Publications by authors named "Neil Smyth"

62 Publications

The duration of embryo culture after mouse IVF differentially affects cardiovascular and metabolic health in male offspring.

Hum Reprod 2020 11;35(11):2497-2514

School of Biological Sciences, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK.

Study Question: Do the long-term health outcomes following IVF differ depending upon the duration of embryo culture before transfer?

Summary Answer: Using a mouse model, we demonstrate that in male but not female offspring, adverse cardiovascular (CV) health was more likely with prolonged culture to the blastocyst stage, but metabolic dysfunction was more likely if embryo transfer (ET) occurred at the early cleavage stage.

What Is Known Already: ART associate with increased risk of adverse CV and metabolic health in offspring, and these findings have been confirmed in animal models in the absence of parental infertility issues. It is unclear which specific ART treatments may cause these risks. There is increasing use of blastocyst, versus cleavage-stage, transfer in clinical ART which does not appear to impair perinatal health of children born, but the longer-term health implications are unknown.

Study Design, Size, Duration: Five mouse groups were generated comprising: (i) natural mating (NM)-naturally mated, non-superovulated and undisturbed gestation; (ii) IV-ET-2Cell-in-vivo derived two-cell embryos collected from superovulated mothers, with immediate ET to recipients; (iii) IVF-ET-2Cell-IVF generated embryos, from oocytes from superovulated mothers, cultured to the two-cell stage before ET to recipients; (iv) IV-ET-BL-in-vivo derived blastocysts collected from superovulated mothers, with immediate ET to recipients; (v) IVF-ET-BL-IVF generated embryos, from oocytes from superovulated mothers, cultured to the blastocyst stage before ET to recipients. Both male and female offspring were analysed for growth, CV and metabolic markers of health. There were 8-13 litters generated for each group for analyses; postnatal data were analysed by multilevel random effects regression to take account of between-mother and within-mother variation and litter size.

Participants/materials, Settings, Methods: C57/BL6 female mice (3-4 weeks old) were used for oocyte production; CBA males for sperm with human tubal fluid medium were used for IVF. Embryos were transferred (ET) to MF1 pseudo-pregnant recipients at the two-cell stage or cultured in synthetic oviductal medium enriched with potassium medium to the blastocyst stage before ET. Control in-vivo embryos from C57BL6 × CBA matings were collected and immediately transferred at the two-cell or blastocyst stage. Postnatal assays included growth rate up to 27 weeks; systolic blood pressure (SBP) at 9, 15 and 21 weeks; lung and serum angiotensin-converting enzyme (ACE) activity at time of cull (27 weeks); glucose tolerance test (GTT; 27 weeks); basal glucose and insulin levels (27 weeks); and lipid accumulation in liver cryosections using Oil Red O imaging (27 weeks).

Main Results And The Role Of Chance: Blastocysts formed by IVF developed at a slower rate and comprised fewer cells that in-vivo generated blastocysts without culture (P < 0.05). Postnatal growth rate was increased in all four experimental treatments compared with NM group (P < 0.05). SBP, serum and lung ACE and heart/body weight were higher in IVF-ET-BL versus IVF-ET-2Cell males (P < 0.05) and higher than in other treatment groups, with SBP and lung ACE positively correlated (P < 0.05). Glucose handling (GTT AUC) was poorer and basal insulin levels were higher in IVF-ET-2Cell males than in IVF-ET-BL (P < 0.05) with the glucose:insulin ratio more negatively correlated with body weight in IVF-ET-2Cell males than in other groups. Liver/body weight and liver lipid droplet diameter and density in IVF-ET-2Cell males were higher than in IVF-ET-BL males (P < 0.05). IVF groups had poorer health characteristics than their in-vivo control groups, indicating that outcomes were not caused specifically by background techniques (superovulation, ET). No consistent health effects from duration of culture were identified in female offspring.

Large Scale Data: N/A.

Limitations, Reasons For Caution: Results from experimental animal models cannot be extrapolated to humans. Nevertheless, they are valuable to develop conceptual models, in this case, in the absence of confounding parental infertility, in assessing the safety of ART manipulations.

Wider Implications Of The Findings: The study indicates that longer duration of embryo culture after IVF up to blastocyst before ET leads to increased dysfunction of CV health in males compared with IVF and shorter cleavage-stage ET. However, the metabolic health of male offspring was poorer after shorter versus longer culture duration. This distinction indicates that the origin of CV and metabolic health phenotypes after ART may be different. The poorer metabolic health of males after cleavage-stage ET coincides with embryonic genome activation occurring at the time of ET.

Study Funding/competing Interest(s): This work was supported through the European Union FP7-CP-FP Epihealth programme (278418) and FP7-PEOPLE-2012-ITN EpiHealthNet programme (317146) to T.P.F., the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/F007450/1) to T.P.F., and the Saudi government, University of Jeddah and King Abdulaziz University to A.A. The authors have no conflicts of interest to declare.
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http://dx.doi.org/10.1093/humrep/deaa205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603862PMC
November 2020

Sensing of Vimentin mRNA in 2D and 3D Models of Wounded Skin Using DNA-Coated Gold Nanoparticles.

