Physician perceptions of the role and value of basic science knowledge in daily clinical practice.

BACKGROUND: The role of basic science education in a clinical setting remains unclear. Research to understand how academic clinicians perceive and use this part of their education can aid curricular development. AIMS: To assess physician's attitudes toward the value of science knowledge in their clinical practice. METHODS: Academic physicians from three medical schools completed a questionnaire about the utility of basic science education in core clinical tasks and in practice-based learning and improvement. RESULTS: A total of 109 clinical faculty returned the survey. Overall, 89% of the respondents indicated that basic science education is valuable to their clinical practice. When asked about the utility of basic science information in relation to direct patient care, greater than 50% of the doctors felt they use this when diagnosing and communicating with patients. This rose to greater than 60% when asked about choosing treatment options for their patients. Individuals also responded that basic science knowledge is valuable when developing evidence-based best practices. Specifically, 89% felt that they draw upon this information when training students/residents and 84% use this information when reading journal articles. CONCLUSIONS: This study shows that basic science education is perceived by responding academic physicians to be important to their clinical work.

ROM-1 potentiates photoreceptor specific membrane fusion processes.

Photoreceptor outer segment (OS) renewal requires a series of tightly regulated membrane fusion events which are mediated by a fusion complex containing protein and lipid components. The best characterized of these components, is a unique photoreceptor specific tetraspanin, peripherin/rds (P/rds, a.k.a., peripherin-2, Rds and Prph). In these studies we investigated the role of peripherin's non-glycosylated homolog, ROM-1, in OS fusion using a COS cell heterologous expression system and a well characterized cell free fusion assay system. Membranes isolated from COS-7 cells transfected with either FLAG-tagged P/rds or HA-tagged ROM-1 or both proteins were assayed for their ability to merge with fluorescently labeled OS plasma membrane (PM). Such membrane merger is one measure of membrane fusogenicity. The highest percent fusion was observed when the proteins were co-expressed. Furthermore detailed analysis of the fusion kinetics between fluorescently labeled PM and proteo-liposomes containing either, pure P/rds, pure ROM-1 or the ROM-1-P/rds complex clearly demonstrated that optimal fusion requires an ROM-1/P/rds complex. Proteo-liposomes composed of ROM-1 alone were not fusogenic. Peptide competition studies suggest that optimization of fusion may be due to the formation of a fusion competent peripherin/rds C-terminus in the presence of ROM-1. These studies provide further support for the hypothesis that a P/rds dependent membrane fusion complex is involved in photoreceptor renewal processes.

Fluorodeoxyuridine modulates cellular expression of the DNA base excision repair enzyme uracil-DNA glycosylase.

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) continues to play a pivotal role in the treatment of cancer. A downstream event of thymidylate synthase inhibition involves the induction of a self-defeating base excision repair process. With the depletion of TTP pools, there is also an increase in dUMP. Metabolism of dUMP to the triphosphate dUTP results in elevated pools of this atypical precursor for DNA synthesis. Under these conditions, there is a destructive cycle of dUMP incorporation into DNA, removal of uracil by the base excision repair enzyme uracil-DNA glycosylase (UDG), and reincorporation of dUMP during the synthesis phase of DNA repair. The end point is DNA strand breaks and loss of DNA integrity, which contributes to cell death. Evidence presented here indicates that both the nuclear and the mitochondrial isoforms of UDG are modulated by FdUrd (and 5-FU) treatment in certain cell lines but not in others. Modulation occurs at the transcriptional and post-translational levels. Under normal conditions, nUDG protein appears in G(1) and is degraded during the S to G(2) phase transition. The present study provides evidence that, in certain cell lines, FdUrd mediates an atypical turnover of nUDG. Additional data indicate that, for cell lines that do not down-regulate nUDG, small interfering RNA-mediated knockdown of nUDG significantly increases resistance to the cytotoxic effects of FdUrd. Results from these studies show that nUDG is an additional determinant in FdUrd-mediated cytotoxicity and bolster the notion that the self-defeating base excision repair pathway, instigated by elevated dUTP (FdUTP) pools, contributes to the cytotoxic consequences of 5-FU chemotherapy.

Proliferation-dependent expression of nuclear uracil-DNA glycosylase is mediated in part by E2F-4.

