Biomedical subjects

W H Colledge

Publications and source records attributed to W H Colledge.

At least 19 recordsLinked to original sources

PubMed2004

Markedly reduced effects of (-)-isoprenaline but not of (-)-CGP12177 and unchanged affinity of beta-blockers at Gly389-beta1-adrenoceptors compared to Arg389-beta1-adrenoceptors.

1. Substitution of arginine by glycine at position 389, a frequent beta(1)-adrenoceptor polymorphism, reduces adenylyl cyclase stimulation by (-)-isoprenaline. beta(1)-Adrenoceptors mediate the effects of catecholamines and nonconventional partial agonists ((-)-CGP12177) through different sites. We investigated the influence of the 389 polymorphism on beta blocker affinity, as well as on the responses to (-)-isoprenaline and the nonconventional partial agonist (-)-CGP12177 on cyclic AMP levels in CHO cells expressing recombinant Arg389-beta(1)-adrenoceptors (101 fmol mg(-1) protein) or Gly389-beta(1)-adrenoceptors (94 fmol mg(-1)). 2. The affinity of beta-blockers and partial agonists, estimated from competition binding with (-)-[(125)I]-cyanopindolol, was not different for Arg389-beta(1)-adrenoceptors and Gly389-beta(1)-adrenoceptors. 3. The maximum cAMP increases by (-)-isoprenaline and (-)-CGP12177 at Gly389-beta(1)-adrenoceptors were reduced by 97 and 46%, but the potencies enhanced 2 and 0.5 log units, respectively, compared to Arg389-beta(1)-adrenoceptors. The intrinsic activity of (-)-CGP12177 with respect to the (-)-isoprenaline was 0.057 at Arg389-beta(1)-adrenoceptors and 1.05 at Gly389-beta(1)-adrenoceptors. 4. We confirm in intact CHO cells that responses to (-)-isoprenaline are markedly reduced at Gly389-beta(1)-adrenoceptors compared to Arg389-beta(1)-adrenoceptors. However, the 389 polymorphism reduces considerably less the agonist responses to (-)-CGP12177, indicating that coupling to G(s) protein is different for beta(1)-adrenoceptors activated by catecholamines than for receptors activated by nonconventional partial agonists. The affinity of beta-blockers is conserved across the Arg389Gly polymorphism.

Adrenergic beta-Antagonists↗

PubMed2004

Mice lacking pro-opiomelanocortin are sensitive to high-fat feeding but respond normally to the acute anorectic effects of peptide-YY(3-36).

Inactivating mutations of the pro-opiomelanocortin (POMC) gene in both mice and humans leads to hyperphagia and obesity. To further examine the mechanisms whereby POMC-deficiency leads to disordered energy homeostasis, we have generated mice lacking all POMC-derived peptides. Consistent with a previously reported model, Pomc(-/-) mice were obese and hyperphagic. They also showed reduced resting oxygen consumption associated with lowered serum levels of thyroxine. Hypothalami from Pomc(-/-) mice showed markedly increased expression of melanin-concentrating hormone mRNA in the lateral hypothalamus, but expression of neuropeptide Y mRNA in the arcuate nucleus was not altered. Provision of a 45% fat diet increased energy intake and body weight in both Pomc(-/-) and Pomc(+/-) mice. The effects of leptin on food intake and body weight were blunted in obese Pomc(-/-) mice whereas nonobese Pomc(-/-) mice were sensitive to leptin. Surprisingly, we found that Pomc(-/-) mice maintained their acute anorectic response to peptide-YY(3-36) (PYY(3-36)). However, 7 days of PYY(3-36) administration had no effect on cumulative food intake or body weight in wild-type or Pomc(-/-) mice. Thus, POMC peptides seem to be necessary for the normal response of energy balance to high-fat feeding, but not for the acute anorectic effect of PYY(3-36) or full effects of leptin on feeding. The finding that the loss of only one copy of the Pomc gene is sufficient to render mice susceptible to the effects of high fat feeding emphasizes the potential importance of this locus as a site for gene-environment interactions predisposing to obesity.

Animals↗

PubMed2004

A LacZ-based transgenic mouse for detection of somatic gene repair events in vivo.