Small 2018 03 21;14(12):e1703489. Epub 2018 Feb 21.

Physics and Astronomy, Faculty of Physical Sciences and Engineering, University of Southampton, Southampton, SO17 1BJ, UK.

Wound healing is a highly complex biological process, which is accompanied by changes in cell phenotype, variations in protein expression, and the production of active biomolecules. Currently, the detection of proteins in cells is done by immunostaining where the proteins in fixed cells are detected by labeled antibodies. However, immunostaining cannot provide information about dynamic processes in living cells, within the whole tissue. Here, an easy method is presented to detect the transition of epithelial to mesenchymal cells during wound healing. The method employs DNA-coated gold nanoparticle fluorescent nanoprobes to sense the production of Vimentin mRNA expressed in mesenchymal cells. Fluorescence microscopy is used to achieve temporal detection of Vimentin mRNA in wounds. 3D light-sheet microscopy is utilized to observe the dynamic expression of Vimentin mRNA spatially around the wounded site in skin tissue. The use of DNA-gold nanoprobes to detect mRNA expression during wound healing opens up new possibilities for the study of real-time mechanisms in complex biological processes.
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http://dx.doi.org/10.1002/smll.201703489DOI Listing
March 2018

Transglutaminases in autoimmune and inherited skin diseases: The phenomena of epitope spreading and functional compensation.

Exp Dermatol 2018 08 26;27(8):807-814. Epub 2018 Feb 26.

Department of Dermatology, University of Münster, Münster, Germany.

Transglutaminases (TGs) are structurally and functionally related enzymes that modify the post-translational structure and activity of proteins or peptides, and thus are able to turn on or switch off their function. Depending on location and activities, TGs are able to modify the signalling, the function and the fate of cells and extracellular connective tissues. Besides mouse models, human diseases enable us to appreciate the function of various TGs. In this study, skin diseases induced by genetic damages or autoimmune targeting of these enzymes will be discussed. TG1, TG3 and TG5 contribute to the cutaneous barrier and thus to the integrity and function of epidermis. TGM1 mutations related to autosomal recessive ichthyosis subtypes, TGM5 mutations to a mild epidermolysis bullosa phenotype and as novelty TGM3 mutation to uncombable hair syndrome will be discussed. Autoimmunity to TG2, TG3 and TG6 may develop in a few of those genetically determined individuals who lost tolerance to gluten, and manifest as coeliac disease, dermatitis herpetiformis or gluten-dependent neurological symptoms, respectively. These gluten responder diseases commonly occur in combination. In autoimmune diseases, the epitope spreading is remarkable, while in some inherited pathologies, a unique compensation of the lost enzyme function is noted.
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http://dx.doi.org/10.1111/exd.13449DOI Listing
August 2018

Deposition of amyloid β in the walls of human leptomeningeal arteries in relation to perivascular drainage pathways in cerebral amyloid angiopathy.

Biochim Biophys Acta 2016 05 29;1862(5):1037-46. Epub 2015 Aug 29.

Faculty of Medicine, University of Southampton, Tremona Road, SO16 6YD, UK. Electronic address:

Deposition of amyloid β (Aβ) in the walls of cerebral arteries as cerebral amyloid angiopathy (CAA) suggests an age-related failure of perivascular drainage of soluble Aβ from the brain. As CAA is associated with Alzheimer's disease and with intracerebral haemorrhage, the present study determines the unique sequence of changes that occur as Aβ accumulates in artery walls. Paraffin sections of post-mortem human occipital cortex were immunostained for collagen IV, fibronectin, nidogen 2, Aβ and smooth muscle actin and the immunostaining was analysed using Image J and confocal microscopy. Results showed that nidogen 2 (entactin) increases with age and decreases in CAA. Confocal microscopy revealed stages in the progression of CAA: Aβ initially deposits in basement membranes in the tunica media, replaces first the smooth muscle cells and then the connective tissue elements to leave artery walls completely or focally replaced by Aβ. The pattern of development of CAA in the human brain suggests expansion of Aβ from the basement membranes to progressively replace all tissue elements in the artery wall. Establishing this full picture of the development of CAA is pivotal in understanding the clinical presentation of CAA and for developing therapies to prevent accumulation of Aβ in artery walls. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.
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http://dx.doi.org/10.1016/j.bbadis.2015.08.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4827375PMC
May 2016

Antagonistic human FcγRIIB (CD32B) antibodies have anti-tumor activity and overcome resistance to antibody therapy in vivo.

