Wild-type Litter Research Articles

Cyclosporin Inhibits Cardiomegaly in the Conditional, Cardiac GCaMP2 Mice

Mice expressing the Ca2+ sensor protein GCaMP2 in cardiac myocytes under the control of a doxocycline -regulated αMHC promoter (ccGC2 mice) develop cardiomegaly by 14 weeks. As the transgene consists of a calmodulin – eGFP chimeric protein, we performed initial experiments to examine the mechanism(s) underlying the development of cardiac hypertrophy. Previous observations suggest that the overexpression of calmodulin can lead to hypertrophic cardiomyopathy (Gruver et al Endocrin 1993), suggesting a possible activation of the calcineurin/nFAT pathway (Dong et al Cardio Res 2003, Molkentin et al Cell 1998). We examined whether inhibiting the activation of this pathway prevented GCaMP2 dependent development of cardiac hypertrophy. Beginning at post natal day 7, wildtype and ccGC2 litter mate controls were injected with either vehicle only or cyclosporin (10–20mg/kg) twice daily for two weeks. Mice were euthanized at 3 weeks of age, at which time heart and body weight were measured. The heart to body weight ratio in 3 week old ccGC2 mice injected with vehicle was 15% greater than vehicle injected wildtype mice or ccGC2 mice injected with cyclosporin A (5.8 ± 0.2 n=5; 5.1 ± 0.2 n=5; 5.2 ± 0.1 n=7 p < 0.02, respectively). Cardiomegaly was inhibited in ccGC2 mice injected with cyclosporin A when compared to cyclosporin A injected wildtype litter mate controls (5.2 ± 0.1 n=7; 5.3 ± 0.1 n=6, respectively). These data suggest that GCaMP2 transgene expression produces cardiac hypertrophy through stimulation of of calcineurin pathway. As the transgene is temporally regulated, ccGC2 mice may be useful in examining the signaling processes leading to the development of cardiomegaly in adult mice. Values are ± SE. NIH grants HL45239 and DK58795.

Decreased Peroxisome Proliferator Activated Receptor α Is Associated with Bile Duct Injury in Cystic Fibrosis Transmembrane Conductance Regulator−/− Mice

Primary sclerosing cholangitis (PSC) is associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. As proof of concept that CFTR dysfunction plays a role in PSC, induction of colitis in cftr mice results in bile duct injury that can be prevented by pretreatment with docosahexaenoic acid (DHA). Determine whether 1) CFTR dysfunction in cftr mice through a reduction in peroxisome proliferator activated receptor (PPAR)alpha or gamma leads to bile duct injury and 2) whether DHA prevents bile duct injury through an increase in PPAR. Cftr and wild-type (WT) mice were treated with dextran sodium sulfate (DSS) to induce colitis with or without pretreatment with oral DHA. PPARalpha and gamma as well as tumor necrosis factor (TNF)alpha were analyzed in liver tissue. PPARalpha mice were also treated with DSS and histology examined. PPARgamma mRNA levels were low, with DSS suppressing mRNA levels equally in WT and cftr mice. PPARalpha levels were no different between cftr and WT litter mates by reverse-transcription polymerase chain reaction. After DSS, WT mice showed a 9.3-fold increase in PPARalpha mRNA levels and increased nuclear localization compared with no DSS (P < 0.05), with no increase seen in cftr mice. This was not caused by changes in TNFalpha. DHA treatment led to 7.0-fold increase in PPARalpha mRNA levels in cftr mice (P < 0.01). PPARalpha mice treated with DSS did not develop bile duct injury, indicating that PPARalpha alone is not sufficient to cause bile duct inflammation. DSS induced bile duct injury in cftr mice is associated with a defect in PPARalpha expression, which is reversed by DHA.

