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Eric Sibley

Publications and source records attributed to Eric Sibley.

12 recordsLinked to original sources
PubMed2006

Lactase gene promoter fragments mediate differential spatial and temporal expression patterns in transgenic mice.

Lactase gene expression is spatiotemporally regulated during mammalian gut development. We hypothesize that distinct DNA control regions specify appropriate spatial and temporal patterning of lactase gene expression. In order to define regions of the lactase promoter involved in mediating intestine-specific and spatiotemporal restricted expression, transgenic mice harboring 100 bp, 1.3- and 2.0- kb fragments of the 5' flanking region of the rat lactase gene cloned upstream of a luciferase reporter were characterized. The 100-bp lactase promoter-reporter transgenic mouse line expressed maximal luciferase activity in the intestine with a posterior shift in spatial restriction and ectopic expression in the stomach and lung. The temporal pattern of expression mediated by the 1.3-kb promoter?reporter transgene increases with postnatal maturation in contrast with the postnatal decline mediated by the 2.0-kb promoter-reporter transgene and the endogenous lactase gene. The differential transgene expression patterns mediated by the lactase promoter fragments suggests that intestine-specific spatial and temporal control elements reside in distinct regions of the DNA sequences upstream of the lactase gene transcription start-site.

5' Flanking Region↗
PubMed2005

Spatio-temporal patterns of intestine-specific transcription factor expression during postnatal mouse gut development.

The small intestine matures from a primitive tube into morphologically and functionally distinct regions during gut development. Maximal expression of the genes encoding the digestive enzymes lactase-phlorizin hydrolase and sucrase-isomaltase is spatially restricted to distinct segments along the anterior-posterior axis of the small intestine and is temporally regulated during postnatal maturation. Transcription factors capable of interacting with the intestinal lactase and sucrase gene promoters are candidate regulators of spatio-temporal patterning during gut development and maturation. We aimed to quantitatively examine and compare the relative expression levels of a set of intestine-specific transcription factors along the anterior-posterior gut axis during postnatal maturation. Our analysis was focused on the transcription factors capable of regulating the intestinal lactase and sucrase-isomaltase genes. A real-time PCR protocol was used to quantitatively examine and compare spatially and temporally the relative transcript abundance levels for intestine-specific factors during postnatal intestinal maturation. Distinct spatial expressions patterns were detected along the length of the small intestine for PDX-1, Cdx-2, GATA-4, GATA-5, GATA-6, HNF-1alpha, HNF-1beta and CDP transcription factor genes. There is a general decline in transcript abundance for the factor genes during postnatal maturation. Defining the spatio-temporal expression patterns for intestine-specific transcription factor genes contributes to investigation of the roles that factor gradients play in mediating gut development and differentiation.

Animals↗
PubMed2005

Proteasome modulating agents induce rAAV2-mediated transgene expression in human intestinal epithelial cells.

Intestinal gene transfer offers promise as a therapeutic option for treatment of both intestinal and non-intestinal diseases. Recombinant adeno-associated virus serotype 2, rAAV2, based vectors have been utilized to transduce lung epithelial cells in culture and in human subjects. rAAV2 transduction of intestinal epithelial cells, however, is limited both in culture and in vivo. Proteasome-inhibiting agents have recently been shown to enhance rAAV2-mediated transgene expression in airway epithelial cells. We hypothesized that similar inhibition of proteasome-related cellular processes can function to induce rAAV2 transduction of intestinal epithelial cells. Our results demonstrate that combined treatment with proteasome-modulating agents MG101 (N-acetyl-L-leucyl-L-leucyl-L-norleucine) and Doxorubicin synergistically induces rAAV2-mediated luciferase transgene expression by >400-fold in undifferentiated Caco-2 cells. In differentiated Caco-2 monolayers, treatment with MG101 and Doxorubicin induces transduction preferentially from the basolateral cell surface. In addition to Caco-2 cells, treatment with MG101 and Doxorubicin also results in enhanced rAAV2 transduction of HT-29, T84, and HCT-116 human intestinal epithelial cell lines. We conclude that MG101 and Doxorubicin mediate generic effects on intestinal epithelial cells that result in enhanced rAAV2 transduction. Use of proteasome-modulating agents to enhance viral transduction may facilitate the development of more efficient intestinal gene transfer protocols.

Caco-2 Cells↗
PubMed2004

Transcriptional regulation of the lactase-phlorizin hydrolase promoter by PDX-1.

Lactase-phlorizin hydrolase gene expression is spatially restricted along the anterior-posterior gut axis. Lactase gene transcription is maximal in the distal duodenum and jejunum in adult mammals and is barely detectable in the proximal duodenum. By contrast, pancreatic duodenal homeobox-1 (PDX-1) protein is expressed maximally in the proximal duodenum. This study aimed to determine the role of PDX-1 in regulating lactase gene promoter activity in intestinal epithelial cells. Caco-2 cells were cotransfected with lactase promoter-reporter constructs in the presence of a PDX-1 expression vector and assayed for luciferase activity. PDX-1 cotransfection results in repression of lactase promoter activity. Sequence analysis of the lactase promoter revealed a putative PDX-1 DNA binding site in the proximal 100-bp lactase gene promoter. EMSAs demonstrated that PDX-1 can interact with the lactase promoter binding site but not with a site in which the core PDX-1 binding sequence TAAT is mutated. Site-directed mutagenesis of the PDX-1 core binding site in the lactase promoter-reporter construct suggests that PDX-1 can function independently of DNA binding to its consensus binding site. Stable overexpression of PDX-1 results in repression of the endogenous human lactase gene in differentiated Caco-2 cells. Given the contrasting spatial expression pattern, PDX-1 may function to specify the anterior boundary of lactase expression in the small intestine and is thus a candidate regulator of anterior spatial restriction in the gut.

