We have studied the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride currents in pancreatic duct cells isolated from a transgenic cf/cf mouse created by targeted insertional mutagenesis. Adenosine 3',5'-cyclic monophosphate (cAMP)-activated CFTR chloride currents were detected in 78% (29/37) of wild-type cells, in 81% (35/43) of heterozygote cells, and in 61% (29/47) of homozygous cf/cf duct cells (P > 0.05, cf/cf vs. wild-type and heterozygote). The CFTR current density measured at membrane potentials of +/- 60 mV averaged 22-26 pA/pF in wild-type and heterozygote groups but only 13 pA/pF in cells derived from cf/cf animals (P < 0.05, cf/cf vs. wild-type and cf/cf vs. heterozygotes). In contrast, duct cells from animals of all three genotypic groups exhibited calcium-activated chloride currents that were of similar magnitude and up to 11-fold larger than the CFTR currents. We speculate that these transgenic insertional null mice do not develop the pancreatic pathology that occurs in cystic fibrosis patients because their duct cells contain 1) some wild-type CFTR generated by exon skipping and aberrant splicing and 2) a separate anion secretory pathway mediated by calcium-activated chloride channels.
The human MUC4 gene is not expressed in normal pancreas; however, its dysregulation results in high levels of expression in pancreatic tumors. To investigate the tumor-associated expression, MUC4 cDNA was cloned from a human pancreatic tumor cell line cDNA expression library using a polyclonal antibody raised against human deglycosylated mucin and RT-PCR. Pancreatic MUC4 cDNA shows differences in 12 amino acid residues in the non-tandem repeat coding region with no structural rearrangement as compared with tracheal MUC4. The full-length MUC4 cDNA includes a leader sequence, a serine and threonine rich non-tandem repeat region, a central large tandem repeat domain containing 48 bp repetitive units, regions rich in potential N-glycosylation sites, two cysteine-rich domains, EGF-like domains, and a transmembrane domain. We also report the presence of a new EGF-like domain in MUC4 cDNA, located in the cysteine-rich region upstream from the first EGF-like domain. Four distinct splice events were identified in the region downstream of the central tandem repeat domain that generate three new MUC4 cDNA sequences (sv4, sv9, and sv10). The deduced amino acid sequences of two of these variants lack the transmembrane domain. Furthermore, two unique forms of MUC4 (MUC4/Y and MUC4/X) generated as a result of alternative splicing lack the salient feature of mucins, the tandem repeat domain. A high degree of polymorphism in the central tandem repeat region of MUC4 was observed in various pancreatic adenocarcinoma cell lines, with allele sizes ranging from 23.5 to 10.0 kb. MUC4 mRNA expression was higher in differentiated cell lines, with no detectable expression in poorly differentiated pancreatic tumor cell lines.
1 Functional characteristics and substrate specificity of the rat proton-coupled amino acid transporter 2 (rat PAT2 (rPAT2)) were determined following expression in Xenopus laevis oocytes using radiolabelled uptake measurements, competition experiments and measurements of substrateevoked current using the two-electrode voltage-clamp technique. The aim of the investigation was to determine the structural requirements and structural limitations of potential substrates for rPAT2. 2 Amino (and imino) acid transport via rPAT2 was pH-dependent, Na þ -independent and electrogenic. At extracellular pH 5.5 (in Na þ -free conditions) proline uptake was saturable (Km 172741 mM), demonstrating that rPAT2 is, relative to PAT1, a high-affinity transporter. 3 PAT2 preferred substrates are L-a-amino acids with small aliphatic side chains (e.g. the methyl group in alanine) and 4-or 5-membered heterocyclic amino and imino acids such as 2-azetidinecarboxylate, proline and cycloserine, where both D-and L-enantiomers are transported. 4 The major restrictions on transport are side chain size (the ethyl group of a-aminobutyric acid is too large) and backbone length, where the separation of the carboxyl and amino groups by only two CH 2 groups, as in b-alanine, is enough to reduce transport. Methylation of the amino group is tolerated (e.g. sarcosine) but increasing methylation, as in betaine, decreases transport. A free carboxyl group is preferred as O-methyl esters show either reduced transport (alanine-O-methyl ester) or are excluded.
Background Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood.ResultsThe genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites.ConclusionsWe are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators.
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