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Lymphatic transport of lipids in apobec-l knockout mice
A662 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
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3624
CLONING AND CHARACTERIZATION OF ARP-l, A NOVEL MEMBER OF THE RNA BINDING CYTIDINE DEAMINASE SUPERGENE FAMILY. V. S. Sankaranand, Shrikant Anant, Susan Kennedy, Nicholas O. Davidson, Washington Univ, St. Louis, MO. Post-transcriptional RNA editing is a process by which the primary sequence encoded by the genomic template is altered, giving rise to an RNA transcript that differs from the structural gene. Two major forms of RNA editing exist in mammals. These are represented by C to U editing, for which the prototype is apolipoprotein B (apoB) mRNA, and A to I editing, which occurs in the calcium gated glutamate receptor subunits and the serotonin 2C receptor. C to U editing of apoB mRNA is mediated by a cytidine deaminase with RNA specificity, referred to as apobec-I and which in humans is expressed exclusively in the luminal GI tract. The sequence spanning the catalytic site of apobec-l was used to screen databases in combination with degenerate PCR to identify and clone candidate genes from human cDNA libraries. These approaches led to the identification of a novel gene, ARP-I, located on human chromosome 6 which demonstrates ~40% identity to apobec-I. ARP-I mRNA is expressed abundantly in skeletal muscle, heart, kidney and small intestine. Its cellular localization, like that of other human cytidine dearninases, is both nuclear and cytoplasmic. The zinc-coordinating residues previously identified in apobec-I span the catalytic site and are required for cytidine deaminase activity. In addition, we have demonstrated directly that recombinant ARP-I exhibits cytidine deaminase activity. Like other cytidine deaminases, enzymatic activity of ARP-I is inhibited by THU and 1,10 phenanthroline. Thus, ARP-I is an authentic cytidine dearninase. In order to determine whether ARP-I is directed to RNA substrates, we performed UV cross linking with different candidate RNA templates, including a 105 nt apo B RNA containing the minimal cis-acting elements required for C to U editing. ARP-l binds apo B RNA with high affinity and was not competed by the addition of apobec-Lsuggesting that ARP-I and apobec-I bind different sites on the apo B RNA transcript. We examined whether ARP-I mediates C to U editing of the apo B RNA using recombinant ARP-I and a source of auxiliary editing activity. ARP-I, over a 20-fold concentration range demonstrated no C to U conversion of an apo B RNA template. Interestingly, the addition of ARP-I in trans to in vitro editing reactions containing apobec-I and auxiliary factors demonstrated a dosedependent inhibition of C to U editing of the apo B RNA. These findings suggest that ARP-l may play a regulatory role in restricting apobec-I mediated RNA editing as well as in cellular nucleoside conservation.
PRE-CHYLOMICRON TRANSPORT VESICLES (PCTV) UTILIZE THE VESICLE ASSOCIATED MEMBRANE PROTEIN 1 (VAMP 1) AND SYNAPTOSOMAL ASSOCIATED PROTEIN 25 (SNAP 25) DEPENDENT PATHWAY. Shadab A. Siddiqi, Charles M. Mansbach, Univ of Tennessee, Memphis,
3623 LYMPHATIC TRANSPORT OF LIPIDS IN APOBEC-l KNOCK· OUT MICE. Vicky Lee Ng, Shuqin Zheng, Nicholas O. Davidson, Patrick Tso, Acad Hosp Med Ctr, Cincinnati, OH; Univ of Cincinnati Coli of Medicine, Cincinnati, OH; Washington Univ Sch of Medicine, St. Louis, MO. Background. Apolipoprotein B (apo B) is obligatory for chylomicron assembly. The apobec-I gene produces an editing enzyme, which generates a translational stop codon (by C to U editing of the apo B RNA) that truncates apo B100 to form apo B48, the predominant isoform in mammalian small intestine. Targeted disruption of apobec-I eliminates apo B48 production yielding mice (KO) that secrete exclusively apo BlOO. Aim. To examine intestinal lipid absorption and lymphatic transport in a congenic mouse strain (C57B1I6J) synthesizing only apo B48 (WT) or apo BlOO (KO) using the lymph fistula mouse model. Methods. Mesenteric lymph fistulae and gastroduodenal feeding tubes were placed in five WT and five KO mice. A lipid emulsion