Cancer Cell 2015 Apr;27(4):473-88

Antibody & Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK. Electronic address:

Therapeutic antibodies have transformed cancer therapy, unlocking mechanisms of action by engaging the immune system. Unfortunately, cures rarely occur and patients display intrinsic or acquired resistance. Here, we demonstrate the therapeutic potential of targeting human (h) FcγRIIB (CD32B), a receptor implicated in immune cell desensitization and tumor cell resistance. FcγRIIB-blocking antibodies prevented internalization of the CD20-specific antibody rituximab, thereby maximizing cell surface accessibility and immune effector cell mediated antitumor activity. In hFcγRIIB-transgenic (Tg) mice, FcγRIIB-blocking antibodies effectively deleted target cells in combination with rituximab, and other therapeutic antibodies, from resistance-prone stromal compartments. Similar efficacy was seen in primary human tumor xenografts, including with cells from patients with relapsed/refractory disease. These data support the further development of hFcγRIIB antibodies for clinical assessment.
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http://dx.doi.org/10.1016/j.ccell.2015.03.005DOI Listing
April 2015

Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo's potential, affecting adult health.

Reprod Fertil Dev 2015 May;27(4):684-92

Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK.

Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences.
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http://dx.doi.org/10.1071/RD14455DOI Listing
May 2015

The effects of sarcolipin over-expression in mouse skeletal muscle on metabolic activity.

Arch Biochem Biophys 2015 Mar 7;569:26-31. Epub 2015 Feb 7.

Centre for Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK. Electronic address:

Studies in sarcolipin knockout mice have led to the suggestion that skeletal muscle sarcolipin plays a role in thermogenesis. The mechanism proposed is uncoupling of the sarcoplasmic reticulum calcium pump. However, in other work sarcolipin was not detected in mouse skeletal tissue. We have therefore measured sarcolipin levels in mouse skeletal muscle using semi-quantitative western blotting and synthetic mouse sarcolipin. Sarcolipin levels were so low that it is unlikely that knocking out sarcolipin would have a measurable effect on thermogenesis by SERCA. In addition, overexpression of neither wild type nor FLAG-tagged variants of mouse sarcolipin in transgenic mice had any major significant effects on body mass, energy expenditure, even when mice were fed on a high fat diet.
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http://dx.doi.org/10.1016/j.abb.2015.01.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362768PMC
March 2015

Epigenetic regulation of histone modifications and Gata6 gene expression induced by maternal diet in mouse embryoid bodies in a model of developmental programming.

BMC Dev Biol 2015 Jan 21;15. Epub 2015 Jan 21.

Centre for Biological Sciences, University of Southampton, Mailpoint 840, Level D Lab & Path Block, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK.

Background: Dietary interventions during pregnancy alter offspring fitness. We have shown mouse maternal low protein diet fed exclusively for the preimplantation period (Emb-LPD) before return to normal protein diet (NPD) for the rest of gestation, is sufficient to cause adult offspring cardiovascular and metabolic disease. Moreover, Emb-LPD blastocysts sense altered nutrition within the uterus and activate compensatory cellular responses including stimulated endocytosis within extra-embryonic trophectoderm and primitive endoderm (PE) lineages to protect fetal growth rate. However, these responses associate with later disease. Here, we investigate epigenetic mechanisms underlying nutritional programming of PE that may contribute to its altered phenotype, stabilised during subsequent development. We use embryonic stem (ES) cell lines established previously from Emb-LPD and NPD blastocysts that were differentiated into embryoid bodies (EBs) with outer PE-like layer.

Results: Emb-LPD EBs grow to a larger size than NPD EBs and express reduced Gata6 transcription factor (regulator of PE differentiation) at mRNA and protein levels, similar to Emb-LPD PE derivative visceral yolk sac tissue in vivo in later gestation. We analysed histone modifications at the Gata6 promoter in Emb-LPD EBs using chromatin immunoprecipitation assay. We found significant reduction in histone H3 and H4 acetylation and RNA polymerase II binding compared with NPD EBs, all markers of reduced transcription. Other histone modifications, H3K4Me2, H3K9Me3 and H3K27Me3, were unaltered. A similar but generally non-significant histone modification pattern was found on the Gata4 promoter. Consistent with these changes, histone deacetylase Hdac-1, but not Hdac-3, gene expression was upregulated in Emb-LPD EBs.

Conclusions: First, these data demonstrate ES cells and EBs retain and propagate nutritional programming adaptations in vitro, suitable for molecular analysis of mechanisms, reducing animal use. Second, they reveal maternal diet induces persistent changes in histone modifications to regulate Gata6 expression and PE growth and differentiation that may affect lifetime health.
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http://dx.doi.org/10.1186/s12861-015-0053-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305257PMC
January 2015

Interactions of skin with gold nanoparticles of different surface charge, shape, and functionality.

Small 2015 Feb 7;11(6):713-21. Epub 2014 Oct 7.

Institute of Life Sciences, Physics and Astronomy, Faculty of Applied and Physical Sciences, University of Southampton, Southampton, SO171BJ, UK.