Somatic gene repair of disease-causing chromosomal mutations is a novel approach for gene therapy. This method would ensure that the corrected gene is regulated by its endogenous promoter and expressed at physiological levels in the appropriate cell types. A reporter mouse, Gtrosa26(tm1Col), was generated by targeting a mutated LacZ gene to the Rosa26 locus in mouse embryonic stem (ES) cells. The LacZ gene contains a G to A point mutation, resulting in a Glu to Lys amino-acid substitution at position 461, which abrogates enzymatic activity. The gene is expressed in ES cells, primary embryonic fibroblasts, and in all tissues examined in the adult mouse, including the lung, liver, kidney, spleen, heart, brain and smooth muscle. This transgenic mouse will allow testing of gene repair strategies in vivo and identification of which cell types can be successfully targeted by chromosomal gene repair. Although low levels of gene repair were achieved in the ES cells used to generate the Gtrosa26(tm1Col) mouse, preliminary attempts at gene repair in vivo were unsuccessful, thus highlighting the difficulties that will have to be overcome to get this approach to work.

Animals↗

PubMed2004

Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice.

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Expression and regulation of the Na+-K+-2Cl- cotransporter NKCC1 in the normal and CFTR-deficient murine colon.

Defective regulation and/or reduced expression of the Na+-K+-2Cl- cotransporter NKCC1 may contribute to the severe secretory defect that is observed in cystic fibrosis, but data concerning the expression and function of NKCC1 in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient cells are equivocal. We therefore investigated NKCC1 mRNA expression, Na+-K+-2Cl- cotransport activity and regulation by cAMP in crypts isolated from the proximal colon of CFTR-containing (CFTR (+/+)) and CFTR-deficient (CFTR (-/-)) mice. mRNA expression levels were determined by semiquantitative PCR, transport rates were measured fluorometrically in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetomethylester (BCECF)-loaded crypts, cytoplasmic volume changes were assessed by confocal microscopy, and [Cl-]i changes were examined by N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) quenching. NKCC1 mRNA expression levels were not significantly reduced in CFTR (-/-) crypts compared to controls. Azosemide-sensitive NH4+ influx (used as a measure of Na+-K+-2Cl- cotransport) was 2.23 +/- 0.72 vs. 1.56 +/- 0.16 mM min-1, and increased by 63.6 % in (+/+) and 87.3 % in (-/-) crypts upon stimulation for 5 min with forskolin. After 20 min of stimulation with forskolin, the Na+-K+-2Cl- cotransport rates in (-/-) and (+/+) crypts were identical. Crypt cross-sectional area and [Cl-]i decreased only in (+/+) crypts upon stimulation. In conclusion, normal NKCC1 expression levels, somewhat reduced Na+-K+-2Cl- cotransport rates, but preserved activation by cAMP were found in colonic crypts from CFTR (-/-) mice, ruling out a severe dysfunction of the Na+-K+-2Cl- cotransporter in the CF intestine. Furthermore, these studies establish the existence of a direct, cell-volume- and [Cl-]i-independent activation of colonic NKCC1 by an increase in intracellular cAMP.

Animals↗

PubMed2003

A comparison of gene repair strategies in cell culture using a lacZ reporter system.

Synthetic oligonucleotides and DNA fragments of less than 1 kilobase (kb) have been shown to cause site-specific genetic alterations in mammalian cells in culture and in vivo. We have used a lacZ reporter gene system to compare the efficiency of episomal and chromosomal gene repair in human embryonic kidney epithelial cells (HEK293), Chinese Hamster Ovary fibroblasts (CHOK1), human bronchial epithelial cells (16HBE), and mouse embryonic stem (ES) cells. The lacZ gene contains a G to A nucleotide change, (Glu to Lys mutation) that abrogates beta-galactosidase activity. We compared the efficiency of different gene repair methods to correct this mutation and restore beta-galactosidase activity. We evaluated PCR-generated double-stranded DNA fragments of 0.52-1.9 kb, single-stranded DNA oligonucleotides of 20, 35, or 80 bases containing internal phosphorothioate links, and a 68 base RNA:DNA oligonucleotide. All of the oligonucleotides and DNA fragments showed some gene repair ability with an episomal plasmid. Short DNA fragments of 0.52 kb or greater gave the highest frequencies of episomal gene repair while single-stranded DNA oligonucleotides gave the highest frequency of chromosomal repair. In the context of a chromosomal target, antisense DNA oligonucleotides gave 5-fold higher frequencies of gene repair than their sense counterparts. The RNA:DNA chimeric oligonucleotide gave little or no gene repair on either a chromosomal or episomal target.

Blotting, Southern↗

PubMed2003

A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo.