containing 41Jomol of [3H] labeled triolein, 0.78 IJomol of [14C] cholesterol, 0.78 IJomol of egg lecithin, and 5.7 IJomol of Na taurocholate in 0.3 ml of phosphate-buffered saline (pH 6.4) was infused at 0.3 mllh for 6h. Lymph was collected during fasting and then hourly for 6h during the lipid infusion. The animals were then re-anesthetized for open laparotomy and extraction of the small intestine to determine luminal lipid content. Results. There was no significant difference in lymph flow rate between the KO and WT mice. [3H] triolein output in lymph equilibrated by the 3'd h of lipid infusion in both groups. The 6th h output was 56.6%:!::1O% (% hourly infused) in the WT mice and 57.5%:!::8% in the KO mice. The 6th h lymphatic outputs of 4C] cholesterol for the WT and KO mice were 26.2%:!::3% and 29.6%:!::3% (% hourly infused) respectively. There were no significant differences in the lymphatic [3H] triolein or 4 C] cholesterol outputs between the two groups. Luminal [3H] triolein recovery was similar in both groups (3.2% of total dose infused) indicating normal digestion and uptake of triglyceride in the KO mice. Luminal cholesterol recovery was significantly higher in the KO mice (17.7%:!::3%) than in the WT mice (7.7%:!::I%) suggesting lower cholesterol uptake in the KO mice (p<0.05). Despite apparent lower cholesterol uptake in the KO mice, lymphatic cholesterol transport was comparable to that in WT mice. Conclusions. The data provide stong evidence that intestinal cells can assemble and secrete chylomicrons containing apo BlOO. From a functional perspective, apo BlOO appears to facilitate lipid transport as efficiently as apo B48, although the role of apo BlOO versus apo B48 in cholesterol uptake remains open. Work is currently underway to determine whether apo B100 chylomicrons are metabolized in a way that is similar to apo B48 chylomicrons.
e
e
TN. Triacylglycerol (TAG) transport from the endoplasmic reticulum (ER) to the Golgi in the intestine is mediated by the PCTV, derived from the ER, which is the rate limiting step in chylomicron export (AlP 276: G378, 1999). Targeting of vesicles requires interactions between vesicle integral membrane proteins, soluble NSF attachment protein receptor (v-SNARE) and target organelles (t-SNARE). The aim of the study was to identify the proteins involved in targeting the PCTV to intestinal cis Golgi. Methods: PCTV were isolated from rat intestine using step and continuous sucrose gradients (AlP 276: 0378, 1999). 2-D gels were performed on PCTV, ER, and cis Golgi membranes obtained by treating the organelles with 100 mM carbonate and collecting the membranes by centrifugation. The pi and Mr of proteins unique to PCTV were used to search a protein data base (Swiss Prot). Results: We identified 32 proteins unique to PCTV membranes. Of these, only one had been associated with secretory proteins, a 25 kDa protein with a pi of 8.86, consistent with VAMP. Western blots showed that both VAMP I and SNAP 25, a protein required for forming the v- and t-SNARE complex, were present in PCTV. Antibodies to VAMP I and SNAP 25 were separately incubated for I h with ER at 4°C. This reduced delivery of TAG from ER to the cis Golgi by 75 % in both cases. Pre-immune serum had no effect. cis Golgi were incubated separately with the immunogenic N-terminal 20 amino acids peptides of VAMP I and SNAP 25. Both peptides inhibited ER-to-Golgi TAG transport by >75 %. Triton-solubilized PCTV membrane proteins were electrophoresed on a native gel, transblotted onto nitrocellulose and the membrane probed with biotinylated cis Golgi membranes. Binding was noted at 120 kDa. Both VAMP I and SNAP 25 antibodies recognized the proteins at this band. On SDS-PAGE of PCTV membranes without boiling, both 120 and 25 kDa proteins were recognized by both antibodies. When the PCTV membranes were boiled 5 min in Laemmli's buffer, only the 25 kDa bands were identified. Conclusions: We conclude that both VAMP I and SNAP 25 are involved in PCTV targeting to the cis Golgi. This is consistent with the secretory protein pathway. We speculate that multimers of VAMP I and SNAP 25 are involved in targeting the PCTV to the intestinal cis Golgi.
