Biomedical subjects

P A Dawson

Publications and source records attributed to P A Dawson.

At least 19 recordsLinked to original sources

PubMed2005

Pathogenetics of the human SLC26 transporters.

Over the past decade, 11 human genes belonging to the solute linked carrier (SLC) 26 family of transporters, have been identified. The SLC26 proteins, which include SAT-1, DTDST, DRA/CLD, pendrin, prestin, PAT-1/CFEX and Tat-1, are structurally related and have been shown to transport one or more of the following substrates: sulfate, chloride, bicarbonate, iodide, oxalate, formate, hydroxyl or fructose. Special interest has focused on four members of the SLC26 family that are associated with distinct recessive diseases: (i) Mutations in SLC26A2 lead to four different chondrodysplasias (diastrophic dysplasia, atelosteogenesis type II, achondrogenesis type IB and multiple epiphyseal dysplasia); (ii) SLC26A3 is associated with congenital chloride diarrhea; (iii) SLC26A4 is associated with Pendred syndrome and non-syndromic deafness, DFNB4; and (iv) SLC26A5 is defective in non-syndromic hearing impairment. This review article summarizes current information on the pathophysiological consequences of mutations in the human SLC26A2 to A5 genes.

Anion Transport Proteins↗

PubMed2002

Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone.

The renal sodium-sulfate cotransporter, NaS(i)-1, a protein implicated to control serum sulfate levels, has been shown to be regulated in vivo by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and tri-iodothyronine (T(3)). Recently, we cloned the mouse NaS(i)-1 gene ( Nas1) and in the present study identified a 1,25-(OH)(2)D(3)- and T(3)-responsive element located within the Nas1 promoter. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T(3)-responsive element (IR0 T(3)RE) at -436 to -425 which conferred 1,25-(OH)(2)D(3) and T(3) responsiveness, respectively. In summary, we have identified responsive elements that mediate the enhanced transcription of Nas1 by 1,25-(OH)(2)D(3) and T(3), and these mechanisms may provide important clues to the physiological control of sulfate homeostasis.

Animals↗

PubMed2001

Absence of dysfunctional ileal sodium-bile acid cotransporter gene mutations in patients with adult-onset idiopathic bile acid malabsorption.

BACKGROUND: A congenital form of idiopathic intestinal bile acid malabsorption (IBAM) has been associated with dysfunctional mutations in the ileal apical sodium-dependent bile acid transporter (ASBT). The aim of this study was to determine whether mutations in the ASBT gene (SLC10A2) predispose to the development of adult-onset idiopathic bile acid malabsorption and chronic watery diarrhea. METHODS: Genomic DNA was obtained from 13 adult IBAM patients previously diagnosed on the basis of clinical data, response to cholestyramine, and abnormal 75Se-homocholic acid taurine (SeHCAT) test values. The ASBT gene was screened for the presence of mutations or polymorphisms by single-stranded conformation polymorphism analysis (SSCP) and DNA sequencing. RESULTS: ASBT gene polymorphisms were detected in 5 of the 13 adult IBAM patients. Four patients were heterozygous for a common polymorphism in exon 3, leading to an alanine to serine substitution at codon 171 (A171S). An additional subject was heterozygous for a polymorphism in exon 1 that causes a valine to isoleucine substitution at codon 98 (V981). These functional polymorphisms were also found in unaffected subjects and do not appear to affect ASBT function. CONCLUSIONS: Adult-onset IBAM is not directly related to dysfunctional mutations in the coding region or intron/exon junctions of the SLC10A2 gene. In the absence of apparent ileal disease or intestinal motility defects, inappropriate down-regulation of the ileal bile acid transporter or defects in ileocyte transfer of bile acids into the portal circulation could explain this form of adult IBAM.

Adult↗

PubMed2001

Identification of a novel human cytokine gene in the interleukin gene cluster on chromosome 2q12-14.

Genes in the interleukin-1 (IL-1) gene cluster on human chromosome 2 play an important role in mediating inflammatory responses and are associated with numerous diseases. We have identified a novel IL-1-like gene, IL-1F10, on human chromosome 2q13-14.1 near the IL-1 receptor antagonist gene (IL-1RN). The IL1F10 gene is encoded by 5 exons spanning over 7.8 kb of genomic DNA. The 1008-bp IL-1F10 cDNA encodes a 152-amino acid protein that shares between 41% and 43% amino acid identity with human IL-1 receptor antagonist (IL-1Ra) and FIL-1delta, respectively. IL-1F10 shares characteristics of the IL-1Ra family, including key amino acid consensus sequences and a similar genomic structure. By multitissue first-strand cDNA PCR analysis, IL-1F10 mRNA is expressed in heart, placenta, fetal liver, spleen, thymus, and tonsil. The expression in a variety of immune tissues and similarity to IL-1Ra suggest a role of IL-1F10 in the inflammatory response.