Animals↗
PubMed2004

PCR-RFLP genotyping assay for a lactase persistence polymorphism upstream of the lactase-phlorizin hydrolase gene.

The majority of the world's human population experiences a decline of lactase gene expression during maturation, so-called lactase nonpersistence. Thus, adults with lactase nonpersistence are susceptible to developing symptoms of lactose intolerance. By contrast, lactase persistence is an autosomal dominant heritable condition that results in a high level of lactase gene expression throughout adulthood and sustained lactose tolerance. Lactase persistence has recently been correlated with a single nucleotide genetic variant (a C --> T mutation) located 13,910 bases upstream from the lactase structural gene. We aimed to develop a restriction fragment length polymorphism (RFLP) method of detecting the C/T variants as a means of identifying individuals genetically inclined toward lactase persistence or nonpersistence. Genomic DNA in a 210-bp region surrounding the -13,910-bp variant site was PCR amplified with unique primers designed to avoid or mutate adjacent restriction sites. The amplified DNA was digested with a restriction enzyme, CviJI, that recognizes the base pair sequence generated by the lactase nonpersistence variant. Restriction digest gel analysis yielded DNA fragments of the expected diagnostic molecular weight sizes for individuals that were homozygote or heterozygote for the lactase persistence and nonpersistence variants. The genotypes predicted by the RFLP-based method were confirmed by DNA sequence analysis. The RFLP-based method provides a quick and noninvasive means of molecular detection of the presence or absence of the lactase persistence variant.

Adult↗
PubMed2004

Carbohydrate intolerance.

PURPOSE OF REVIEW: The small intestinal mucosa is highly specialized for terminal digestion of nutrient polysaccharides and disaccharides and absorption of monosaccharides. However, in the case of digestive or absorptive deficiency, symptoms of carbohydrate intolerance result. Significant progress has been made toward defining the molecular genetic mechanisms responsible for several carbohydrate intolerances. RECENT FINDINGS: This review summarizes monosaccharide and disaccharide intolerance conditions and recent clinical and basic science reports related to carbohydrate digestion and membrane transport. Genetic polymorphisms closely associated with lactase persistence/nonpersistence have been identified. Lactose intolerance is capable of preventing the achievement of adequate peak bone mass in susceptible young adults and may predispose to osteoporosis. Recent studies support previous reports that fructose malabsorption is associated with unexplained gastrointestinal symptoms. GLUT2 may be recruited from the basolateral to the apical membrane of enterocytes to facilitate small intestinal fructose absorption. SUMMARY: Knowledge regarding the clinical aspects of and the physiologic mechanisms responsible for specific carbohydrate intolerances has allowed for improved diagnostic and treatment options and has contributed to continuing investigation of intestinal gene expression.

Journal Article↗
PubMed2004

Genetic variation and lactose intolerance: detection methods and clinical implications.

The maturational decline in lactase activity renders most of the world's adult human population intolerant of excessive consumption of milk and other dairy products. In conditions of primary or secondary lactase deficiency, the lactose sugars in milk pass through the gastrointestinal tract undigested or are partially digested by enzymes produced by intestinal bacterial flora to yield short chain fatty acids, hydrogen, carbon dioxide, and methane. The undigested lactose molecules and products of bacterial digestion can result in symptoms of lactose intolerance, diarrhea, gas bloat, flatulence, and abdominal pain. Diagnosis of lactose intolerance is often made on clinical grounds and response to an empiric trail of dietary lactose avoidance. Biochemical methods for assessing lactose malabsorption in the form of the lactose breath hydrogen test and direct lactase enzyme activity performed on small intestinal tissue biopsy samples may also be utilized. In some adults, however, high levels of lactase activity persist into adulthood. This hereditary persistence of lactase is common primarily in people of northern European descent and is attributed to inheritance of an autosomal-dominant mutation that prevents the maturational decline in lactase expression. Recent reports have identified genetic polymorphisms that are closely associated with lactase persistence and nonpersistence phenotypes. The identification of genetic variants associated with lactase persistence or nonpersistence allows for molecular detection of the genetic predisposition towards adult-onset hypolactasia by DNA sequencing or restriction fragment length polymorphism analysis. The role for such genetic detection in clinical practice seems limited to ruling out adult-onset hypolactasia as a cause of intolerance symptoms but remains to be fully defined. Attention should be paid to appropriate interpretation of genetic detection in order to avoid potentially harmful reduction in dairy intake or misdiagnosis of secondary lactase deficiency.