There are two isoforms of the prototypical human uracil-DNA glycosylase: one mitochondrial (UDG1) and one nuclear (UDG1A). Results presented here reveal a novel genetic organization of UDG1. Specifically, the UDG1 5' UTR is composed of two non-coding exons and the promoter region is located much farther upstream than previously recognized. We also examine the proliferation-dependent expression of UDG1A and demonstrate that the protein disappears rapidly as cells transit from the cell cycle into G0. Ribonuclease protection assays reveal that UDG1A mRNA levels are greatly reduced during G0 as well. To begin to characterize the mechanisms contributing to this regulation, we identified two overlapping candidate E2F binding sites (denoted A and B) in the UDG1A 5' UTR. EMSA analysis of this region shows a unique protein complex present only in extracts derived from G0 cells. In vitro studies using purified E2F-4 and mutant competitors demonstrate that binding occurs in a proliferation-dependent manner exclusively to E2F site A. Two approaches were then used to assess the in vivo role of the candidate E2F sites. First, chromatin immunoprecipitation (ChIP) analysis demonstrates that E2F-4 binds to the UDG1A 5' UTR exclusively in G0 cells. Secondly, using transient transfection analysis, we show that mutating these sites abolishes the proliferation-dependent response of UDG1A.

Proteolytic degradation of the nuclear isoform of uracil-DNA glycosylase occurs during the S phase of the cell cycle.

Uracil-DNA glycosylases are enzymes that remove uracil from DNA and initiate base-excision repair. These enzymes play a key role in maintaining genomic integrity by reducing the mutagenic events caused by G:C to A:T transition mutations. The recent finding that a family of RNA editing enzymes (APOBECs) can deaminate cytosine in DNA has raised the interest in these base-excision repair enzymes. This research focuses on the regulation of the nuclear isoform of uracil-DNA glycosylase, a 36000 Da protein that contains a unique 44 amino acid N-terminus. In synchronized HeLa cells, UDG1A protein levels decrease to barely detectable levels during the S phase of the cell cycle. Immunoblot analysis of immunoprecipitated or affinity-isolated UDG1A reveals ubiquitin-conjugated UDG1A when proteolysis is inhibited using N-acetyl-leu-leu-norleu-al or MG132, inhibitors of proteosomal dependent protein degradation. Transient transfection experiments, with histidine-tagged ubiquitin, were used to confirm that endogenous UDG1A is ubiquitinated in vivo. Addition of the nuclear export inhibitor, leptomycin B, prevents ubiquitination and degradation of UDG1A. This indicates that translocation from the nucleus may be a step in UDG1A turnover. Finally, UDG1A protein degradation is prevented when cells are incubated with the cyclin-dependent kinase inhibitor, roscovitine. These results suggest that protein phosphorylation and/or nuclear export participate in the post-translational regulation of UDG1A protein levels.

The transcription factor, NFI/CTF plays a positive regulatory role in expression of the hSMUG1 gene.

SMUG1 is a recently discovered uracil-DNA glycosylase with the ability to remove uracil from single-stranded as well as double-stranded DNA. SMUG1 also has the capacity to excise oxidized pyrimidine bases such as 5-hydroxymethyluracil and 5-formyluracil from DNA. Very little is known about the regulation of this enzyme. Therefore, we undertook this study to begin to elucidate the mechanisms of hSMUG1 gene expression. Northern blot analysis performed on mRNAs derived from different cell lines reveals that the steady-state levels of hSMUG1 transcript are about 10-fold lower relative to UDG. In addition to the 1.6kb transcript known to encode a functional hSMUG1 protein, an alternate 0.7kb transcript was uncovered that contains an open reading frame. Interestingly, this alternate transcript is missing a carboxy-terminal domain which is necessary for catalytic activity. Utilizing a luciferase reporter assay system we show that significant promoter activity is associated with a 2000bp region, located immediately upstream of the first transcribed, non-translated exon. 5' deletion analysis of this 2000bp region reveals that there are both negative and positive regulatory elements that control expression of SMUG1. Using electrophoretic mobility shift analysis we show that a number of DNA-protein complexes are formed within the region (-705 to -604) of positive regulation. At least two of these complexes contain the transcription factor NFI/CTF as demonstrated by oligonucleotide competition studies with NFI/CTF consensus sequence containing both protein-binding half-sites. We further demonstrate that purified NFI-C protein will bind to this positive regulatory region within the hSMUG1 gene. DNase I footprint analysis reveals that the 3' half-site is protected when using crude nuclear extract as a protein source. However, the introduction of mutations into either or both of the half-sites indicates that the individual half-sites contribute to NFI/CTF binding. Overexpression of NFI-C in NIH-3T3 cells results in an increase in SMUG1 enzyme activity. Collectively, these data indicate that the NFI/CTF consensus site may function as a cis-element in the SMUG1 promoter and that this transcription factor contributes to the positive regulation of SMUG1 gene expression.