Electrical stimulation promotes peripheral axon regeneration by enhanced neuronal neurotrophin signaling

Electrical stimulation of cut peripheral nerves at the time of their surgical repair results in an enhancement of axon regeneration. Regeneration of axons through nerve allografts was used to evaluate whether this effect is due to an augmentation of cell autonomous neurotrophin signaling in the axons or signaling from neurotrophins produced in the surrounding environment. In the thy-1-YFP-H mouse, a single 1 h application of electrical stimulation at the time of surgical repair of the cut common fibular nerve results in a significant increase in the proportion of YFP+ dorsal root ganglion neurons, which were immunoreactive for BDNF or trkB, as well as an increase in the length of regenerating axons through allografts from wild type litter mates, both 1 and 2 weeks later. Axon growth through allografts from neurotrophin-4/5 knockout mice or grafts made acellular by repeated cycles of freezing and thawing is normally very poor, but electrical stimulation results in a growth of axons through these grafts, which is similar to that observed through grafts from wild type mice after electrical stimulation. When cut nerves in NT-4/5 knockout mice were electrically stimulated, no enhancement of axon regeneration was found. Electrical stimulation thus produces a potent enhancement of the regeneration of axons in cut peripheral nerves, which is independent of neurotrophin production by cells in their surrounding environment but is dependent on stimulation of trkB and its ligands in the regenerating axons themselves. © 2006 Wiley Periodicals, Inc. Develop Neurobiol 67: 158–172, 2007.

The effect of morphine on mast cell–mediated mucosal permeability

The late phase of post-traumatic multisystem organ failure is associated with sepsis from organisms that normally reside within the gut's lumen. Morphine, a commonly employed analgesic in injured patients, is associated with intestinal stasis, bacterial overgrowth, and translocation when administered to rats. N-formyl-methionyl-leucyl-phenylalanine (FMLP), a toxic product of gram-negative organisms, provokes an increase in mucosal permeability when infused into the ileal lumen of this species. The current study was designed to examine the effects of morphine on FMLP perturbation of the mucosal barrier of the ileum of rats and mice to an impermeant macromolecule, dextran 4400. The potential role of mucosal mast cells in the response to either agent alone or in combination was examined. Intact and isolated segments of distal ileum of naïve and sensitized (Trichinella spiralis and egg albumin) Sprague-Dawley rats were exposed to FMLP with or without morphine or doxantrazole, a mast cell-stabilizing agent. Isolated segments of distal ileum of mast cell-deficient mice also were studied. Mucosal exposure of distal ileal mucosa (intact and isolated, and naive and sensitized) to FMLP was associated with an increase in permeability to dextran 4400, which was completely ablated by morphine and doxantrazole. Sensitization was associated with a prolongation of the FMLP response. Ilea of mast cell-deficient mice (but not their wild type litter mates) were unresponsive to FMLP. Morphine antagonizes the provocative effect of FMLP on the mucosal barrier to dextran 4400 of the ilea of rats and mice. Intestinal mucosal mast cells play a central role in the process.

The role of prenatal versus postnatal environment on postnatal growth

GROWTH SHANNON CLARK (F), MONICA LONGO, JOSJE LANGENVELD, G. D. V. HANKINS, GARLAND ANDERSON, GEORGE SAADE, University of Texas Medical Branch at Galveston, Obstetrics and Gynecology, Galveston, Texas, University Hospital, Maastricht, Obstetrics and Gynecology, Maastricht, Netherlands OBJECTIVE: It has been shown that an altered uterine environment affects postnatal growth. Our aim was to determine the role of cross-fostering on the altered postnatal growth in an established transgenic animal model of fetal programming induced by an abnormal uterine environment secondary to lack of endothelial nitric oxide synthase (NOS3). STUDY DESIGN: Homozygous NOS3 knockout (C57BL/6J-NOS3 / ) and wild-type mice (NOS3) were cross-bred to obtain 2 heterozygous litters that are genomically-similar but with normal (paternally-derived; NOS3 ) or abnormal (maternally-derived; NOS3 ) uterine environment. Within 24 hours of delivery, the NOS3 mat were switched to a wild-type foster mother (NOS3 ) and NOS3 pat litters to a knockout foster mother (NOS3 ). Pups from each litter were jointly counted and weighed for 21 days (weaning time), then the litters were separated by sex and weighed weekly until week 8 (adult time). The cross-fostered litters were compared with similar naturally-fostered heterozygous litters. One-way ANOVA and Newman-Keuls post hoc tests were used for statistical analysis. RESULTS: The growth curve of NOS3 pat litters was significantly higher than NOS3 , NOS3 , and NOS3 pat litters from day 13 to 21 (p ! 0.001). The growth of male NOS3 pat pups from week 4 to 8 was also significantly better compared with the other groups (p!0.05). No differences were observed between NOS3 mat and NOS3 pat litters, however these litters had significantly decreased growth at 7-8 weeks when compared to naturally-fostered NOS3 mat and NOS3 pat litters (p ! 0.05). CONCLUSION: An adverse uterine environment has a deleterious effect on postnatal growth, and its effect predominates over the postnatal environment. While an adverse postnatal environment can worsen growth, an improvement in the postnatal environment has only a slight beneficial effect. Our findings confirm the primary importance of the uterine environment and fetal programming in health and disease in later life.

