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author:("dalhoff, A.")
1.  Electrostatic anchoring precedes stable membrane attachment of SNAP25/SNAP23 to the plasma membrane 
eLife  null;6:e19394.
The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.
DOI: http://dx.doi.org/10.7554/eLife.19394.001
eLife digest
Cells often communicate with each other by releasing chemicals that normally are stored in small membrane-bound compartments called vesicles. For example, when a neuron is stimulated, vesicles merge with its cell membrane and release their content into a gap between itself and other neurons. This complicated process involves many steps and molecules, including proteins called SNAREs.
Some SNARE proteins reside at the inner side of the cell membrane and help vesicles to fuse with this membrane. Two SNARE proteins called SNAP25 and SNAP23 are produced in the liquid inside the cell and initially float freely. Eventually, these proteins become directly anchored to the cell membrane, however, not much is known about what happens to these proteins in between these stages, or how they first attach to the membrane before anchoring to it.
Electrostatic forces between oppositely charged molecules are known to be important for them to bind with each other. Here, electrostatic forces are less likely to occur because SNAP25 and SNAP23 are both mostly negatively charged, and should therefore be repelled from the cell membrane, which also typically has a negative charge. However, both SNAP25 and SNAP23 have a small cluster of positively charged amino acids (the building blocks of proteins) near the attachment site, and Weber et al. have now tested whether this charge is sufficient to overcome the predicted repulsion.
The approach involved making mutant proteins with either more or less positively charged attachment regions. Mutant SNAP25 or SNAP23 proteins with more positive charges may stick more tightly but not necessarily more permanently to the membrane. However, when the number of positive charges was lowered, more of the proteins remained floating freely in the liquid inside the cell. These results suggest that even a small number of positively charged amino acids is sufficient to help a protein bind to a cell membrane for further processing.
The findings of Weber et al. reveal an early step in the life cycle of SNAP25 and SNAP23 before they anchor to the cell membrane. They suggest that finely tuned protein electrostatics can regulate how long a protein spends at a specific site and thereby indirectly determine its fate. Such fine-tuned protein electrostatics are difficult to recognize and could represent an underestimated regulatory mechanism in all types of cells.
DOI: http://dx.doi.org/10.7554/eLife.19394.002
doi:10.7554/eLife.19394
PMCID: PMC5362264  PMID: 28240595
protein electrostatics; membrane targeting; phosphoinositides; SNAREs; liposome; post-translational modification; Rat
2.  3D structured illumination microscopy of mammalian embryos and spermatozoa 
Background
Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass.
Results
Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa.
Conclusions
Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa.
doi:10.1186/s12861-015-0092-7
PMCID: PMC4661982  PMID: 26610350
Super-resolution fluorescence microscopy; Mammalian embryos
3.  ERBB3 is required for tumor promotion in a mouse model of skin carcinogenesis 
Molecular Oncology  2015;9(9):1825-1833.
The epidermal growth factor receptor (EGFR) plays a key role in skin inflammation, wound healing, and carcinogenesis. Less is known about the functions of the structurally related receptor ERBB3 (HER3) in the skin. We assessed the requirement of ERBB3 for skin homeostasis, wound healing, and tumorigenesis by crossing mice carrying a conditional Erbb3 allele with animals expressing cre under the control of the keratin 5 promoter. Erbb3del mice, lacking ERBB3 specifically in keratinocytes, showed no obvious abnormalities. The EGFR was upregulated in Erbb3del skin, possibly compensating the loss of ERBB3. Nonetheless, healing of full‐thickness excisional wounds was negatively affected by ERBB3 deficiency. To analyze the function of ERBB3 during tumorigenesis, we employed the established DMBA/TPA multi‐stage chemical carcinogenesis protocol. Erbb3del mice remained free of papillomas for a longer time and had significantly reduced tumor burden compared to control littermates. Tumor cell proliferation was considerably reduced in Erbb3del mice, and loss of ERBB3 also impaired keratinocyte proliferation after a single application of TPA. In human skin tumor samples, upregulated ERBB3 expression was observed in squamous cell carcinoma, condyloma, and malignant melanoma. Thus, we conclude that ERBB3, while dispensable for the development and the homeostasis of the epidermis and its appendages, is required for proper wound healing and for the progression of skin tumors during multi‐stage chemical carcinogenesis in mice. ERBB3 may also be important for human skin cancer progression. The latter effects most probably reflect a key role for ERBB3 in increasing cell proliferation after stimuli as wounding or carcinogenesis.