A novel single nucleotide polymorphism within the 5' tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional activity.

Thymidylate synthase (TS) gene expression is modulated by a polymorphism in the 5' regulatory region of the gene. The polymorphism consists mainly of either two repeats (2R) or three repeats (3R) of a 28-bp sequence, yielding greater TS gene expression and protein levels with a 3R genotype. Two USF family E-box consensus elements are found within the tandem repeats of the 3R genotype, and one is found within the 2R genotype. These elements bind USF proteins in vitro by electrophoretic mobility shift analysis and in vivo by chromatin immunoprecipitation assay. We show that the additional USF consensus element within the 3R construct confers greater transcriptional activity relative to the 2R construct. Mutagenesis of the USF sites shows that the transcriptional regulation of TS is dependent, in part, on USF proteins binding within the tandem repeats. In addition, we identified a novel G-->C single nucleotide polymorphism in the second repeat of 3R alleles within the USF consensus element that alters the ability of USF proteins to bind and thus alters the transcriptional activation of TS gene constructs bearing this genotype. Through RFLP analysis, we determined the respective frequencies of the C allele (3RC) among all 3R alleles in non-Hispanic whites, Hispanic whites, African Americans, and Singapore Chinese to be 56%, 47%, 28%, and 37%, respectively. Based on our findings, this novel single nucleotide polymorphism should be considered when the 5' tandem repeat polymorphism is being used as a predictor of clinical outcome to TS inhibitors.

hSMUG1 can functionally compensate for Ung1 in the yeast Saccharomyces cerevisiae.

There are at least four distinct families of enzymes that recognize and remove uracil from DNA. Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro. Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system. These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair. When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs. The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair. When uracil-DNA glycosylase is deleted (deltaung1), cells continue through S-phase and arrest at G(2)/M, presumably due to the effects of stable uracil misincorporation in DNA. Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair. As a result, these cells experience early protection from antifolate-induced cytotoxicity. When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest. The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE. Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.

Deletional analysis of the rod photoreceptor cell peripherin/RDS carboxy-terminal region.

The C-terminal region of peripherin/rds contains three predicted alpha-helical domains. One of these domains, corresponding to amino acids 311-322, form an amphiphilic alpha-helix previously shown to promote membrane fusion. The present studies were conducted to determine how the additional alpha-helical regions of the peripherin/rds C-terminus affect complex formation with rom-1, glycosylation, intracellular localization and membrane fusion properties. Bovine peripherin/rds and rom-1 were epitope tagged with an amino-terminal FLAG-tag or amino-terminal hemagglutinin (HA)-tag, respectively, and cloned into the pCI-neo expression vector for transient transfection into COS cells. Similarly, four C-terminal peripherin/rds truncation mutants (Delta1, Delta2, Delta3 and Delta4), corresponding to deletions of -19, -29, -39 and -59 amino acids were designed to disrupt the alpha-helical domains. Immunofluorescence microscopy and enzymatic digestions demonstrated that full-length peripherin/rds and the four C-terminal deletion mutants were localized to intracellular membranes and were all Endo-H sensitive. Western blotting and immunoprecipitation studies showed that the FLAG-tagged bovine peripherin/rds (full-length) was expressed as a 76kDa dimer, which associates with HA-tagged rom-1 to form a higher order complex. The deletion mutants were also able to associate with rom-1. However, when analyzed using non-denaturing tricine electrophoresis, full-length peripherin/rds and the Delta1, Delta2 and Delta3 mutants formed homo-oligomeric complexes, while the Delta4 mutant appeared to form only homodimers suggesting a region upstream of amino acid 300 may be involved in C-terminal interactions. Membrane fusion was then evaluated using fluorescence resonance energy transfer (RET) techniques. Intracellular COS cell membranes containing full-length peripherin/rds fused with rod outer segment plasma membrane vesicles. This fusion was inhibited with the addition of a synthetic peptide (PP-5) corresponding to the fusion domain of peripherin/rds. In contrast, fusion was negligible with any of the C-terminal truncation mutants. Collectively, these results suggest that in addition to the fusion domain, other regions of the peripherin/rds C-terminus are required for fusion. Most interesting is the observation that the last 19amino acids, a region downstream of the fusion peptide that is deleted in the Delta1 mutant, appear to be necessary for fusion. This region corresponds to the epitope for anti-peripherin/rds monoclonal antibody 2B6, which is shown to partially inhibit peripherin/rds mediated membrane fusion.