Polyethylenimine (PEI), a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. We have defined the optimal conditions for PEI-based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore Cl(-) channel activity in a CF mouse model. Three forms of PEI, a linear 22 kDa (ExGen 500) form and branched 25 or 50 kDa forms were evaluated. All forms of PEI significantly increased luciferase reporter gene expression compared to the liposome DCChol/DOPE in a human bronchial epithelial cell line (16HBE) irrespective of the extent of cell confluency. With subconfluent cells, gene expression was around 1000-, 200- and 25-fold higher than liposomes using linear 22, 25 and 50 kDa PEI, respectively. The transfection efficiency was reduced in confluent and polarized epithelial cells but linear 22 kDa PEI showed the smallest decrease and gave 8000-fold better transfection in polarized cells compared to liposomes. A comparison of linear 22 or 25 kDa PEI with DCChol/DOPE for airway delivery in vivo via intranasal instillation was also performed. Linear 22 kDa PEI gave significantly better luciferase reporter gene expression of 350-fold in the lung, 180-fold in the nose and 85-fold in the trachea compared to liposome. In contrast, the 25 kDa form of PEI was no better than DCChol/DOPE. Repeat dosing with linear 22 kDa PEI failed to give reporter gene delivery comparable to the initial dose. To establish that PEI can be used to deliver a physiologically relevent gene in vivo, we used it to restore Cl(-) secretion by CFTR gene delivery in the airways of a CF mouse model.

Animals↗

PubMed2002

cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1.

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.

Animals↗

PubMed2002

Optimisation of real-time quantitative RT-PCR for the evaluation of non-viral mediated gene transfer to the airways.

Naked plasmid DNA and DNA/liposome complexes are currently being considered as gene therapy treatments for cystic fibrosis (CF) pulmonary disease. Current methods of gene delivery to the airways result only in transient correction of the CF ion transport defect, and disease treatment is likely to require repeated administrations of vector. However, it is unclear if repeat administration will be tolerated by CF individuals. Technologies including TaqMan (Applied Biosystems) real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) can be used to determine the efficacy of gene transfer formulations. TaqMan RT-PCR assays were designed and optimised to detect plasmid vector-derived and endogenous gene expression. Subsequently, these assays were used to quantify vector-derived mRNA after delivery of naked DNA and DNA/liposome formulations expressing human and murine cystic fibrosis transmembrane conductance regulator (CFTR) to the mouse airways. Vector-derived mRNA was detected in samples following the delivery of naked DNA or DNA/liposomes to the mouse airways, and no reduction in vector-derived mRNA was observed upon repeat administration, a finding that is consistent with the murine and human CFTR being tolerated by the mouse. Although it remains to be seen if CF patients can tolerate long-term expression of wild-type CFTR, these data demonstrate that TaqMan RT-PCR is an effective tool to accurately quantify transgene expression in the airways.

Animals↗

PubMed2001

Ultrastructural changes in exocrine tissues of a DeltaF-508 CFTR mouse model.

Cystic fibrosis (CF) is characterized by abnormal secretion from epithelial cells. We wanted to detect changes in the ultrastructural characteristics of cells within a number of exocrine tissues, including the colon, submandibular and parotid salivary glands of DeltaF-508 CFTR animals. Therefore, in the present study a DeltaF-508 CFTR mouse model was compared to control, by applying conventional and complex carbohydrates staining techniques to tissue sections at the electron microscope level. The colon of DeltaF-508 CFTR mice contained thick mucous secretions that harbored many bacteria, along with cytoplasmic fragments and leukocytes. Leukocytes were also seen to infiltrate the cytoplasm of goblet cells. Tissues were taken before, 10 min after isoprenaline, and 30 min after a further injection of methacholine. In the submandibular gland, there is limited secretory activity after isoprenaline treatment, and this increases further with methacholine treatment. Depletion of the secretory granules of acinar cells is observed, following the combined isoprenaline and methacholine treatment, but no significant changes in granule numbers occurred in granular tubule cells. Glycogen, abundant before treatment, is reduced within 10 min of isoprenaline treatment and is completely exhausted by 30 min, especially in the convoluted granular tubule cells. A few secretory granules in acinar and in granular tubule cells of the DeltaF-508 CFTR submandibular glands displayed two electron densities. The secretory responses of the parotid gland cells were similar to those in submandibular gland cells, except that in these DeltaF-508 CFTR cells, secretory granules appeared more polymorphic in structure than those found in control animals.

Animals↗

PubMed2001

Effect of acute saline volume expansion in the anaesthetised DeltaF508 cystic fibrosis mouse.