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varying the charge, shape, and functionality of gold nanoparticles, the nanoparticle penetration through the different skin layers is assessed. The penetration is evaluated both qualitatively and quantitatively using a variety of complementary techniques. Inductively coupled plasma optical emission spectrometry (ICP-OES) is used to quantify the total number of particles which penetrate the skin structure. Transmission electron microscopy (TEM) and two photon photoluminescence microscopy (TPPL) on skin cross sections provide a direct visualization of nanoparticle migration within the different skin substructures. These studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R7 are found in the skin in larger quantities than polyethylene glycol-functionalized nanoparticles, and are able to enter deep into the skin structure. The systematic studies presented in this work may be of strong interest for developments in transdermal administration of drugs and therapy.
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http://dx.doi.org/10.1002/smll.201401913DOI Listing
February 2015

FHL2 regulates the resolution of tissue damage in chronic inflammatory arthritis.

Ann Rheum Dis 2015 Dec 14;74(12):2216-23. Epub 2014 Aug 14.

Institute of Experimental Musculoskeletal Medicine, University Hospital Muenster, Muenster, Germany.

Objective: We analysed the role of the adaptor molecule four-and-a-half Lin11, Isl-1 & Mec-3 (LIM) domain protein 2 (FHL2) in the activation of fibroblast-like synoviocytes in human rheumatoid arthritis (RA) and tumour necrosis factor α (TNFα)-dependent animal models of the disease.

Methods: Synovial tissues of patients with RA and osteoarthritis (OA) as well as hind paw sections from arthritic human TNFα transgenic (hTNFtg) mice and synovial fibroblasts from these were analysed. The effects of cytokines on the expression of FHL2 and disease-relevant matrixmetalloproteases (MMPs) were determined. Analyses of human tissue specimens from patients treated with anti-TNFα as well as anti-TNFα treatment of hTNFtg mice were performed to substantiate the TNFα effects on FHL2 levels. FHL2(-/-) mice and hTNFtg mice (with constitutive or inducible transgene expression) were crossbred to generate TNFα overexpressing FHL2-deficient animals. Signalling pathways were analysed in cells from these mice and in human cells after knock down of FHL2 by western blot.

Results: FHL2 levels were higher in RA than in OA and in hTNFtg than in wild-type mice. Surprisingly, while transforming growth factor (TGF)β-induced FHL2 expression, TNFα suppressed FHL2. In vivo, anti-TNFα treatment led to higher FHL2 levels both in RA patients and hTNFtg mice. The loss of FHL2 increased joint destruction in hTNFtg mice, which was accompanied by elevated MMP-13. In vitro, TNFα-mediated MMP-13 was significantly higher in FHL2(-/-) cells and after knock down of FHL2, which was caused by prolonged p38 MAPK activation.

Conclusions: These data suggest that FHL2 serves as a protective factor and that, rather than promoting the pathology, the upregulation of FHL2 in RA occurs in frame of a regenerative attempt.
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http://dx.doi.org/10.1136/annrheumdis-2013-205061DOI Listing
December 2015

Mouse early extra-embryonic lineages activate compensatory endocytosis in response to poor maternal nutrition.

Development 2014 Mar 6;141(5):1140-50. Epub 2014 Feb 6.

Centre for Biological Sciences, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UK.

Mammalian extra-embryonic lineages perform the crucial role of nutrient provision during gestation to support embryonic and fetal growth. These lineages derive from outer trophectoderm (TE) and internal primitive endoderm (PE) in the blastocyst and subsequently give rise to chorio-allantoic and visceral yolk sac placentae, respectively. We have shown maternal low protein diet exclusively during mouse preimplantation development (Emb-LPD) is sufficient to cause a compensatory increase in fetal and perinatal growth that correlates positively with increased adult-onset cardiovascular, metabolic and behavioural disease. Here, to investigate early mechanisms of compensatory nutrient provision, we assessed the influence of maternal Emb-LPD on endocytosis within extra-embryonic lineages using quantitative imaging and expression of markers and proteins involved. Blastocysts collected from Emb-LPD mothers within standard culture medium displayed enhanced TE endocytosis compared with embryos from control mothers with respect to the number and collective volume per cell of vesicles with endocytosed ligand and fluid and lysosomes, plus protein expression of megalin (Lrp2) LDL-family receptor. Endocytosis was also stimulated using similar criteria in the outer PE-like lineage of embryoid bodies formed from embryonic stem cell lines generated from Emb-LPD blastocysts. Using an in vitro model replicating the depleted amino acid (AA) composition found within the Emb-LPD uterine luminal fluid, we show TE endocytosis response is activated through reduced branched-chain AAs (leucine, isoleucine, valine). Moreover, activation appears mediated through RhoA GTPase signalling. Our data indicate early embryos regulate and stabilise endocytosis as a mechanism to compensate for poor maternal nutrient provision.
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http://dx.doi.org/10.1242/dev.103952DOI Listing
March 2014

Anti-angiogenic effect of the basement membrane protein nidogen-1 in a mouse model of choroidal neovascularization.

Exp Eye Res 2014 Jan 23;118:80-8. Epub 2013 Nov 23.

Department of Ophthalmology, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany.

