Characterization of the Cobalamin (Vitamin B12) Biosynthetic Genes ...
Cubilin and mac2-binding protein are two interacting proteins involved in intrinsic factor-cobalamin...
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S1769 Low and High Vitamin Intake both Increase Intestinal Neoplasia and Crypt Fission in the Min Mouse Omar Bashir, Anthony J. Fitzgerald, Robert A Goodlad Background: The multiple intestinal neoplasia (Min) mouse is a very useful model for the study of the actions of dietary factors on gut biology and turnout progression as it has the same genetic defect, loss of APC, that is seen in sporadic tumours and familial adenomatons polyposis (FAP) in man. Methods: 90 Min mice C57BK/6J-ApcMIn) and 90 wild type littermates, 4 weeks old, were divided into 6 groups and fed three diets for 8 weeks. The control diet was a semisythetic one and the test diets were the semisythetic diet with either a low or high vitamin content. The vitamin content of the low diet was lowered to a third of the RDA and the high diet had five times the RDA (except for retinal and folate, which were only doubled). The number and size of polyps in the small and large intestines were scored, as was the number of native mitoses (per crypt) and crypt fission. Results: The small intestines of the low and high vitamin groups were heavier than the controls. There were significantly more polyps and the tumour burden was higher in the both the low and the high v~tamin groups (P<0.02). Proliferation was slightly reduced by the vitamin alteration and crypt fission was significantly increased in the Min mice when compared to the wild type, (p<0.00]) Fission indices were decreased by vitamni alteration in the small intestine, but increased in the colon. The effects of vitamin alteration on polyp number were most pronounced in the proximal intestine, which is also the site of maximum crypt fission. Conclusion: Both vitamin deficiency and over-supplementation can markedly enhance polyp number and tumour burden, and this may be related to altered crypt fission. S1770 Cubilin and Mac2-Binding Protein Are Two Interacting Proteins Involved in Intrinsic Factor-Cobalamin Binding Defects of Grasbeck-Imerslund Syndrome Celine Chery, Jean-Louis Gueant, Veronique Regrlauh, Hakon Leffler, Thierry Pillot, Gp Sinha, Mark Caswell, Martin Wnstinger, Fares Namour Selective congenital intestinal malabsorption of cobalamin (Grasbeck-lmerslund syndrome, GIS) is associated with defective excretion of a hydrophobic inmnsic factor receptor (IFCR). and a FM1 mutation of cubiiin gene decreasing intrinsic lactor/cubifin binding affinity by 5-fold. We investigated the hypothesis that IFCR corresponds to the association of cubilin w~th a hydrophobic protein. IFCR was detected in concentrated urines from patients and controls by phenyl-Sepharose-R1A and affinity chromatography, cubilin by Western blot. Mutations of exons encoding for the binding domains of cubilin and mac-2 binding protein were searched by genomic DNA sequencing Maximum binding of IFCR and detection of cubilin were respectively : patient SF : 192 fmol/mL, cubilin undetectable; LP : 5 fmol/mL, cubilin present; OM : 6.6 fmoi/ml, cubilin undetectable; TB and TM : undetectable IFCR, cubilin present; controls : 26.1fmol/ml +/- 8.2, unconstant detection of cubilin. IFCR affinity was unchanged in patients OM and LP and decreased in patient SF. Agamse-Cbl- IF affinity chromatography of urine concentrate isolated two IF-Cbl bmding proteins, cubilin and mac-2 binding protein, a hydrophobic 70 kDa ligand of galectin-3 lectin. The binding affinity ol purified mac-2 binding protein for IF and cubilin was evidenced by plasmon resonance surface analysis (Biacore). Assuming a binding complex ABj <-> A+B] where A is IF or cubilin and B is mac-2 binding protein, the two koff kinetic dissociation constants were evaluated to 015 +/- 0.03 s-1 and 4.7 X 10-3 +/- 0.3 X 10-3 s-1, and to 0.14 +/- 0.04 s-1 and 3.7 X 10-3 +/- 0.4 X 10-3 s-l, respectively. Mac-2 binding protein concentration in urines behaved similarly to IFCR binding, with the highest level observed in patient SF. No mutation was detected in genomic DNA In conclusion, mac-2 binding protein and cubilin are two interacting proteins