Amino Acid Sequence↗

PubMed2001

Analysis of the ileal bile acid transporter gene, SLC10A2, in subjects with familial hypertriglyceridemia.

Familial hypertriglyceridemia (FHTG), a disease characterized by elevated plasma very low density lipoprotein triglyceride levels, has been associated with impaired intestinal absorption of bile acids. The aim of this study was to test the hypothesis that defects in the active ileal absorption of bile acids are a primary cause of FHTG. Single-stranded conformation polymorphism analysis was used to screen the ileal Na(+)/bile acid cotransporter gene (SLC10A2) for FHTG-associated mutations. Analysis of 20 hypertriglyceridemic patients with abnormal bile acid metabolism revealed 3 missense mutations (V98I, V159I, and A171S), a frame-shift mutation (646insG) at codon 216, and 4 polymorphisms in the 5' flanking sequence of SLC10A2. The SLC10A2 missense mutations and 5' flanking sequence polymorphisms were not correlated with bile acid production or turnover in the hypertriglyceridemic patients and were equally prevalent in the unaffected control subjects. In transfected COS cells, the V98I, V159I, and A171S isoforms all transported bile acids similar to the wild-type SLC10A2. The 646insG frame-shift mutation abolished bile acid transport activity in transfected COS cells but was found in only a single FHTG patient. These findings indicate that the decreased intestinal bile acid absorption in FHTG patients is not commonly associated with inherited defects in SLC10A2.

Adult↗

PubMed2000

Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts.

Ribozymes are a promising agent for the gene therapy of dominant negative genetic disorders by allele-specific mRNA suppression. To test allele-specific mRNA suppression in cells, we used fibroblasts from a patient with osteogenesis imperfecta (OI). These cells contain a mutation in one alpha1(I) collagen allele which both causes the skeletal disorder and generates a novel ribozyme cleavage site. In a preliminary in vitro assay, ribozymes cleaved mutant RNA substrate whereas normal substrate was left intact. For the studies in cell culture we generated cell lines stably expressing active (AR) and inactive (IR) ribozymes targeted to mutant alpha1(I) collagen mRNA. Quantitative competitive RT-PCR analyses of type I collagen mRNA, normalized to beta-actin expression levels, revealed that the level of mutant alpha1(I) collagen mRNA was significantly decreased by approximately 50% in cells expressing AR. Normal alpha1(I) collagen mRNA showed no significant reduction when AR or IR was expressed from the pHbetaAPr-1-neo vector and a small (10-20%) but significant reduction when either ribozyme was expressed from the pCI.neo vector. In clonal lines derived from cells expressing AR the level of ribozyme expression correlated with the extent of reduction in the mutant:normal alpha1(I) mRNA ratio, ranging from 0.33 to 0.96. Stable expression of active ribozyme did not affect cell viability, as assessed by growth rates. Ribozyme cleavage of mutant mRNA results in a reduction in mutant type I collagen protein, as demonstrated by SDS-urea-PAGE. This is the first report of ribozymes causing specific suppression of an endogenous mutant mRNA in cells derived from a patient with a dominant negative genetic disorder.

Alleles↗

PubMed2000

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties.

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na(+)-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the approximately 19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Alternative Splicing↗

PubMed2000

Expression, transport properties, and chromosomal location of organic anion transporter subtype 3.

The rat and mouse organic anion-transporting polypeptides (oatp) subtype 3 (oatp3) were cloned to further define components of the intestinal bile acid transport system. In transfected COS cells, oatp3 mediated Na(+)-independent, DIDS-inhibited taurocholate uptake (Michaelis-Menten constant approximately 30 microM). The oatp3-mediated uptake rates and affinities were highest for glycine-conjugated dihydroxy bile acids. In stably transfected, polarized Madin-Darby canine kidney (MDCK) cells, oatp3 mediated only apical uptake of taurocholate. RT-PCR analysis revealed that rat oatp3, but not oatp1 or oatp2, was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. An antibody directed to the carboxy terminus localized oatp3 to the apical brush-border membrane of rat jejunal enterocytes. The mouse oatp3 gene was localized to a region of mouse chromosome 6. This region is syntenic with human chromosome 12p12, where the human OATP-A gene was mapped, suggesting that rodent oatp3 is orthologous to the human OATP-A. These transport and expression properties suggest that rat oatp3 mediates the anion exchange-driven absorption of bile acids previously described for the proximal small intestine.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Membrane insertion scanning of the human ileal sodium/bile acid co-transporter.

