Differences in effectiveness and use of laparoscopic surgery in locally advanced colon cancer patients.

Patients with locally advanced colon cancer have worse outcomes. Guidelines of various organizations are conflicting about the use of laparoscopic colectomy (LC) in locally advanced colon cancer. We determined whether patient outcomes of LC and open colectomy (OC) for locally advanced (T4) colon cancer are comparable in all colon cancer patients, T4a versus T4b patients, obese versus non-obese patients, and tumors located in the ascending, descending, and transverse colon. We used data from the 2013-2015 American College of Surgeons' National Surgical Quality Improvement Program. Patients were diagnosed with nonmetastatic pT4 colon cancer, with or without obstruction, and underwent LC (n = 563) or OC (n = 807). We used a composite outcome score (mortality, readmission, re-operation, wound infection, bleeding transfusion, and prolonged postoperative ileus); length of stay; and length of operation. Patients undergoing LC exhibited a composite outcome score that was 9.5% lower (95% CI - 15.4; - 3.5) versus those undergoing OC. LC patients experienced a 11.3% reduction in postoperative ileus (95% CI - 16.0; - 6.5) and an average of 2 days shorter length of stay (95% CI - 2.9; - 1.0). Patients undergoing LC were in the operating room an average of 13.5 min longer (95% CI 1.5; 25.6). We found no evidence for treatment heterogeneity across subgroups (p > 0.05). Patients with locally advanced colon cancer who receive LC had better overall outcomes and shorter lengths of stay compared with OC patients. LC was equally effective in obese/nonobese patients, in T4a/T4b patients, and regardless of the location of the tumor.

Differences in Effectiveness and Use of Robotic Surgery in Patients Undergoing Minimally Invasive Colectomy.

BACKGROUND: We compared patient outcomes of robot-assisted surgery (RAS) and laparoscopic colectomy without robotic assistance for colon cancer or nonmalignant polyps, comparing all patients, obese versus nonobese patients, and male versus female patients. METHODS: We used the 2013-2015 American College of Surgeons National Surgical Quality Improvement Program data to examine a composite outcome score comprised of mortality, readmission, reoperation, wound infection, bleeding transfusion, and prolonged postoperative ileus. We used propensity scores to assess potential heterogeneous treatment effects of RAS by patient obesity and sex. RESULTS: In all, 17.1% of the 10,844 of patients received RAS. Males were slightly more likely to receive RAS. Obese patients were equally likely to receive RAS as nonobese patients. In comparison to nonRAS, RAS was associated with a 3.1% higher adverse composite outcome score. Mortality, reoperations, wound infections, sepsis, pulmonary embolisms, deep vein thrombosis, myocardial infarction, blood transfusions, and average length of hospitalization were similar in both groups. Conversion to open surgery was 10.1% lower in RAS versus nonRAS patients, but RAS patients were in the operating room an average of 52.4 min longer. We found no statistically significant differences (p > 0.05) by obesity status and gender. CONCLUSIONS: Worse patient outcomes and no differential improvement by sex or obesity suggest more cautious adoption of RAS.

The utility of Google Trends data to examine interest in cancer screening.

OBJECTIVES: We examined the utility of January 2004 to April 2014 Google Trends data from information searches for cancer screenings and preparations as a complement to population screening data, which are traditionally estimated through costly population-level surveys. SETTING: State-level data across the USA. PARTICIPANTS: Persons who searched for terms related to cancer screening using Google, and persons who participated in the Behavioral Risk Factor Surveillance System (BRFSS). PRIMARY AND SECONDARY OUTCOME MEASURES: (1) State-level Google Trends data, providing relative search volume (RSV) data scaled to the highest search proportion per week (RSV100) for search terms over time since 2004 and across different geographical locations. (2) RSV of new screening tests, free/low-cost screening for breast and colorectal cancer, and new preparations for colonoscopy (Prepopik). (3) State-level breast, cervical, colorectal and prostate cancer screening rates. RESULTS: Correlations between Google Trends and BRFSS data ranged from 0.55 for ever having had a colonoscopy to 0.14 for having a Pap smear within the past 3 years. Free/low-cost mammography and colonoscopy showed higher RSV during their respective cancer awareness months. RSV for Miralax remained stable, while interest in Prepopik increased over time. RSV for lung cancer screening, virtual colonoscopy and three-dimensional mammography was low. CONCLUSIONS: Google Trends data provides enormous scientific possibilities, but are not a suitable substitute for, but may complement, traditional data collection and analysis about cancer screening and related interests.