Swimming behaviour and post-swimming activity in Vitamin D receptor knockout mice

Animal experiments have shown that Vitamin D plays a role in both brain development and adult brain function. The adult Vitamin D receptor null mutant mouse (VDR −/−) is reported to be less active and more anxious than wild-type litter mate controls and to have poor swimming ability. However, an “anxious” behavioural phenotype is inferred from differences in locomotor behaviour. This is a general problem in behavioural phenotyping where a neurological phenotype is inferred from changes in locomotion which will be affected by non-neurological factors, such as muscle fatigue. In this study of VDR −/−, we conducted a detailed examination of one form of motor behaviour, swimming, compared to wildtype littermate controls. Swimming was assessed using a forced swim test, a laneway swimming test and a watermaze test using a visible platform. Post-swimming activity was assessed by comparing grooming and rearing behaviour before, and 5 min after, the forced swimming test. We replicated previous findings in which VDR −/− mice demonstrate more sinking episodes than wildtype controls in the forced swim test but they were similar to controls in the time taken to swim a 1 m laneway, and in the time taken to reach a visible platform in the watermaze. Thus, the VDR −/− mice were able to swim but were not able to float. Grooming and rearing behaviour of the VDR −/− mice was similar to wildtype controls before the forced swim but the VDR −/− were much less active after the swim compared with wildtype mice which displayed high levels of grooming and rearing. We conclude that VDR −/− mice have muscular and motor impairments that do not affect their ability to swim but significantly alters the ability to float as well as their post-swimming activity. Differences in muscle strength may confound tests of activity that are used to infer an “anxious” phenotype.

A Global Role for EKLF in Definitive and Primitive Erythropoiesis.

Erythroid Kruppel-like factor (EKLF or Klf1) is an erythroid specific C2H2 zinc-finger transcription factor which is essential for definitive erythropoiesis and β-globin gene expression. The absence of EKLF results in fatal anaemia but correction of globin chain imbalance does result in rescue, suggesting the existence of additional EKLF target genes. The aim of this study was to search for such genes by expression profiling. We performed profiling on fetal livers from wild-type versus EKLF null litter mates, and also EKLF null erythroid cell lines containing an inducible EKLF-ERTM fusion construct. Hybridisations were performed on microarray slides printed with a 23K oligo library from Compugen. Target gene validation was performed by real-time RT-PCR, chromatin immuno-precipitation (ChIP) and promoter-reporter assays. A large number of genes were down regulated in the absence of EKLF but few were up regulated, suggesting EKLF acts primarily as a transcriptional activator in vivo. One hundred genes were EKLF dependent in both systems. These include heme synthesis enzymes, red cell surface proteins including Rh and the transferrin receptor, and erythroid transcription factors. Two interesting highly EKLF-dependent genes are α-haemoglobin stabilising protein (AHSP), a key chaperone for free a-globin chains, and dematin (band 4.9) which links the cytoskeleton to the red cell membrane. A search for EKLF binding sites within the dematin and AHSP genes demonstrated a number of phylogenetically conserved CACC sites, and ChIP demonstrated in vivo EKLF occupancy at some but not all of these. Promoter-reporter assays showed EKLF directly activates dematin gene transcription through two promoters containing these sites. Lastly, investigation of EKLF target genes in the yolk sac lead to the discovery of unexpected defects in the embryonic red cell membrane and cytoskeleton. In conclusion, EKLF regulates global erythroid gene expression which is critical for development of primitive as well as definitive red cells.