involved in a IFCR complex, which may be distinctly implicated in different subsets of GIS S1771 The Therapeutic Role of Antioxidant Vitamins: C and E in Radiation-lnduced Rectal Injury Chenf M E1 Younis, Ovadia Abu[afia Purpose: The increasing use of pelwc radiation as an accepted modality in the treatment of pelvic cancer has led to an increase in the incidence of radiation proctitis. The mechanisms underlying the ischemic process that characterizes radiation proctins are not clear. Some studies suggested increased mucosal levels of cytokines and oxidative stress as possible initiating mechanisms. An earlier report suggested a beneficial effect of antioxidant vitamin C and vitamin E in symptomatic radiation proctitis. Methods: 9 patients (age: 43-72) were referred for evaluation of symptoms that developed (8 months to 4 years) following pelvic radiation ( 6 patients with cervical carcinoma and 3 patients with prostate carcinoma.). All patients had endoscopicafiy documented rectal involvement ( proctitis: 6, ulcerations: 3, stricture: 1). All patients failed steroid enema (8 patients), and local thermal coagulation (2 patients). Multiple blood transfusion was required in two patients. All patients were placed on vitamin C 500 mg po rid and vitamin E 400 units po tid The patients were followed over 3-8 months. Results: All symptoms subsided at 6 -12 weeks of the treatment None of the patients placed on the treatment required anymore blood transfusion. Conclusion: This favorable outcome in this small number of patients further suggests the beneficial effect of the antioxidant vitamins. Larger open labeled study may further characterize this observed benefit. The potential use of vitamins C and E to prevent radiation-induced injury also deserves evaination $1772 Regulation of the Murine PHEX Gene by Vitamin D3 Eric R. Hines, Ogla I. Kolek, Marci D. Jones, Samantha Serey, Nafisseh Baradaran, James F. Collins, Fayez K. Ghishan Introduction: 1, 25-(OH)2 vitamin D3 (D3) increases intestinal phosphate (Pi) absorption and renal Pi reabsorption via increased expression of type II sodium-phosphate (NaPi-ll) cotransporters. The PHEX gene (Phosphate encoding gene with Homologies to Endopepti- dases on the X chromosome), expressed predominantly in osteoblasts, exerts effects on renal Pi reabsorption via the NaPi-lla cotransporter thru an unidentified, circulating hormonal factor termed "phosphatonin'?. It also seems likely that "phosphatonin" regulates intestinal Pi absorption, although this possibility remains largely unexplored. Previous studies have also shown the D3 downregulates PHEX in primary and cultured mouse osteobfasts. We previously cloned the mouse PHEX gene promoter and demonstrated its functionality in mouse osteoblast-fike (UMR106) cells. The present study was undertaken to determine if D3 regulates PHEX at a transcriptional level and if so, to determine the promoter region responsible for this regulation. The overall goal of this project is to decipher the role that PHEX and "phosphotonin" play in intestinal Pi absorption. Methods: PHEX promoter constructs were cloned into the pl3-Gal basic vector (Clontech) and transfected into UMR- 106 cells. Cells were treated with D3 or vehicle, and then either total RNA was isolated for Northern blot analysis or cell extracts were prepared for [3-galactosidase reporter gene assays. In some experiments, cells were treated with 3 ng/ml actinomycin D (Act D) prior to addition of and during treatment with D> Results: Northern blot analysis revealed a 40% reduction ni PHEX mRNA expression in UMR-106 cells after 24 treatment with 10 .7 M D> with a maximum reduction of 75% - 80% after 48 hours of treatment. A similar 40% downregulation of reporter gene expression was seen with 24 hour D3treatment of cells transfected with the -1061/+ 104 bp PHEX promoter construct. Act D treatment blunted the down regulation by D> Furthermore, -1061/+ 104, -542/+ 104 and -133/+ 104 bp PHEX promoter con- structs all responded similarly to D~ treatment Conclusions: These studies demonstrate that PHEX is regulated partially at the transcriptional level by D3 in a time dependent manner. We have further shown that the -133 to + 104 bp region of the PHEX promoter is