Postoperative 30-day mortality in patients undergoing surgery for colorectal cancer: development of a prognostic model using administrative claims data.

PURPOSE: To develop a prognostic model to predict 30-day mortality following colorectal cancer (CRC) surgery using the Surveillance, Epidemiology, and End Results (SEER)-Medicare-linked data and to assess whether race/ethnicity, neighborhood, and hospital characteristics influence model performance. METHODS: We included patients aged 66 years and older from the linked 2000-2005 SEER-Medicare database. Outcome included 30-day mortality, both in-hospital and following discharge. Potential prognostic factors included tumor, treatment, sociodemographic, hospital, and neighborhood characteristics (census-tract-poverty rate). We performed a multilevel logistic regression analysis to account for nesting of CRC patients within hospitals. Model performance was assessed using the area under the receiver operating characteristic curve (AUC) for discrimination and the Hosmer-Lemeshow goodness-of-fit test for calibration. RESULTS: In a model that included all prognostic factors, important predictors of 30-day mortality included age at diagnosis, cancer stage, and mode of presentation. Race/ethnicity, census-tract-poverty rate, and hospital characteristics were independently associated with 30-day mortality, but they did not influence model performance. Our SEER-Medicare model achieved moderate discrimination (AUC = 0.76), despite suboptimal calibration. CONCLUSIONS: We developed a prognostic model that included tumor, treatment, sociodemographic, hospital, and neighborhood predictors. Race/ethnicity, neighborhood, and hospital characteristics did not improve model performance compared with previously developed models.

Hospital and geographic variability in two colorectal cancer surgery outcomes: complications and mortality after complications.

BACKGROUND: The purpose of this study was to describe hospital and geographic variation in 30-day risk of surgical complications and death among colorectal cancer (CRC) patients and the extent to which patient-, hospital-, and census-tract-level characteristics increased risk of these outcomes. METHODS: We included patients at least 66 years old with first primary stage I-III CRC from the 2000-2005 National Cancer Institute's Surveillance, Epidemiology, and End Results data linked with 1999-2005 Medicare claims. A multilevel, cross-classified logistic model was used to account for nesting of patients within hospitals and within residential census tracts. Outcomes were risk of complications and death after a complication within 30 days of surgery. RESULTS: Data were analyzed for 35,946 patients undergoing surgery at 1,222 hospitals and residing in 12,187 census tracts; 27.2 % of patients developed complications, and of these 13.4 % died. Risk-adjusted variability in complications across hospitals and census tracts was similar. Variability in mortality was larger than variability in complications, across hospitals and across census tracts. Specific characteristics increased risk of complications (e.g., census-tract-poverty rate, emergency surgery, and being African-American). No hospital characteristics increased complication risk. Specific characteristics increased risk of death (e.g. census-tract-poverty rate, being diagnosed with colon (versus rectal) cancer, and emergency surgery), while hospitals with at least 500 beds showed reduced death risk. CONCLUSIONS: Large, unexplained variations exist in mortality after surgical complications in CRC across hospitals and geographic areas. The potential exists for quality improvement efforts targeted at the hospital and/or census-tract levels to prevent complications and augment hospitals' ability to reduce mortality risk.

Dextran sodium sulfate inhibition of real-time polymerase chain reaction amplification: a poly-A purification solution.