Mammalian sodium-dependent bile acid transporters (SBATs) responsible for bile salt uptake across the liver sinusoidal or ileal/renal brush border membrane have been identified and share approximately 35% amino acid sequence identity. Programs for prediction of topology and localization of transmembrane helices identify eight or nine hydrophobic regions for the SBAT sequences as membrane spanning. Analysis of N-linked glycosylation has provided evidence for an exoplasmic N-terminus and a cytoplasmic C-terminus, indicative of an odd number of transmembrane segments. To determine the membrane topography of the human ileal SBAT (HISBAT), an in vitro translation/translocation protocol was employed using three different fusion protein constructs. Individual HISBAT segments were analyzed for signal anchor or stop translocation (stop transfer) activity by insertion between a cytoplasmic anchor (HK M0) or a signal anchor segment (HK M1) and a glycosylation flag (HK beta). To examine consecutive HISBAT sequences, sequential hydrophobic sequences were inserted into the HK M0 vector or fusion vectors were made that included the glycosylated N-terminus of HISBAT, sequential hydrophobic sequences, and the glycosylation flag. Individual signal anchor (SA) and stop transfer (ST) properties were found for seven out of the nine predicted hydrophobic segments (H1, H2, H4, H5, H6, H7, and H9), supporting a seven transmembrane segment model. However, the H3 region was membrane inserted when translated in the context of the native HISBAT flanking sequences. Furthermore, results from translations of sequential constructs ending after H7 provided support for integration of H8. These data provide support for a SBAT transmembrane domain model with nine integrated segments with an exoplasmic N-terminus and a cytoplasmic C-terminus consistent with a recent predictive analysis of this transporter topology.

Amino Acid Sequence↗

PubMed1999

Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells.

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.

Animals↗

PubMed1999

Intestinal cholesterol absorption.

The strong association between intestinal cholesterol absorption and total plasma cholesterol level has renewed interest in the absorptive process and stimulated the generation of new animal models. Increasingly, new studies suggest that cholesterol absorption is genetically controlled and supports a protein-mediated mechanism for cholesterol uptake into the intestinal mucosal cell. Insights into potential mechanisms are predicted to lead to novel pharmacological approaches to inhibit cholesterol absorption.

Animals↗

PubMed1999

Extension of phenotype associated with structural mutations in type I collagen: siblings with juvenile osteoporosis have an alpha2(I)Gly436 --> Arg substitution.

Mutations in the type I collagen genes have been identified as the cause of all four types of osteogenesis imperfecta (OI). We now report a mutation that extends the phenotype associated with structural abnormalities in type I collagen. Two siblings presented with a history of back pain and were diagnosed with juvenile osteoporosis, based on clinical and radiological examination. Radiographs showed decreased lumbar bone density and multiple compression fractures throughout the thoracic and lumbar spines of both patients. One child has moderate short stature and mild neurosensory hearing loss. However, neither child has incurred the long bone fractures characteristic of OI. Protein studies demonstrated electrophoretically abnormal type I collagen in samples from both children. Enzymatic cleavage of RNA:RNA hybrids identified a mismatch in type I collagen alpha2 (COL1A2) mRNA. DNA sequencing of COL1A2 cDNA subclones defined the mismatch as a single-base mutation (1715G --> A) in both children. This mutation predicts the substitution of arginine for glycine at position 436 (G436R) in the helical domain of the alpha2(I) chain. Analysis of genomic DNA identified the mutation in the asymptomatic father, who is presumably a germ-line mosaic carrier. The presence of the same heterozygous mutation in two siblings strongly suggests that the probands display the full phenotype. Taken together, the clinical, biochemical, and molecular findings of this study extend the phenotype associated with type I collagen mutations to cases with only spine manifestations and variable short stature into adolescence.

Amino Acid Substitution↗

PubMed1998

Bile acid uptake via the human apical sodium-bile acid cotransporter is electrogenic.