Highlights
The EGFR‐related ERBB3 receptor is dispensable for skin development and homeostasis.ERBB3 is required for wound healing and for the progression of skin tumors in mice.ERBB3 may also be important for human skin cancer progression.ERBB3 is an attractive drug target for skin cancer therapy.
doi:10.1016/j.molonc.2015.06.007
PMCID: PMC5528713  PMID: 26194695
EGFR; ERBB3; Mouse; Skin; Carcinogenesis; Wound healing
4.  Effects of the glucagon-like peptide-1 receptor agonist liraglutide in juvenile transgenic pigs modeling a pre-diabetic condition 
Background
The glucagon-like peptide-1 receptor (GLP1R) agonist liraglutide improves glycemic control and reduces body weight of adult type 2 diabetic patients. However, efficacy and safety of liraglutide in adolescents has not been systematically investigated. Furthermore, possible pro-proliferative effects of GLP1R agonists on the endocrine and exocrine pancreas need to be further evaluated. We studied effects of liraglutide in adolescent pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) in the beta-cells, leading to a pre-diabetic condition including disturbed glucose tolerance, reduced insulin secretion and progressive reduction of functional beta-cell mass.
Methods
Two-month-old GIPRdn transgenic pigs were treated daily with liraglutide (0.6-1.2 mg per day) or placebo for 90 days. Glucose homeostasis was evaluated prior to and at the end of the treatment period by performing mixed meal and intravenous glucose tolerance tests (MMGTT and IVGTT). Finally animals were subjected to necropsy and quantitative-stereological analyses were performed for evaluation of alpha- and beta-cell mass, beta-cell proliferation as well as acinus-cell proliferation.
Results
MMGTT at the end of the study revealed 23% smaller area under the curve (AUC) for glucose, a 36% smaller AUC insulin, and improved insulin sensitivity, while IVGTT showed a 15% smaller AUC glucose but unchanged AUC insulin in liraglutide- vs. placebo-treated animals. Liraglutide led to marked reductions in body weight gain (-31%) and food intake (-30%) compared to placebo treatment, associated with reduced phosphorylation of insulin receptor beta (INSRB)/insulin-like growth factor-1 receptor beta (IGF1RB) and protein kinase B (AKT) in skeletal muscle. Absolute alpha- and beta-cell mass was reduced in liraglutide-treated animals, but alpha- and beta-cell mass-to-body weight ratios were unchanged. Liraglutide neither stimulated beta-cell proliferation in the endocrine pancreas nor acinus-cell proliferation in the exocrine pancreas, excluding both beneficial and detrimental effects on the pig pancreas.
Conclusions
Although plasma liraglutide levels of adolescent transgenic pigs treated in our study were higher compared to human trials, pro-proliferative effects on the endocrine or exocrine pancreas or other liraglutide-related side-effects were not observed.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0431-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12967-015-0431-2
PMCID: PMC4362632  PMID: 25890210
Transgenic pig model; Type 2 diabetes; GIP receptor; GLP1 receptor agonist; Incretin-based therapeutics; Liraglutide; Adolescents; Beta-cell mass
5.  Overexpression of Epigen during Embryonic Development Induces Reversible, Epidermal Growth Factor Receptor-Dependent Sebaceous Gland Hyperplasia 
Molecular and Cellular Biology  2014;34(16):3086-3095.
The epidermal growth factor receptor (EGFR) system is a key regulator of epithelial development and homeostasis. Its functions in the sebaceous gland (SG), however, remain poorly characterized. In this study, using a transgenic mouse line with tissue-specific and inducible expression of the EGFR ligand epigen, we showed that increased activation of the EGFR in skin keratinocytes results in enlarged SGs and increased sebum production. The phenotype can be reverted by interrupting transgene expression and is EGFR dependent, as gland size and sebum levels return to normal values after crossing to the EGFR-impaired mouse line Wa5. Intriguingly, however, the SG enlargement appears only if EGFR activation occurs before birth. Importantly, the enlarged sebaceous glands are associated with an increased expression of the transcription factor MYC and of the transmembrane proteins LRIG1, an established negative-feedback regulator of the EGFR/ERBB tyrosine kinase receptors and a stem cell marker. Our findings identify EGFR signaling as a major pathway determining SG activity and suggest a functional relationship between the EGFR/ERBB system and MYC/LRIG1 in the commitment of stem cells toward specific progenitor cell types, with implications for our understanding of their role in tissue development, homeostasis, and disease.
doi:10.1128/MCB.00302-14
PMCID: PMC4135600  PMID: 24891618
6.  Methyl-donor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levelsa 
Molecular Metabolism  2014;3(5):565-580.
Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.
doi:10.1016/j.molmet.2014.04.010
PMCID: PMC4099513  PMID: 25061561
Obesity; Hepatic steatosis; One-carbon metabolism; AMP-activated protein kinase; β-oxidation; Acyl-carnitines; ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ANT, adenine nucleotide translocase; Bhmt, betaine-homocysteine methyltransferase; C1, one-carbon; CACT, carnitine-acylcarnitine transporter; Cpt1a, carnitine palmitoyltransferase-1a; Cbs, cystathionine β-synthase; C, control diet; CMS, methyl-donor supplemented control diet; DIO, diet-induced obesity; Fasn, fatty acid synthase; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; GNMT, glycine N-methyltransferase; HSP90, heat shock protein 90; HF, high-fat diet; HFMS, methyl-donor supplemented high-fat diet; HMW adiponectin, high molecular weight adiponectin; Hcy, homocysteine; β-HAD, β-hydroxyacyl CoA dehydrogenase; 3-HB, β-hydroxybutyrate; Hprt1, hypoxanthine phosphoribosyltransferase 1; LDL, low density lipoprotein; MAT, methionine adenosyltransferase; MCD, malonyl-CoA decarboxylase; MDS, methyl-donor supplementation; MTR, methionine synthase; NAFLD, non-alcoholic fatty liver disease; NEFA, non-esterified fatty acids; PC, phosphatidylcholine; Pemt, phosphatidylethanolamine methyltransferase; PGC1α, peroxisome proliferator-activated receptor-γ co-activator-1α; PL, phospholipids; PPARα, peroxisome proliferator-activated receptor-α; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SM, sphingomyelin; SREBP1c, sterol regulatory element-binding protein-1c; TG, triacylglycerol; VAT, visceral adipose tissue; VLDL, very low density lipoprotein
7.  PLIN2, the major perilipin regulated during sebocyte differentiation, controls sebaceous lipid accumulation in vitro and sebaceous gland size in vivo 
Biochimica et biophysica acta  2013;1830(10):4642-4649.
Background
Lipid synthesis and storage are accomplished by lipid droplets (LDs). The perilipin family of LD-associated proteins, comprising 5 members (PLIN1-PLIN5), has been well characterized in adipocytes but not in sebocytes, epithelial cells in which LD formation is a key feature of the cellular differentiation.
Methods
Perilipin expression in the sebaceous gland cell line SZ95 and in human sebaceous glands was studied by qRT-PCR, Western blots, and immunohistochemistry. Lipid accumulation was evaluated by Nile red staining and mass spectrometry.
Results
PLIN2 and PLIN3 are the most abundant perilipins in undifferentiated sebocytes. Induction of lipogenesis by linoleic acid (LA) resulted in increased transcript levels of all perilipins except for PLIN3 and in a time-dependent increase of PLIN2 protein. Nile red staining revealed that siRNA-mediated downregulation of PLIN2 significantly impaired basal and LA-induced lipid accumulation. Mass spectrometry revealed PLIN2 deficiency to cause a reduction in the amount of several specific lipid fractions, including di- and triacyl-glycerol esters, phosphatidylcholine lipids, and ceramides in sebocytes under basal conditions. In contrast, PLIN2 downregulation exerted a statistically significant inhibitory effect only on the accumulation of specific LA-induced triglycerides. PLIN2-deficient mice showed normal morphology of sebaceous glands. However, their sebaceous glands were significantly reduced in size and showed less cell proliferation.
Conclusions
PLIN2 is the major perilipin regulated during sebocyte differentiation in vitro. PLIN2 is also important for sebaceous lipid accumulation in vitro and regulates sebaceous gland size in vivo.