In patients with age-related macular degeneration disruption of the integrity of the retinal pigment epithelium (RPE) and Bruch's membrane (BrM), precedes choroidal neovascularization (CNV). We investigated the role of the basement membrane (BM) proteins nidogen-1 and nidogen-2 for the development of experimental CNV. Laser-induced CNV was studied in Nid1(-/-) and Nid2(-/-) mice and wild type (WT) controls by fluorescein angiography, by immune histochemistry of flat-mounts or paraffin sections to analyze expression pattern of nidogen-1 and -2 and nidogen binding BM proteins, and by western blotting. The influence of VEGF and bFGF on the mRNA expression of nidogen-1 was studied in vitro. Nidogen-1 protein is present in the BM of the inner limiting membrane (ILM), the retinal capillaries, and the choroid/sclera and CNV. Nidogen-2 protein is also found in these BMs but with a weaker expression in the ILM. In the retina the absence of nidogen-1 does not influence the expression of nidogen-2 and vice versa and does not influence the expression of the BM components collagen IV, laminin γ1, and perlecan. In Nid1(-/-) mice, CNV lesions showed increased vessel leakage during angiography and the CNV area was larger than in WT or nidogen-2 deficient mice. Laser treatment led to up-regulation of nidogen-1 protein expression in the sclera/choroid of nidogen-2 deficient or WT mice. The treatment of HUVECs with VEGF leads to a reduced expression of nidogen-1 mRNA whereas its expression remained unchanged in RPE cells. In conclusion, nidogen-1 produced by the endothelial cells acts as a factor to help stabilizing the BM, thus preventing the sprouting of new vessels or the infiltration of endothelial cells. In this sense nidogen-1 is essential to provide an anti-angiogenic environment of differentiated vessels.
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http://dx.doi.org/10.1016/j.exer.2013.11.006DOI Listing
January 2014

Reduced inflammatory threshold indicates skin barrier defect in transglutaminase 3 knockout mice.

J Invest Dermatol 2014 Jan 24;134(1):105-111. Epub 2013 Jul 24.

Department of Dermatology, Venereology and Dermato-oncology, Semmelweis University, Budapest, Hungary. Electronic address:

Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.
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http://dx.doi.org/10.1038/jid.2013.307DOI Listing
January 2014

Epidermal transglutaminase (TGase 3) is required for proper hair development, but not the formation of the epidermal barrier.

PLoS One 2012 4;7(4):e34252. Epub 2012 Apr 4.

Center for Biochemistry, University of Cologne, Cologne, North Rhine-Westphalia, Germany.

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034252PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319564PMC
August 2012

A function for Rac1 in the terminal differentiation and pigmentation of hair.

J Cell Sci 2012 Feb 24;125(Pt 4):896-905. Epub 2012 Jan 24.

Department of Dermatology, University of Cologne, Kerpener Strasse 62, D-50924 Cologne, Germany.

The small GTPase Rac1 is ubiquitously expressed in proliferating and differentiating layers of the epidermis and hair follicles. Previously, Rac1 was shown to regulate stem cell behaviour in these compartments. We have asked whether Rac1 has, in addition, a specific, stem-cell-independent function in the regulation of terminal hair follicle differentiation. To address this, we have expressed a constitutively active mutant of Rac1, L61Rac1, only in the basal epidermal layer and outer root sheath of mice possessing an epidermis-specific deletion of endogenous Rac1, which experience severe hair loss. The resulting 'rescue' mice exhibited a hair coat throughout their lives. Therefore, expression of Rac1 activity in the keratin-14-positive compartment of the skin is sufficient for the formation of hair follicles and hair in normal quantities. The quality of hair formed in rescue mice was, however, not normal. Rescue mice showed a grey, dull hair coat, whereas that of wild-type and L61Rac1-transgenic mice was black and shiny. Hair analysis in rescue mice revealed altered structures of the hair shaft and the cuticle and disturbed organization of medulla cells and pigment distribution. Disorganization of medulla cells correlates with the absence of cortical, keratin-filled spikes that normally protrude from the cortex into the medulla. The desmosomal cadherin Dsc2, which normally decorates these protrusions, was found to be reduced or absent in the hair of rescue mice. Our study demonstrates regulatory functions for Rac1 in the formation of hair structure and pigmentation and thereby identifies, for the first time, a role for Rac1 in terminal differentiation.
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http://dx.doi.org/10.1242/jcs.091868DOI Listing
February 2012

Reducing expression of NAD+ synthesizing enzyme NMNAT1 does not affect the rate of Wallerian degeneration.

FEBS J 2011 Aug 14;278(15):2666-79. Epub 2011 Jun 14.

The Babraham Institute, Cambridge, UK.

NAD(+) synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein Wld(S), which delays axon degeneration by 10-fold. NMNAT1 activity is necessary but not sufficient for Wld(S) neuroprotection in mice and 70 amino acids at the N-terminus of Wld(S), derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro and partially in Drosophila. However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet. To address this question we disrupted the Nmnat1 locus by gene targeting. Homozygous Nmnat1 knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss. Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurodegeneration or axon pathology. Wallerian degeneration after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration.
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http://dx.doi.org/10.1111/j.1742-4658.2011.08193.xDOI Listing
August 2011

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain.