BACKGROUND: Dextran sulfate sodium (DSS) induces experimental colitis and promotes colitis-associated cancer in rodents. Here we document potent inhibition of real-time quantitative polymerase chain reaction (qPCR) using cDNA from DSS-exposed mouse tissues, which complicates gene expression analysis. METHODS: We characterize DSS inhibition of qPCR in-vitro and in a wide array of murine tissues following ingestion of DSS. We examine different approaches to RNA purification prior to cDNA synthesis in order to optimize real-time polymerase chain reaction amplification and gene expression analysis. RESULTS: DSS inhibits qPCR amplification of cDNA between 1 and 10 nM. Orally administered DSS interferes with qPCR amplification of cDNA derived from multiple tissues. Poly-A purification of DSS-exposed RNA allows reliable and cost-effective gene expression analysis in DSS-exposed tissue. CONCLUSIONS: DSS is a potent inhibitor of real-time qPCR amplification and interferes with tissue-specific gene expression analysis in DSS-exposed mice. Poly-A purification of tissue-derived RNA results in reliable and cost-effective gene expression analysis in DSS-exposed mice.

Disruption of hedgehog signalling in ApoE - /- mice reduces plasma lipid levels, but increases atherosclerosis due to enhanced lipid uptake by macrophages.

Embryonic pathways are often re-expressed in adult pathology. Here we investigated the role of the morphogen hedgehog (hh), which we found to be re-expressed in atherosclerotic plaques. Male ApoE - /- mice were treated for 12 weeks with an anti-hh antibody (5E1) or a control IgG (1E6) starting at the age of 6 or 18 weeks. Inhibition of hh signalling induced a significant increase in total plaque area in the aortic arch, a result of an increase (54% and 36%, respectively) in the area of advanced plaques (atheromata). In mice treated with anti-hh, plaques contained large (18-35% > ctrl), lipid-filled, sometimes multinucleated macrophage foam cells. Plasma cholesterol levels decreased after anti-hh treatment. In bone marrow-derived macrophages, foam cell formation was enhanced after inhibition of hh signalling. Anti-hh treatment caused a 54-75% increase in early oxLDL uptake (10-240 min), which was scavenger receptor-mediated. After 3-24 h of oxLDL incubation, intense Oil red O staining as well as increased amounts of cholesterol esters were present in these macrophages after anti-hh treatment. Activation of the HH-signalling cascade by recombinant Shh induced a decrease in oxLDL uptake. Here we show that the hh-signalling pathway is one of the morphogenic pathways that regulate plasma lipid levels and atherosclerosis development and progression.

Apo(a) promotes thrombosis in a vascular injury model by a mechanism independent of plasminogen.

OBJECTIVE: Structural similarity between apolipoprotein(a) [apo(a)], the unique apoprotein of lipoprotein(a), and plasminogen (Plg), the zymogen for plasmin, results in inhibition of functions of Plg by apo(a) in vitro. The objective of this study was to evaluate the interaction of Plg and apo(a) in vivo. METHODS AND RESULTS: Vascular injury was induced in the carotid artery with a perivascular cuff in: (i) wild-type (WT); (ii) Plg deficient (Plg-/-); (iii) apo(a) (6 KIV construct) transgenic [apo(a)tg]; and (iv) apo(a) transgenic and Plg deficient [apo(a):Plg-/-] mice. At 10 days after cuff placement, the media and adventitia area were increased in the injured carotids compared with the uninjured carotids, and collagen deposition was greater in apo(a)tg, Plg-/- and apo(a):Plg-/- mice compared with WT mice. The incidence of a thrombus was greater (P < 0.05) in apo(a):Plg-/- mice (83%) than WT (20%), Plg-/- (12%), and apo(a)tg mice (9%). In the thrombi from apo(a)tg and apo(a):Plg-/- mice, P-selectin and von Willebrand factor immunostaining, indicating a platelet-rich thrombi, was greater than in WT and Plg-/- mice. The presence of fibrin(ogen) in the thrombi was greater in Plg-/- and apo(a):Plg-/- mice than apo(a)tg and WT mice. Of the four genotypes, only the apo(a):Plg-/- mice had both increased platelet and increased fibrin(ogen) deposition. CONCLUSIONS: The major finding of this study is the high incidence of thrombosis after vascular injury in apo(a)transgenic mice in a Plg deficient background, providing strong evidence for a prothrombotic role of apo(a) independent of Plg in vivo.

ARCD-1, an apobec-1-related cytidine deaminase, exerts a dominant negative effect on C to U RNA editing.