It has been suggested that CFTR Cl(-) channels in the renal inner medullary collecting duct may be involved in mediating increased renal salt excretion during extracellular fluid volume expansion. To investigate this hypothesis, in-vivo clearance experiments were performed comparing wild-type (WT) and DeltaF508-CFTR transgenic mice (cftr (tm2Cam)). Control animals were given a 0.1-ml bolus of 0.9% saline, followed by I.V. infusion at 0.3 ml x h(-1). Volume expansion was applied by infusing a 1-ml bolus of 0.9% saline followed by infusion at 0.6 ml x h(-1). No significant differences in renal NaCl handling between WT mice ( C(Na)=1.2 +/- 0.3 microl x min(-1), C(Cl)=4.0 +/- 0.5 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=1.7 +/- 0.5 microl x min(-1), C(Cl)=4.1 +/- 0.8 microl x min(-1)) were observed under control conditions. Volume expansion resulted in large significant increases in NaCl clearance in both WT mice ( C(Na)=7.0 +/- 0.9 microl x min(-1), C(Cl)=12.0 +/- 0.6 microl x min(-1)) and DeltaF508-CFTR mice ( C(Na)=7.2 +/- 1.6 microl x min(-1), C(Cl)=11.0 +/- 2.2 microl x min(-1)). However, there was no significant difference between WT and DeltaF508-CFTR mice. In conclusion, renal NaCl excretion is not significantly different under basal conditions and during saline volume expansion in DeltaF508-CFTR mice. The data suggest that CFTR is not a physiologically important mediator of volume natriuresis.

Anesthesia↗

PubMed2001

Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium.

Nonviral vectors have been shown to be a safe and valid alternative to recombinant viruses for gene therapy of cystic fibrosis (CF). Nevertheless, gene transfer efficiency needs to be increased before clinical efficacy is likely in man. One barrier to increased efficacy is normal airway mucus. Using an ex vivo model of sheep tracheal epithelium, we show that this barrier can, in part, be overcome by treatment with the mucolytic agents, Nacystelyn or N-acetylcysteine using either a cationic lipid or a cationic polymer as the gene transfer agent. Further, in vivo application of either Nacystelyn or the anticholinergic glycopyrrolate, both clinically used agents, resulted in increased reporter gene expression in the mouse lung, but no significant correction of the bioelectric defect in CF null mice. These results, whilst unlikely to be sufficient in themselves to achieve clinically relevant gene therapy, may be a further useful step in the attainment of this goal.

Acetylcysteine↗

PubMed2001

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter.

For effective gene therapy of chronic disease, persistent transgene expression at therapeutic levels is required. Clinical studies of airway gene transfer in patients with cystic fibrosis (CF) have resulted in short-lived transgene expression. We used intra-nasal dosing of naked plasmid DNA to the murine lung as a model for investigating the duration of airway gene transfer from a series of reporter expression plasmids. Transgene expression was transient when mediated by the viral promoters CMV, RSV and SV40, falling to less than 10% of peak expression after 2 weeks, although the presence of the adenoviral E4ORF3 gene in cis, resulted in extended duration of reporter activity from the CMV promoter. Transient expression from these promoters was not due to loss of the vector as determined by quantitative TaqMan PCR analysis. However, use of the promoters from the human polybiquitin C (UbC) and the elongation factor 1alpha (EF1alpha) genes resulted in persistent gene expression in the mouse lung. The UbC promoter directed high-level reporter activity which was maintained for up to 8 weeks and was still detectable 6 months after a single administration. Such persistent airway transgene expression from a nonviral vector without the concomitant expression of a potential antigen has not been reported previously. Thus, despite the persistence of vector DNA in vivo, attenuation of promoter function may lead to silencing of transgene expression and careful selection of promoter sequences is recommended for in vivo gene transfer.

Administration, Inhalation↗

PubMed2001

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo.

Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.

Animals↗

PubMed2001

Inflammation in cystic fibrosis airways: relationship to increased bacterial adherence.

It is unclear whether inflammation in the cystic fibrosis (CF) lung relates predominantly to bacterial infection, or occurs as a direct consequence of mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. Interleukin (IL)-8 secretion from CF and non-CF cell lines, and from CF and non-CF human primary nasal epithelial cells incubated with or without Pseudomonas aeruginosa, was measured. Activation of nuclear factor-kappaB (NF-kappaB) in unstimulated CF and non-CF nasal epithelial cells, cell lines and murine tissues was measured by gel-shift assays. No significant difference in basal IL-8 production or NF-kappaB activation was observed between CF and non-CF primary nasal cells. However, CF cells exhibited a significantly (p<0.01) increased IL-8 secretion following P. aeruginosa stimulation. Equalization of the increased P. aeruginosa adherence observed in CF cells, to non-CF levels, resulted in comparable IL-8 secretion. Further, IL-8 production did not differ with mutations which result in either correctly localized CFTR, or in partial/total mislocalization of this protein. Similar levels of NF-kappaB activation were observed in a number of organs of wildtype and CF mice. Finally, IL-8 secretion and NF-kappaB activity were not consistently increased in CF cell lines. Cos-7 cell transfection with plasmids expressing deltaF508 or G551D mutant CFTR protein resulted in increased activation of a p50-containing NF-kappaB complex, but IL-8 secretion was similar to wild-type cells. The authors conclude that the stimulus produced by Pseudomonas aeruginosa is the predominant inflammatory trigger in their models.