J Biol Chem 2011 Jan 17;286(3):1911-8. Epub 2010 Nov 17.

Department of Dermatology, University Hospital of Cologne, 50937 Cologne, Germany.

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.
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http://dx.doi.org/10.1074/jbc.M110.149864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023487PMC
January 2011

Computational modeling of laminin N-terminal domains using sparse distance constraints from disulfide bonds and chemical cross-linking.

Proteins 2010 Dec 11;78(16):3409-27. Epub 2010 Oct 11.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

Basement membranes are thin extracellular protein layers, which separate endothelial and epithelial cells from the underlying connecting tissue. The main noncollagenous components of basement membranes are laminins, trimeric glycoproteins, which form polymeric networks by interactions of their N-terminal (LN) domains; however, no high-resolution structure of laminin LN domains exists so far. To construct models for laminin β(1) and γ(1) LN domains, 14 potentially suited template structures were determined using fold recognition methods. For each target/template-combination comparative models were created with Rosetta. Final models were selected based on their agreement with experimentally obtained distance constraints from natural cross-links, that is, disulfide bonds as well as chemical cross-links obtained from reactions with two amine-reactive cross-linkers. We predict that laminin β(1) and γ(1) LN domains share the galactose-binding domain-like fold.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079110PMC
http://dx.doi.org/10.1002/prot.22848DOI Listing
December 2010

A novel disulfide pattern in laminin-type epidermal growth factor-like (LE) modules of laminin β1 and γ1 chains.

Biochemistry 2010 Sep;49(38):8359-66

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, D-06120 Halle (Saale), Germany.

In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin β1 and γ1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal β1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin γ1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns.
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http://dx.doi.org/10.1021/bi101187fDOI Listing
September 2010

The cystatin M/E-cathepsin L balance is essential for tissue homeostasis in epidermis, hair follicles, and cornea.

FASEB J 2010 Oct 21;24(10):3744-55. Epub 2010 May 21.

Department of Dermatology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Cystatin M/E (CST6) is a nonredundant, epithelium-specific protease inhibitor with a presumed role in epidermal differentiation and tumor suppression. We have previously reported that cystatin M/E deficiency in Cst6(-/-) mice causes neonatal lethality because of excessive transepidermal water loss. Biochemical evidence suggests that cystatin M/E controls the activity of legumain, cathepsin L, cathepsin V, and transglutaminase-3. Using a genetic approach we sought to define the role of cystatin M/E in epithelial biology by identification of its target proteases and their downstream functions. Ablation of cathepsin L in a Cst6(-/-) background (Cst6(-/-)Ctsl(-/-) double-knockout mice) restored viability and resulted in normalization of stratum corneum morphology. Ablation of legumain or transglutaminase-3 in Cst6(-/-) mice, however, did not rescue the lethal phenotype. Intriguingly, both Cst6(-/-)Ctsl(-/-) and Cst6(-/-)Ctsl(+/-) mice were viable, but the absence of cystatin M/E caused scarring alopecia in adult animals. In the cornea of Cst6(-/-)Ctsl(+/-) mice, we observed keratitis, hyperplasia, and transition to a cornified epithelium. Evidence is provided that activation of cathepsin D and transglutaminase-1 are downstream events, dependent of cathepsin L activity. We conclude that a tightly regulated balance between cathepsin L and cystatin M/E is essential for tissue integrity in epidermis, hair follicles, and corneal epithelium.
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http://dx.doi.org/10.1096/fj.10-155879DOI Listing
October 2010

Impaired wound healing in mice lacking the basement membrane protein nidogen 1.

Matrix Biol 2010 Jan 18;29(1):15-21. Epub 2009 Sep 18.

Department of Dermatology, University Hospital Cologne, Cologne, Germany.

Nidogens 1 and 2 are ubiquitous basement membrane (BM) components, whose interactions in particular with laminin, collagen IV and perlecan have been considered important for BM formation. Genetic deletion of either NID gene does not reveal BM alterations suggesting compensatory roles for nidogens 1 and 2. However, neurological deficits in nidogen 1 null mice, not seen in the absence of nidogen 2, also suggest isoform specific functions. To test this further, skin wound healing which requires BM reformation was studied in adult nidogen 1 deficient mice. Although re-epithelialization was not altered, the newly formed epidermis showed marked hyperproliferation and a delay in differentiation at day 10 post injury. Distinct to control wounds, there was also considerable alpha-smooth muscle actin staining in the dermis of nidogen 1 deficient wounds at this time point. Further, laminin deposition and distribution of the beta1 and beta4 integrin chains were also significantly altered whereas the deposition of other BM components, including nidogen 2, was unchanged. Surprisingly, these differences between control and mutant wounds at day 10 post wounding did not affect the ultrastructural appearance of the dermo-epidermal BM suggesting a non-structural role for nidogen 1 in wound repair.
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http://dx.doi.org/10.1016/j.matbio.2009.09.004DOI Listing
January 2010

Basement membrane protein nidogen-1 shapes hippocampal synaptic plasticity and excitability.

