Intestinal smooth muscle dysfunction develops postnatally in cystic fibrosis mice.

OBJECTIVES: Intestinal dysmotility is one of the effects of cystic fibrosis (CF), but when and how this develops is not well understood. The goal of the present study was to use the Cftr knockout mouse to determine when in development circular smooth muscle of the small intestine becomes dysfunctional. METHODS: Wild-type (WT) and CF mice were used at postnatal day 5 (P5) through adult. Pieces of small intestine were used to measure contractile activity of the circular muscle. Bacterial overgrowth was measured by quantitative polymerase chain reaction (PCR) of the bacterial 16S gene. Intestinal gene expression was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Prostaglandin E2 (PGE2) and its metabolites were measured by enzyme immunoassay. RESULTS: CF circular muscle response to cholinergic stimulation was similar to WT at P5, became somewhat impaired at P7, and was severely impaired by P14. In the CF intestine, bacterial overgrowth occurred by P4 and was maintained into adulthood. Eicosanoid metabolic gene expression in the CF intestine did not differ from WT shortly after birth. The phospholipase A2 genes, Pla2g4c and Pla2g5 exhibited increased expression in CF mice at P24. Prostaglandin degradative genes, Hpgd and Ptgr1, showed lower expression in CF as compared with WT at P16 and P24, respectively. PGE2 levels were significantly greater in CF mice at most ages from P7 through adulthood. CONCLUSIONS: The results clearly demonstrate that lack of CFTR itself does not cause smooth muscle dysfunction, because the circular muscle from P5 CF mice had normal activity and dysfunction developed between P7 and P14.

Transcription factor binding to a DNA zip code controls interchromosomal clustering at the nuclear periphery.

Active genes in yeast can be targeted to the nuclear periphery through interaction of cis-acting "DNA zip codes" with the nuclear pore complex. We find that genes with identical zip codes cluster together. This clustering was specific; pairs of genes that were targeted to the nuclear periphery by different zip codes did not cluster together. Insertion of two different zip codes (GRS I or GRS III) at an ectopic site induced clustering with endogenous genes that have that zip code. Targeting to the nuclear periphery and interaction with the nuclear pore is a prerequisite for gene clustering, but clustering can be maintained in the nucleoplasm. Finally, we find that the Put3 transcription factor recognizes the GRS I zip code to mediate both targeting to the NPC and interchromosomal clustering. These results suggest that zip-code-mediated clustering of genes at the nuclear periphery influences the three-dimensional arrangement of the yeast genome.

Compartmentalization of the nucleus.

The nucleus is a spatially organized compartment. The most obvious way in which this is achieved is at the level of chromosomes. The positioning of chromosomes with respect to nuclear landmarks and with respect to each other is both non-random and cell-type specific. This suggests that cells possess molecular mechanisms to influence the folding and disposition of chromosomes within the nucleus. The localization of many proteins is also heterogeneous within the nucleus. Therefore, chromosome folding and the localization of proteins leads to a model in which individual genes are positioned in distinct protein environments that can affect their transcriptional state. We focus here on the spatial organization of the nucleus and how it impacts upon gene expression.

Mast cells and gastrointestinal dysmotility in the cystic fibrosis mouse.

BACKGROUND: Cystic fibrosis (CF) has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr(tm1UNC), Cftr knockout). METHODOLOGY: Wild type (WT) and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole) or were treated acutely with a mast cell activator (compound 48/80). Gastrointestinal transit was measured using gavage of a fluorescent tracer. RESULTS: In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice. CONCLUSIONS: The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis.

Altered eicosanoid metabolism in the cystic fibrosis mouse small intestine.

OBJECTIVES: Imbalances in essential fatty acid levels have been reported in cystic fibrosis (CF), which may relate to elevated proinflammatory eicosanoid generation. The aim of this work was to better define eicosanoid metabolism in the CF intestine. MATERIALS AND METHODS: We used the small intestine of the cystic fibrosis transmembrane conductance regulator knockout mouse (CF mouse) to measure eicosanoid metabolic gene expression by quantitative reverse transcription polymerase chain reaction and Western blot, and eicosanoid levels by enzyme immunoassay, as compared with wild-type (WT) littermates. RESULTS: In the CF small intestine, expression of the secretory phospholipase A2 Pla2g5 mRNA was upregulated to 980% of WT levels. The following were downregulated: leukotriene C4 synthase Ltc4s (mRNA 55% of WT); omega-hydroxylase cytochrome P450s Cyp2c40 (mRNA 54% of WT), and Cyp4a10 (mRNA 4% of WT); and the major prostaglandin degradative enzymes prostaglandin dehydrogenase Hpgd (mRNA 27% of WT) and leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase Ltb4dh (mRNA 64% and protein 30% of WT). The prostaglandins PGE2 and PGF2alpha were increased to 400% to 600% of WT levels in the CF mouse intestine, and the hydroxyeicosatetraenoic acids (HETEs) 12-, 15-, and 20-HETE were decreased to 3% to 20% of WT levels. CONCLUSIONS: There are changes in eicosanoid metabolic gene expression that are accompanied by significant changes in specific eicosanoid levels. These changes are expected to play important roles in the pathophysiology of CF in the intestine.