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At least 595 records · Page 33Linked to original sources
PubMed2006

RNAi components are required for nuclear clustering of Polycomb group response elements.

Drosophila Polycomb group (PcG) proteins silence homeotic genes through binding to Polycomb group response elements (PREs). Fab-7 is a PRE-containing regulatory element from the homeotic gene Abdominal-B. When present in multiple copies in the genome, Fab-7 can induce long-distance gene contacts that enhance PcG-dependent silencing. We show here that components of the RNA interference (RNAi) machinery are involved in PcG-mediated silencing at Fab-7 and in the production of small RNAs at transgenic Fab-7 copies. In general, these mutations do not affect the recruitment of PcG components, but they are specifically required for the maintenance of long-range contacts between Fab-7 copies. Dicer-2, PIWI, and Argonaute1, three RNAi components, frequently colocalize with PcG bodies, and their mutation significantly reduces the frequency of PcG-dependent chromosomal associations of endogenous homeotic genes. This suggests a novel role for the RNAi machinery in regulating the nuclear organization of PcG chromatin targets.

Animals↗
PubMed2004

The DNA binding protein H-NS binds to and alters the stability of RNA in vitro and in vivo.

H-NS is an abundant prokaryotic transcription factor that preferentially binds to intrinsically bent DNA. Although H-NS has been shown to reduce the transcription of over 100 genes, evidence suggests that H-NS can also affect the translation of some genes. One such gene, rpoS, specifies a sigma factor, RpoS. The ability of H-NS to bind to the rpoS mRNA and the non-coding RNA regulator, DsrA, was tested. Electrophoretic mobility-shift assays yielded an apparent binding affinity of H-NS binding to curved DNA of approximately 1 microM, whereas binding to rpoS mRNA or DsrA RNA was approximately 3 microM. This RNA binding was not prevented by an excess of competitor yeast RNA, suggesting that H-NS specifically bound these RNAs. Footprint analysis with a single strand-specific ribonuclease was used to identify the H-NS binding site(s) on DsrA and rpoS mRNA. Surprisingly, H-NS appeared to enhance the cleavage of DsrA and rpoS mRNA. The enhanced cleavage was at sites that were predicted to be single-stranded and did not result from contaminating nucleases in the H-NS protein preparation or non-specific effects of the nuclease. Quantitative RT-PCR of RNA isolated from wild-type and hns- strains revealed that H-NS also affects the stability of DsrA in vivo. Thus H-NS appears to modulate RNA stability in vivo and in vitro.

Bacterial Proteins↗
PubMed1997

Adenovirus-mediated expression of AMPA-type glutamate receptor channels in PC12 cells.

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor channels are expressed ubiquitously in brain neurons and mediate fast excitatory neurotransmission. They are composed of four subunits, GluR1, GluR2, GluR3 and/or GluR4. We constructed recombinant adenoviruses encoding rat AMPA receptor subunit cDNAs, GluR1 (AxCAGluR1) and silently mutated GluR2 (AxCAGluR2X) with modified chicken beta-actin promoter and cytomegalovirus immediate-early enhancer. Using these adenoviral vectors, we transferred the GluR1 and GluR2 genes into PC12 cells that possess no functional AMPA receptor channels. PC12 cells infected with these viruses expressed GluR1 and GluR2 RNAs. Immunoblot analysis indicated that the expressed GluR1 and GluR2 proteins were equivalent to those of the rat brain. Functional expression of the AMPA receptor channels was examined using the whole-cell patch clamp technique. In AxCAGluR1-infected cells, the current-voltage (I-V) relationship of response to kainate, a non-desensitizing agonist of AMPA receptors, exhibited a strong inward rectification, indicating the formation of functional GluR1-homomeric channels. In cells infected with both AxCAGluR1 and AxCAGluR2X, the I-V relationship of kainate responses exhibited an outward rectification, indicating the formation of heteromeric GluR1/R2 channels. Immunocytochemical analysis revealed that the AMPA receptor subunit genes were transferred in more than 95% of the infected PC12 cells.

Adenoviridae↗
PubMed2001

Large-scale evaluation of imprinting status in the Prader-Willi syndrome region: an imprinted direct repeat cluster resembling small nucleolar RNA genes.

Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader-Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive. Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established. Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS. Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15. Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval. This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion. Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs). Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, Xenopus and Drosophila. These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.

Base Sequence↗
PubMed2005

Replication of a recombinant hepatitis E virus genome tagged with reporter genes and generation of a short-term cell line producing viral RNA and proteins.

Hepatitis E virus (HEV) replication has been demonstrated in HepG2 cells transfected with full-length in vitro transcripts of an infectious cDNA clone. This cDNA clone was modified to generate several subgenomic HEV replicons with fused reporter genes. In vitro-transcribed capped RNAs generated from these were transfected into HepG2 cells. Negative-strand RNA was detected, indicating the occurrence of replication. The replicon containing an in-frame fusion of HEV ORF2 with enhanced green fluorescent protein (EGFP) was positive for fluorescence, whereas no signal was observed when the replicase domain was deleted. An HEV ORF3-EGFP in-frame fusion did not yield fluorescence. Deletions introduced into ORF2 did not affect the replication competency of the viral RNA. To explore the possibility of using a reporter-gene assay to monitor the synthesis of plus- and minus-strand RNA, the EGFP gene fused to the encephalomyocarditis virus internal ribosome entry site (IRES) was inserted into partially deleted ORF2 of HEV, in both the sense [HEV-IRES-EGFP(+)] and antisense [HEV-IRES-EGFP(-)] orientations. HepG2 cells transfected with HEV-IRES-EGFP(+) and HEV-IRES-EGFP(-) vectors were positive for EGFP fluorescence. To quantify HEV replication, EGFP was replaced with Renilla luciferase (RLuc). HEV-IRES-RLuc(+) showed approximately 10-fold higher luminescence than HEV-IRES-RLuc(-). There was complete loss of activity when the helicase-replicase domain in HEV-IRES-RLuc(-) was deleted. A short-term HepG2 cell line containing the full-length viral genome in the pcDNA3 vector was established. Viral RNA and proteins (RdRp, pORF2 and pORF3) could be detected in the geneticin-resistant cells, even after the seventh passage. In the absence of a reliable cell-culture system to study HEV biology, these reporter replicons, as well as the cell line, bestow immense utility.

Cell Line, Tumor↗
PubMed2002

Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis.

bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c-myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of bic, particularly its ability to cooperate with c-myc in oncogenesis, was tested directly by expressing c-myc and bic, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c-myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c-myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.

Animals↗
PubMed2005

Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism.

It is controversial whether the accessory human immunodeficiency virus type 1 (HIV-1) Nef protein inhibits or enhances apoptosis. To address this issue, we investigated the effect of Nef on programmed cell death with vectors or proviral HIV-1 constructs coexpressing Nef and green fluorescent protein from single bicistronic RNAs. This approach allows us to readily identify transfected or infected cells and to correlate cell death directly with Nef expression levels. We demonstrate that Nef does not significantly affect apoptosis in transfected or HIV-1-infected Jurkat T cells or primary human peripheral blood mononuclear cells. Unexpectedly, however, both nef+ and nef-defective HIV-1 infection blocked apoptosis in cells treated with UV light or etoposide but not cell death induced by CD95 antibody, TRAIL, Ly294002, or serum starvation. Our results show that HIV-1 infection inhibits DNA damage-induced but not death receptor-dependent cell death by a Nef-independent mechanism.

Apoptosis↗
PubMed1990

Translational regulation of the immunoglobulin heavy-chain binding protein mRNA.

Translation of the mRNA encoding the immunoglobulin heavy-chain binding protein (BiP) is enhanced in poliovirus-infected cells at a time when translation of host cell mRNAs is inhibited. To test whether the mRNA of BiP is translated by internal ribosome binding, like picornaviral RNAs, we constructed plasmids for the expression of dicistronic hybrid RNAs containing the 5' noncoding region (5'NCR) of BiP as an intercistronic spacer element between two cistrons. Expression of these dicistronic mRNAs in mammalian cells resulted in efficient translation of both cistrons, demonstrating that the 5'NCR of BiP can confer internal ribosome binding to a heterologous RNA. This result suggests that the mRNA encoding BiP is bifunctional and can be translated by an internal ribosome-binding mechanism, in addition to the conventional cap-dependent scanning mechanism. This is the first demonstration of a cellular mRNA that can be translated by internal ribosome binding, and implies that this may be a mechanism for cellular translational regulation.