Mammalian apolipoprotein B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme containing a single catalytic subunit, apobec-1. We have characterized an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously expressed; phylogenetic analysis reveals it to be a distant member of the RNA editing family. Recombinant ARCD-1 demonstrates cytidine deaminase and apoB RNA binding activity but does not catalyze C to U RNA editing, either in vitro or in vivo. Although not competent itself to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to both the nucleus and cytoplasm of transfected cells. Together, the data suggest that ARCD-1 is a novel cytidine deaminase that interacts with apobec-1 and ACF to inhibit apoB mRNA editing, possibly through interaction with other protein components of the apoB RNA editing holoenzyme.

Isolation, characterization and developmental regulation of the human apobec-1 complementation factor (ACF) gene.

Mammalian apolipoprotein B (apo B) mRNA undergoes site-specific C to U deamination which is mediated by a multicomponent enzyme complex containing a minimal core composed of apobec-1 and a complementation factor, ACF. We have isolated and characterized the human ACF gene and examined its tissue-specific and developmental expression. The ACF gene spans approximately 80 kb and contains 15 exons, three of which are non-coding. Multiple alternative splice acceptor sites were found, generating at least nine different transcripts. Of these, the majority (approximately 75-89%) encode functional protein. In order to examine the role of ACF mRNA expression in the regulation of apo B mRNA editing, we examined a panel of fetal intestinal and hepatic mRNAs as well as RNA from an intestinal cell line. A developmental increase in C to U RNA editing has been previously noted in the human intestine. In both instances, the pattern of alternative splicing and overall abundance of ACF mRNA was relatively constant during development in both liver and small intestine. Taken together, the data demonstrate a complex pattern of differential, tissue-specific splicing of ACF mRNA, but suggest that other mechanisms are responsible for the developmental increase noted in intestinal apo B mRNA editing in humans.

Mutagenesis of apobec-1 complementation factor reveals distinct domains that modulate RNA binding, protein-protein interaction with apobec-1, and complementation of C to U RNA-editing activity.

C to U editing of apolipoprotein B (apoB) RNA requires a multicomponent holoenzyme complex in which minimal constituents include apobec-1 and apobec-1 complementation factor (ACF). We have examined the predicted functional domains in ACF in binding apoB RNA, interaction with apobec-1, and complementation of RNA editing. We demonstrate that apoB RNA binding and apobec-1-interacting domains are defined by two partially overlapping regions containing the NH(2)-terminal RNA recognition motifs of ACF. Both apoB RNA binding and apobec-1 interaction are required for editing complementation activity. ACF is a nuclear protein that upon cotransfection with apobec-1 results in nuclear colocalization and redistribution of apobec-1 from the cytoplasm. ACF constructs with deletions or mutations in the putative nuclear localization signal (NLS) still localize in the nucleus of transfected cells but do not colocalize with apobec-1, the latter remaining predominantly cytoplasmic. These observations suggest that the putative NLS motif in ACF is not responsible for its nucleo-cytoplasmic trafficking. By contrast, protein-protein interaction is important for the nuclear import of apobec-1. Taken together, these data suggest that functional complementation of C to U RNA editing by apobec-1 involves the NH(2)-terminal 380 residues of ACF.

Novel role for RNA-binding protein CUGBP2 in mammalian RNA editing. CUGBP2 modulates C to U editing of apolipoprotein B mRNA by interacting with apobec-1 and ACF, the apobec-1 complementation factor.

Mammalian apolipoprotein B (apoB) mRNA editing is mediated by a multicomponent holoenzyme containing apobec-1 and ACF. We have now identified CUGBP2, a 54-kDa RNA-binding protein, as a component of this holoenzyme. CUGBP2 and ACF co-fractionate in bovine liver S-100 extracts, and addition of recombinant apobec-1 leads to assembly of a holoenzyme. Immunodepletion of CUGBP2 co-precipitates ACF, and these proteins co-localize the nucleus of transfected cells, suggesting that CUGBP2 and ACF are bound in vivo. CUGBP2 binds apoB RNA, specifically an AU-rich sequence located immediately upstream of the edited cytidine. ApoB RNA from McA cells, bound to CUGBP2, was more extensively edited than the unbound fraction. However, addition of recombinant CUGBP2 to a reconstituted system demonstrated a dose-dependent inhibition of C to U RNA editing, which was rescued with either apobec-1 or ACF. Antisense CUGBP2 knockout increased endogenous apoB RNA editing, whereas antisense knockout of either apobec-1 or ACF expression eliminated apoB RNA editing, establishing the absolute requirement of these components of the core enzyme. These data suggest that CUGBP2 plays a role in apoB mRNA editing by forming a regulatory complex with the three components of the minimal editing enzyme, apobec-1, ACF, and apoB RNA.