responsible for D3 regulation. Supported by NIH grant R37 DK33209. $1773 Pyridoxin Deficiency Correlates with Disease Activity in Pediatric and Young Adult Patients with Crohn's Disease Yoram Bujanover, Batia Weiss, Akiva Fradkm, Ilana Weintraub, Ben Ami Sela Background:Nearly 100 enzymatic reactions have been reported in which pyridoxin plays a coenzyme role.This vitamin has a significant impact on immune functinn.Deficiency of pyridoxin was reported previously in two inflammatory conditions-rheumatoid arthritis and cardiovascular disease. Aim:To evaluate pyridoxin status in active and nonactive Crohn's disease(CD). Methods:Fifty CD patients were studied,34 male, 16 female,age range ll-25yr(M 15yr).Patient's disease activity was assessed using PCDAI and CDAI.The following parame- ters-CBC,ESR,CRP,ALB,Fe,Zn,Folic acid,B12 and AST were determined by conventional laboratory methods.Serum pyridoxin levels were determined by HPLC.A group of 80 age matched healthy children and young adults served as a control group. Results:Serum pyri- doxin levels were low in 38% of the CD patients studied kevds were significantly lower in the CD group 5-18Onmol/l.(M 41)rdative to controls 28-222nmol/L (M 68)p<0.05.Acuve CD patients had significantly lower levels 5-27.5nmol/k(M 19)compared with nonactive or low active CD patients 27-180nmol/L(M 45) P<0.02.In 15 patients changes in disease activity were followed by increase or decrease in pyridoxin levels.Pyridoxin deficiency was frequendy associated with low AST and Fe deficiency. Conclusions:Pyridoxin deficiency is common in CD patients.It is more prevalent in those with active disease. Chronic inflammation may be the cause for pyridoxin consumption.Determination of pyridoxin status should be part of the nutritional evaluation of CD patients. S1774 Analysis of the Minimal Promoter Region of the Human Thiamin Transporter SLC19A2 and in viva Confirmation of Activity in Transgenic Mice Jack C. Reidling, Hamid Said Background: The molecular mechanism and regulation of thiamin transport is not well understood. Prevaons studies have established the possibility that a human thiamin trans- porter, SLC19A2, plays a role m thiamin uptake. To examine the regulation of SLC19A2 expression we reported cloning the promoter and determining the activity in human intestinal Caco-2 cells. In this study we further characterize the promoter of SLC19A2 and its expression by determining the activity in other tissue specific cell lines, identifying putative cis-regulatory elements, and confirming promoter activity in viva. Methods: Promoter-luciferase reporter gene activity assay wath mutational analysis, electrophoretic mobility shift assays (EMSA) with competition and supershift assays, RT-PCR, and Luciferase assay on transgenic mouse tissues. Results: Human liver HepG2 cells have a higher SLCIgA2 promoter activity level than human intestinal Caco-2 cells which in turn have a higher activity level compared to human kidney HEK293 cells. The activity levels agree with endogenous SLC19A2 RNA levels in the different cell types. Mutational analysis determined that a GKLF, NF-1, and SP-1 site are each important for promoter activity. Using EMSA we identified four specific DNA/ protein complexes for the minimal promoter region. Competition with oligonucleotides for NF-1 and SP-1 consensus cis-elemem sequences eliminated two of the complexes. Specific antibodies to GKLF, NF-1 or SP-1 each individually caused a supershifted band to appear. To characterize the promoter region in viva we established transgenic mouse lines carrying either a full-length or minimal promoter-luciferase construct. We examined the tissue distri- bution of the luciferase activity in the transgenic mice and found the activity to mimic the expression pattern reported for the human SLC19A2 gene. Complimenting our in vitro studies, higher levels of luciferase activity were observed in the mouse liver compared to the intestine and kidney for both the minimal region and fufiqength promoter-fuciferase constructs. In summary these data confirm the activity of the SLC19A2 promoter in v~vo AGA Abstracts A-264
Transcript of Cubilin and mac2-binding protein are two interacting proteins involved in intrinsic factor-cobalamin...
