You spend months, if not years, plugging away at what seems like an interminable project in the marathon that is your doctoral program. But oh, the feeling of absolute accomplishment once you complete it and stand before your committee to defend it! Finally, you have crossed the finish line and move on to postdoctoral life. Most of the time, the dissertation is written in phases across a long period of time—time that is usually split among your other competing interests.
The co-localization of these macro-molecules within the nucleus and the modulation of hepatic apoB mRNA editing activity have been described following a variety of metabolic perturbations, but the mechanism that regulates editosome assembly is unknown. Ethanol stimulated apoB mRNA editing was associated with a 2- to 3-fold increase in ACF phosphorylation relative to that in control primary hepatocytes.
Significantly, phosphorylated ACF was restricted to nuclear extracts where it co-sedimented with 27S editing competent complexes. Two-dimensional phosphoamino acid analysis of ACF immunopurified from hepatocyte nuclear extracts demonstrated phosphorylation of serine residues that was increased by ethanol treatment.
Consequently, two apoB protein variants are expressed, full-length apoB and the truncated protein apoB48, both of which participate in lipid transport, but with markedly different roles as atherogenic risk factors 1. ACF is the mooring sequence-specific RNA binding protein apob editing services directs site-specific editing 6 — 9 Hepatic editing is modulated by fasting and refeeding in part due to an insulin-dependent increase in APOBEC-1 expression Hepatic editing is also regulated independently of changes in APOBEC-1 expression levels by developmental, hormonal and nutritional perturbations 17 — The mechanism for this form of editing activity regulation has not been defined, but involves the nuclear trafficking of editing factors 24 — Under in vitro editing conditions, 60S complexes reorganized to active 27S complexes on reporter RNAs 6 In support of trafficking as a regulatory mechanism, ethanol, insulin and thyroid hormone stimulation of hepatocyte editing activity were associated with an an apob editing services in nuclear localization of ACF 2429 Induction of editing by ethanol occurred within minutes 212329and withdrawal of the stimulus both reduced the abundance of ACF in the nucleus and suppressed editing activity 23 Ethanol induced editing is not dependent on de novo protein or RNA synthesis 33 suggesting that modulation of pre-existing editing factors is sufficient to support enhanced editing activity.
These observations substantiated the possibility that cytoplasmic 60S complexes may serve as a reservoir of editing factors necessary for rapid assembly of nuclear 27S editosomes. Protein phosphorylation is one of the most common protein modifications known and its importance in the regulation of protein activity has been well documented In fact phosphorylation has been implicated as having a role in apoB mRNA editing although its mechanism remains unclear PhosphoACF was only detected in the nucleus, and was selectively recovered with active 27S editosomes.
Protein phosphatase inhibitor studies suggest that protein phosphatase 1 is involved in regulating editing activity, ACF phosphorylation and ACF subcellular distribution. Louis, MO ad libitum and euthanized between 9 and 10 a. In vivo phosphorylation of ACF In vivo32P labeling was performed by intraperitoneal injection of rats with After 4 h, rats were sacrificed and hepatic cytoplasmic and nuclear extracts prepared.
Primary hepatocyte cultures in 60 mm dishes were incubated in phosphate-free Minimum Essential Eagle Media Sigma containing 0. Cultures were labeled for 4 h prior to extract preparation. Subcellular extract preparations Livers were perfused in situ with 0.
Cytoplasmic and nuclear extracts 37 were supplemented with 10 mM NaF and fractionated through glycerol gradients. Protein phosphatase inhibitor studies Primary hepatocytes were treated for 6 h with cantharidin, endothall or okadaic acid at concentrations encompassing their respective in vivo IC50 values as described by the manufacturer Calbiochem, La Jolla, CA and their suggested references.
The effect of nM cantharidin on ACF subcellular distribution was investigated by treating primary hepatocyte cultures for 4 h followed by nuclear and cytoplasmic extract preparation.