Molecular mechanisms of apolipoprotein B mRNA editing.

A site-specific post-transcriptional cytidine to uridine deamination reaction is responsible for the production of apolipoprotein B48 in the mammalian small intestine. The molecular machinery responsible for apolipoprotein B RNA editing consists of apobec-1, an RNA-specific cytidine deaminase that functions in conjunction with a recently identified protein referred to as ACF/ASP. These proteins together represent the minimal editing enzyme, although other proteins may associate with the enzyme complex. Apobec-1 is a member of a supergene family of cytidine deaminases, with several homologs recently identified in the human genome. ACF/ASP is novel, and emerging information reveals interesting clues to its role in the apolipoprotein B RNA editing enzyme complex.

Identification of GRY-RBP as an apolipoprotein B RNA-binding protein that interacts with both apobec-1 and apobec-1 complementation factor to modulate C to U editing.

C to U editing of apolipoprotein B (apoB) mRNA involves the interaction of a multicomponent editing enzyme complex with a requisite RNA sequence embedded within an AU-rich context. This enzyme complex includes apobec-1, an RNA-specific cytidine deaminase, and apobec-1 complementation factor (ACF), a novel 65-kDa RNA-binding protein, that together represent the minimal core of the editing enzyme complex. The precise composition of the holo-enzyme, however, remains unknown. We have previously isolated an enriched fraction of S100 extracts, prepared from chicken intestinal cells, that displays apoB RNA binding and which, following supplementation with apobec-1, permits efficient C to U editing. Peptide sequencing of this most active fraction reveals the presence of ACF as well as GRY-RBP, an RNA-binding protein with approximately 50% homology to ACF. GRY-RBP was independently isolated from a two-hybrid screen of chicken intestinal cDNA. GRY-RBP binds to ACF, to apobec-1, and also binds apoB RNA. Experiments using recombinant proteins demonstrate that GRY-RBP binds to ACF and inhibits both the binding of ACF to apoB RNA and C to U RNA editing. This competitive inhibition is rescued by addition of ACF, suggesting that GRY-RBP binds to and sequesters ACF. As further evidence of the role of GRY-RBP, rat hepatoma cells treated with an antisense oligonucleotide to GRY-RBP demonstrated an increase in C to U editing of endogenous apoB RNA. ACF and GRY-RBP colocalize in the nucleus of transfected cells and, in cotransfection experiments with apobec-1, each appears to colocalize in a predominantly nuclear distribution. Taken together, the results indicate that GRY-RBP is a member of the ACF gene family that may function to modulate C to U RNA editing through binding either to ACF or to apobec-1 or, alternatively, to the target RNA itself.

Escherichia coli sepsis increases hepatic apolipoprotein B secretion by inhibiting degradation.

Sepsis leads to hypertriglyceridemia in both humans and animals. Previously, we reported that plasma very low density lipoprotein apolipoprotein (apo) B and hepatic production of apoB increased during Escherichia coli sepsis. The present experiments were undertaken to determine whether the altered hepatic secretion of apoB was associated with an increase in synthesis or a decrease in degradation rate. Sepsis was induced in male, Lewis rats (225-275 g) by intravenous injection of 3.8 x 10(8) live E. coli colonies/100 g body. Twenty-four hours later rats were sacrificed, and primary hepatocytes were prepared and incubated overnight with 35S-methionine. Hepatocytes from E. coli-treated rats secreted twice as much apoB-48 and total apoB than the hepatocytes from control rats. Escherichia coil sepsis increased cellular triglyceride mass by 86%, which was due to a stimulation in triglyceride synthesis from newly synthesized fatty acids, measured by 3H2O incorporation into triglycerides. The apoB synthesis rate, apoB mRNA levels, and apoB mRNA editing were not altered during E. coil sepsis. The pulse-chase experiments showed that the rate of apoB degradation decreased in E. coli-treated rats. These findings demonstrate that the secretion of apoB is regulated posttranslationally during E. coli sepsis by decreasing the degradation of newly synthesized apoB, which contributes to the development of hypertriglyceridemia.

