidase, and anaphylotoxin inactivator). We demonstrate that SDF-1alpha in serum and plasma lacks the carboxy terminal lysine, and depletion of CPN from serum and plasma significantly reduces the SDF-1alpha carboxypeptidase activity. Purified CPN effectively and specifically removes the carboxy-terminal lysine from SDF-1alpha and significantly reduces the chemokine's biologic activity as a pre-B-cell growth factor and chemoattractant. Thus, in addition to its role as a regulator of the biologic activity of kinins and anaphylatoxins, CPN is an important regulator of the biologic activity of SDF-1alpha by reducing the chemokine-specific activity.

Animals↗
PubMed2005

Increasing warfarin dosage reductions associated with concurrent warfarin and repeated cycles of 5-Fluorouracil therapy.

Concurrent use of warfarin and 5-fluorouracil has resulted in elevated international normalized ratios (INRs). Although this drug interaction is well established in the literature, we found no documented cases that address its effects on anticoagulation parameters in patients requiring repeated cycles of 5-fluorouracil and continuous warfarin therapy. We describe the effect of multiple cycles of 5-fluorouracil administration in a patient receiving warfarin therapy. The patient's INR increased 11-14 days after each cycle of chemotherapy with 5-fluorouracil and leucovorin. In addition, she required additive reductions of 33-42% in her weekly warfarin dose with each chemotherapy cycle to maintain a therapeutic INR (goal range 2.0-3.0). After three cycles of chemotherapy over a 10-week period, the patient's dosage requirements returned to her baseline level (before treatment with 5-fluorouracil and leucovorin had started). Pertinent literature was reviewed to provide supporting evidence for the mechanism and clinical implications of the drug interaction. Based on this report and previous case reports, caution is advised when concurrent warfarin and 5-fluorouracil are prescribed. In addition, patients should be closely monitored for a possible delayed interaction that may occur with each repeated cycle of 5-fluorouracil chemotherapy.

Adenocarcinoma↗
PubMed2004

Small-molecule dimerization inhibitors of wild-type and mutant HIV protease: a focused library approach.

We demonstrate that a focused library based on truncated, cross-linked interfacial peptides of HIV-1 protease produces effective dimerization inhibitors of the enzyme. By combining individual changes of the library into a single compound, we obtained a significantly more potent agent and found that an additive increase in inhibitor efficacy was obtained. The good activity of library members against an active-site drug-resistant protease mutant bodes well for dimerization inhibition as a complementary method to targeting the active site.

Binding Sites↗
PubMed2004

Solving the information overload problem: a letter from Canada.

Doctors are inundated with medical information, some inadequately evidence-based, much of it captured in clinical practice guidelines (CPGs). The Ontario Guidelines Advisory Committee (GAC) selects topic areas, searches for all CPGs on the topic, and reviews them using the AGREE Instrument. Based in large part on the AGREE score, the GAC summarises one guideline in each topic area and mounts it on its website, with links to other information (eg, clinical algorithms) where possible. Two topic areas have been selected for implementation--the reduction of unnecessary preoperative testing and the rational management of acute low back pain. Implementation strategies include performance feedback, training of opinion leaders, development of algorithms and reminders, and communication through journals and continuing medical education activities.

Diagnostic Tests, Routine↗
PubMed2003

Angiolymphoid hyperplasia with eosinophilia responsive to pulsed dye laser.

Angiolymphoid hyperplasia with eosinophilia is an uncommon, benign, but potentially disfiguring vascular proliferation. Angiolymphoid hyperplasia with eosinophilia commonly affects women in the third decade. Although a benign process, significant morbidity can occur including bleeding, pain, and disfigurement. Several therapies have been attempted including curettage, cryotherapy, retinoids, excision, and various lasers. We report on a case of angiolymphoid hyperplasia with eosinophilia responding to pulsed dye laser therapy (585 nm) (SPTL-1a, Candela Corp, Wayland, Mass).

Adult↗
PubMed2003

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) contains hypoxia response elements: relevance to lytic induction by hypoxia.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is an etiologic agent of KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease. We recently demonstrated that hypoxia can induce lytic replication of KSHV in PEL cell lines. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF), and we hypothesized that the KSHV genome may respond to hypoxia through functional hypoxia response elements (HREs). Here, we demonstrate the presence of at least two promoters within the KSHV genome that are activated by hypoxia or hypoxia mimics. One is in the promoter region of the gene for Rta, the main lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three putative consensus HREs oriented in the direction of the gene. Dissection and site-directed mutagenesis studies confirmed that one of the HREs of the ORF34 promoter is functional. Under conditions of hypoxia, the ORF34 promoter was strongly upregulated by HIF-1 alpha and HIF-2 alpha. By contrast, the promoter of the gene for Rta appeared to be preferentially upregulated by HIF-2 alpha. Reverse transcription-PCR analysis revealed that specific messages for ORF34 and ORF50 are upregulated in BCBL-1 cells exposed to hypoxia. An HIF-1 binding and competition assay demonstrated that the HRE sequence from the ORF34 promoter can compete for HIF-1 alpha binding to an erythropoietin HRE oligonucleotide while a mutant sequence cannot. Thus, we demonstrated that a viral gene can be activated by hypoxia through activation of a functional viral HRE. To our knowledge, this is the first example of a functional HRE in a viral promoter.

Binding, Competitive↗
PubMed2003

Reversible oxidative modification as a mechanism for regulating retroviral protease dimerization and activation.

Human immunodeficiency virus protease activity can be regulated by reversible oxidation of a sulfur-containing amino acid at the dimer interface. We show here that oxidation of this amino acid in human immunodeficiency virus type 1 protease prevents dimer formation. Moreover, we show that human T-cell leukemia virus type 1 protease can be similarly regulated through reversible glutathionylation of its two conserved cysteine residues. Based on the known three-dimensional structures and multiple sequence alignments of retroviral proteases, it is predicted that the majority of retroviral proteases have sulfur-containing amino acids at the dimer interface. The regulation of protease activity by the modification of a sulfur-containing amino acid at the dimer interface may be a conserved mechanism among the majority of retroviruses.

Amino Acid Sequence↗