Developmental and reproductive performance in circadian mutant mice

Genes underlying circadian rhythm generation are expressed in many tissues. We explore a role for circadian rhythms in the timing and efficacy of mouse reproduction and development using a genetic approach. We compare fecundity in Clock(Delta19) mutant mice (a dominant-negative protein essential for circadian rhythm activity) and in Vipr2-/- null mutant mice (affecting the generation and output of the circadian rhythm of the hypothalamic suprachiasmatic nucleus) with wild type (WT) litter mates under both a 12 h:12 h light:dark cycle and continuous darkness. Uteri from Clock(Delta19) mice show no circadian rhythm and Vipr2-/- mice show a phase-advanced rhythm compared to WT uteri. In neither mutant line were homozygous or heterozygous fetuses lethal. Sexually mature adults of both mutant lines showed mildly reduced male in vivo (but not in vitro) fertility and irregular estrous cycles exacerbated by continuous darkness. However, pregnancy rates and neonatal litter sizes were not affected. The Clock(Delta19) mutant line was distinguishable from the Vipr2-/- null mutant line in showing more peri-natal delivery problems and very poor survival of offspring to weaning. A fully functional central and peripheral circadian clock is not essential for reproduction and development to term, but has critical roles peri-natally and post-partum.

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). l-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p < 0.05) and a ∼3 fold greater Ca 2+-dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53–7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.

Lysophosphatidic Acid Inhibits Adipocyte Differentiation via Lysophosphatidic Acid 1 Receptor-dependent Down-regulation of Peroxisome Proliferator-activated Receptor γ2

Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue.

MHC class I expression regulates susceptibility to spontaneous autoimmune disease in (NZBxNZW)F1 mice

(NZBxNZW)F1 mice spontaneously develop with age an autoimmune disease that resembles the human disease, systemic lupus erythematosus (SLE). Previous studies have demonstrated that susceptibility to experimentally induced SLE depended on the expression of MHC class I molecules: mice deficient in beta2-microglobulin did not express cell surface class I and were resistant to the induction of experimental SLE. Furthermore, the spontaneous SLE-like disease of (NZBxNZW)F1 mice was ameliorated by treatment with an agent that reduces MHC class I expression, methimazole (MMI). In the present study, the role of MHC class I has been examined in (NZBxNZW)F1 mice deficient in beta2-microglobulin expression. Homozygous (NZBxNZW)F1 beta2m-/- mice do not express class I or develop CD8+ T cells. Surprisingly, they show an increased susceptibility to disease. In sharp contrast, heterozygous (NZBxNZW)F1 beta2m+/- express class I, albeit at reduced levels, develop normal levels of CD8+ T cells and are less susceptible to autoimmune disease, relative to their wild-type litter mates. Taken together, these findings suggest that class I expression regulates the development of disease, both positively and negatively. We speculate that MHC class I expression itself confers susceptibility to disease through presentation of self-peptides, while also selecting for a CD8+ suppressor T cell population that mitigates disease.

PET imaging of brain with the β-amyloid probe, [11C]6-OH-BTA-1, in a transgenic mouse model of Alzheimer’s disease