Carrier Proteins↗
PubMed2026

EV-B 3D polymerase remodels viral populations through 5'UTR recombination to subvert cardiac antiviral innate immunity.

Viral myocarditis, a leading cause of morbidity in young populations, is strongly linked to Coxsackievirus B (CV-B) infections harboring dominant 5'-terminally deleted (5'TD) and minor full-length (FL) CV-B RNA populations in cardiac tissues. Here, we demonstrate how viral RNA-dependent RNA polymerase (3Dpol)-driven recombination in the 5'UTR orchestrates viral RNA populations dynamics and subverts type I interferon responses. In primary human cardiomyocytes (HCMs), 3Dpol-mediated copy-choice recombination enhances 5'TD RNA replication while suppressing FL populations. Infection of immunocompetent mice with recombination-deficient CV-B3 (3Dpol Y276H) shifted 5'TD populations ratios toward immune-sensing viral RNAs, elevating cardiac IFN-β/ISG15 and accelerating viral clearance. Transfection experiments confirmed that 50-nt 5'TD RNAs (TD50) evade innate immunity, whereas shorter deletions (9-36-nt, TD15) restore type I interferon responses in HCMs. Our findings establish 3Dpol-driven recombination as a critical mechanism sustaining pathogenic 5'TD RNAs that subvert antiviral innate immunity, highlighting recombination inhibition as a promising therapeutic strategy against CV-B myocarditis.

Animals↗
PubMed2004

Role for plasma membrane-related Ca2+-ATPase-1 (ATP2C1) in pancreatic beta-cell Ca2+ homeostasis revealed by RNA silencing.

Changes in intracellular Ca(2+) concentration play a key role in the regulation of insulin secretion by glucose and other secretagogues. Here, we explore the importance of the secretory pathway Ca(2+)-ATPase, plasma membrane-related Ca(2+)-ATPase-1 (PMR1; human orthologue ATP2C1) in intracellular Ca(2+) homeostasis in pancreatic islet beta-cells. Endogenous PMR1 mRNA and protein were detected in both isolated rat islets and beta-cell-derived lines (MIN6 and INS1). Subcellular fractionation of the cell lines revealed PMR1 immunoreactivity in both microsomal and dense-core secretory vesicle-enriched fractions. Correspondingly, depletion of cellular PMR1 with small interfering RNAs inhibited Ca(2+) uptake into the endoplasmic reticulum and secretory vesicles by approximately 20%, as assessed using organelle-targeted aequorins in permeabilized INS1 cells. In intact cells, PMR1 depletion markedly enhanced flux though L-type Ca(2+) channels and augmented glucose-stimulated, but not basal, insulin secretion. Whereas average cytosolic [Ca(2+)] increases in response to 30.0 mmol/l glucose were unaffected by PMR1 depletion, [Ca(2+)] oscillation shape, duration, and decay rate in response to glucose plus tetraethylammonium were modified in PMR1-depleted single cells, imaged using fluo-3-acetoxymethylester. PMR1 thus plays an important role, which is at least partially nonoverlapping with that of sarco(endo-)plasmic reticulum Ca(2+)-ATPases, in the control of beta-cell Ca(2+) homeostasis and insulin secretion.

ATP-Binding Cassette Transporters↗
PubMed1994

Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF-1) activates erythropoietin gene transcription in Hep3B cells subjected to hypoxia. HIF-1 activity is also induced by hypoxia in non-erythropoietin-producing cells, suggesting a more general regulatory role. We now report that RNAs encoding the glycolytic enzymes aldolase A (ALDA), phosphoglycerate kinase 1 (PGK1), and pyruvate kinase M were induced by exposure of Hep3B or HeLa cells to inducers of HIF-1 (1% O2, cobalt chloride, or desferrioxamine), whereas cycloheximide blocked induction of glycolytic RNAs and HIF-1 activity. Oligonucleotides from the ALDA, PGK1, enolase 1, lactate dehydrogenase A, and phosphofructokinase L (PFKL) genes, containing sequences similar to the HIF-1 binding site in the erythropoietin enhancer, specifically bound HIF-1 present in crude nuclear extracts or affinity-purified preparations. Sequences from the ALDA, PFKL, and PGK1 genes containing HIF-1 binding sites mediated hypoxia-inducible transcription in transient expression assays. These results support the role of HIF-1 as a mediator of adaptive responses to hypoxia that underlie cellular and systemic oxygen homeostasis.