Hippocampus 2010 May;20(5):608-20

Center for Biochemistry and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Köln, Germany.

The basement membrane (BM) is a specialized form of extracellular matrix (ECM) underlying epithelia and endothelia and surrounding many types of mesenchymal cells. Nidogen, along with collagen IV and laminin, is a major component of BMs. Although certain ECM proteins such as laminin or reelin influence neuronal function via interactions with cell-surface receptors such as integrins, behavioral neurological impairments due to deficits of BM components have been recognized only recently. Here, alterations in neuronal network function underlying these behavioral changes are revealed. Using nidogen-1 knockout mice, with or without additional heterozygous nidogen-2 knockout (NID1(-/-)/NID2(+/+) or NID1(-/-)/NID2(+/-)), we demonstrate that nidogen is essential for normal neuronal network excitability and plasticity. In nidogen-1 knockouts, seizurelike behavior occurs, and epileptiform spiking was seen in hippocampal in vivo EEG recordings. In vitro, hippocampal field potential recordings revealed that lack of nidogen-1, while not causing conspicuous morphological changes, led to the appearance of spontaneous and evoked epileptiform activity, significant increase of the input/output ratio of synaptically evoked responses in CA1 and dentate gyrus, as well as of paired pulse accentuation, and loss of perforant-path long-term synaptic potentiation. Nidogen-1 is thus essential for normal network excitability and plasticity.
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http://dx.doi.org/10.1002/hipo.20660DOI Listing
May 2010

Plastin 1 binds to keratin and is required for terminal web assembly in the intestinal epithelium.

Mol Biol Cell 2009 May 25;20(10):2549-62. Epub 2009 Mar 25.

Center for Biochemistry, Medical Faculty, University of Cologne, D-50931 Cologne, Germany.

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.
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http://dx.doi.org/10.1091/mbc.e08-10-1030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682596PMC
May 2009

PLRG1 is an essential regulator of cell proliferation and apoptosis during vertebrate development and tissue homeostasis.

Mol Cell Biol 2009 Jun 23;29(11):3173-85. Epub 2009 Mar 23.

Institute for Genetics, Department of Mouse Genetics and Metabolism, University of Cologne, Cologne, Germany.

PLRG1, an evolutionarily conserved component of the spliceosome, forms a complex with Pso4/SNEV/Prp19 and the cell division and cycle 5 homolog (CDC5L) that is involved in both pre-mRNA splicing and DNA repair. Here, we show that the inactivation of PLRG1 in mice results in embryonic lethality at 1.5 days postfertilization. Studies of heart- and neuron-specific PLRG1 knockout mice further reveal an essential role of PLRG1 in adult tissue homeostasis and the suppression of apoptosis. PLRG1-deficient mouse embryonic fibroblasts (MEFs) fail to progress through S phase upon serum stimulation and exhibit increased rates of apoptosis. PLRG1 deficiency causes enhanced p53 phosphorylation and stabilization in the presence of increased gamma-H2AX immunoreactivity as an indicator of an activated DNA damage response. p53 downregulation rescues lethality in both PLRG1-deficient MEFs and zebrafish in vivo, showing that apoptosis resulting from PLRG1 deficiency is p53 dependent. Moreover, the deletion of PLRG1 results in the relocation of its interaction partner CDC5L from the nucleus to the cytoplasm without general alterations in pre-mRNA splicing. Taken together, the results of this study identify PLRG1 as a critical nuclear regulator of p53-dependent cell cycle progression and apoptosis during both embryonic development and adult tissue homeostasis.
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http://dx.doi.org/10.1128/MCB.01807-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682009PMC
June 2009

Eukaryotic expression and purification of recombinant extracellular matrix proteins carrying the strep II tag.

Methods Mol Biol 2009 ;522:63-72

University of Cologne, Cologne, Germany.

For recombinant expression of extracellular matrix (ECM) proteins or their individual domains, the use of transformed mammalian cells offers two major advantages. First, eukaryotic expression can be expected under optimum conditions to produce a large proportion of correctly folded molecules. ECM proteins are made from a group of 25 structurally known (Rev. Biophys. 29:119-167, 1996) and about 200 cDNA derived domains many of which regularly reappear in the different proteins. These have often a complex secondary structure, maintained by multiple disulfide bonds. Whereas by denaturing and then carefully renaturing, an approximation to the native structure may be obtained using prokaryotic expression systems, and the best that may be expected is that a small percentage of the protein folds into such a conformation. Second, most ECM proteins are at least to some extent glycosylated and often heavily so, and the use of the mammalian system offers the best approximation to the sugar structures present in the native form of the molecule.
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http://dx.doi.org/10.1007/978-1-59745-413-1_4DOI Listing
April 2009

Lack of laminin gamma1 in embryonic stem cell-derived cardiomyocytes causes inhomogeneous electrical spreading despite intact differentiation and function.