The purpose of this study was to evaluate the capacity of [11C]6-OH-BTA-1 and positron emission tomography (PET) to quantify beta-amyloid (Abeta) plaques in the Tg2576 mouse model of Alzheimer's disease (AD). PET imaging was performed with the NIH ATLAS small animal scanner in six elderly transgenic mice (Tg2576; age 22.0+/-1.8 months; 23.6+/-2.6 g) overexpressing a mutated form of human beta-amyloid precursor protein (APP) known to result in the production of Abeta plaques, and in six elderly wild-type litter mates (age 21.8+/-1.6 months; 29.5+/-4.7 g). Dynamic PET scans were performed for 30 min in each mouse under 1% isoflurane inhalation anesthesia after a bolus injection of 13-46 MBq of [11C]6-OH-BTA-1. PET data were reconstructed with 3D OSEM. On the coronal PET image, irregular regions of interest (ROIs) were placed on frontal cortex (FR), parietal cortex (PA), striatum (ST), thalamus (TH), pons (PO), and cerebellum (CE), guided by a mouse stereotaxic atlas. Time-activity curves (TACs) (expressed as percent injected dose per gram normalized to body weight: % ID-kg/g) were obtained for FR, PA, ST, TH, PO, and CE. ROI-to-CE radioactivity ratios were also calculated. Following PET scans, sections of mouse brain prepared from anesthetized and fixative-perfused mice were stained with thioflavin-S. TACs for [11C]6-OH-BTA-1 in all ROIs peaked early (at 30-55 s), with radioactivity washing out quickly thereafter in both transgenic and wild-type mice. Peak uptake in all regions was significantly lower in transgenic mice than in wild-type mice. During the later part of the washout phase (12-30 min), the mean FR/CE and PA/CE ratios were higher in transgenic than in wild-type mice (1.06+/-0.04 vs 0.98+/-0.07, p=0.04; 1.06+/-0.09 vs 0.93+/-0.08 p=0.02) while ST/CE, TH/CE, and PO/CE ratios were not. Ex vivo staining revealed widespread Abeta plaques in cortex, but not in cerebellum of transgenic mice or in any brain regions of wild-type mice. Marked reductions in brain uptake of this radioligand in transgenic mice may be due to reduced cerebral blood flow relative to that in wild-type mice. Specific [11C]6-OH-BTA-1 binding to Abeta plaques, if any, is probably very low, as reflected in the small FR/CE and PA/CE ratio differences. FR/CE and PA/CE ratios are considerably higher in AD patients while Abeta plaque densities in 22-month-old transgenic mice may be expected to show essentially the same density as is observed in the AD brain. This implies that the absence of tracer retention in 22-month-old transgenic mice may be due to the smaller number of Abeta plaque binding sites and/or to lower affinity of the binding sites for [11C]6-OH-BTA-1 as compared with AD patients. [11C]6-OH-BTA-1 shows excellent brain uptake in mice.

Inhibition of Tumor Necrosis Factor-α Improves Physiological Angiogenesis and Reduces Pathological Neovascularization in Ischemic Retinopathy

The present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-alpha can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-alpha-/- and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-alpha-/- and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNF-alpha-/- and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-alpha significantly improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy.

ClC-3 Chloride Channels Facilitate Endosomal Acidification and Chloride Accumulation

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or alpha2-macroglobulin (alpha2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in alpha2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in alpha2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.

The bacterial flora of α-Gal knockout mice express the α-Gal epitope comparable to wild type mice

The human genome possesses pseudogenes for the enzyme α1,3 galactosyltransferase and hence, human cells and tissues do not express the Galα terminated trisaccharide structure Galα1-3Galβ1-4GlcNAc, the so-called α-Gal epitope. Circulating antibodies specific for this carbohydrate epitope are, however, present in high amounts in humans. It has previously been hypothesized that the antibody production is induced by the presence of the α-Gal epitope in the cell walls of the enteric flora, especially Enterobacteriaceae spp. However, in mice, in which the epitope has been deleted by targeted mutation of the gal-transferase gene, α-Gal antibodies do not appear without prior immunization, although the mice through their growth probably have been exposed to a normal bacterial flora of e.g. Enterobacteriaceae spp. It is unknown whether there are different types of immune reactions to antigenic carbohydrate expressing bacteria and whether there are discrepancies in the enteric flora between these knockout mice and their wild type litter mates. In this study the enteric flora of α-Gal knockout and wild type mice was compared both in relation to the prevalence of different types of bacteria in the two groups of mice, as well as in relation to the expression of the epitope on the surface of Enterobacteriaceae spp. Our results showed that the enteric flora did not differ significantly between knockout and wild type mice and that it was comparable to the flora known to be present in the intestines of other mice. All Enterobacteriaceae spp. examined expressed the α-Gal epitope no matter whether they were isolated from knockout or wild type mice. It is, therefore, discussed whether it is more reasonable to assume that α-Gal antibodies in mammals that do not produce α1,3 galactosyltransferase such as in the knock mice and in humans are the result of another antigen stimulant than these common representatives of the enteric flora, that we isolated from the two types of mice. Possible candidates for a carrier in humans could be bacteria or viruses not isolated from barrier-bred mice.