Base Sequence↗
PubMed1975

Block to multiplication of adenovirus serotype 2 in monkey cells.

The block to adenovirus 2 (Ad2) multiplication in monkey cells can be overcome by coinfection with simian virus 40 (SV40). To identify this block we have compared the synthesis of Ad2 proteins in monkey cells infected with Ad2 alone (unenhanced) or with Ad2 plus SV40 (enhanced). Synthesis of viral proteins in enhanced cells was virtually identical to that found for permissive infection of human cells by Ad2 alone. In contrast, the unenhanced cells were strikingly deficient in the production of the IV (fiber) and 11.5K proteins whereas the synthesis of 100K and IVa2 was normal. Synthesis of a number of other proteins such as II, V, and P-VII was partially reduced. A similar specific reduction in synthesis of these proteins was found when their messages were assayed by cell-free translation. This result suggests that the block to Ad2 protein synthesis is at the RNA level rather than with the translational machinery of monkey cells. Analysis of the complexity and the concentration of Ak2-specific RNAs, using hybridization of restriction endonuclease fragments of the Ad2 genome to increasing concentrations of RNA, shows that although all species of late Ad2 mRNA are present, the concentration of several species is reduced sevenfold or more in unenhanced monkey cells as compared with enhanced cells. These species come from regions of the genome known to encode the deficient proteins. A model for the failure of adenovirus to multiply in monkey cells, based on abnormal processing of specific adenovirus messages, is presented.

Adenoviridae↗
PubMed1996

Enhancement of hammerhead ribozyme catalysis by glyceraldehyde-3-phosphate dehydrogenase.

A specific tumour necrosis factor alpha ribozyme (TNF-alpha-Rz) binding activity has been purified and identified by N-terminal microsequencing as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The purified protein as well as commercial GAPDH binds tightly to TNF-alpha ribozyme compared to a variety of other ribozymes and RNAs. Binding of GAPDH to the TNF-alpha-Rz and its derivatives was inhibited by NAD+ and ATP, suggesting that the GAPDH Rossmann fold structure is a part of the ribozyme binding site. Interestingly, GAPDH increased the in vitro cleavage rates of hammerhead ribozymes by up to 25-fold, while no significant stimulation was observed with the lactate dehydrogenase (LDH). This effect was found to be due to the unfolding activity of GAPDH. In fact, pulse-chase experiments demonstrate directly that GAPDH has the capacity to accelerate the ribozyme/substrate association, especially of ribozymes and/or substrates whose predicted secondary structure might interfere with the association step. Under our conditions, the presumed unfolding activity of GAPDH also enhances the turnover of ribozymes by increasing the rate of product dissociation, although only for short cleavage products. Longer duplexes required more incubation time to dissociate. In vitro non-specific interaction of the GAPDH with hammerhead ribozymes and RNA substrates was found to be adequate for the cleavage enhancement effect to occur. However, an analysis of the ability of various prototypical ribozymes to inhibit the expression of interleukin-2 suggests that the addition of a sequence having a high affinity for GAPDH improves the efficacy of ribozymes in the cells. Thus the characterization of cellular proteins with unfolding activity, which specifically bind to hammerhead ribozyme, should facilitate the design of a more effective ribozyme in vivo.

Base Sequence↗
PubMed2005

The human herpesvirus 6 G protein-coupled receptor homolog U51 positively regulates virus replication and enhances cell-cell fusion in vitro.

Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G protein-coupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.

Antibodies, Viral↗
PubMed2000

Characterization of a maternal T-Box gene in Ciona intestinalis.