Stem Cells 2009 Jan;27(1):88-99

Institute of Physiology I, Life and Brain Center, University of Bonn, Germany.

Laminins form a large family of extracellular matrix (ECM) proteins, and their expression is a prerequisite for normal embryonic development. Herein we investigated the role of the laminin gamma1 chain for cardiac muscle differentiation and function using cardiomyocytes derived from embryonic stem cells deficient in the LAMC1 gene. Laminin gamma1 (-/-) cardiomyocytes lacked basement membranes (BM), whereas their sarcomeric organization was unaffected. Accordingly, electrical activity and hormonal regulation were found to be intact. However, the inadequate BM formation led to an increase of ECM deposits between adjacent cardiomyocytes, and this resulted in defects of the electrical signal propagation. Furthermore, we also found an increase in the number of pacemaker areas. Thus, although laminin and intact BM are not essential for cardiomyocyte development and differentiation per se, they are required for the normal deposition of matrix molecules and critical for intact electrical signal propagation.
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http://dx.doi.org/10.1634/stemcells.2008-0335DOI Listing
January 2009

A synaptic nidogen: developmental regulation and role of nidogen-2 at the neuromuscular junction.

Neural Dev 2008 Sep 25;3:24. Epub 2008 Sep 25.

Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA 02138, USA.

Background: The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.

Results: In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.

Conclusion: All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.
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http://dx.doi.org/10.1186/1749-8104-3-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567315PMC
September 2008

Determination of disulfide bond patterns in laminin beta1 chain N-terminal domains by nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry.

Rapid Commun Mass Spectrom 2008 Jun;22(12):1933-40

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle, Germany.

The disulfide bonding patterns in the N-terminal (LN) domains of the basement membrane protein laminin beta1 have not been investigated so far. We report an in-depth mass spectrometric analysis using offline nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) for determining the disulfide bond patterns in the LN-domain of recombinant mouse laminin beta1 chain for the first time. Mass spectra were recorded and the putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide-linked peptides for characteristic 66-amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin beta1 chain N-terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN-domain of the laminin beta1 chain was observed with a (1-2, 3-4, 5-6, 7-8) connectivity of cysteines. The identical disulfide-bonding pattern was found in E4, the N-terminal laminin beta1 chain fragment derived by elastase digestion of mouse tumor laminin-111, confirming that this pattern also occurs in native laminin.
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http://dx.doi.org/10.1002/rcm.3576DOI Listing
June 2008

Basement membranes in skin are differently affected by lack of nidogen 1 and 2.

J Invest Dermatol 2008 Sep 20;128(9):2259-67. Epub 2008 Mar 20.

Department of Dermatology, University of Cologne, Cologne, Germany.

Nidogens have been proposed to play a key role in basement membrane (BM) formation. However, recent findings using genetic approaches and organotypic coculture models demonstrated distinct tissue requirements thus changing the classical view of BM assembly. Toward this end, we have analyzed the dermo-epidermal junction and the microvasculature in skin of nidogen-deficient mice for their BM composition and structural assembly. Histology of nidogen double-null embryos at embryonic day (E)18.5 revealed overall normal skin morphology with a regularly differentiated epidermis. However, in the dermis, numerous erythrocytes had extravasated out of the microvasculature. Residual composition and ultrastructure of the dermo-epidermal BM are not altered in the absence of nidogens, demonstrating that the deposition of laminin, collagen IV, and perlecan occurs and allows cutaneous BM formation. In contrast, in capillaries, BM formation is severely impaired in the absence of nidogens, showing an irregular, patchy distribution and a dramatically reduced deposition of collagen IV, perlecan, and particularly laminin-411. Ultrastructure revealed thin fragile walls in the small blood vessels next to the epidermis, completely lacking a distinct endothelial BM. In summary, our results indicate that in skin the laminin composition of the various BMs determines whether nidogens are required for their assembly and stabilization.
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http://dx.doi.org/10.1038/jid.2008.65DOI Listing
September 2008

Nidogens-Extracellular matrix linker molecules.

Microsc Res Tech 2008 May;71(5):387-95

Center for Biochemistry and Center for Molecular Medicine, Medical Faculty, University of Cologne, D-50924 Cologne, Germany.

Nidogens/entactins are a family of highly conserved, sulfated glycoproteins. Biochemical studies have implicated them as having a major structural role in the basement membrane. However despite being ubiquitous components of this specialized extracellular matrix and having a wide spectrum of binding partners, genetic analysis has shown that they are not required for the overall architecture of the basement membrane. Rather in development they play an important role in its stabilization especially in tissues undergoing rapid growth or turnover. Nidogen breakdown has been implicated as a key event in the basement membrane degradation occurring in mammary gland involution. A number of studies, most compellingly those in C. elegans, demonstrated that nidogens may have other nonstructural roles and be involved in axonal pathfinding and synaptic transmission.
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http://dx.doi.org/10.1002/jemt.20567DOI Listing
May 2008