S1769
Low and High Vitamin Intake both Increase Intestinal Neoplasia and Crypt Fission in the Min Mouse Omar Bashir, Anthony J. Fitzgerald, Robert A Goodlad
Background: The multiple intestinal neoplasia (Min) mouse is a very useful model for the study of the actions of dietary factors on gut biology and turnout progression as it has the same genetic defect, loss of APC, that is seen in sporadic tumours and familial adenomatons polyposis (FAP) in man. Methods: 90 Min mice C57BK/6J-ApcMIn) and 90 wild type littermates, 4 weeks old, were divided into 6 groups and fed three diets for 8 weeks. The control diet was a semisythetic one and the test diets were the semisythetic diet with either a low or high vitamin content. The vitamin content of the low diet was lowered to a third of the RDA and the high diet had five times the RDA (except for retinal and folate, which were only doubled). The number and size of polyps in the small and large intestines were scored, as was the number of native mitoses (per crypt) and crypt fission. Results: The small intestines of the low and high vitamin groups were heavier than the controls. There were significantly more polyps and the tumour burden was higher in the both the low and the high v~tamin groups (P<0.02). Proliferation was slightly reduced by the vitamin alteration and crypt fission was significantly increased in the Min mice when compared to the wild type, (p<0 .00] ) Fission indices were decreased by vitamni alteration in the small intestine, but increased in the colon. The effects of vitamin alteration on polyp number were most pronounced in the proximal intestine, which is also the site of maximum crypt fission. Conclusion: Both vitamin deficiency and over-supplementation can markedly enhance polyp number and tumour burden, and this may be related to altered crypt fission.
S1770
Cubilin and Mac2-Binding Protein Are Two Interacting Proteins Involved in Intrinsic Factor-Cobalamin Binding Defects of Grasbeck-Imerslund Syndrome Celine Chery, Jean-Louis Gueant, Veronique Regrlauh, Hakon Leffler, Thierry Pillot, Gp Sinha, Mark Caswell, Martin Wnstinger, Fares Namour
Selective congenital intestinal malabsorption of cobalamin (Grasbeck-lmerslund syndrome, GIS) is associated with defective excretion of a hydrophobic inmnsic factor receptor (IFCR). and a FM1 mutation of cubiiin gene decreasing intrinsic lactor/cubifin binding affinity by 5-fold. We investigated the hypothesis that IFCR corresponds to the association of cubilin w~th a hydrophobic protein. IFCR was detected in concentrated urines from patients and controls by phenyl-Sepharose-R1A and affinity chromatography, cubilin by Western blot. Mutations of exons encoding for the binding domains of cubilin and mac-2 binding protein were searched by genomic DNA sequencing Maximum binding of IFCR and detection of cubilin were respectively : patient SF : 192 fmol/mL, cubilin undetectable; LP : 5 fmol/mL, cubilin present; OM : 6.6 fmoi/ml, cubilin undetectable; TB and TM : undetectable IFCR, cubilin present; controls : 26.1fmol/ml +/- 8.2, unconstant detection of cubilin. IFCR affinity was unchanged in patients OM and LP and decreased in patient SF. Agamse-Cbl- IF affinity chromatography of urine concentrate isolated two IF-Cbl bmding proteins, cubilin and mac-2 binding protein, a hydrophobic 70 kDa ligand of galectin-3 lectin. The binding affinity ol purified mac-2 binding protein for IF and cubilin was evidenced by plasmon resonance surface analysis (Biacore). Assuming a binding complex ABj < - > A+B] where A is IF or cubilin and B is mac-2 binding protein, the two koff kinetic dissociation constants were evaluated to 015 +/- 0.03 s-1 and 4.7 X 10-3 +/- 0.3 X 10-3 s-1, and to 0.14 +/- 0.04 s-1 and 3.7 X 10-3 +/- 0.4 X 10-3 s-l, respectively. Mac-2 binding protein concentration in urines behaved similarly to IFCR binding, with the highest level observed in patient SF. No mutation was detected in genomic DNA In conclusion, mac-2 binding protein and cubilin are two interacting proteins involved in a IFCR complex, which may be distinctly implicated in different subsets of GIS
S1771