THE ABSENCE OF AN EXOCRINE SPECIFIC TRANSCRIPTION FACTOR, MIST1, LEADS TO INCREASED SEVERITY IN SECRETAGOGUE INDUCED PANCREATITIS.

Gallstone migration into the common duct and alcohol abuse are the most common causes of pancreatitis. There is also evidence for genetic basis for the susceptibility to the disease. Unfortunately, with the exception of the cationic trypsinogen gene and SPINK, there is a lack of knowledge of the factors leading to increased disease susceptibility and severity. In an effort to link factors that regulate pancreatic development and function to disease susceptibility, we have examined of the role of Mist1 in pancreatitis. Mist1 is a basic helix-loop-helix (bHLH) transcription factor, highly expressed in acinar cells and the absence of Mist1 in mice (Mist1KO) results in incomplete maturation of acinar cells. In addition, Mist1KO mice exhibit premature enzyme activation and an increased presence of stellate cells, which are hallmarks of pancreatitis yet these mice do not develop the disease. We hypothesize that Mist1KO mice will develop more severe pancreatitis than wild type litter mates. To address this hypothesis, we have injected supramaximal amounts of caerulein, a cholecystokinin analogue. Analysis of serum following initiation of pancreatitis reveal higher levels of amylase in Mist1KO mice suggesting a more severe form of pancreatitis. Histological analysis confirms this suggestion as Mist1KO pancreatic tissue exhibits a progressive degeneration characterized by distended duct formation, vacuolization, pronounced fibrosis and a decrease in cells undergoing apoptosis. To identify additional factors that may play a role in disease susceptibility, widespread analysis by gene microarray was used to compare the molecular profile of wild type and Mist1KO mice. We have identified a number of genes that are differentially expressed in wild type and Mist1KO mice that may be linked to either protection or severity to pancreatitis. Current studies are underway to determine the importance of these factors in the disease process and their potential regulation by Mist1. Our results indicate that the transcription factor, Mist1 plays a role in dictating the severity of pancreatitis making the Mist1KO mouse a good model for studying the factors involved in the modulation of the disease process.

Food‐Reinforced Operant Behavior in Dopamine Transporter Knockout Mice: Enhanced Resistance to Extinction

Dopamine (DA) plays roles in circuits that are important for brain reward and in striatal brain regions that are important for certain types of habit learning. These processes in wildtype, heterozygous, and homozygous dopamine transporter knockout (DAT-KO) mice, which were mildly food deprived and allowed to make nose-poke responses for food reinforcement, were studied. The mice were given 20-min sessions of daily (a) baseline exposure to the operant chambers, (b) acquisition of nose-poke responses in which responses were reinforced under a fixed ratio (FR5) schedule, (c) a progressive ratio schedule in which the number of responses required to obtain food was gradually increased, and (d) extinction of responses in which nose pokes were not followed by food. Neither heterozygous nor homozygous DAT-KO mice differed from their wildtype litter mates in the number of nose pokes displayed during baseline exposures to the chambers, the number of sessions required for acquisition, the number of responses under the FR5 schedule, or the number of responses under the progressive ratio schedule. Interestingly, however, in the five extinction sessions in which food was no longer delivered by nose poking, homozygous DAT-KO mice exerted significantly more responses than mice of either of the other two genotypes. These lines of evidence suggest a greater resistance of DAT-KO mice to the elimination of the response and support roles of dopaminergic systems in habit memory.