A new T-box gene, CiVegTR, was isolated in the ascidian Ciona intestinalis. CiVegTR maternal RNAs become localized to the vegetal cytoplasm of fertilized eggs and are incorporated into muscle lineages derived from the B4.1 blastomere. The CiVegTR protein binds to specific sequences within a minimal, 262-bp enhancer that mediates Ci-snail expression in the tail muscles. Mutations in these binding sites abolish expression from an otherwise normal lacZ reporter gene in electroporated embryos. In addition to the previously identified AC-core E-box sequences, T-box recognition sequences are conserved in the promoter regions of many genes expressed in B4.1 lineages in both Ciona and the distantly related ascidian Halocynthia. These results suggest that CiVegTR encodes a component of the classical muscle determinant that was first identified in ascidians nearly 100 years ago.

Amino Acid Sequence↗
PubMed2005

Unusual structure of ribosomal DNA in the copepod Tigriopus californicus: intergenic spacer sequences lack internal subrepeats.

Eukaryotic nuclear ribosomal DNA (rDNA) is typically arranged as a series of tandem repeats coding for 18S, 5.8S, and 28S ribosomal RNAs. Transcription of rDNA repeats is initiated in the intergenic spacer (IGS) region upstream of the 18S gene. The IGS region itself typically consists of a set of subrepeats that function as transcriptional enhancers. Two important evolutionary forces have been proposed to act on the IGS region: first, selection may favor changes in the number of subrepeats that adaptively adjust rates of rDNA transcription, and second, coevolution of IGS sequence with RNA polymerase I transcription factors may lead to species specificity of the rDNA transcription machinery. To investigate the potential role of these forces on population differentiation and hybrid breakdown in the intertidal copepod Tigriopus californicus, we have characterized the rDNA of five T. californicus populations from the Pacific Coast of North America and one sample of T. brevicornicus from Scotland. Major findings are as follows: (1) the structural genes for 18S and 28S are highly conserved across T. californicus populations, in contrast to other nuclear and mitochondrial DNA (mtDNA) genes previously studied in these populations. (2) There is extensive differentiation among populations in the IGS region; in the extreme, no homology is observed across the IGS sequences (>2 kb) from the two Tigriopus species. (3) None of the Tigriopus IGS sequences have the subrepeat structure common to other eukaryotic IGS regions. (4) Segregation of rDNA in laboratory crosses indicates that rDNA is located on at least two separate chromosomes in T. californicus. These data suggest that although IGS length polymorphism does not appear to play the adaptive role hypothesized in some other eukaryotic systems, sequence divergence in the rDNA promoter region within the IGS could lead to population specificity of transcription in hybrids.

Copepoda↗
PubMed2005

Silencing of endogenous IGFBP-5 by micro RNA interference affects proliferation, apoptosis and differentiation of neuroblastoma cells.

Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.

Apoptosis↗
PubMed1996

Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver.

BACKGROUND & AIMS: While the lobular heterogeneity of hepatocytes is established, limited data suggest that bile duct epithelial cells, of cholangiocytes, are heterogeneous along the normal intrahepatic biliary tree. Thus, we tested the hypothesis that cholangiocytes are structurally and functionally heterogeneous in the biliary tract of normal rats. METHODS: A series of in situ and in vitro experiments was performed in normal rats using complementary morphometric, molecular, and functional approaches. RESULTS: In situ morphometry showed that (1) intrahepatic bile ducts are heterogeneous in external diameter (5-200 mum); (2) individual cholangiocytes lining bile ducts are heterogeneous in area (3-80 mum2); and (3) a significant relationship exists between bile duct diameter and cholangiocyte area. Using a novel approach developed by us, we isolated three subpopulations of small, medium, and large cholangiocytes and compared them at the molecular and functional level. The expression of two cholangiocyte-specific genes (gamma-glutamyl transpeptidase and cytokeratin 19) was similar among the three subpopulations. In contrast, secretion receptor, Cl-/HCO3- exchanger, and cystic fibrosis transmembrane conductance regulator messenger RNAs were differentially expressed, being present on medium and large but not small cholangiocytes. At the functional level, adenosine 3', 5'-cyclic monophosphate and intracellular pH responses and Cl-/HCO3- exchanger activity was enhanced by secretion in medium and large but not small cholangiocytes. CONCLUSIONS: These data indicate that cholangiocytes are morphologically and functionally heterogeneous along the normal intrahepatic biliary tree and suggest that secretion-regulated transport of water and electrolytes occurs principally in medium and large ducts.

Animals↗