The Therapeutic Role of Antioxidant Vitamins: C and E in Radiation-lnduced Rectal Injury Chenf M E1 Younis, Ovadia Abu[afia
Purpose: The increasing use of pelwc radiation as an accepted modality in the treatment of pelvic cancer has led to an increase in the incidence of radiation proctitis. The mechanisms underlying the ischemic process that characterizes radiation proctins are not clear. Some studies suggested increased mucosal levels of cytokines and oxidative stress as possible initiating mechanisms. An earlier report suggested a beneficial effect of antioxidant vitamin C and vitamin E in symptomatic radiation proctitis. Methods: 9 patients (age: 43-72) were referred for evaluation of symptoms that developed (8 months to 4 years) following pelvic radiation ( 6 patients with cervical carcinoma and 3 patients with prostate carcinoma.). All patients had endoscopicafiy documented rectal involvement ( proctitis: 6, ulcerations: 3, stricture: 1). All patients failed steroid enema (8 patients), and local thermal coagulation (2 patients). Multiple blood transfusion was required in two patients. All patients were placed on vitamin C 500 mg po rid and vitamin E 400 units po tid The patients were followed over 3-8 months. Results: All symptoms subsided at 6 -12 weeks of the treatment None of the patients placed on the treatment required anymore blood transfusion. Conclusion: This favorable outcome in this small number of patients further suggests the beneficial effect of the antioxidant vitamins. Larger open labeled study may further characterize this observed benefit. The potential use of vitamins C and E to prevent radiation-induced injury also deserves evaination
$1772
Regulation of the Murine PHEX Gene by Vitamin D3 Eric R. Hines, Ogla I. Kolek, Marci D. Jones, Samantha Serey, Nafisseh Baradaran, James F. Collins, Fayez K. Ghishan
Introduction: 1, 25-(OH)2 vitamin D3 (D3) increases intestinal phosphate (Pi) absorption and renal Pi reabsorption via increased expression of type II sodium-phosphate (NaPi-ll) cotransporters. The PHEX gene (Phosphate encoding gene with Homologies to Endopepti- dases on the X chromosome), expressed predominantly in osteoblasts, exerts effects on renal Pi reabsorption via the NaPi-lla cotransporter thru an unidentified, circulating hormonal factor termed "phosphatonin'?. It also seems likely that "phosphatonin" regulates intestinal Pi absorption, although this possibility remains largely unexplored. Previous studies have also shown the D 3 downregulates PHEX in primary and cultured mouse osteobfasts. We previously cloned the mouse PHEX gene promoter and demonstrated its functionality in mouse osteoblast-fike (UMR106) cells. The present study was undertaken to determine if D3 regulates PHEX at a transcriptional level and if so, to determine the promoter region responsible for this regulation. The overall goal of this project is to decipher the role that PHEX and "phosphotonin" play in intestinal Pi absorption. Methods: PHEX promoter constructs were cloned into the pl3-Gal basic vector (Clontech) and transfected into UMR- 106 cells. Cells were treated with D3 or vehicle, and then either total RNA was isolated for Northern blot analysis or cell extracts were prepared for [3-galactosidase reporter gene assays. In some experiments, cells were treated with 3 ng/ml actinomycin D (Act D) prior to addition of and during treatment with D> Results: Northern blot analysis revealed a 40% reduction ni PHEX mRNA expression in UMR-106 cells after 24 treatment with 10 .7 M D> with a maximum reduction of 75% - 80% after 48 hours of treatment. A similar 40% downregulation of reporter gene expression was seen with 24 hour D3treatment of cells transfected with the -1061/+ 104 bp PHEX promoter construct. Act D treatment blunted the down regulation by D> Furthermore, -1061/+ 104, -542/+ 104 and -133/+ 104 bp PHEX promoter con- structs all responded similarly to D~ treatment Conclusions: These studies demonstrate that PHEX is regulated partially at the transcriptional level by D3 in a time dependent manner. We have further shown that the -133 to + 104 bp region of the PHEX promoter is responsible for D3 regulation. Supported by NIH grant R37 DK33209.