Homozygous deletion of early growth response 1 gene and critical limb ischemia after vascular ligation in mice: Evidence for a central role in vascular homeostasis

Background The early growth response 1 gene (Egr1) encodes for an immediate to early response transcription factor that is upregulated by changes in vascular strain and hypoxia and in turn upregulates the downstream expressions of a number of angiogenic growth factors. We therefore hypothesized that early growth response 1 may be a critical early messenger governing revascularization in the setting of acute vascular occlusions. Methods C57 BL/6 mice deficient in the Egr1 gene (knockout) and their wild-type litter mates underwent ligation and excision of the femoral artery with or without the previous administration of 2.7 × 10 9 particle units of an adenoviral vector coding for the vascular endothelial growth factor gene (VEGF) or Egr1. Distal hind limb perfusion was serially measured in these animals with laser Doppler perfusion imaging. Results Wild-type mice (n = 9) had nearly complete restitution of hind limb perfusion by day 35 after ligation. In contrast, all noninjected Egr1 knockout mice (n = 5) had severe ipsilateral limb necrosis develop between 1 and 4 days after ligation ( P < .0001). Egr1 knockout mice injected with VEGF vector (n = 4) demonstrated significantly improved perfusion relative to baseline by postligation day 28, which persisted to postligation day 35 ( P < .05). Egr1 knockout animals injected with Egr1 vector (n = 7) demonstrated a partial recovery of hind limb perfusion relative to VEGF vector–treated knockout animals at postligation day 4 ( P < .01), which persisted to day 35. Conclusions These findings suggest that early growth response 1 plays a pivotal role in reperfusion responses to vascular occlusion in mice and possibly other mammals.

The role of the tachykinin NK 1 receptor in airway changes in a mouse model of allergic asthma

Tachykinins are present in sensory nerves and in nonneuronal cells like macrophages. Human data suggest a role for these peptides in asthma, but the exact role of tachykinins and their receptors in allergic airway inflammation is still a matter of debate. The aim of this study was to determine the role of the tachykinin NK1 receptor in allergic airway responses in a mouse model. Tachykinin NK1 receptor wild-type and knockout animals were sensitized intraperitoneally to ovalbumin and subsequently exposed from days 14 to 21 to aerosolized ovalbumin (1% ). On day 22, the immunologic and histologic changes were evaluated, and lung function measurements were performed. Mice lacking the tachykinin NK1 receptor and their wild-type litter mates developed inflammatory cell infiltrates in the airways and ovalbumin-specific IgE on sensitization and exposure to ovalbumin compared with saline-exposed controls. No differences were detected between wild-type and knockout mice. The substance P content of alveolar macrophages was not influenced by ovalbumin or by the lack of the NK1 receptor. Ovalbumin-induced hyperresponsiveness was not observed, but at baseline, the knockout mice were more reactive despite similar morphology. Ovalbumin induced more goblet cell hyperplasia in wild-type animals compared with knockout animals. No differences in airway wall thickness were observed. These data suggest that tachykinin NK1 receptors do not affect allergic airway inflammation or endogenous substance P content of alveolar macrophages but influence baseline responsiveness and promote features of remodeling such as goblet cell hyperplasia.

Lipid metabolism in phosphatidylinositol transfer protein α-deficient vibrator mice

Mice that are homozygous for the vibrator mutation express 65–85% less phosphatidylinositol transfer protein α (PITPα) than their wild type litter mates. By postnatal day 10–12 (P10–12) they exhibit signs of neurodegeneration and die prematurely by P40. In the present study, we examine the lipid content of brain, liver, and mammary glands from these animals. Lipid-mediated signal transduction is evaluated in primary fibroblast cultures. With respect to the lipid make-up of brain and liver, we report that there is a significant increase (2- to 4-fold) in the neutral lipids present in the livers of vb/ vb animals when compared with wild type (+/+) litter mates. No significant changes are observed in the brains of these animals. The mammary glands of vb/ vb mice are underdeveloped with respect to ductal and alveolar structures, and the fat pad is composed of predominantly brown adipose tissue rather than the white adipose tissue characteristic of age-matched wild type litter mates. No differences are observed in any aspect of lipid-mediated signal transduction.