3625 UP-REGULATION OF INDUCIBLE CO-STIMULATOR (ICOS) EXPRESSION AND ITS REGULATION OF CYTOKINE PRODUCTION IN INFLAMMATORY BOWEL DISEASE. Toshirou Sato, Mamoru Watanabe, Takanori Kanai, Akira Okazawa, Hanae Takagi, Atushi Nakazawa, Hiromasa Ishii, Richard Kroczek, Toshifumi Hibi, Sch of Med, Keio Univ, Tokyo, Japan; Keio Cancer Ctr, Tokyo, Japan; Robert-Koch Institute, Berlin, Germany. Background: Activation via CD28 on the surface of T cells provides a potent amplification signal for several cytokine productions. We have demonstrated that a ligand for CD28, CD86 expressed on colonic epithelial cells are involved in the activation of mucosal T cells (Gastroenterology 117: 536, 1999). Recently, a third member of CD28 family of molecules, inducible co-stimulator (ICOS) has been identified (Nature 397: 263, 1999). Herein, we investigated whether ICOS signaling participates in mucosal immune responses in ulcerative colitis (UC) and Crohn's disease (CD). Methods: I) Expression of ICOS on LPLs isolated from normal controls (NL), UC, and CD was assessed by flow cytometry. 2) Expression of ICOS on LPLs was also analyzed after stimulation with anti-CD3 mAb. 3) Tissue sections of intestinal specimens were stained CD28 or ICOS. 4) LPLs were stimulated by anti-CD3 in the presence of anti-CD28, antiICOS, or control mAbs. Culture supernatants were collected and assayed for various cytokines using ELISA. Results: I) ICOS expression was significantly up-regulated on freshly isolated LPLs from inflamed mucosa of UC and CD, as compared with those from NL. 2) LPLs from inflamed mucosa of UC and CD showed increased ICOS expression after stimulation with anti-CD3 mAb for 48 hrs. 3) Co-stimulation by anti-CD3 and anti-CD28 mAbs up-regulated the secretion of IL-2, IFN-g and IL-IO in LPL cultures from NL, UC and CD. However, co-stimulation by anti-CD3 and anti-ICOS mAbs failed to up-regulate the secretion of lL-2. Interestingly, co-stimulation by anti-CD3 and anti-ICOS mAbs predominantly induced IL-IO in LPL cultures from UC, but not those from NL and CD. Conclusion: These findings suggest that up-regulation of ICOS expression may be involved in chronic intestinal inflammation of inflammatory bowel disease, and that inhibition of ICOS signaling may lead to therapeutic effects in these patients.
GASTROENTEROLOGY Vol. 118, No.4
3622
3624
CLONING AND CHARACTERIZATION OF ARP-l, A NOVEL MEMBER OF THE RNA BINDING CYTIDINE DEAMINASE SUPERGENE FAMILY. V. S. Sankaranand, Shrikant Anant, Susan Kennedy, Nicholas O. Davidson, Washington Univ, St. Louis, MO. Post-transcriptional RNA editing is a process by which the primary sequence encoded by the genomic template is altered, giving rise to an RNA transcript that differs from the structural gene. Two major forms of RNA editing exist in mammals. These are represented by C to U editing, for which the prototype is apolipoprotein B (apoB) mRNA, and A to I editing, which occurs in the calcium gated glutamate receptor subunits and the serotonin 2C receptor. C to U editing of apoB mRNA is mediated by a cytidine deaminase with RNA specificity, referred to as apobec-I and which in humans is expressed exclusively in the luminal GI tract. The sequence spanning the catalytic site of apobec-l was used to screen databases in combination with degenerate PCR to identify and clone candidate genes from human cDNA libraries. These approaches led to the identification of a novel gene, ARP-I, located on human chromosome 6 which demonstrates ~40% identity to apobec-I. ARP-I mRNA is expressed abundantly in skeletal muscle, heart, kidney and small intestine. Its cellular localization, like that of other human cytidine dearninases, is both nuclear and cytoplasmic. The zinc-coordinating residues previously identified in apobec-I span the catalytic site and are required for cytidine deaminase activity. In addition, we have demonstrated directly that recombinant ARP-I exhibits cytidine deaminase activity. Like other cytidine deaminases, enzymatic activity of ARP-I is inhibited by THU and 1,10 phenanthroline. Thus, ARP-I is an authentic cytidine dearninase. In order to determine whether ARP-I is directed to RNA substrates, we performed UV cross linking with different candidate RNA templates, including a 105 nt apo B RNA containing the minimal cis-acting elements required for C to U editing. ARP-l binds apo B RNA with high affinity and was not competed by the addition of apobec-Lsuggesting that ARP-I and apobec-I bind different sites on the apo B RNA transcript. We examined whether ARP-I mediates C to U editing of the apo B RNA using recombinant ARP-I and a source of auxiliary editing activity. ARP-I, over a 20-fold concentration range demonstrated no C to U conversion of an apo B RNA template. Interestingly, the addition of ARP-I in trans to in vitro editing reactions containing apobec-I and auxiliary factors demonstrated a dosedependent inhibition of C to U editing of the apo B RNA. These findings suggest that ARP-l may play a regulatory role in restricting apobec-I mediated RNA editing as well as in cellular nucleoside conservation.