$1773
Pyridoxin Deficiency Correlates with Disease Activity in Pediatric and Young Adult Patients with Crohn's Disease Yoram Bujanover, Batia Weiss, Akiva Fradkm, Ilana Weintraub, Ben Ami Sela
Background:Nearly 100 enzymatic reactions have been reported in which pyridoxin plays a coenzyme role.This vitamin has a significant impact on immune functinn.Deficiency of pyridoxin was reported previously in two inflammatory conditions-rheumatoid arthritis and cardiovascular disease. Aim:To evaluate pyridoxin status in active and nonactive Crohn's disease(CD). Methods:Fifty CD patients were studied,34 male, 16 female,age range ll-25yr(M 15yr).Patient's disease activity was assessed using PCDAI and CDAI.The following parame- ters-CBC,ESR,CRP,ALB,Fe,Zn,Folic acid,B12 and AST were determined by conventional laboratory methods.Serum pyridoxin levels were determined by HPLC.A group of 80 age matched healthy children and young adults served as a control group. Results:Serum pyri- doxin levels were low in 38% of the CD patients studied kevds were significantly lower in the CD group 5-18Onmol/l.(M 41)rdative to controls 28-222nmol/L (M 68)p<0.05.Acuve CD patients had significantly lower levels 5-27.5nmol/k(M 19)compared with nonactive or low active CD patients 27-180nmol/L(M 45) P<0.02.In 15 patients changes in disease activity were followed by increase or decrease in pyridoxin levels.Pyridoxin deficiency was frequendy associated with low AST and Fe deficiency. Conclusions:Pyridoxin deficiency is common in CD patients.It is more prevalent in those with active disease. Chronic inflammation may be the cause for pyridoxin consumption.Determination of pyridoxin status should be part of the nutritional evaluation of CD patients.
S1774
Analysis of the Minimal Promoter Region of the Human Thiamin Transporter SLC19A2 and in viva Confirmation of Activity in Transgenic Mice Jack C. Reidling, Hamid Said
Background: The molecular mechanism and regulation of thiamin transport is not well understood. Prevaons studies have established the possibility that a human thiamin trans- porter, SLC19A2, plays a role m thiamin uptake. To examine the regulation of SLC19A2 expression we reported cloning the promoter and determining the activity in human intestinal Caco-2 cells. In this study we further characterize the promoter of SLC19A2 and its expression by determining the activity in other tissue specific cell lines, identifying putative cis-regulatory elements, and confirming promoter activity in viva. Methods: Promoter-luciferase reporter gene activity assay wath mutational analysis, electrophoretic mobility shift assays (EMSA) with competition and supershift assays, RT-PCR, and Luciferase assay on transgenic mouse tissues. Results: Human liver HepG2 cells have a higher SLCIgA2 promoter activity level than human intestinal Caco-2 cells which in turn have a higher activity level compared to human kidney HEK293 cells. The activity levels agree with endogenous SLC19A2 RNA levels in the different cell types. Mutational analysis determined that a GKLF, NF-1, and SP-1 site are each important for promoter activity. Using EMSA we identified four specific DNA/ protein complexes for the minimal promoter region. Competition with oligonucleotides for NF-1 and SP-1 consensus cis-elemem sequences eliminated two of the complexes. Specific antibodies to GKLF, NF-1 or SP-1 each individually caused a supershifted band to appear. To characterize the promoter region in viva we established transgenic mouse lines carrying either a full-length or minimal promoter-luciferase construct. We examined the tissue distri- bution of the luciferase activity in the transgenic mice and found the activity to mimic the expression pattern reported for the human SLC19A2 gene. Complimenting our in vitro studies, higher levels of luciferase activity were observed in the mouse liver compared to the intestine and kidney for both the minimal region and fufiqength promoter-fuciferase constructs. In summary these data confirm the activity of the SLC19A2 promoter in v~vo
AGA Abstracts A-264