Cultures were harvested and subcellular extracts prepared. Immuno-absorbed material to be treated with alkaline phosphatase was washed with 50 mM Tris, pH 8. ACF-antibody complexes were eluted with 3 M sodium thiocyanate, acetone precipitated and analyzed by For ACF immunoprecipitation from gradient fractions an equal volume of pooled gradient fractions from sedimentation zones of interest were reacted with sub-saturating amounts of ACF CT antibody to ensure that the recovery of phosphoACF was not simply a reflection of ACF abundance in each zone.
Proteins were resolved through a Two-dimensional phosphoamino acid analysis ACF was immunopurified using the ACF CT antibody from 27S enriched nuclear extracts of 32P-labeled primary hepatocytes cultured in basal media 0.
Lyophilized hydrolysates were spiked with unlabeled phosphoserine, phosphothreonine and phosphotyrosine Sigma and resolved on thin layer chromatography plates Merck, Germany by 2D electrophoresis using an HTLE peptide mapping system CBS Scientific Co.
Del Mar, CA The migration of the unlabeled standards and ACF radiolabeled amino acid s were visualized by ninhydrin staining and PhosphorImager Scanning densitometry, respectively. As an interaction between ACF and APOBEC-1 is critical for editing activity 42 we investigated the effect of alkaline phosphatase treatment on in vitro editing activity of hepatocyte extracts.
Although our aforementioned data are consistent with this possibility, the low level of endogenous APOBEC-1 expression has prohibited in vivo validation of this finding. Furthermore, in vivo studies carried out in our laboratory using exogenous rat APOBEC-1 were unable to detect APOBEC-1 phosphorylation data not shown and the phosphorylation sites suggested by the authors are not conserved between rat and human To evaluate whether endogenous ACF is phosphorylated in vivo, rats were radiolabeled for 4 h via an intraperitoneal injection of orthophosphoric acid in HEPES-buffered saline.
A single band was detected by autoradiography that super-imposed with ACF-specific immunoblot reactivity. To investigate the complexity of physiologically relevant ACF phosphorylation sites, liver extracts prepared from control rats were resolved by equilibrium 2D gel electrophoresis and immunoblotted with ACF NT antibody The predicted isoelectric point pI of ACF is 8.
The major isoform migrated at pI 8.Anti-Apolipoprotein B Antibody is an antibody against Apolipoprotein B for use in IP & WB. Find MSDS or SDS, a COA, data sheets and more information. blockers and custom services.
The shorter apoB protein is produced after RNA editing of the apoB transcript at residue (CAA->UAA), resulting in the creation of a stop codon.
Abstract. Apolipoprotein B (apoB) mRNA editing is a nuclear event that minimally requires the RNA substrate, APOBEC-1 and APOBEC-1 Complementation Factor (ACF).The co-localization of these macro-molecules within the nucleus and the modulation of hepatic apoB mRNA editing activity have been described following a variety of metabolic perturbations, but the mechanism that regulates editosome.
Developmental regulation of the catalytic subunit of the apolipoprotein B mRNA editing enzyme apoB mRNA editing in both human fetal small intestine Research Interchange tissue procurement services or during the course of surgical intestinal resection. The. Creative Biogene provides kits, reagents, and services that help researchers explore questions about gene discovery, regulation, and function More Creative Biogene offers challenging job opportunities for people looking for career growth in an entrepreneurial environment that .
ApoB minigenes were constructed by ligating the adenovirus major late promoter to a fragment of apoB DNA containing the editing site and used for the transcription-editing reaction.
We defined the sequence specificity of the editing reaction using site-specific single and multiple base mutants constructed by the polymerase chain reaction. The APOB gene provides instructions for making two versions of the apolipoprotein B protein, a short version called apolipoprotein B and a longer version known as apolipoprotein B Both of these proteins are components of lipoproteins, which are particles that carry fats and fat-like substances (such as cholesterol) in the blood.