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N O Davidson

Publications and source records attributed to N O Davidson.

At least 19 recordsLinked to original sources
PubMed2005

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.

Animals↗
PubMed2001

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.

APOBEC-1 Deaminase↗
PubMed2001

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.

APOBEC-1 Deaminase↗
PubMed2001

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.

APOBEC-1 Deaminase↗
PubMed2001

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.

Apolipoproteins B↗
PubMed2001

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.

APOBEC-1 Deaminase↗
PubMed2000

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.

APOBEC-1 Deaminase↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

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.

3' Untranslated Regions↗
PubMed2000

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.

Animals↗
PubMed1999

Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.

We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.

APOBEC-1 Deaminase↗
PubMed1999

Sequence elements required for apolipoprotein B mRNA editing enhancement activity from chicken enterocytes.

Mammalian intestinal apolipoprotein B (apoB) mRNA edits codon 2153 from CAA in apoB100 mRNA to a stop codon (UAA) in apoB48 mRNA. By contrast, chicken intestinal apoB mRNA contains a CAA codon at the corresponding site, but is not edited. Chicken enterocyte S100 extracts fail to edit mammalian apoB RNA, but contain factor(s) which enhance the mammalian enterocytes editing activity. By converting the chicken apoB mooring sequences to the conserved mammalian sequences, the study confirmed that this 11-nucleotide stretch was necessary and sufficient for minimal RNA editing. Using rat and chicken apoB chimeric constructs, the study revealed that mammalian apoB sequences were required for editing enhancement. In concert with the 29-nucleotide conserved cassette, the 5' rat apoB element (nucleotides 6615-6629) increased editing at C-6666, and was necessary for editing enhancement of chicken enterocyte S100 extracts. Similarly, the 3' rat apoB element (nucleotides 6726-6752) was required for editing enhancement of chicken enterocyte S100 extracts, but to a lesser extent in efficiency, compared to the 5' region. In conclusion, this study identified the sequences required for editing enhancement activity from chicken enterocyte S100 extracts.

Animals↗
PubMed1999

Psoriasis upregulated phorbolin-1 shares structural but not functional similarity to the mRNA-editing protein apobec-1.

Earlier studies of psoriatic and normal primary keratinocytes treated with phorbol 12-myristate-1-acetate identified two low-molecular-weight proteins, termed phorbolin-1 (20 kDa; pI 6.6) and phorbolin-2 (17.6 kDa; pI 6.5). As a first step towards elucidating the role of these proteins in psoriasis, we report here the molecular cloning and chromosomal mapping of phorbolin-1 and a related cDNA that codes for a protein exhibiting a similar amino acid sequence. The phorbolins were mapped to position 22q13 immediately centromeric to the c-sis proto-oncogene. Transient expression of the phorbolin-1 cDNA in COS cells and by in vitro transcription/translation, yielded polypeptides that comigrated with phorbolins-1 and -2. Comparative sequence analysis revealed 22% overall identity and a similarity of 44% of the phorbolins to apobec-1, the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme; however, recombinant-expressed phorbolin-1 exhibited no cytidine deaminase activity, using either a monomeric nucleoside or apolipoprotein B cRNA as substrate, and failed to bind an AU-rich RNA template. Whereas the precise function of the phorbolins remains to be elucidated, the current data suggest that it is unlikely to include a role in the post-transcriptional modification of RNA in a manner analogous to that described for apobec-1.

APOBEC-1 Deaminase↗
PubMed1998

Apolipoprotein(a) synthesis and secretion from hepatoma cells is coupled to triglyceride synthesis and secretion.

Apolipoprotein(a) (apo(a)) is synthesized and secreted from liver cells and represents one of the two major protein components of the atherogenic lipoprotein, Lp(a). Little is known, however, of the factors that regulate the secretion of this protein. We have undertaken an analysis of the response to oleate supplementation in stable clones of HepG2 and McA-RH7777 cells expressing either a 6 K-IV or 17 K-IV isoform of apo(a). These cell lines were examined by pulse-chase analysis and each demonstrated an increase (range 2-6-fold) in apo(a) secretion following supplementation with 0.8 mM oleate. Microsomal membranes, prepared from HepG2 cells expressing a 6 K-IV apo(a) isoform, demonstrated that oleate supplementation increased the apparent protection of apo(a) from protease digestion, suggesting that alterations in the translocation efficiency of apo(a) may accompany the addition of oleate. Cells incubated with brefeldin A demonstrated increased recovery of the precursor form of apo(a) with oleate supplementation, suggesting that alterations in post-translational degradation may also contribute to the observed increase in apo(a) secretion following oleate addition. To further characterize the oleate-dependent increase in apo(a) secretion, cells were incubated with an inhibitor of the microsomal triglyceride transfer protein. These experiments demonstrated a dose-dependent decrease in apo(a) secretion from both cell lines. Furthermore, addition of either the microsomal triglyceride transfer protein inhibitor or triacsin C, an inhibitor of acyl-CoA synthase, completely abrogated the oleate-dependent increase in apo(a) secretion. Taken together, these data provide evidence that apo(a) secretion from hepatoma cells may be linked to elements of cellular triglyceride assembly and secretion.

Apolipoproteins↗