General significance
Our study provides the first systematic analysis of LD-associated proteins in sebocytes.
doi:10.1016/j.bbagen.2013.05.016
PMCID: PMC3998206  PMID: 23688400
Sebaceous glands; Lipid droplets; Perilipins
8.  Differences in the Aerobic Capacity of Flight Muscles between Butterfly Populations and Species with Dissimilar Flight Abilities 
PLoS ONE  2014;9(1):e78069.
Habitat loss and climate change are rapidly converting natural habitats and thereby increasing the significance of dispersal capacity for vulnerable species. Flight is necessary for dispersal in many insects, and differences in dispersal capacity may reflect dissimilarities in flight muscle aerobic capacity. In a large metapopulation of the Glanville fritillary butterfly in the Åland Islands in Finland, adults disperse frequently between small local populations. Individuals found in newly established populations have higher flight metabolic rates and field-measured dispersal distances than butterflies in old populations. To assess possible differences in flight muscle aerobic capacity among Glanville fritillary populations, enzyme activities and tissue concentrations of the mitochondrial protein Cytochrome-c Oxidase (CytOx) were measured and compared with four other species of Nymphalid butterflies. Flight muscle structure and mitochondrial density were also examined in the Glanville fritillary and a long-distance migrant, the red admiral. Glanville fritillaries from new populations had significantly higher aerobic capacities than individuals from old populations. Comparing the different species, strong-flying butterfly species had higher flight muscle CytOx content and enzymatic activity than short-distance fliers, and mitochondria were larger and more numerous in the flight muscle of the red admiral than the Glanville fritillary. These results suggest that superior dispersal capacity of butterflies in new populations of the Glanville fritillary is due in part to greater aerobic capacity, though this species has a low aerobic capacity in general when compared with known strong fliers. Low aerobic capacity may limit dispersal ability of the Glanville fritillary.
doi:10.1371/journal.pone.0078069
PMCID: PMC3885395  PMID: 24416122
10.  Increased Plasma Citrulline in Mice Marks Diet-Induced Obesity and May Predict the Development of the Metabolic Syndrome 
PLoS ONE  2013;8(5):e63950.
In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the “diabetic fingerprint” of plasma amino acid changes observed in humans. Additionally, citrulline may serve as an early indicator of the obesity-dependent metabolic impairments.
doi:10.1371/journal.pone.0063950
PMCID: PMC3653803  PMID: 23691124
11.  Hepatic Methionine Homeostasis Is Conserved in C57BL/6N Mice on High-Fat Diet Despite Major Changes in Hepatic One-Carbon Metabolism 
PLoS ONE  2013;8(3):e57387.
Obesity is an underlying risk factor in the development of cardiovascular disease, dyslipidemia and non-alcoholic fatty liver disease (NAFLD). Increased hepatic lipid accumulation is a hallmark in the progression of NAFLD and impairments in liver phosphatidylcholine (PC) metabolism may be central to the pathogenesis. Hepatic PC biosynthesis, which is linked to the one-carbon (C1) metabolism by phosphatidylethanolamine N-methyltransferase, is known to be important for hepatic lipid export by VLDL particles. Here, we assessed the influence of a high-fat (HF) diet and NAFLD status in mice on hepatic methyl-group expenditure and C1-metabolism by analyzing changes in gene expression, protein levels, metabolite concentrations, and nuclear epigenetic processes. In livers from HF diet induced obese mice a significant downregulation of cystathionine β-synthase (CBS) and an increased betaine-homocysteine methyltransferase (BHMT) expression were observed. Experiments in vitro, using hepatoma cells stimulated with peroxisome proliferator activated receptor alpha (PPARα) agonist WY14,643, revealed a significantly reduced Cbs mRNA expression. Moreover, metabolite measurements identified decreased hepatic cystathionine and L-α-amino-n-butyrate concentrations as part of the transsulfuration pathway and reduced hepatic betaine concentrations, but no metabolite changes in the methionine cycle in HF diet fed mice compared to controls. Furthermore, we detected diminished hepatic gene expression of de novo DNA methyltransferase 3b but no effects on hepatic global genomic DNA methylation or hepatic DNA methylation in the Cbs promoter region upon HF diet. Our data suggest that HF diet induces a PPARα-mediated downregulation of key enzymes in the hepatic transsulfuration pathway and upregulates BHMT expression in mice to accommodate to enhanced dietary fat processing while preserving the essential amino acid methionine.
doi:10.1371/journal.pone.0057387
PMCID: PMC3589430  PMID: 23472083
12.  Consolidation of Remote Fear Memories Involves Corticotropin-Releasing Hormone (CRH) Receptor Type 1-Mediated Enhancement of AMPA Receptor GluR1 Signaling in the Dentate Gyrus 
Neuropsychopharmacology  2011;37(3):787-796.