PRE-CHYLOMICRON TRANSPORT VESICLES (PCTV) UTILIZE THE VESICLE ASSOCIATED MEMBRANE PROTEIN 1 (VAMP 1) AND SYNAPTOSOMAL ASSOCIATED PROTEIN 25 (SNAP 25) DEPENDENT PATHWAY. Shadab A. Siddiqi, Charles M. Mansbach, Univ of Tennessee, Memphis,
3623 LYMPHATIC TRANSPORT OF LIPIDS IN APOBEC-l KNOCK· OUT MICE. Vicky Lee Ng, Shuqin Zheng, Nicholas O. Davidson, Patrick Tso, Acad Hosp Med Ctr, Cincinnati, OH; Univ of Cincinnati Coli of Medicine, Cincinnati, OH; Washington Univ Sch of Medicine, St. Louis, MO. Background. Apolipoprotein B (apo B) is obligatory for chylomicron assembly. The apobec-I gene produces an editing enzyme, which generates a translational stop codon (by C to U editing of the apo B RNA) that truncates apo B100 to form apo B48, the predominant isoform in mammalian small intestine. Targeted disruption of apobec-I eliminates apo B48 production yielding mice (KO) that secrete exclusively apo BlOO. Aim. To examine intestinal lipid absorption and lymphatic transport in a congenic mouse strain (C57B1I6J) synthesizing only apo B48 (WT) or apo BlOO (KO) using the lymph fistula mouse model. Methods. Mesenteric lymph fistulae and gastroduodenal feeding tubes were placed in five WT and five KO mice. A lipid emulsion containing 41Jomol of [3H] labeled triolein, 0.78 IJomol of [14C] cholesterol, 0.78 IJomol of egg lecithin, and 5.7 IJomol of Na taurocholate in 0.3 ml of phosphate-buffered saline (pH 6.4) was infused at 0.3 mllh for 6h. Lymph was collected during fasting and then hourly for 6h during the lipid infusion. The animals were then re-anesthetized for open laparotomy and extraction of the small intestine to determine luminal lipid content. Results. There was no significant difference in lymph flow rate between the KO and WT mice. [3H] triolein output in lymph equilibrated by the 3'd h of lipid infusion in both groups. The 6th h output was 56.6%:!::1O% (% hourly infused) in the WT mice and 57.5%:!::8% in the KO mice. The 6th h lymphatic outputs of 4C] cholesterol for the WT and KO mice were 26.2%:!::3% and 29.6%:!::3% (% hourly infused) respectively. There were no significant differences in the lymphatic [3H] triolein or 4 C] cholesterol outputs between the two groups. Luminal [3H] triolein recovery was similar in both groups (3.2% of total dose infused) indicating normal digestion and uptake of triglyceride in the KO mice. Luminal cholesterol recovery was significantly higher in the KO mice (17.7%:!::3%) than in the WT mice (7.7%:!::I%) suggesting lower cholesterol uptake in the KO mice (p<0.05). Despite apparent lower cholesterol uptake in the KO mice, lymphatic cholesterol transport was comparable to that in WT mice. Conclusions. The data provide stong evidence that intestinal cells can assemble and secrete chylomicrons containing apo BlOO. From a functional perspective, apo BlOO appears to facilitate lipid transport as efficiently as apo B48, although the role of apo BlOO versus apo B48 in cholesterol uptake remains open. Work is currently underway to determine whether apo B100 chylomicrons are metabolized in a way that is similar to apo B48 chylomicrons.