Adult↗
PubMed2003

Lactase persistence DNA variant enhances lactase promoter activity in vitro: functional role as a cis regulatory element.

Lactase persistence is a heritable, autosomal dominant, condition that results in a sustained ability to digest the milk sugar lactose throughout adulthood. The majority of the world's human population experiences a decline in production of the digestive enzyme lactase-phlorizin hydrolase during maturation. However, individuals with lactase persistence continue to express high levels of the lactase gene into adulthood. Lactase persistence has been strongly correlated with single nucleotide genetic variants, C/T_(13910) and G/A_(22018), located 13.9 and 22 kb upstream from the lactase structural gene. We aimed to characterize a functional role for the polymorphisms in regulating lactase gene transcription. DNA in the region of the C/T_(13910) or G/A_(22018) human lactase variants was cloned upstream of the 3.0 kb rat lactase gene promoter in a luciferase reporter construct. Human intestinal Caco-2 cells were transfected with the lactase variant/promoter-reporter constructs and assayed for promoter activity. A 200 bp region surrounding the C_(13910) variant, associated with lactase non-persistence, results in a 2.2-fold increase in lactase promoter activity. The T_(13910) variant, associated with lactase persistence, results in an even greater 2.8-fold increase. The DNA sequence of the C/T_(13910) variants differentially interacts with intestinal cell nuclear proteins on EMSAs. AP2 co-transfection results in a similar repression of the C/T_(13910) variant/promoter-reporter constructs. The DNA region of the C/T_(13910) lactase persistence/non-persistence variant functions in vitro as a cis element capable of enhancing differential transcriptional activation of the lactase promoter. Such differential regulation by the C and T variants is consistent with a causative role in the mechanism specifying the lactase persistence/non-persistence phenotypes in humans.

Base Sequence↗
PubMed2002

Regulation of intestine-specific spatiotemporal expression by the rat lactase promoter.

Lactase gene transcription is spatially restricted to the proximal and middle small intestine of the developing mouse. To identify regions of the lactase gene involved in mediating the spatiotemporal expression pattern, transgenic mice harboring 0.8-, 1.3-, and 2.0-kb fragments of the 5'-flanking region cloned upstream of a firefly-luciferase reporter were generated. Transgene expression was assessed noninvasively in living mice using a sensitive low light imaging system. Two independent, 1.3- and 2.0-kb, lactase promoter-reporter transgenic lines expressed appropriate high levels of luciferase activity in the small intestine (300-3,000 relative light units/microg) with maximal expression in the middle segments. Post-weaned 30-day transgenic offspring also demonstrated an appropriate 4-fold maturational decline in luciferase expression in the small intestine. The pattern of the 2.0-kb promoter transgene mRNA abundance most closely mimicked that of the endogenous lactase gene with respect to spatiotemporal restriction. In contrast, a 0.8-kb promoter-reporter construct expressed low level luciferase activity (<25 relative light units/microg) in multiple organs and throughout the gastrointestinal tract in transgenic mice. Thus, a distinct 5'-region of the lactase promoter directs intestine-specific expression in the small intestine of transgenic mice, and regulatory sequences have been localized to a 1.2-kb region upstream of the lactase transcription start site. In addition, we have demonstrated that in vivo bioluminescence imaging can be utilized for assessment of intestinal expression patterns of a luciferase reporter gene driven by lactase promoter regions in transgenic mice.

Animals↗
PubMed2002

Lactase gene transcription is activated in response to hypoxia in intestinal epithelial cells.

Lactase-phlorizin hydrolase, a brush-border membrane disaccharidase, is a marker of intestinal epithelial cell differentiation and digestive function. The intestine is susceptible to conditions of hypoxia resulting from vascular perfusion deficits. We hypothesized that lactase gene induction may provide a mechanism to efficiently increase nutrient energy substrates during gut hypoxia. These studies sought to characterize expression of the lactase gene in response to hypoxia and to characterize a role for hypoxia-inducible factor (HIF-1) in mediating the hypoxic response. Microarray analysis and confirmatory RT-PCR identified a 4-fold induction of lactase mRNA abundance in intestinal epithelial Caco-2 cells exposed to hypoxia. Lactase promoter activity was similarly induced by hypoxia in cells stably transfected with a 2.0-kb 5' flanking region of the rat lactase gene linked to a reporter gene. Transient cotransfection with HIF-1alpha and beta stimulated lactase promoter activity 2.4- and 3.5-fold under conditions of normoxia and hypoxia, respectively. We conclude that HIF-1 can activate the lactase promoter in intestinal epithelial cells exposed to hypoxia. Induction of lactase transcription may represent an adaptive response to gut hypoxia.

Animals↗
PubMed2002

Subarachnoid hemorrhage and isolated atresia of the aortic arch.

The case of a 14 year old boy with subarachnoid hemorrhage and atresia of the aorta without patent ductus arteriosus or intracardiac shunt is described. This case calls attention to the possibility of aortic obstruction in adolescents or young adults with hypertensive stroke. The clinical symptoms, radiographic findings, and surgical repair of isolated aortic interruption, including atresia, are discussed.

Adolescent↗