Persistent dreadful memories and hyperarousal constitute prominent psychopathological features of posttraumatic stress disorder (PTSD). Here, we used a contextual fear conditioning paradigm to demonstrate that conditional genetic deletion of corticotropin-releasing hormone (CRH) receptor 1 within the limbic forebrain in mice significantly reduced remote, but not recent, associative and non-associative fear memories. Per os treatment with the selective CRHR1 antagonist DMP696 (3 mg/kg) attenuated consolidation of remote fear memories, without affecting their expression and retention. This could be achieved, if DMP696 was administered for 1 week starting as late as 24 h after foot shock. Furthermore, by combining electrophysiological recordings and western blot analyses, we demonstrate a delayed-onset and long-lasting increase in AMPA receptor (AMPAR) GluR1-mediated signaling in the dentate gyrus (DG) of the dorsal hippocampus 1 month after foot shock. These changes were absent from CRHR1-deficient mice and after DMP696 treatment. Inactivation of hippocampal GluR1-containing AMPARs by antisense oligonucleotides or philantotoxin 433 confirmed the behavioral relevance of AMPA-type glutamatergic neurotransmission in maintaining the high levels of remote fear in shocked mice with intact CRHR1 signaling. We conclude that limbic CRHR1 receptors enhance the consolidation of remote fear memories in the first week after foot shock by increasing the expression of Ca2+-permeable GluR1-containing AMPARs in the DG. These findings suggest both receptors as rational targets for the prevention and therapy, respectively, of psychopathology associated with exaggerated fear memories, such as PTSD.
doi:10.1038/npp.2011.256
PMCID: PMC3260988  PMID: 22030710
CRHR1; GluR1; PTSD; therapy; CRF; CRF receptor 1; biological psychiatry; glutamate; animal models; mood/anxiety/stress disorders; GluR1; PTSD; dentate gyrus; CRH; CRF
13.  In Vivo Evidence for Epidermal Growth Factor Receptor (EGFR)-mediated Release of Prolactin from the Pituitary Gland 
The Journal of Biological Chemistry  2011;286(45):39297-39306.
Background: The epidermal growth factor receptor (EGFR) regulates mammary gland development and function.
Results: Overexpression of betacellulin in transgenic mice induces a lactation-like phenotype due to increased circulating levels of prolactin.
Conclusion: The EGFR system also regulates mammary gland activity by modulating prolactin release.
Significance: We provide the first in vivo evidence that the EGFR system regulates prolactin release from the pituitary gland.
Members of the epidermal growth factor receptor (EGFR/ERBB) system are essential local regulators of mammary gland development and function. Emerging evidence suggests that EGFR signaling may also influence mammary gland activity indirectly by promoting the release of prolactin from the pituitary gland in a MAPK and estrogen receptor-α (ERα)-dependent manner. Here, we report that overexpression of the EGFR ligand betacellulin (BTC) causes a lactating-like phenotype in the mammary gland of virgin female mice including the major hallmarks of lactogenesis. BTC transgenic (BTC-tg) females showed reduced levels of prolactin in the pituitary gland and increased levels of the hormone in the circulation. Furthermore, treatment of BTC-tg females with bromocriptine, an inhibitor of prolactin secretion, blocked the development of the lactation-like phenotype, suggesting that it is caused by central release of prolactin rather than by local actions of BTC in the mammary gland. Introduction of the antimorphic Egfr allele Wa5 also blocked the appearance of the mammary gland alterations, revealing that the phenotype is EGFR-dependent. We detected an increase in MAPK activity, but unchanged phosphorylation of ERα in the pituitary gland of BTC-tg females as compared with control mice. These results provide the first functional evidence in vivo for a role of the EGFR system in regulating mammary gland activity by modulating prolactin release from the pituitary gland.