e
e
TN. Triacylglycerol (TAG) transport from the endoplasmic reticulum (ER) to the Golgi in the intestine is mediated by the PCTV, derived from the ER, which is the rate limiting step in chylomicron export (AlP 276: G378, 1999). Targeting of vesicles requires interactions between vesicle integral membrane proteins, soluble NSF attachment protein receptor (v-SNARE) and target organelles (t-SNARE). The aim of the study was to identify the proteins involved in targeting the PCTV to intestinal cis Golgi. Methods: PCTV were isolated from rat intestine using step and continuous sucrose gradients (AlP 276: 0378, 1999). 2-D gels were performed on PCTV, ER, and cis Golgi membranes obtained by treating the organelles with 100 mM carbonate and collecting the membranes by centrifugation. The pi and Mr of proteins unique to PCTV were used to search a protein data base (Swiss Prot). Results: We identified 32 proteins unique to PCTV membranes. Of these, only one had been associated with secretory proteins, a 25 kDa protein with a pi of 8.86, consistent with VAMP. Western blots showed that both VAMP I and SNAP 25, a protein required for forming the v- and t-SNARE complex, were present in PCTV. Antibodies to VAMP I and SNAP 25 were separately incubated for I h with ER at 4°C. This reduced delivery of TAG from ER to the cis Golgi by 75 % in both cases. Pre-immune serum had no effect. cis Golgi were incubated separately with the immunogenic N-terminal 20 amino acids peptides of VAMP I and SNAP 25. Both peptides inhibited ER-to-Golgi TAG transport by >75 %. Triton-solubilized PCTV membrane proteins were electrophoresed on a native gel, transblotted onto nitrocellulose and the membrane probed with biotinylated cis Golgi membranes. Binding was noted at 120 kDa. Both VAMP I and SNAP 25 antibodies recognized the proteins at this band. On SDS-PAGE of PCTV membranes without boiling, both 120 and 25 kDa proteins were recognized by both antibodies. When the PCTV membranes were boiled 5 min in Laemmli's buffer, only the 25 kDa bands were identified. Conclusions: We conclude that both VAMP I and SNAP 25 are involved in PCTV targeting to the cis Golgi. This is consistent with the secretory protein pathway. We speculate that multimers of VAMP I and SNAP 25 are involved in targeting the PCTV to the intestinal cis Golgi.
3625 UP-REGULATION OF INDUCIBLE CO-STIMULATOR (ICOS) EXPRESSION AND ITS REGULATION OF CYTOKINE PRODUCTION IN INFLAMMATORY BOWEL DISEASE. Toshirou Sato, Mamoru Watanabe, Takanori Kanai, Akira Okazawa, Hanae Takagi, Atushi Nakazawa, Hiromasa Ishii, Richard Kroczek, Toshifumi Hibi, Sch of Med, Keio Univ, Tokyo, Japan; Keio Cancer Ctr, Tokyo, Japan; Robert-Koch Institute, Berlin, Germany. Background: Activation via CD28 on the surface of T cells provides a potent amplification signal for several cytokine productions. We have demonstrated that a ligand for CD28, CD86 expressed on colonic epithelial cells are involved in the activation of mucosal T cells (Gastroenterology 117: 536, 1999). Recently, a third member of CD28 family of molecules, inducible co-stimulator (ICOS) has been identified (Nature 397: 263, 1999). Herein, we investigated whether ICOS signaling participates in mucosal immune responses in ulcerative colitis (UC) and Crohn's disease (CD). Methods: I) Expression of ICOS on LPLs isolated from normal controls (NL), UC, and CD was assessed by flow cytometry. 2) Expression of ICOS on LPLs was also analyzed after stimulation with anti-CD3 mAb. 3) Tissue sections of intestinal specimens were stained CD28 or ICOS. 4) LPLs were stimulated by anti-CD3 in the presence of anti-CD28, antiICOS, or control mAbs. Culture supernatants were collected and assayed for various cytokines using ELISA. Results: I) ICOS expression was significantly up-regulated on freshly isolated LPLs from inflamed mucosa of UC and CD, as compared with those from NL. 2) LPLs from inflamed mucosa of UC and CD showed increased ICOS expression after stimulation with anti-CD3 mAb for 48 hrs. 3) Co-stimulation by anti-CD3 and anti-CD28 mAbs up-regulated the secretion of IL-2, IFN-g and IL-IO in LPL cultures from NL, UC and CD. However, co-stimulation by anti-CD3 and anti-ICOS mAbs failed to up-regulate the secretion of lL-2. Interestingly, co-stimulation by anti-CD3 and anti-ICOS mAbs predominantly induced IL-IO in LPL cultures from UC, but not those from NL and CD. Conclusion: These findings suggest that up-regulation of ICOS expression may be involved in chronic intestinal inflammation of inflammatory bowel disease, and that inhibition of ICOS signaling may lead to therapeutic effects in these patients.