Homeodomain factor Nkx2-3 controls regional expression of leukocyte homing coreceptor MAdCAM-1 in specialized endothelial cells of the viscera.

Regulated emigration of blood-borne leukocytes plays a defining role in lymphoid organ development, immune surveillance, and inflammatory responses. We report here that mice deficient in the homeobox gene Nkx2-3, expressed in developing visceral mesoderm, show a complex intestinal malabsorption phenotype and striking abnormalities of gut-associated lymphoid tissue and spleen suggestive of deranged leukocyte homing. Mutant Peyer's patches were reduced in number and size, intestinal villi contained few IgA(+) plasma cells, and mutant spleens were small and often atrophic, showing fused periarterial lymphoid sheaths, partially merged T and B cell zones, an absent marginal zone, and a dearth of macrophages in red pulp. Semiquantitative RT-PCR analysis and immunohistochemistry revealed down-regulation of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in endothelial cells in which Nkx2-3 is normally expressed. MAdCAM-1 is a member of the immunoglobulin superfamily, acting as an endothelial cell ligand for leukocyte homing receptors L-selectin and alpha4beta7 integrin. Our data suggest a role for a homeodomain factor in establishing the developmental and positional cues in endothelia that regulate leukocyte homing through local control of cellular adhesion and identify MAdCAM-1 as a candidate target gene of Nkx2-3.

APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and presecretory degradation.

Apolipoprotein (apo)B circulates in two distinct forms, apoB100 and apoB48. Human liver secretes apoB100, the product of a large mRNA encoding 4536 residues. The small intestine of all mammals secretes apoB48, which arises following C-to-U deamination of a single cytidine base in the nuclear apoB transcript, introducing a translational stop codon. This process, referred to as apoB RNA editing, operates through a multicomponent enzyme complex that contains a single catalytic subunit, apobec-1, in addition to other protein factors that have yet to be cloned. ApoB RNA editing also exhibits stringent cis-acting requirements that include both structural and sequence-specific elements-specifically efficiency elements that flank the minimal cassette, an AU-rich RNA context, and an 11-nucleotide mooring sequence-located in proximity to a suitably positioned (usually upstream) cytidine. C-to-U RNA editing may become unconstrained under circumstances where apobec-1 is overexpressed, in which case multiple cytidines in apoB RNA, as well as in other transcripts, undergo C-to-U editing. ApoB RNA editing is eliminated following targeting of apobec-1, establishing that there is no genetic redundancy in this function. Under physiological circumstances, apoB RNA editing exhibits developmental, hormonal, and nutritional regulation, in some cases related to transcriptional regulation of apobec-1 mRNA. ApoB and the microsomal triglyceride transfer protein (MTP) are essential for the assembly and secretion of apoB-containing lipoproteins. MTP functions by transferring lipid to apoB during its translation and by transporting triglycerides into the endoplasmic reticulum to form apoB-free lipid droplets. These droplets fuse with nascent apoB-containing particles to form mature, very low-density lipoproteins or chylomicrons. In cultured hepatic cells, lipid availability dictates the rate of apoB production. Unlipidated or underlipidated forms of apoB are subjected to presecretory degradation, a process mediated by retrograde transport from the lumen of the endoplasmic reticulum to the cytosol, coupled with multiubquitination and proteasomal degradation. Although control of lipid secretion in vivo is primarily achieved at the level of lipoprotein particle size, regulation of apoB production by presecretory degradation may be relevant in some dyslipidemic states.

An AU-rich sequence element (UUUN[A/U]U) downstream of the edited C in apolipoprotein B mRNA is a high-affinity binding site for Apobec-1: binding of Apobec-1 to this motif in the 3' untranslated region of c-myc increases mRNA stability.

Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a K(d) of approximately 435 nM. A series of AU-rich templates was used to identify a high-affinity ( approximately 50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3' untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA-specific cytidine deaminase.