Intestinal absorption of bile acids depends on a sodium-bile acid cotransport protein in the apical membrane of the ileal epithelial cell. Transport is Na+-dependent, but the Na+-bile acid stoichiometry and electrogenicity of transport are not known. Studies in whole intestine, isolated cells, and ileal membrane vesicles have been unable to resolve this issue because transport currents are small and can be obscured by other ionic conductances and transport proteins present in these membranes. In this study, the human apical sodium-bile acid transporter was expressed in stably transfected Chinese hamster ovary cells that lack other bile acid transporters. The Na+-dependent transport of a fluorescent bile acid analog, chenodeoxycholyl-Nepsilon-nitrobenzoxadiazol-lysine, was monitored by fluorescence microscopy in single, voltage-clamped cells. Bile acid movement was bidirectional and voltage-dependent with negative intracellular voltage-stimulating influx. A 3-fold reduction in extracellular Na+ produced a negative 52 mV shift of the flux-voltage relationship, consistent with a 2:1 Na+:bile acid coupling stoichiometry. No Na+- or voltage-dependent uptake was observed in nontransfected Chinese hamster ovary cells. These results indicate that the cotransport of bile acids and Na+ by human apical sodium-bile acid transporter is electrogenic and bidirectional and is best explained by a 2:1 Na+:bile acid coupling stoichiometry. These results suggest that membrane potential may regulate bile acid transport rates under physiological and pathophysiological conditions.

Animals↗

PubMed1998

Mutational analysis of the cystathionine beta-synthase gene: a splicing mutation, two missense mutations and an insertion in patients with homocystinuria. Mutations in brief no. 120. Online.

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA.

Alternative Splicing↗

PubMed1998

New insights into bile acid transport.

The enterohepatic circulation of bile acids is maintained by a series of membrane transport proteins. Recent studies of the cloned sodium bile acid cotransporters have provided new insights into their tissue expression, regulation, and their relationship to cholesterol homeostasis and human diseases such as primary bile acid malabsorption.

Animals↗

PubMed1998

Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter.

The enterohepatic circulation of bile acids is maintained by Na(+)-dependent transport mechanisms. To better understand these processes, a full-length human ileal Na(+)-bile acid cotransporter cDNA was identified using rapid amplification of cDNA ends and genomic cloning techniques. Using Northern blot analysis to determine its tissue expression, we readily detected the ileal Na(+)-bile acid cotransporter mRNA in terminal ileum and kidney. Direct cloning and mapping of the transcriptional start sites confirmed that the kidney cDNA was identical to the ileal Na(+)-bile acid cotransporter. In transiently transfected COS cells, ileal Na(+)-bile acid cotransporter-mediated taurocholate uptake was strictly Na+ dependent and chloride independent. Analysis of the substrate specificity in transfected COS or CHO cells showed that both conjugated and unconjugated bile acids are efficiently transported. When the inhibition constants for other potential substrates such as estrone-3-sulfate were determined, the ileal Na(+)-bile acid cotransporter exhibited a narrower substrate specificity than the related liver Na(+)-bile acid cotransporter. Whereas the multispecific liver Na(+)-bile acid cotransporter may participate in hepatic clearance of organic anion metabolites and xenobiotics, the ileal and renal Na(+)-bile acid cotransporter retains a narrow specificity for reclamation of bile acids.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of a novel peptidase from rat and human ileum.

A novel 100-kDa ileal brush border membrane protein (I100) has been purified by anionic glycocholate affinity chromatography. Polyclonal antibodies raised against this protein were utilized to clone and characterize I100 in rats. A partial length human I100 cDNA was identified by hybridization screening. In the rat, the I100 protein is a 746-amino acid glycosylated (calculated core molecular mass of 80 kDa) type II integral membrane protein found on the apical surface of ileal villus enterocytes. Its 2.6-kilobase mRNA is expressed in distal small intestine in rats and in humans. The I100 cDNA is homologous to but distinct from human prostate-specific membrane antigen and rat brain N-acetylaspartylglutamate peptidase. It is expressed on both the basolateral and apical surfaces of stably transfected Madin Darby canine kidney cells. Analysis of these stably transfected Madin Darby canine kidney cells and I100 immunoprecipitates of rat ileal brush border membrane vesicles reveals that it has dipeptidyl peptidase IV activity. Future invesitgations will need to determine the exact substrate specificity of this novel peptidase.

Amino Acid Sequence↗

PubMed1997

Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter.

Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.

Animals↗