doi:10.1074/jbc.M111.243493
PMCID: PMC3234754  PMID: 21914800
Epidermal Growth Factor Receptor (egfr); Lactation; Mammary Gland; Mouse; Prolactin
14.  Search for lepton flavour violation in the eμ continuum with the ATLAS detector in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\sqrt{s} = 7~\mbox{TeV}$\end{document}pp collisions at the LHC 
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This paper presents a search for the t-channel exchange of an R-parity violating scalar top quark (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\tilde{t}$\end{document}) in the e±μ∓ continuum using 2.1 fb−1 of data collected by the ATLAS detector in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\sqrt{s}=7~\mbox{TeV}$\end{document}pp collisions at the Large Hadron Collider. Data are found to be consistent with the expectation from the Standard Model backgrounds. Limits on R-parity-violating couplings at 95 % C.L. are calculated as a function of the scalar top mass (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$m_{\tilde{t}}$\end{document}). The upper limits on the production cross section for pp→eμX, through the t-channel exchange of a scalar top quark, ranges from 170 fb for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$m_{\tilde{t}}=95~\mbox{GeV}$\end{document} to 30 fb for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$m_{\tilde{t}}=1000~\mbox{GeV}$\end{document}.
doi:10.1140/epjc/s10052-012-2040-z
PMCID: PMC4370899  PMID: 25814838
15.  Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo 
PLoS Pathogens  2012;8(6):e1002728.
There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.
Author Summary
All herpesviruses show a precisely regulated gene expression profile, including true-late genes, which are turned on only after the onset of DNA replication. We used this intrinsic viral mechanism to generate a versatile conditional gene expression system that exploits the activity of the murine cytomegalovirus (MCMV) viral origin of lytic replication (oriLyt). Upon virus infection, replication of the viral genome also led to the replication and activation of the oriLyt-coupled episomal transgene. The oriLyt-based replicons were silenced in all stable cell lines and transgenic mice; however, virus infection liberated the plasmids from histone-deacetylase-induced inactivation. As maximum gene expression relied on relief from silencing via replication of the episomal constructs, very strong induction of the reporter gene was achieved. We showed that this system can be used for trans-complementation of late, toxic viral genes, to block virus production by activating dominant-negative (DN) transgenes, and to provide a new tool to study the principles of viral replication.
doi:10.1371/journal.ppat.1002728
PMCID: PMC3369935  PMID: 22685399
16.  C57Bl/6 N mice on a western diet display reduced intestinal and hepatic cholesterol levels despite a plasma hypercholesterolemia 
BMC Genomics  2012;13:84.
Background
Small intestine and liver greatly contribute to whole body lipid, cholesterol and phospholipid metabolism but to which extent cholesterol and phospholipid handling in these tissues is affected by high fat Western-style obesogenic diets remains to be determined.
Methods
We therefore measured cholesterol and phospholipid concentration in intestine and liver and quantified fecal neutral sterol and bile acid excretion in C57Bl/6 N mice fed for 12 weeks either a cholesterol-free high carbohydrate control diet or a high fat Western diet containing 0.03% (w/w) cholesterol. To identify the underlying mechanisms of dietary adaptations in intestine and liver, changes in gene expression were assessed by microarray and qPCR profiling, respectively.
Results
Mice on Western diet showed increased plasma cholesterol levels, associated with the higher dietary cholesterol supply, yet, significantly reduced cholesterol levels were found in intestine and liver. Transcript profiling revealed evidence that expression of numerous genes involved in cholesterol synthesis and uptake via LDL, but also in phospholipid metabolism, underwent compensatory regulations in both tissues. Alterations in glycerophospholipid metabolism were confirmed at the metabolite level by phospolipid profiling via mass spectrometry.
Conclusions
Our findings suggest that intestine and liver react to a high dietary fat intake by an activation of de novo cholesterol synthesis and other cholesterol-saving mechanisms, as well as with major changes in phospholipid metabolism, to accommodate to the fat load.
doi:10.1186/1471-2164-13-84
PMCID: PMC3319424  PMID: 22394543
17.  A Key Role for E-cadherin in Intestinal Homeostasis and Paneth Cell Maturation 
PLoS ONE  2010;5(12):e14325.
Background
E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease.
Methods and Findings
To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.
Conclusion
These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.
doi:10.1371/journal.pone.0014325
PMCID: PMC3001873  PMID: 21179475

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