Adolescent↗

PubMed2001

Acetylcholine induces cytosolic Ca2+ mobilization in isolated distal colonic crypts from normal and cystic fibrosis mice.

In intestinal biopsies from cystic fibrosis (CF) patients acetylcholine fails to elicit a chloride secretion response, and this observation can be explained by a defect in the Ca2+ signalling pathway in CF secretory cells. We tested the hypothesis that in CF intestine, the generation of an intracellular Ca2+ signal upon cholinergic stimulation is absent. A transgenic CF mouse model was used. Electrical measurements on intact jejunum and unstripped colon were performed in Ussing chambers. Intact distal colonic crypts were isolated, and the intracellular Ca2+ concentration was monitored using the Ca2+-sensitive dye fura-2. Acetylcholine increased the short-circuit current generated by wild-type jejunum and colon, but failed to induce a response in CF tissues. Acetylcholine caused a transient elevation in the intracellular Ca2+ concentration in colonic crypts from both wild-type and CF mice; the amplitude and timing of the response in CF crypts was indistinguishable from that in wild-type crypts. The response to acetylcholine was also observed in the absence of extracellular calcium, indicating intracellular stores as the source from which the cytosolic Ca2+ concentration increased. We conclude that the absence of a cholinergically-induced secretory response in CF intestine is not due to a defect in the generation of a Ca2+ signal in intestinal cells upon cholinergic stimulation.

Acetylcholine↗

PubMed2000

Evidence for cystic fibrosis transmembrane conductance regulator-dependent sodium reabsorption in kidney, using Cftr(tm2cam) mice.

The aims of this study were to investigate (a) if renal Na(+) handling was normal in Cftr(tm2cam) delta F508 cystic fibrosis mice, (b) whether adaptation to dietary salt depletion was preserved and (c) whether Cftr(tm2cam) delta F508 mice exhibited enhanced amiloride-sensitive Na(+) absorption. In Na(+)-replete animals (maintained on a 0.32 % NaCl diet) given a 150 mM NaCl i.v. maintenance infusion, there was no difference in fractional Na(+) excretion (FE(Na)) between wild-type (0. 42 +/- 0.06 %, n = 12) and Cftr(tm2cam) delta F508 mice (0.47 +/- 0.13 %, n = 7). Amiloride infusion significantly increased FE(Na) in both wild-type (3.14 +/- 0.83 %, n = 6) and Cftr(tm2cam) delta F508 mice (3. 47 +/- 0.63 %, n = 9), though with no significant difference between genotypes. A 14 day dietary salt restriction (animals maintained on a 0.03 % NaCl diet) and maintenance infusion with a 15 mM NaCl vehicle caused a reduction in FE(Na) to 0.14 +/- 0.05 %, n = 8 in wild-type mice and 0.14 +/- 0.04 %, n = 8 in Cftr(tm2cam) delta F508 mice. No significant difference in the ability to adapt to low salt conditions was apparent comparing the two genotypes. Treatment of salt-restricted mice with amiloride resulted in a blunted natriuresis in both wild-type mice (FE(Na) = 1.10 +/- 0.16 %, n = 7) and Cftr(tm2cam) delta F508 mice (FE(Na) = 1.97 +/- 0.29 %, n = 9). The natriuresis induced by amiloride was significantly greater in Cftr(tm2cam) delta F508 mice than in wild-type controls. In conclusion, Cftr(tm2cam) delta F508 mice exhibit normal renal salt excretion when either salt replete or salt restricted. Enhanced amiloride-sensitive FE(Na) is consistent with increased Na(+) absorption via the amiloride-sensitive sodium channel ENaC, in cystic fibrosis kidney, but this was only observed during salt restriction.

Amiloride↗

PubMed2000

Eomesodermin is required for mouse trophoblast development and mesoderm formation.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Amino Acid Sequence↗